U.S. patent application number 15/838066 was filed with the patent office on 2018-04-12 for compositions and methods for managing weight.
This patent application is currently assigned to Unigen, Inc.. The applicant listed for this patent is Unigen, Inc., Unigen, Inc.. Invention is credited to Lidia Alfaro Brownell, Byong-II Choi, Brandon Corneliusen, Mei-Feng Hong, Ji-Hye Hwang, Eu-Jin Hyun, QI Jia, Ping Jiao, Hyun-Jin Kim, Mi-Ran Kim, Tae-Woo Kim, Bo-Su Lee, Young-Chul Lee, Jeong-Bum Nam, Mi-Sun Oh, Mesfin Yimam.
Application Number | 20180099019 15/838066 |
Document ID | / |
Family ID | 49674047 |
Filed Date | 2018-04-12 |
United States Patent
Application |
20180099019 |
Kind Code |
A1 |
Brownell; Lidia Alfaro ; et
al. |
April 12, 2018 |
Compositions and Methods for Managing Weight
Abstract
The present disclosure provides Diels-Alder adducts of chalcone
and prenylphenyl moieties capable of modulating the activity of
cannabinoid receptors, and to oligomers of flavan-3-ol capable of
modulating fat absorption and storage. Such Diels-Alder adducts of
chalcone and prenylphenyl moieties or oligomers of flavan-3-ol can
optionally be used in combination with other weight management
agents, such as anorectic agents, a lipase inhibitors, other
cannabinoid receptor modulators, psychotropic agents, insulin
sensitizers, stimulants, or satiety agents, as well as to methods
of use thereof such as treating or preventing weight gain or
obesity, promoting weight loss, appetite suppression, modifying
satiety, or the like.
Inventors: |
Brownell; Lidia Alfaro;
(Tacoma, WA) ; Choi; Byong-II; (Cheongwon-gun,
KR) ; Corneliusen; Brandon; (Tenino, WA) ;
Hong; Mei-Feng; (Lacey, WA) ; Hyun; Eu-Jin;
(Cheonan-si, KR) ; Jia; QI; (Olympia, WA) ;
Jiao; Ping; (Lacey, WA) ; Kim; Hyun-Jin;
(Asan-si, KR) ; Kim; Mi-Ran; (Cheonan-si, KR)
; Kim; Tae-Woo; (Ulsan, KR) ; Lee; Bo-Su;
(Pohang-si, KR) ; Lee; Young-Chul; (Daejeon,
KR) ; Nam; Jeong-Bum; (Cheongwon-gun, KR) ;
Yimam; Mesfin; (Kent, WA) ; Hwang; Ji-Hye;
(Jecheon-si, KR) ; Oh; Mi-Sun; (Cheonan-si,
KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Unigen, Inc.
Unigen, Inc. |
Seattle
Cheonan-si |
WA |
US
KR |
|
|
Assignee: |
Unigen, Inc.
Seattle
WA
Unigen, Inc.
Cheonan-si
|
Family ID: |
49674047 |
Appl. No.: |
15/838066 |
Filed: |
December 11, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13904851 |
May 29, 2013 |
9844576 |
|
|
15838066 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 36/752 20130101;
A61K 36/67 20130101; A61K 36/346 20130101; A61K 36/38 20130101;
A61K 36/575 20130101; A61K 36/37 20130101; A61K 36/605 20130101;
A61P 25/00 20180101; A61K 36/56 20130101; A61K 31/12 20130101; A61K
36/889 20130101; A61K 31/343 20130101; A61P 3/04 20180101; A61K
36/10 20130101; A61K 45/06 20130101; A61K 36/53 20130101; A61P 3/06
20180101; A61K 31/352 20130101; A61K 36/185 20130101; A61K 36/258
20130101; A61K 31/352 20130101; A61K 2300/00 20130101; A61K 31/343
20130101; A61K 2300/00 20130101; A61K 31/12 20130101; A61K 2300/00
20130101 |
International
Class: |
A61K 36/575 20060101
A61K036/575; A61K 36/889 20060101 A61K036/889; A61K 45/06 20060101
A61K045/06; A61K 31/12 20060101 A61K031/12; A61K 31/343 20060101
A61K031/343; A61K 31/352 20060101 A61K031/352; A61K 36/10 20060101
A61K036/10; A61K 36/185 20060101 A61K036/185; A61K 36/258 20060101
A61K036/258; A61K 36/346 20060101 A61K036/346; A61K 36/37 20060101
A61K036/37; A61K 36/38 20060101 A61K036/38; A61K 36/53 20060101
A61K036/53; A61K 36/56 20060101 A61K036/56; A61K 36/605 20060101
A61K036/605; A61K 36/67 20060101 A61K036/67; A61K 36/752 20060101
A61K036/752 |
Claims
1. A composition, comprising a mixture of a Morus extract enriched
for one or more Diels-Alder adducts of a chalcone and a
prenylphenyl moiety, a Magnolia extract, and a Yerba Mate
extract.
2. The composition according to claim 1, wherein the Morus extract
is from Morus alba, the Magnolia extract is from Magnolia
officinalis, and the Yerba Mate extract is from Ilex
paraguayensis.
3. The composition according to claim 1, wherein the one or more
Diels-Alder adducts of a chalcone and a prenylphenyl moiety are
compounds having a structure of Formula Ib or IIb: ##STR00127## or
a pharmaceutically or nutraceutically acceptable salt, tautomer,
glycoside, prodrug or stereoisomer thereof, wherein: R.sup.4a,
R.sup.5a, R.sup.6a and R.sup.7a are each independently H, hydroxyl,
halogen, sulfhydryl, amino, aldehyde, C.sub.1-12 alkyl, C.sub.1-12
alkoxy, C.sub.1-12 alkthio, C.sub.1-12 alkyamino, aryl, heteroaryl,
aralkyl, alkyl carbonyl, aralkylcarbonyl or one of R.sup.4a,
R.sup.5a, R.sup.6a or R.sup.7a joins with another of R.sup.4a,
R.sup.5a, R.sup.6a or R.sup.7a to form a heterocyclic or
heteroaromatic; R.sup.4b, R.sup.5b, R.sup.6b and R.sup.7b are each
independently H, hydroxyl, halogen, sulfhydryl, amino, aldehyde,
C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.1-12 alkthio, C.sub.1-12
alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl,
aralkylcarbonyl or one of R.sup.4b, R.sup.5b, R.sup.6b, R.sup.7b
joins with another of R.sup.4b, R.sup.5b, R.sup.6b, R.sup.7b to
form a heterocyclic or heteroaromatic ring; R.sup.8a, R.sup.8b,
R.sup.9a, R.sup.9b, R.sup.10a, R.sup.10b, R.sup.11a and R.sup.11b
are each independently H, hydroxyl, halogen, sulfhydryl, amino,
aldehyde, C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.1-12 alkthio,
C.sub.1-12 alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl,
aralkylcarbonyl or one of R.sup.8a, R.sup.9a, R.sup.10a or
R.sup.11a or one of R.sup.8b, R.sup.9b, R.sup.10b or R.sup.11b
joins with another of R.sup.8a, R.sup.9a, R.sup.10a or R.sup.11a or
another one of R.sup.8b, R.sup.9b, R.sup.10b or R.sup.11b,
respectively, to form a heterocyclic or heteroaromatic ring;
R.sup.12a, R.sup.13a, R.sup.14a and R.sup.15a are each
independently H, hydroxyl, halogen, sulfhydryl, amino, aldehyde,
C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.1-12 alkthio, C.sub.1-12
alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl,
aralkylcarbonyl or one of R.sup.12a, R.sup.13a, R.sup.14a,
R.sup.15a joins with another of R.sup.12a, R.sup.13a, R.sup.14a or
R.sup.15a to form a heterocyclic or heteroaromatic ring; and
R.sup.12b, R.sup.13b, R.sup.14b and R.sup.15b are each
independently H, hydroxyl, halogen, sulfhydryl, amino, aldehyde,
C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.1-12 alkthio, C.sub.1-12
alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl,
aralkylcarbonyl or one of R.sup.12b, R.sup.13b, R.sup.14b,
R.sup.15b joins with another of R.sup.12b, R.sup.13b, R.sup.14b or
R.sup.15b to form a heterocyclic or heteroaromatic ring.
4. The composition of claim 1, wherein at least two of R.sup.4a,
R.sup.4b, R.sup.5a, R.sup.5b, R.sup.6a, R.sup.6b, R.sup.7a,
R.sup.7b, R.sup.8a, R.sup.8b, R.sup.9a, R.sup.9b, R.sup.10a,
R.sup.10b, R.sup.11a, R.sup.11b, R.sup.12a, R.sup.12b, R.sup.13a,
R.sup.13b, R.sup.14a, R.sup.14b, R.sup.15a and R.sup.15b are
hydroxyl.
5. The composition according to claim 1, wherein the Morus extract
is enriched for Albanin G, Kuwanon G, Morusin, or any combination
thereof.
6. The composition according to claim 1, wherein the Magnolia
extract is enriched for magnolol, honokiol, or both.
7. The composition according to claim 1, wherein the Yerba Mate
extract is enriched for caffeine, dicaffeoylquinic acid, or
both.
8. The composition according to claim 1, wherein the composition
further comprises a pharmaceutically or nutraceutically acceptable
carrier, diluent, or excipient, wherein the pharmaceutical or
nutraceutical formulation comprises from about 0.5 weight percent
(wt %) to about 90 wt % of active ingredients of the extract
mixture.
9. The composition according to claim 8, wherein the composition is
formulated as a tablet, capsule, powder, or granule.
10. A composition, comprising a mixture of a Morus extract enriched
for Diels-Alder adducts of a chalcone and a prenylphenyl moiety, a
Magnolia extract, and a Mutamba extract.
11. The composition according to claim 10, wherein the Morus
extract is from Morus alba, the Magnolia extract is from Magnolia
officinalis, and the Mutamba extract is from Guazuma ulmifolia.
12. The composition according to claim 10, wherein the one or more
Diels-Alder adducts of a chalcone and a prenylphenyl moiety are
compounds having a structure of Formula Ib or IIb: ##STR00128## or
a pharmaceutically or nutraceutically acceptable salt, tautomer,
glycoside, prodrug or stereoisomer thereof, wherein: R.sup.4a,
R.sup.5a, R.sup.6a and R.sup.7a are each independently H, hydroxyl,
halogen, sulfhydryl, amino, aldehyde, C.sub.1-12 alkyl,
C.sub.1-alkoxy, C.sub.1-12 alkthio, C.sub.1-12 alkyamino, aryl,
heteroaryl, aralkyl, alkyl carbonyl, aralkylcarbonyl or one of
R.sup.4a, R.sup.5a, R.sup.6a or R.sup.7a joins with another of
R.sup.4a, R.sup.5a, R.sup.6a or R.sup.7a to form a heterocyclic or
heteroaromatic; R.sup.4b, R.sup.5b, R.sup.6b and R.sup.7b are each
independently H, hydroxyl, halogen, sulfhydryl, amino, aldehyde,
C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.1-12 alkthio, C.sub.1-12
alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl,
aralkylcarbonyl or one of R.sup.4b, R.sup.5b, R.sup.6b, R.sup.7b
joins with another of R.sup.4b, R.sup.5b, R.sup.6b, R.sup.7b to
form a heterocyclic or heteroaromatic ring; R.sup.8a, R.sup.8b,
R.sup.9a, R.sup.9b, R.sup.10a, R.sup.10b, R.sup.11a and R.sup.11b
are each independently H, hydroxyl, halogen, sulfhydryl, amino,
aldehyde, C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.1-12 alkthio,
C.sub.1-12 alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl,
aralkylcarbonyl or one of R.sup.8a, R.sup.9a, R.sup.10a or
R.sup.11a or one of R.sup.8b, R.sup.9b, R.sup.10b or R.sup.11b
joins with another of R.sup.8a, R.sup.9a, R.sup.10a or R.sup.11a or
another one of R.sup.8b, R.sup.9b, R.sup.10b or R.sup.11b,
respectively, to form a heterocyclic or heteroaromatic ring;
R.sup.12a, R.sup.13a, R.sup.14a and R.sup.15a are each
independently H, hydroxyl, halogen, sulfhydryl, amino, aldehyde,
C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.1-12 alkthio, C.sub.1-12
alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl,
aralkylcarbonyl or one of R.sup.12a, R.sup.13a, R.sup.14a,
R.sup.15a joins with another of R.sup.12a, R.sup.13a, R.sup.14a or
R.sup.15a to form a heterocyclic or heteroaromatic ring; and
R.sup.12b, R.sup.13b, R.sup.14b and R.sup.15b are each
independently H, hydroxyl, halogen, sulfhydryl, amino, aldehyde,
C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.1-12 alkthio, C.sub.1-12
alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl,
aralkylcarbonyl or one of R.sup.12b, R.sup.13b, R.sup.14b,
R.sup.15b joins with another of R.sup.12b, R.sup.13b, R.sup.14b or
R.sup.15b to form a heterocyclic or heteroaromatic ring.
13. The composition of claim 10, wherein at least two of R.sup.4a,
R.sup.4b, R.sup.5a, R.sup.5b, R.sup.6a, R.sup.6b, R.sup.7a,
R.sup.7b, R.sup.8a, R.sup.8b, R.sup.9a, R.sup.9b, R.sup.10a,
R.sup.10b, R.sup.11a, R.sup.11b, R.sup.12a, R.sup.12b, R.sup.13a,
R.sup.13b, R.sup.14a, R.sup.14b, R.sup.15a and R.sup.15b are
hydroxyl.
14. The composition according to claim 10, wherein the Morus
extract is enriched for Albanin G, Kuwanon G, Morusin, or any
combination thereof.
15. The composition according to claim 10, wherein the Magnolia
extract is enriched for magnolol, honokiol, or both.
16. The composition according to claim 10, wherein the Mutamba
extract comprises one or more oligomers comprising from two to
thirty subunits, wherein the subunits have, at each occurrence,
independently the following structure (I): ##STR00129## or a
pharmaceutically acceptable salt, stereoisomer or tautomer thereof,
wherein: R.sup.1a and R.sup.1h are, at each occurrence,
independently H, hydroxyl, halo, a gallic acid ester, a glycoside,
sulfhydryl, amino, aldehyde, C.sub.1-12 alkyl, C.sub.1-12 alkoxy,
C.sub.1-12 alkthio, C.sub.1-12 alkyamino, aryl, heteroaryl,
aralkyl, alkyl carbonyl, aralkylcarbonyl, or a direct bond to an
adjacent subunit; R.sup.2 is, at each occurrence, independently H
or an ether bond to an adjacent subunit; R.sup.3 is, at each
occurrence, independently H or a direct bond to an adjacent
subunit; R.sup.4 is, at each occurrence, OH or an ether bond to an
adjacent subunit; and R.sup.5a, R.sup.5b, R.sup.5c, R.sup.5d and
R.sup.5e are, at each occurrence, independently H, hydroxyl, halo,
a gallic acid ester, a glycoside, sulfhydryl, amino, aldehyde,
C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.1-12 alkthio, C.sub.1-12
alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl or
aralkylcarbonyl, wherein at least one of R.sup.1a, R.sup.1b,
R.sup.2, R.sup.3 or R.sup.4 is a direct bond or ether bond to an
adjacent subunit.
17. The composition according to claim 10, wherein the Mutamba
extract is enriched for procyanidins, procyanidin dimers,
procyanidin trimers, procyanidin tetramers, procyanidin pentamers,
procyanidin hexamers, condensed tannins, oligomers of catechin or
epicatechin, epicatechin, or any combination thereof.
18. The composition according to claim 10, wherein the composition
further comprises a pharmaceutically or nutraceutically acceptable
carrier, diluent, or excipient, wherein the pharmaceutical or
nutraceutical formulation comprises from about 0.5 weight percent
(wt %) to about 90 wt % of active ingredients of the extract
mixture.
19. The composition according to claim 18, wherein the composition
is formulated as a tablet, capsule, powder, or granule.
20. A composition, comprising a mixture of a Morus extract enriched
for Diels-Alder adducts of a chalcone and a prenylphenyl moiety, a
Rosemary extract, and a Yerba Mate extract.
21. The composition according to claim 20, wherein the Morus
extract is from Morus alba, the Rosemary extract is from Rosmarinus
officinalis, and the Yerba Mate extract is from Ilex
paraguayensis.
22. The composition according to claim 20, wherein the one or more
Diels-Alder adducts of a chalcone and a prenylphenyl moiety are
compounds having a structure of Formula Ib or IIb: ##STR00130## or
a pharmaceutically or nutraceutically acceptable salt, tautomer,
glycoside, prodrug or stereoisomer thereof, wherein: R.sup.4a,
R.sup.5a, R.sup.6a and R.sup.7a are each independently H, hydroxyl,
halogen, sulfhydryl, amino, aldehyde, C.sub.1-12 alkyl, C.sub.1-12
alkoxy, C.sub.1-12 alkthio, C.sub.1-12 alkyamino, aryl, heteroaryl,
aralkyl, alkyl carbonyl, aralkylcarbonyl or one of R.sup.4a,
R.sup.5a, R.sup.6a or R.sup.7a joins with another of R.sup.4a,
R.sup.5a, R.sup.6a or R.sup.7a to form a heterocyclic or
heteroaromatic; R.sup.4b, R.sup.5b, R.sup.6b and R.sup.7b are each
independently H, hydroxyl, halogen, sulfhydryl, amino, aldehyde,
C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.1-12 alkthio, C.sub.1-12
alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl,
aralkylcarbonyl or one of R.sup.4b, R.sup.5b, R.sup.6b, R.sup.7b
joins with another of R.sup.4b, R.sup.5b, R.sup.6b, R.sup.7b to
form a heterocyclic or heteroaromatic ring; R.sup.8a, R.sup.8b,
R.sup.9a, R.sup.9b, R.sup.10a, R.sup.10b, R.sup.11a and R.sup.11b
are each independently H, hydroxyl, halogen, sulfhydryl, amino,
aldehyde, C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.1-12 alkthio,
C.sub.1-12 alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl,
aralkylcarbonyl or one of R.sup.8a, R.sup.9a, R.sup.10a or
R.sup.11a or one of R.sup.8b, R.sup.9b, R.sup.10b or R.sup.11b
joins with another of R.sup.8a, R.sup.9a, R.sup.10a or R.sup.11a or
another one of R.sup.8b, R.sup.9b, R.sup.10b or R.sup.11b,
respectively, to form a heterocyclic or heteroaromatic ring;
R.sup.12a, R.sup.13a, R.sup.14a and R.sup.15a are each
independently H, hydroxyl, halogen, sulfhydryl, amino, aldehyde,
C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.1-12 alkthio, C.sub.1-12
alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl,
aralkylcarbonyl or one of R.sup.12a, R.sup.13a, R.sup.14a,
R.sup.15a joins with another of R.sup.12a, R.sup.13a, R.sup.14a or
R.sup.15a to form a heterocyclic or heteroaromatic ring; and
R.sup.12b, R.sup.13b, R.sup.14b and R.sup.15b are each
independently H, hydroxyl, halogen, sulfhydryl, amino, aldehyde,
C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.1-12 alkthio, C.sub.1-12
alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl,
aralkylcarbonyl or one of R.sup.12b, R.sup.13b, R.sup.14b,
R.sup.15b joins with another of R.sup.12b, R.sup.13b, R.sup.14b or
R.sup.15b to form a heterocyclic or heteroaromatic ring.
23. The composition of claim 20, wherein at least two of R.sup.4a,
R.sup.4b, R.sup.5a, R.sup.5b, R.sup.6a, R.sup.6b, R.sup.7a,
R.sup.7b, R.sup.8a, R.sup.8b, R.sup.9a, R.sup.9b, R.sup.10a,
R.sup.10b, R.sup.11a, R.sup.11b, R.sup.12a, R.sup.12b, R.sup.13a,
R.sup.13b, R.sup.14a, R.sup.14b, R.sup.15a and R.sup.15b are
hydroxyl.
24. The composition according to claim 20, wherein the Morus
extract is enriched for Albanin G, Kuwanon G, Morusin, or any
combination thereof.
25. The composition according to claim 20, wherein the Rosemary
extract is enriched for carnosol, carnosoic acid, ursolic acid, or
any combination thereof.
26. The composition according to claim 20, wherein the Yerba Mate
extract is enriched for caffeine, dicaffeoylquinic acid, or
both.
27. The composition according to claim 20, wherein the composition
further comprises a pharmaceutically or nutraceutically acceptable
carrier, diluent, or excipient, wherein the pharmaceutical or
nutraceutical formulation comprises from about 0.5 weight percent
(wt %) to about 90 wt % of active ingredients of the extract
mixture.
28. The composition according to claim 27, wherein the composition
is formulated as a tablet, capsule, powder, or granule.
29. A method for maintaining body weight in a mammal comprising
administering an effective amount of a composition according to
claim 1.
30. A method for promoting a healthy lipid profile by lowering LDL
cholesterol, lowering total cholesterol, lowering triglyceride, or
increasing HDL in a mammal comprising administering an effective
amount of a composition according to claim 10.
31. A method for treating, preventing, or managing weight gain in a
mammal comprising administering an effective amount of a
composition according to claim 20.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. .sctn.
119(e) of U.S. Provisional Patent Application No. 61/652,807, filed
May 29, 2012, and U.S. Provisional Patent Application No.
61/783,729, filed Mar. 14, 2013, where these two provisional
applications are incorporated herein by reference in their
entireties.
BACKGROUND
Technical Field
[0002] The present disclosure relates to compositions and methods
for weight management and, more particularly, to Diels-Alder
adducts of chalcone and prenylphenyl moieties, to oligomers of
flavan-3-ol, or both, optionally in combination with other weight
management agents, such as anorectic agents, a lipase inhibitors,
cannabinoid receptor modulators, psychotropic agents, insulin
sensitizers, stimulants, or satiety agents, as well as to methods
of use thereof such as treating or preventing weight gain or
obesity, promoting weight loss, appetite suppression, modifying
satiety, or the like.
Description of the Related Art
[0003] Obesity is a food problem. In industrialized countries,
affluence provides abundant and variable food items to the general
public. Food, with the associated taste and olfactory pleasures, is
an indulgence, not just for basic survival. As a result, obesity
and obesity-related health issues are increasing rapidly and there
is a strong need for dietary supplements that help with weight
control. The market size for food supplements that decrease body
weight is large and there are few effective products.
[0004] For many years, Cannabis sativa (marijuana) has been known
to stimulate food consumption through the action of its active
component, delta-9-tetrahydrocannabinol (THC), an exogenous
cannabinoid. This effect prompted research into its mechanism of
action. The binding sites for THC were eventually cloned and named
CB1 and CB2. These receptors belong to the G-protein coupled family
characterized by seven trans-membrane loop domains. Both receptors
belong to G.sub.i/0 subclass and signal by negatively regulating
cyclic AMP levels. CB1 was also shown to activate potassium
channels. CB2 receptor is present in immune cells and is not
involved in regulation of food consumption. CB1, the cannabinoid
receptor involved in feeding behavior, is widely expressed both in
brain and peripheral tissues, including adipose tissue, skeletal
muscles, liver, and gastrointestinal (GI) tract.
[0005] Most of the published CB1 receptor antagonists might be
better termed "inverse agonists" as they are capable of inhibiting
constitutive activity of non-occupied CB1 receptors. The major
clinical indications for this group of compounds are obesity and
substance abuse. In the past, five CB1 compounds have been tested
in clinical studies. They include Rimonabant (Sanofi-Aventis,
launched in 2006), MK-0364 (Merck, Phase III), Surinabant and
AVE-1625 (Sanofi-Aventis, Phase II), and SLV-319 (Solvay, Phase
II). Rimonabant (marketed as Acomplia.RTM., Rimoslim.TM. or
Zimulti.RTM.) was the first selective CB1 antagonist discovered in
1994. It was approved in 37 countries, but it has since been
withdrawn from obesity treatment due to neurological side
effects.
[0006] Another product on the market is tetrahydrolipstatin
(orlistat, sold as Alli.RTM. or Xenical.RTM.). Orlistat was
identified from a chemical library based on its inhibition of fatty
acid synthase, but was developed as a pancreatic triglyceride
lipase inhibitor. But, orlistat has been placed on a list of drugs
having a potential signal of serious risk due to cases of liver
toxicity, which has led to a change in the product labeling and the
FDA is continuing to evaluate this issue to determine the need for
any further regulatory action (see
www.fda.gov/Drugs/GuidanceComplianceRegulatorylnformationiSurvcillance/Ad-
verscDrugEffe cts/ucm161063.htm).
[0007] From the foregoing, a need is apparent for improved
compositions and methods for weight management.
BRIEF SUMMARY
[0008] In brief, the present disclosure is directed to compounds
and compositions useful for weight management and related methods,
including stereoisomers, pharmaceutically or nutraceutically
acceptable salts, tautomers, glycosides and prodrugs of the
disclosed compounds.
[0009] In certain embodiments, this disclosure provides a
composition comprising a mixture of a Morus extract enriched for
one or more Diels-Alder adducts of a chalcone and a prenylphenyl
moiety, a Magnolia extract, and a Yerba Mate extract. In further
embodiments, this disclosure provides a composition comprising a
mixture of a Morus extract enriched for Diels-Alder adducts of a
chalcone and a prenylphenyl moiety, a Magnolia extract, and a
Mutamba extract. In further embodiments, this disclosure provides a
composition comprising a mixture of a Morus extract enriched for
Diels-Alder adducts of a chalcone and a prenylphenyl moiety, a
Rosemary extract, and a Yerba Mate extract.
[0010] Exemplary Diels-Alder adducts of a chalcone and a
prenylphenyl moiety include compounds having a structure of Formula
I or II:
##STR00001##
or a pharmaceutically or nutraceutically acceptable salt, tautomer,
glycoside or stereoisomer thereof, wherein the substituents are as
defined herein.
[0011] In another aspect, the present disclosure provides methods
for managing weight. In certain embodiments, the compositions of
this disclosure can be used in methods for treating, preventing, or
managing weight gain or obesity or excess weight, promoting or
managing weight loss, appetite suppression, reducing food craving,
reducing eating between meals and in the evening hours, modifying
satiety, modifying fat uptake or fat absorption, increasing
metabolism to promote weight loss or prevent weight gain,
maintaining body weight, promoting fat burn, increasing lipolysis,
reducing body fat or fatty tissues, increasing muscle or lean body
mass, reducing hepato-steatosis, improving fatty liver, improving
one or more liver NASH scores, enhancing fat metabolism, reducing
the release of pro-inflammatory adipokines, increasing adiponectin
from fat tissues, promoting a healthy lipid profile (by, e.g.,
lowering LDL cholesterol, lowering total cholesterol, lowering
triglyceride, or increasing HDL), promoting glucose metabolism,
reducing fasting glucose levels, block absorption of carbohydrates,
maintaining healthy glucose levels, reducing caloric intake,
improving caloric efficiency, reducing food intake, reducing
visceral fat, reducing waist circumference, reducing body-to-mass
index (BMI), or any combination thereof.
[0012] These and other aspects of the invention will be apparent
upon reference to the following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows a graph of mean, body weights for mice on a
high fat diet that had been treated for 8 weeks with one of the
following: Magnolia extract, Morus alba extract, Mutamba extract,
Rosemary extract, or Yerba Mate extract. The negative controls
included mice on the high fat diet that received no treatment (HFD)
and mice kept on a normal diet control (NC), while the positive
control was mice on a high fat diet treated with orlistat.
[0014] FIG. 2 shows a graph of mean body weights for mice on a
high, fat diet that had been treated for 8 weeks with one of the
following combination of extracts: (1) Composition 1A of Example 63
(3 components)--Magnolia (100 mg/kg): Morus alba (200 mg/kg): Yerba
Mate (500 mg/kg): (2) Composition 2A (3 components)--Magnolia (100
mg/kg): Morus alba (200 mg/kg): Mutamba (500 mg/kg); (3)
Composition 10 of Example 68 (3 components)--Magnolia (100 mg/kg):
Yerba Mate (500 mg/kg): Mutamba (500 mg/kg); (4) Composition 12 (3
components)--Morus alba (200 mg/kg): Yerba Mate (500 mg/kg):
Mutamba (500 mg/kg); and (5) Composition 11 of Example 73 (4
components)--Magnolia (100 mg/kg): Morus alba (200 mg/kg): Yerba
Mate (500 mg/kg): Mutamba (500 mg/kg). The negative controls
included mice on the high fat diet that received no treatment (HFD)
and mice kept on, a normal diet (ND), while the positive control
was mice on a high fat diet treated with orlistat (ORI).
[0015] FIG. 3 shows MALDI-TOF positive ion mode mass spectrum of
Mutamba fraction 84/F6 generated from Mutamba stem bark EtOH
extract 84.
DETAILED DESCRIPTION
[0016] The present disclosure provides compositions of Diels-Alder
adducts of a chalcone and a prenylphenyl moiety and at least one
other weight management agent. The Diels-Alder adducts of a
chalcone and a prenylphenyl moiety can be obtained or enriched from
certain plants or certain plant parts, such as Morus alba root
bark, and can be used as a cannabinoid receptor (e.g., CB1, CB2)
modulator. Modulation of cannabinoid receptor activity can be
helpful in managing weight or diabetes. Exemplary weight management
agents for use with the Diels-Alder adducts of a chalcone and a
prenylphenyl moiety include anorectic agents, lipase inhibitors,
other cannabinoid receptor modulators, psychotropic agents, insulin
sensitizers, stimulants, satiety agents, or any combination
thereof. Furthermore, the Diels-Alder adducts of a chalcone and a
prenylphenyl moiety of the present disclosure, as well as
compositions thereof, can be used in methods to treat or prevent
weight associated disorders.
[0017] In the following description, certain specific details are
set forth in order to provide a thorough understanding of various
embodiments of this disclosure. However, one skilled in the art
will understand that the invention may be practiced without these
details.
[0018] In the present description, any concentration range,
percentage range, ratio range, or integer range is to be understood
to include the value of any integer within the recited range and,
when appropriate, fractions thereof (such as one tenth and one
hundredth of an integer), unless otherwise indicated. Also, any
number range recited herein relating to any physical feature, such
as polymer subunits, size or thickness, are to be understood to
include any integer within the recited range, unless otherwise
indicated. As used herein, the terms "about" and "consisting
essentially of" mean.+-.20% of the indicated range, value, or
structure, unless otherwise indicated. It should be understood that
the terms "a" and "an" as used herein refer to "one or more" of the
enumerated components. The use of the alternative (e.g., "or")
should be understood to mean either one, both, or any combination
thereof of the alternatives. Unless the context requires otherwise,
throughout the present specification and claims, the word
"comprise" and variations thereof, such as, "comprises" and
"comprising," as well as synonymous terms like "include" and "have"
and variants thereof, are to be construed in an open, inclusive
sense; that is, as "including, but not limited to."
[0019] Reference throughout this specification to "one embodiment"
or "an embodiment" means that a particular feature, structure or
characteristic described in connection with the embodiment is
included in at least one embodiment of the present invention. Thus,
the appearances of the phrases "in one embodiment" or "in an
embodiment" in various places throughout this specification are not
necessarily all referring to the same embodiment. Furthermore, the
particular features, structures, or characteristics may be combined
in any suitable manner in one or more embodiments.
[0020] "Amino" refers to the --NH.sub.2 radical.
[0021] "Cyano" refers to the --CN radical.
[0022] "Hydroxy" or "hydroxyl" refers to the --OH radical.
[0023] "Imino" refers to the .dbd.NH substituent.
[0024] "Nitro" refers to the --NO.sub.2 radical.
[0025] "Oxo" refers to the .dbd.O substituent.
[0026] "Thioxo" refers to the .dbd.S substituent.
[0027] "Alkyl" refers to a straight or branched hydrocarbon chain
radical consisting solely of carbon and hydrogen atoms, which is
saturated or unsaturated (i.e., contains one or more double or
triple bonds), having from one to twelve carbon atoms
(C.sub.1-C.sub.12 alkyl), or one to eight carbon atoms
(C.sub.1-C.sub.8 alkyl) or one to six carbon atoms (C.sub.1-C.sub.6
alkyl), and which is attached to the rest of the molecule by a
single bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl
(iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl),
3-methylhexyl, 2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl,
pent-1-enyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl,
pentynyl, hexynyl, and the like. Unless stated otherwise
specifically in the specification, an alkyl group may be optionally
substituted.
[0028] "Alkylene" or "alkylene chain" refers to a straight or
branched divalent hydrocarbon chain linking the rest of the
molecule to a radical group, consisting solely of carbon and
hydrogen, which is saturated or unsaturated (i.e., contains one or
more double or triple bonds), and having from one to twelve carbon
atoms, e.g., methylene, ethylene, propylene, n-butylene,
ethenylene, propenylene, n-butenylene, propynylene, n-butynylene,
and the like. The alkylene chain is attached to the rest of the
molecule through a single or double bond and to the radical group
through a single or double bond. The points of attachment of the
alkylene chain to the rest of the molecule and to the radical group
can be through one carbon or any two carbons within the chain.
Unless stated otherwise specifically in the specification, an
alkylene chain may be optionally substituted.
[0029] "Alkoxy" refers to a radical of the formula --OR.sub.a where
R.sub.a is an alkyl radical as defined above containing one to
twelve carbon atoms. Unless stated otherwise specifically in the
specification, an alkoxy group may be optionally substituted.
[0030] "Alkylamino" refers to a radical of the formula --NHR.sub.a
or --NR.sub.aR.sub.a where each R is, independently, an alkyl
radical as defined above containing one to twelve carbon atoms.
Unless stated otherwise specifically in the specification, an
alkylamino group may be optionally substituted.
[0031] "Thioalkyl" refers to a radical of the formula --SR.sub.a
where R.sub.a is an alkyl radical as defined above containing one
to twelve carbon atoms. Unless stated otherwise specifically in the
specification, a thioalkyl group may be optionally substituted.
[0032] "Aryl" refers to a hydrocarbon ring system radical
comprising hydrogen, 6 to 18 carbon atoms and at least one aromatic
ring. For purposes of this disclosure, the aryl radical may be a
monocyclic, bicyclic, tricyclic or tetracyclic ring system, which
may include fused or bridged ring systems. Aryl radicals include
aryl radicals derived from aceanthrylene, acenaphthylene,
acephenanthrylene, anthracene, azulene, benzene, chrysene,
fluoranthene, fluorene, as-indacene, s-indacene, indane, indene,
naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and
triphenylene. Unless stated otherwise specifically in the
specification, the term "aryl" or the prefix "ar-" (such as in
"aralkyl") is meant to include aryl radicals that are optionally
substituted.
[0033] "Aralkyl" refers to a radical of the formula
--R.sub.b--R.sub.c where R.sub.b is an alkylene chain as defined
above and R, is one or more aryl radicals as defined above, for
example, benzyl, diphenylmethyl and the like. Unless stated
otherwise specifically in the specification, an aralkyl group may
be optionally substituted.
[0034] "Cycloalkyl" or "carbocyclic ring" refers to a stable
non-aromatic monocyclic or polycyclic hydrocarbon radical
consisting solely of carbon and hydrogen atoms, which may include
fused or bridged ring systems, having from three to fifteen carbon
atoms, or having from three to ten carbon atoms, and which is
saturated or unsaturated and attached to the rest of the molecule
by a single bond. Monocyclic radicals include, for example,
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and
cyclooctyl. Polycyclic radicals include, for example, adamantyl,
norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the
like. Unless otherwise stated specifically in the specification, a
cycloalkyl group may be optionally substituted.
[0035] "Cycloalkylalkyl" refers to a radical of the formula
--R.sub.bR.sub.d where R.sub.b is an alkylene chain as defined
above and R.sub.d is a cycloalkyl radical as defined above. Unless
stated otherwise specifically in, the specification, a
cycloalkylalkyl group may be optionally substituted.
[0036] "Fused" refers to any ring structure described herein which
is fused to an existing ring structure in the compounds of this
disclosure. When the fused ring is a heterocyclyl ring or a
heteroaryl ring, any carbon atom on the existing ring structure
which becomes part of the fused beterocyclyl ring or the fused
heteroaryl ring may be replaced with a nitrogen atom.
[0037] "Halo" or "halogen" refers to bromo, chloro, fluoro or
iodo.
[0038] "Haloalkyl" refers to an alkyl radical, as defined above,
that is substituted by one or more halo radicals, as defined above,
e.g., trifluoromethyl, difluoromethyl, trichloromethyl,
2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl,
1,2-dibromoethyl, and the like. Unless stated otherwise
specifically in the specification, a haloalkyl group may be
optionally substituted.
[0039] "Heterocyclyl" or "heterocyclic ring" refers to a stable 3-
to 18-membered non-aromatic ring radical which consists of two to
twelve carbon atoms and from one to six heteroatoms selected from
the group consisting of nitrogen, oxygen and sulfur. Unless stated
otherwise specifically in the specification, the heterocyclyl
radical may be a monocyclic, bicyclic, tricyclic or tetracyclic
ring system, which may include fused or bridged ring systems; and
the nitrogen, carbon or sulfur atoms in the heterocyclyl radical
may be optionally oxidized; the nitrogen atom may be optionally
quaternized; and the heterocyclyl radical may be partially or fully
saturated. Examples of such heterocyclyl radicals include
dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl,
imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl,
morpholinyl, octahydroindolyl, octahydroisoindolyl,
2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl,
oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl,
pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl,
tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl,
thiamorpholinyl, 1-oxo-thiomorpholinyl, and
1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in
the specification, Unless stated otherwise specifically in the
specification, a heterocyclyl group may be optionally
substituted.
[0040] "N-heterocyclyl" refers to a beterocyclyl radical as defined
above containing at least one nitrogen and where the point of
attachment of the heterocyclyl radical to the rest of the molecule
is through a nitrogen atom in the heterocyclyl radical. Unless
stated otherwise specifically in the specification, a
N-heterocyclyl group may be optionally substituted.
[0041] "Heterocyclylalkyl" refers to a radical of the formula
--R.sub.bR.sub.e where R.sub.b is an alkylene chain as defined
above and R.sub.e is a heterocyclyl radical as defined above, and
if the heterocyclyl is a nitrogen-containing heterocyclyl, the
heterocyclyl may be attached to the alkyl radical at the nitrogen
atom. Unless stated otherwise specifically in the specification, a
heterocyclylalkyl group may be optionally substituted.
[0042] "Heteroaryl" refers to a 5- to 14-membered ring system
radical comprising hydrogen atoms, one to thirteen carbon atoms,
one to six heteroatoms selected from the group consisting of
nitrogen, oxygen and sulfur, and at least one aromatic ring. For
purposes of this disclosure, the heteroaryl radical may be a
monocyclic, bicyclic, tricyclic or tetracyclic ring system, which
may include fused or bridged ring systems; and the nitrogen, carbon
or sulfur atoms in the heteroaryl radical may be optionally
oxidized; the nitrogen atom may be optionally quaternized. Examples
include azepinyl, acridinyl, benzimidazolyl, benzothiazolyl,
benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl,
benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl,
1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl,
benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl,
benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl),
benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl,
cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl,
isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl,
isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl,
isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl,
oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl,
1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl,
phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl,
pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl,
pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl,
isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl,
triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e. thienyl).
Unless stated otherwise specifically in the specification, a
heteroaryl group may be optionally substituted.
[0043] "N-heteroaryl" refers to a heteroaryl radical as defined
above containing at least one nitrogen and where the point of
attachment of the heteroaryl radical to the rest of the molecule is
through a nitrogen atom in the heteroaryl radical. Unless stated
otherwise specifically in the specification, an N-heteroaryl group
may be optionally substituted.
[0044] "Heteroarylalkyl" refers to a radical of the formula
--R.sub.bR.sub.f where R.sub.b is an, alkylene chain as defined
above and R.sub.f is a heteroaryl radical as defined above. Unless
stated otherwise specifically in the specification, a
heteroarylalkyl group may be optionally substituted.
[0045] The term "substituted" used herein means any of the above
groups (i.e., alkyl, alkylene, alkoxy, alkylamino, thioalkyl, aryl,
aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl,
N-heterocyclyl, heterocyclylalkyl, heteroaryl. N-heteroaryl or
heteroarylalkyl), wherein at least one hydrogen atom is replaced by
a bond to a non-hydrogen atoms such as a halogen atom such as F,
Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups,
alkoxy groups, and ester groups; a sulfur atom in groups such as
thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups,
and sulfoxide groups; a nitrogen atom in groups such as amines,
amides, alkylamines, dialkylamines, arylamines, alkylarylamines,
diarylamines, N-oxides, imides, and enamines; silicon atom in
groups such as trialkylsilyl groups, dialkylarylsilyl groups,
alkyldiarylsilyl groups, and triarylsilyl groups; and other
heteroatoms in various other groups. "Substituted" also means any
of the above groups in which one or more hydrogen atoms are
replaced by a higher-order bond (e.g., a double- or triple-bond) to
a heteroatom such as oxygen in oxo, carbonyl, carboxyl, and ester
groups; and nitrogen in groups such as imines, oximes, hydrazones,
and nitriles. For example, "substituted" includes any of the above
groups in which one or more hydrogen atoms are replaced with
--NR.sub.gR.sub.h, --NR.sub.gC(.dbd.O)R.sub.h,
--NR.sub.gC(.dbd.O)NR.sub.gR.sub.h, --NR.sub.gC(.dbd.O)OR.sub.h,
--NR.sub.gSO.sub.2R.sub.h, --OC(.dbd.O)NR.sub.gR.sub.h, --OR.sub.g,
--SR.sub.g, --SOR.sub.g, --SO.sub.2R.sub.g, --OSO.sub.2R.sub.g,
--SO.sub.2OR.sub.g, .dbd.NSO.sub.2R.sub.g, and
--SO.sub.2NR.sub.gR.sub.h. "Substituted also means any of the above
groups in which one or more hydrogen atoms are replaced with
--C(.dbd.O)R.sub.g, --C(.dbd.O)OR.sub.g,
--C(.dbd.O)NR.sub.gR.sub.h, --CH.sub.2SO.sub.2R.sub.g,
--CH.sub.2SO.sub.2NR.sub.gR.sub.h. In the foregoing, R.sub.g and
R.sub.h are the same or different and independently hydrogen,
alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl,
cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl,
heterocyclylalkyl, heteroaryl, N-heteroaryl or heteroarylalkyl,
"Substituted" further means any of the above groups in which one or
more hydrogen atoms are replaced by a bond to an amino, cyano,
hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkoxy,
alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl,
haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl,
heteroaryl, N-heteroaryl or heteroarylalkyl group. In addition,
each of the foregoing substituents may also be optionally
substituted with one or more of the above substituents.
[0046] "Glycoside" refers to a molecule in which a sugar group is
bonded through its anomeric carbon to another group via a
glycosidic bond. Exemplary sugars include glucose, rhamnose,
manose, galactose, arabinose, glucuronide and others. Glycosides
can be linked by an O-- (an O-glycoside), N-- (a glycosylamine),
S-(a thioglycoside), or C-- (a C-glycoside) glycosidic bond.
Compounds of this disclosure can form glycosides at any suitable
attachment point.
[0047] "Prenyl" refers to the
##STR00002##
radical. Prenyl includes isoprenyl, which refers to the
##STR00003##
radical (cis or trans). Prenyl groups may be substituted or
unsubstituted.
[0048] "Prenylphenyl" refers to a phenyl moiety connected to a
prenyl moiety as defined above. Prenylphenyls include substituted
phenyls such as flavonoids and other substituted phenyls and
heteroaryls, provided there is at least one prenyl group in the
molecule. In the case of substituted phenyls and heteroaryl, the
prenyl moiety need not be directly attached to the phenyl ring, but
can be attached at any point in the molecule.
[0049] "Chalcone" refers to a compound comprising the following
core structure:
##STR00004##
Chalcones can be variously substituted at any of the above carbon
atoms.
[0050] "Prodrug" is meant to indicate a compound that may be
converted under physiological conditions or by solvolysis to a
biologically active compound of this disclosure. Thus, the term
"prodrug" refers to a metabolic precursor of a compound of this
disclosure that is pharmaceutically and nutraceutically acceptable.
A prodrug may be inactive when administered to a subject in need
thereof, but is converted in vivo to an active compound of this
disclosure. Prodrugs are typically rapidly transformed in vivo to
yield the parent compound of this disclosure, for example, by
hydrolysis in blood. The prodrug compound often offers advantages
of solubility, tissue compatibility or delayed release in a
mammalian organism (see, Bundgard, H., Design of Prodrugs (1985),
pp. 7-9, 21-24 (Elsevier, Amsterdam)). A discussion of prodrugs is
provided in Higuchi, T., et al., A.C.S. Symposium Series, Vol. 14,
and in Bioreversible Carriers in Drug Design, Ed. Edward B. Roche,
American Pharmaceutical and Nutraceutical Association and Pergamon
Press, 1987.
[0051] The term "prodrug" is also meant to include any covalently
bonded carriers, which release the active compound of this
disclosure in vivo when such prodrug is administered to a mammalian
subject. Prodrugs of a compound of this disclosure may be prepared
by modifying functional groups present in the compound of this
disclosure in such a way that the modifications are cleaved, either
in routine manipulation or in vivo, to the parent compound of this
disclosure. Prodrugs include compounds of this disclosure wherein a
hydroxy, amino or mercapto group is bonded to any group that, when
the prodrug of the compound of this disclosure is administered to a
mammalian subject, cleaves to form a free hydroxy, free amino or
free mercapto group, respectively. Examples of prodrugs include
acetate, formate and benzoate derivatives of alcohol or amide
derivatives of amine functional groups in the compounds of this
disclosure and the like.
[0052] The instant disclosure is also meant to encompass all
pharmaceutically or nutraceutically acceptable compounds of
structure (1) being isotopically-labelled by having one or more
atoms replaced by an atom having a different atomic mass or mass
number. Examples of isotopes that can be incorporated into the
disclosed compounds include isotopes of hydrogen, carbon, nitrogen,
oxygen, phosphorous, fluorine, chlorine, and iodine, such as
.sup.2H, .sup.3H, .sup.11C, .sup.13C, .sup.14C, .sup.13N, .sup.15N,
.sup.15O, .sup.17O, .sup.18O, .sup.31P, .sup.32P, .sup.35S,
.sup.18F, .sup.36Cl, .sup.123I, and .sup.125I, respectively. These
radiolabelled compounds could be useful to help determine or
measure the effectiveness of the compounds, by characterizing, for
example, the site or mode of action, or binding affinity to
pharmacologically important site of action. Certain
isotopically-labelled compounds of structure (1), for example,
those incorporating a radioactive isotope, are useful in drug or
substrate tissue distribution studies. The radioactive isotopes
tritium, i.e. .sup.3H, and carbon-14, i.e. .sup.14C, are
particularly useful for this purpose in view of their ease of
incorporation and ready means of detection.
[0053] Substitution with heavier isotopes such as deuterium, i.e.
H, may afford certain therapeutic advantages resulting from greater
metabolic stability, for example, increased in vive half-life or
reduced dosage requirements, and hence may be preferred in some
circumstances.
[0054] Substitution with positron emitting isotopes, such as
.sup.11C, .sup.18F, .sup.15O and .sup.13N, can be useful in
Positron Emission Topography (PET) studies for examining substrate
receptor occupancy. Isotopically-labeled compounds of structure (I)
can generally be prepared by conventional techniques known to those
skilled in the art or by processes analogous to those described in
the Preparations and Examples as set out below using an appropriate
isotopically-labeled reagent in place of the non-labeled reagent
previously employed.
[0055] The instant disclosure is also meant to encompass the in
vivo metabolic products of the disclosed compounds. Such products
may result from, for example, the oxidation, reduction, hydrolysis,
amidation, esterification, and the like of the administered
compound, primarily due to enzymatic processes. Accordingly, this
disclosure includes compounds produced by a process comprising
administering a compound of this invention to a mammal for a period
of time sufficient to yield a metabolic product thereof. Such
products are typically identified by administering a radiolabelled
compound of this disclosure in a detectable dose to an animal, such
as rat, mouse, guinea pig, monkey, or to human, allowing sufficient
time for metabolism to occur, and isolating its conversion products
from the urine, blood or other biological samples.
[0056] "Stable compound" and "stable structure" are meant to
indicate a compound that is sufficiently robust to survive
isolation to a useful degree of purity from a reaction mixture, and
formulation into an efficacious therapeutic agent.
[0057] "Mammal" includes humans and both domestic animals, such as
laboratory animals or household pets (e.g., cats, dogs, swine,
cattle, sheep, goats, horses, rabbits), and non-domestic animals,
such as wildlife or the like.
[0058] "Optional" or "optionally" means that the subsequently
described element, component, event or circumstances may or may not
occur, and that the description includes instances where the
element, component, event or circumstance occur and instances in
which they do not. For example, "optionally substituted aryl" means
that the aryl radical may or may not be substituted and that the
description includes both substituted aryl radicals and aryl
radicals having no substitution.
[0059] "Pharmaceutically or nutraceutically acceptable carrier,
diluent or excipient" includes any adjuvant, carrier, excipient,
glidant, sweetening agent, diluent, preservative, dye/colorant,
flavor enhancer, surfactant, wetting agent, dispersing agent,
suspending agent, stabilizer, isotonic agent, solvent, or
emulsifier which has been approved by the United States Food and
Drug Administration as being acceptable for use in humans or
domestic animals.
[0060] "Pharmaceutically or nutraceutically acceptable salt"
includes both acid and base addition salts.
[0061] "Pharmaceutically or nutraceutically acceptable acid
addition salt" refers to those salts which retain the biological,
effectiveness and properties of the free bases, which are not
biologically or otherwise undesirable, and which are formed with
inorganic acids such as hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid, phosphoric acid and the like, and
organic acids such as acetic acid, 2,2-dichloroacetic acid, adipic
acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic
acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid,
camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid,
carbonic acid, cinnamic acid, citric acid, cyclamic acid,
dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic
acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid,
galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid,
glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid,
glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric
acid, lactic acid, lactobionic acid, lauric acid, maleic acid,
malic acid, malonic acid, mandelic acid, methanesulfonic acid,
mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic
acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid,
orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic
acid, pyroglutamic acid, pyruvic acid, salicylic acid,
4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,
tartaric acid, thiocyanic acid, p-toluenesulfonic acid,
trifluoroacetic acid, undecylenic acid, and the like.
[0062] "Pharmaceutically or nutraceutically acceptable base
addition salt" refers to those salts which retain the biological
effectiveness and properties of the free acids, which are not
biologically or otherwise undesirable. These salts are prepared
from addition of an inorganic base or an organic base to the free
acid. Salts derived from inorganic bases include the sodium,
potassium, lithium, ammonium, calcium, magnesium, iron, zinc,
copper, manganese, aluminum salts and the like. In certain
embodiments, the inorganic salts are ammonium, sodium, potassium,
calcium, or magnesium salts. Salts derived from organic bases
include salts of primary, secondary, and tertiary amines,
substituted amines including naturally occurring substituted
amines, cyclic amines and basic ion exchange resins, such as
ammonia, isopropylamine, trimethylamine, diethylamine,
triethylamine, tripropylamine, diethanolamine, ethanolamine,
deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol,
dicyclohexylamine, lysine, arginine, histidine, procaine,
hydrabamine, choline, betaine, benethamine, benzathine,
ethylenediamine, glucosamine, methylglucamine, theobromine,
triethanolamine, tromethamine, purines, piperazine, piperidine,
N-ethylpiperidine, polyamine resins and the like. Particularly
useful organic bases are isopropylamine, diethylamine,
ethanolamine, trimethylamine, dicyclohexylamine, choline and
caffeine.
[0063] Often crystallizations produce a solvate of the compound of
this disclosure. As used herein, the term "solvate" refers to an
aggregate that comprises one or more molecules of a compound of
this disclosure with one or more molecules of solvent. The solvent
may be water, in which case the solvate may be a hydrate.
Alternatively, the solvent may be an organic solvent. Thus, the
compounds of the present invention may exist as a hydrate,
including a monohydrate, dihydrate, hemihydrate, sesquihydrate,
trihydrate, tetrahydrate and the like, as well as the corresponding
solvated forms. The compound of this disclosure may be true
solvates, while in other cases, the compound of this disclosure may
merely retain adventitious water or be a mixture of water plus some
adventitious solvent.
[0064] A "pharmaceutical composition" or "nutraceutical
composition" refers to a formulation of a compound of this
disclosure and a medium generally accepted in the art for the
delivery of the biologically active compound to mammals, e.g.,
humans. For example, a pharmaceutical composition of the present
disclosure may be formulated or used as a stand alone composition,
or as a component in a prescription drug, an over the counter (OTC)
medicine, a botanical drug, an herbal medicine, a homeopathic
agent, or any other form of health care product reviewed and
approved by a government agency. Exemplary nutraceutical
compositions of the present disclosure may be formulated or used as
a stand alone composition, or as a nutritional or bioactive
component in food, a functional food, a beverage, a bar, a food
flavor, a medical food, a dietary supplement, or an herbal product.
A medium generally accepted in the art includes all
pharmaceutically or nutraceutically acceptable carriers, diluents
or excipients therefor.
[0065] As used herein, "enriched for" refers to a plant extract or
other preparation having at least a two-fold up to about a
1000-fold increase of one or more active compounds as compared to
the amount of one or more active compounds found in the weight of
the plant material or other source before extraction or other
preparation. In certain embodiments, the weight of the plant
material or other source before extraction or other preparation may
be dry weight, wet weight, or a combination thereof.
[0066] As used herein, "major active ingredient" or "major active
component" refers to one or more active compounds found in a plant
extract or other preparation, or enriched for in a plant extract or
other preparation, which is capable of at least one biological
activity. In certain embodiments, a major active ingredient of an
enriched extract will be the one or more active compounds that were
enriched in that extract. Generally, one or more major active
components will impart, directly or indirectly, most (i.e., greater
than 50%) of one or more measurable biological activities or
effects as compared to other extract components. In certain
embodiments, a major active ingredient may be a minor component by
weight percentage of an extract (e.g., less than 50%, 25%, or 10%
of the components contained in an extract) but still provide most
of the desired biological activity. Any composition of this
disclosure containing a major active ingredient may also contain
minor active ingredients that may or may not contribute to the
pharmaceutical or nutraceutical activity of the enriched
composition, but not to the level of major active components, and
minor active components alone may not be effective in the absence
of a major active ingredient.
[0067] "Effective amount" or "therapeutically effective amount"
refers to that amount of a compound or composition of this
disclosure which, when administered to a mammal, such as a human,
is sufficient to effect treatment, including any one or more of:
(1) treating or preventing weight gain in a mammal; (2) promoting
weight loss; (3) suppressing appetite in a mammal; (4) modifying
satiety in a mammal; (5) treating or preventing obesity in a
mammal; (6) modifying fat uptake in a mammal; and (7) increasing
metabolism to promote weight loss or prevent weight gain in a
mammal. The amount of a compound or composition of this disclosure
that constitutes a "therapeutically effective amount" will vary
depending on the compound, the condition being treated and its
severity, the manner of administration, the duration of treatment,
or the age of the subject to be treated, but can be determined
routinely by one of ordinary skill in the art having regard to his
own knowledge and to this disclosure.
[0068] "Dietary supplements" as used herein are a product that
improves, promotes, increases, manages, controls, maintains,
optimizes, modifies, reduces, inhibits, or prevents a particular
condition associated with a natural state or biological process
(i.e., are not used to diagnose, treat, mitigate, cure, or prevent
disease). For example, with regard to weight-related conditions,
dietary supplements may be used to promote weight loss, manage
weight gain, maintain weight, modify satiety, reduce caloric
intake, increase muscle mass, or the like. Exemplary dietary
supplements include one or more of a dietary ingredient such as a
vitamin, a mineral, an herb or other botanical, an amino acid, or
any other substance used to supplement the diet by increasing total
dietary intake, or a concentrate, metabolite, constituent, extract,
or any combination thereof. In certain embodiments, dietary
supplements are a special category of food and are not a drug.
[0069] "Treating" or "treatment" as used herein refers to the
treatment of the disease or condition of interest in a mammal, such
as a human, having the disease or condition of interest, and
includes: (i) preventing the disease or condition from occurring in
a mammal, in particular, when such mammal is predisposed to the
condition but has not yet been diagnosed as having it; (ii)
inhibiting the disease or condition, i.e., arresting its
development; (iii) relieving the disease or condition, i.e.,
causing regression of the disease or condition; or (iv) relieving
the symptoms resulting from the disease or condition, (e.g.,
relieving pain, reducing inflammation, causing weight loss) without
addressing the underlying disease or condition. As used herein, the
terms "disease" and "condition" may be used interchangeably or may
be different in that the particular malady or condition may not
have a known causative agent (so that etiology has not yet been
worked out) and it is therefore not yet recognized as a disease but
only as an undesirable condition or syndrome, wherein a more or
less specific set of symptoms have been identified by
clinicians.
[0070] As used herein, "statistical significance" refers to a p
value of 0.050 or less when calculated using the Students t-test
and indicates that it is unlikely that a particular event or result
being measured has arisen by chance.
[0071] The compounds of this disclosure, or their pharmaceutically
or nutraceutically acceptable salts may contain one or more
asymmetric centers and may thus give rise to enantiomers,
diastercomers, and other stereoisomeric forms that may be defined,
in terms of absolute stereochemistry, as (R)- or (S)- or, as (D)-
or (L). For example, the compounds of structure I or II may have
chiral centers at least at the positions noted with * in the
structures below.
##STR00005##
Accordingly, in, certain embodiments the positions marked with an *
above (and various other positions within compounds I and II) can
each independently exist as either R or S isomers, and the present
invention is meant to include all such possible isomers, as well as
their racemic and optically pure forms. Optically active (+) and
(-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using
chiral synthons or chiral reagents, or resolved using conventional
techniques, for example, chromatography and fractional
crystallization. Conventional techniques for the
preparation/isolation of individual enantiomers include chiral
synthesis from a suitable optically pure precursor or resolution of
the racemate (or the racemate of a salt or derivative) using, for
example, chiral high pressure liquid chromatography (HPLC). When
the compounds described herein contain olefinic double bonds or
other centres of geometric asymmetry, and unless specified
otherwise, it is intended that the compounds include both E and Z
geometric isomers. Likewise, all tautomeric forms are also intended
to be included.
[0072] A "stereoisomer" refers to a compound made up of the same
atoms bonded by the same bonds but having different
three-dimensional structures, which are not interchangeable. The
present invention contemplates various stereoisomers and mixtures
thereof and includes "enantiomers", which refers to two
stereoisomers whose molecules are nonsuperimposeable mirror images
of one another.
[0073] A "tautomer" refers to a proton shift from one atom of a
molecule to another atom of the same molecule. The present
invention includes tautomers of any said compounds.
[0074] The chemical naming protocol and structure diagrams used
herein are a modified form of the I.U.P.A.C. nomenclature system,
using the ACD/Name Version 9.07 software program or ChemDraw Ultra
Version 11.0 software naming program (CambridgeSoft), wherein the
compounds of this disclosure are named herein as derivatives of the
central core structure, e.g., the imidazopyridine structure. For
complex chemical names employed herein, a substituent group is
named before the group to which it attaches. For example,
cyclopropylethyl comprises an ethyl backbone with cyclopropyl
substituent. Except as described below, all bonds are identified in
the chemical structure diagrams herein, except for some carbon
atoms, which are assumed to be bonded to sufficient hydrogen atoms
to complete the valency.
[0075] As noted herein, in certain embodiments, the present
disclosure provides a composition comprising a Diels-Alder adduct
of a chalcone and a prenylphenyl moiety, and at least one other
weight management agent, wherein the weight management agent is an
anorectic agent, a lipase inhibitor, a cannabinoid receptor
modulator, a psychotropic agent, an insulin sensitizer, a
stimulant, or a satiety agent, wherein the Diels-Alder adduct of a
chalcone and a prenylphenyl moiety is a compound having a structure
of Formula I or II:
##STR00006##
or a pharmaceutically or nutraceutically acceptable salt, tautomer,
glycoside, prodrug or stereoisomer thereof, wherein:
[0076] R.sup.1a and R.sup.1b are each independently C.sub.1-12
alkyl;
[0077] R.sup.2a and R.sup.2b are each independently H or R.sup.2a
or R.sup.2b joins with one of R.sup.4a, R.sup.5a, R.sup.6a,
R.sup.7a, R.sup.12a, R.sup.13a, R.sup.14a or R.sup.15a or R.sup.4b,
R.sup.5b, R.sup.6b, R.sup.7b, R.sub.12b, R.sup.13b, R.sup.14b or
R.sup.15b, respectively, to form an ether bond;
[0078] R.sup.3a and R.sup.3b are each independently H, hydroxyl or
oxo;
[0079] R.sup.4a, R.sup.5a, R.sup.6a and R.sup.7a are each
independently H, hydroxyl, halogen, sulfhydryl, amino, aldehyde,
C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.1-12 alkthio, C.sub.1-12
alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl,
aralkylcarbonyl or one of R.sup.4a, R.sup.5a, R.sup.6a or R.sup.7a
joins with another of R.sup.4a, R.sup.5a, R.sup.6a or R.sup.7a to
form a heterocyclic or heteroaromatic ring or one of R.sup.4a,
R.sup.5a, R.sup.6a or R.sup.7a joins with X.sup.1 to form a direct
bond:
[0080] R.sup.4b, R.sup.5b, R.sub.6b and R.sup.7b are each
independently H, hydroxyl, halogen, sulfhydryl, amino, aldehyde,
C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.1-12 alkthio, C.sub.1-12
alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl,
aralkylcarbonyl or one of R.sup.4a, R.sup.5b, R.sup.6b, R.sup.7b
joins with another of R.sup.4b, R.sup.5b, R.sup.6b, R.sup.7b to
form a heterocyclic or heteroaromatic ring or one of R.sup.4b,
R.sup.5b, R.sup.6b or R.sup.7b joins with X.sup.2 to form a direct
bond;
[0081] R.sup.8a, R.sup.8b, R.sup.9a, R.sup.9b, R.sup.10a,
R.sup.10b, R.sup.11a and R.sup.11b are each independently H,
hydroxyl, halogen, sulfhydryl, amino, aldehyde, C.sub.1-12 alkyl,
C.sub.1-12 alkoxy, C.sub.1-12 alkthio, C.sub.1-12 alkyamino, aryl,
heteroaryl, aralkyl, alkyl carbonyl, aralkylcarbonyl or one of
R.sup.8a, R.sup.9a, R.sup.10a or R.sup.11a or one of R.sup.8b,
R.sup.9b, R.sup.10b or R.sup.11b joins with another of R.sup.8a,
R.sup.9a, R.sup.10a or R.sup.11a or another one of R.sup.8b,
R.sup.9b, R.sup.10b or R.sup.11b, respectively, to form a
heterocyclic or heteroaromatic ring;
[0082] R.sup.12a, R.sup.13a, R.sup.14a and R.sup.15a are each
independently H, hydroxyl, halogen, sulfhydryl, amino, aldehyde,
C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.1-12 alkthio, C.sub.1-12
alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl,
aralkylcarbonyl or one of R.sup.12a, R.sup.13a, R.sup.14a,
R.sup.15a joins with another of R.sup.12a, R.sup.13a, R.sup.14a or
R.sup.15a to form a heterocyclic or heteroaromatic ring or one of
R.sup.12a, R.sup.13a, R.sup.14a or R.sup.15a joins with Y.sup.1 to
form an ether bond;
[0083] R.sup.12b, R.sup.13b, R.sup.14b and R.sup.15b are each
independently H, hydroxyl, halogen, sulfhydryl, amino, aldehyde,
C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.1-12 alkthio, C.sub.1-12
alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl,
aralkylcarbonyl or one of R.sup.12b, R.sup.13b, R.sup.14b,
R.sup.15b joins with another of R.sup.12b, R.sup.13b, R.sup.14b or
R.sup.15b to form a heterocyclic or heteroaromatic ring;
[0084] X.sup.1 joins with one of R.sup.4a, R.sup.5a, R.sup.6a or
R.sup.7a to form a direct bond or X.sup.1 joins with Y.sup.1 to
form an oxo moiety;
[0085] X.sup.2 joins with one of R.sup.4b, R.sup.5b, R.sup.6b or
R.sup.6b to form a direct bond or X.sup.2 joins with Y.sup.2 to
form an oxo moiety;
[0086] Y.sup.1 is H or Y.sup.1 joins with one of R.sup.12a,
R.sup.13a, R.sup.14a or R.sup.15a to form an ether bond or Y.sup.1
joins with an adjacent aliphatic carbon to form, an oxirane
ring;
[0087] Y.sup.2 is H or Y.sup.2 joins with an adjacent aliphatic
carbon to form an oxirane ring; and
[0088] a dashed bond represents an optional double bond such that
all valences are satisfied.
[0089] In further embodiments of the foregoing, R.sup.1a and
R.sup.1b are each independently methyl,
##STR00007##
[0090] In some embodiments, R.sup.2a and R.sup.2b are H. In other
embodiments, R.sup.3a and R.sup.3b are H.
[0091] In yet other embodiments, the compound has the following
structure (Ia) or (IIa):
##STR00008##
[0092] In still other embodiments, the compound has the following
structure (Ib) or (IIb):
##STR00009##
[0093] In other aspects of the foregoing composition, R.sup.4a,
R.sup.4b, R.sup.5a, R.sup.5b, R.sup.6a, R.sup.6b, R.sup.7a,
R.sup.7b, R.sup.8a, R.sup.8b, R.sup.9a, R.sup.9b, R.sup.10a,
R.sup.10b, R.sup.11a, R.sup.11b, R.sup.12a, R.sup.12b, R.sup.13a,
R.sup.13b, R.sup.14a, R.sup.14b, R.sup.15a and R.sup.15b are each
independently H, hydroxyl, halogen, C.sub.1-12 alkoxy, C.sub.1-12
alkyl or heteroaryl, or one of R.sup.4a, R.sup.4b, R.sup.5a,
R.sup.5b, R.sup.6a, R.sup.6b, R.sup.7a, R.sup.7b, R.sup.8a,
R.sup.8b, R.sup.9a, R.sup.9b, R.sup.10a, R.sup.10b, R.sup.11a,
R.sup.11b, R.sup.12a, R.sup.12b, R.sup.13a, R.sup.13b, R.sup.14a,
R.sup.14b, R.sup.15a or R.sup.15b joins with another of R.sup.4a,
R.sup.4b, R.sup.5a, R.sup.5b, R.sup.6a, R.sup.6b, R.sup.7a,
R.sup.7b, R.sup.8a, R.sup.8b, R.sup.9a, R.sup.9b, R.sup.10a,
R.sup.10b, R.sup.11a, R.sup.11b, R.sup.12a, R.sup.12b, R.sup.13a,
R.sup.13b, R.sup.14a, R.sup.14b, R.sup.15a or R.sup.15b on the same
ring to form a heteroaromatic ring.
[0094] In some embodiments, one of R.sup.4a, R.sup.4b, R.sup.5a,
R.sup.5b, R.sup.6a, R.sup.6b, R.sup.7a, R.sup.7b, R.sup.8a,
R.sup.8b, R.sup.9a, R.sup.9b, R.sup.10a, R.sup.10b, R.sup.11a,
R.sup.11b, R.sup.12a, R.sup.12b, R.sup.13a, R.sup.13b, R.sup.14a,
R.sup.14b, R.sup.15a or R.sup.15b joins with another of R.sup.4a,
R.sup.4b, R.sup.5a, R.sup.5b, R.sup.6a, R.sup.6b, R.sup.7a,
R.sup.7b, R.sup.8a, R.sup.8b, R.sup.9a, R.sup.9b, R.sup.10a,
R.sup.10b, R.sup.11a, R.sup.11b, R.sup.12a, R.sup.12b, R.sup.13a,
R.sup.13b, R.sup.14a, R.sup.14b, R.sup.15a or R.sup.15b on the same
ring to form a heteroaromatic ring. For example, in some
embodiments the foregoing R groups may join to form a
dioxymethylene group and the heteroaromatic ring thus formed is an
optionally substituted benzodiaoxazole.
[0095] In still other embodiments, the compound has one of the
following structures (Ic), (Id), (Ie), (If), (Ig), (IIc), (IId),
(IIe), (IIf) or (IIg):
##STR00010## ##STR00011## ##STR00012## [0096] wherein R is, at each
occurrence, independently H, hydroxyl or C.sub.1-12 alkyl.
[0097] In further embodiments, of the foregoing, the compound has
one of the following structures (Ih), (Ii), (Ij), (Ik), (Il),
(IIh), (IIi), (IIj), (IIk) or (IIl):
##STR00013## ##STR00014## ##STR00015##
[0098] In still, other embodiments, at least one R is hydroxyl, and
in other embodiments at least one R is
##STR00016##
[0099] In other related embodiments, at least two of R.sup.4a,
R.sup.4b, R.sup.5a, R.sup.5b, R.sup.6a, R.sup.6b, R.sup.7a,
R.sup.7b, R.sup.8a, R.sup.8b, R.sup.9a, R.sup.9b, R.sup.10a,
R.sup.10b, R.sup.11a, R.sup.11b, R.sup.12a, R.sup.12b, R.sup.13a,
R.sup.13b, R.sup.14a, R.sup.14b, R.sup.15a or R.sup.15b are
hydroxyl.
[0100] In some exemplary compositions, the compound has one of the
following structures (Im), (In), (IIm) or (IIn):
##STR00017##
[0101] In some embodiments, at least one of R.sup.4a, R.sup.4b,
R.sup.5a, R.sup.5b, R.sup.6a, R.sup.6b, R.sup.7a, R.sup.7b,
R.sup.8a, R.sup.8b, R.sup.9a, R.sup.9b, R.sup.10a, R.sup.10b,
R.sup.11a, R.sup.11b, R.sup.12a, R.sup.12b, R.sup.13a, R.sup.13b,
R.sup.14a, R.sup.14b, R.sup.15a or R.sup.15b is
##STR00018##
In other embodiments, at least one of R.sup.4a, R.sup.4b, R.sup.5a,
R.sup.5b, R.sup.6a, R.sup.6b, R.sup.7a, R.sup.7b, R.sup.8a,
R.sup.8b, R.sup.9a, R.sup.9b, R.sup.10a, R.sup.10b, R.sup.11a,
R.sup.11b, R.sup.12a, R.sup.12b, R.sup.13a, R.sup.13b, R.sup.14a,
R.sup.14b, R.sup.15a or R.sup.15b is heteroaryl. For example, in
some embodiments, the heteroaryl is selected from:
##STR00019##
wherein R is, at each occurrence, independently H, hydroxyl or
C.sub.1-12 alkyl. For example, in some embodiments at least one R
is hydroxyl, and in other embodiments at least one R is
##STR00020##
[0102] In still other embodiments, X.sup.1 joins with one of
R.sup.4a, R.sup.5a, R.sup.6a or R.sup.7a to form a direct bond or
X.sup.2 joins with one of R.sup.12b, R.sup.13b, R.sup.14b or
R.sup.15b to form a direct bond and the compound has one of the
following structures (Io) or (IIo):
##STR00021##
For example, in some further embodiments, the compound has one of
the following structures (Ip) or (IIp):
##STR00022##
In still some other further embodiments, Y.sup.1 joins with one of
R.sup.12a, R.sup.13a, R.sup.14a or R.sup.15a to form an ether bond
and the compound h of the following structures (Iq) or (Ir):
##STR00023##
In some more specific embodiments, the compound is selected from
any of the compounds provided in Table A.
TABLE-US-00001 TABLE A List of Exemplary Compounds Molecular
Structure Name Species Formula M.W. ##STR00024## Albafuran C Morus
alba C.sub.34H.sub.28O.sub.9 580.590 ##STR00025## Albafuran C; 2-
Epimer Morus australis C.sub.34H.sub.28O.sub.9 580.590 ##STR00026##
Albanin F Morus alba, also from Morus australis, Morus bombycis,
and Morus lhou C.sub.40H.sub.36O.sub.11 692.718 ##STR00027##
Albanin F (Moracenin D); 12,13-Dihydro, 13-hydroxy Morus sp.
C.sub.40H.sub.38O.sub.12 710.733 ##STR00028## Albanin G (Kuwanon H.
Moracenin A.) Morus alba; also isol. from Morus australis, Morus
bombycis, and Morus lhou C.sub.45H.sub.44O.sub.11 760.836
##STR00029## Albanin G; 2'''- Deoxy (Mongolicin D) Morus mongolica
C.sub.45H.sub.44O.sub.10 744.837 ##STR00030## Albanol A
(Mulberrofuran G.) Morus lhou C.sub.34H.sub.26O.sub.8 562.575
##STR00031## Albanol A; 3''- (3-Methyl-2- butenyl), Mulberrofuran F
Morus lhou C.sub.39H.sub.34O.sub.8 630.693 ##STR00032## Albanol B
Morus alba C.sub.34H.sub.22O.sub.8 558.543 ##STR00033## Artonin C
Artocarpus heterophyllus (jackfruit) C.sub.40H.sub.38O.sub.10
678.734 ##STR00034## Artonin D Artocarpus heterophyllus (jackfruit)
C.sub.40H.sub.36O.sub.10 676.718 ##STR00035## Artonin I Morus
heterophyllus C.sub.40H.sub.36O.sub.11 692.718 ##STR00036##
Australisin B Morus australis C.sub.39H.sub.34O.sub.9 646.692
##STR00037## Australisin C; 2-Epimer Morus australis
C.sub.34H.sub.28O.sub.9 ##STR00038## Brosimone B Brosimopsis
oblongifolia (preferred genus name Brosimum)
C.sub.40H.sub.38O.sub.10 678.734 ##STR00039## Brosimone D
Brosimopsis oblongifolia (preffered genus name Brosimum)
C.sub.45H.sub.44O.sub.13 760.836 ##STR00040## Cathayanon A Morus
cathayana C.sub.40H.sub.36O.sub.12 708.717 ##STR00041## Cathayanon
A; 14-Epimer Morus cathayana C.sub.40H.sub.36O.sub.12 708.717
##STR00042## Cathayanon E Morus cathayana C.sub.40H.sub.36O.sub.12
708.717 ##STR00043## Chalcomoracin Morus alba and Morus mongolica
C.sub.39H.sub.36O.sub.9 648.708 ##STR00044## Chalcomoracin;
3'',5''-Diepimer Sorocea muriculata C.sub.39H.sub.36O.sub.9 648.708
##STR00045## Chalcomoracin; 3''-Epimer Morus mongolica
C.sub.39H.sub.36O.sub.9 648.708 ##STR00046## Dorstenone Dorstenia
barteri C.sub.40H.sub.38O.sub.8 646.735 ##STR00047## Guangsangon C
Morus macroura C.sub.35H.sub.30O.sub.10 610.616 ##STR00048##
Guangsangon D Morus macroura C.sub.35H.sub.30O.sub.10 610.616
##STR00049## Guangsangon D; 2'-Deoxy, 4',6'-dihydroxy Morus
macroura C.sub.35H.sub.30O.sub.11 626.615 ##STR00050## Guangsangon
D; 3-Deoxy, 4'- hydroxy Morus macroura and Morus wittiorum
C.sub.35H.sub.30O.sub.10 610.616 ##STR00051## Guangsangon D;
2-Epimer, 3- deoxy, 4'- hydroxy Morus macroura
C.sub.36H.sub.30O.sub.10 610.616 ##STR00052## Guangsangon E Morus
macroura C.sub.39H.sub.36O.sub.9 648.708 ##STR00053## Guangsangon
E; 3''-Epimer, 2'''',3''''-dihydro, 3''''-hydroxy Morus macroura
C.sub.39H.sub.38O.sub.10 666.723 ##STR00054## Guangsangon F Morus
macroura C.sub.40H.sub.36O.sub.10 676.718 ##STR00055## Guangsangon
G Morus macroura C.sub.35H.sub.28O.sub.10 608.600 ##STR00056##
Guangsangon G; 1''-Epimer, 2'-hydroxy Morus macroura
C.sub.35H.sub.28O.sub.11 624.600 ##STR00057## Guangsangon G;
2'-Hydroxy Morus macroura C.sub.35H.sub.28O.sub.11 624.600
##STR00058## Guangsangon G; 5-Hydroxy Morus wittiorum
C.sub.35H.sub.28O.sub.11 625.600 ##STR00059## Guangsangon H Morus
macroura C.sub.40H.sub.38O.sub.10 678.734 ##STR00060## Guangsangon
J Morus macroura C.sub.39H.sub.36O.sub.9 648.708 ##STR00061##
Guangsangon L Morus alba C.sub.27H.sub.24O.sub.8 476.482
##STR00062## Isobavachromene dimer Dorstenia zenkeri
C.sub.40H.sub.38O.sub.8 646.735 ##STR00063## Kuwanol A Morus
bombycis C.sub.34H.sub.28O.sub.8 564.590 ##STR00064## Kuwanol B
Morus bombycis C.sub.34H.sub.26O.sub.8 562.575 ##STR00065## Kuwanol
E Morus alba (white mulberry) C.sub.39H.sub.38O.sub.9 650.724
##STR00066## Kuwanol E; 2''',3'''-Dihydro, 3'''-hydroxy Sorocea
ilicifolia C.sub.39H.sub.40O.sub.10 668.739 ##STR00067## Kuwanon J
Morus alba and from Morus bombycus and Morus nigra
C.sub.40H.sub.39O.sub.10 678.734 ##STR00068## Kuwanon J; 16''-Deoxy
Morus alba (white mulberry) C.sub.40H.sub.38O.sub.9 662.735
##STR00069## Kuwanon J; 2- Deoxy Morus alba (white mulberry)
C.sub.40H.sub.38O.sub.9 662.735 ##STR00070## Kuwanon J,
.DELTA.21'',22''- Isomer, 2-deoxy Morus alba (white mulberry)
C.sub.40H.sub.38O.sub.9 662.735 ##STR00071## Kuwanon J;
2,16''-Dideoxy Morus alba (white mulberry) C.sub.40H.sub.38O.sub.8
646.735 ##STR00072## Kuwanon J; 2',3'-Dihydro Morus mongolica
C.sub.40H.sub.40O.sub.10 680.750 ##STR00073## Kuwanon J; 1''-
Epimer Morus alba and Morus bombycus C.sub.40H.sub.38O.sub.10
678.734 ##STR00074## Kuwanon J; .DELTA.21'',22''- Isomer, 2-deoxy
(Artonin X.) Artocarpus heterophyllus (jackfruit)
C.sub.40H.sub.38O.sub.9 662.735 ##STR00075## Kuwanon L Morus alba
(white mulberry) C.sub.35H.sub.30O.sub.11 626.615 ##STR00076##
Kuwanon L; 2,3-Didehydro, 3-(3-methyl-2- butenyl) Morus alba (white
mulberry) C.sub.40H.sub.36O.sub.11 692.718 ##STR00077## Kuwanon N
Morus lhou C.sub.45H.sub.44O.sub.11 760.836 ##STR00078## Kuwanon O
Morus lhou C.sub.40H.sub.38O.sub.11 694.734 ##STR00079## Kuwanon P
Morus lhou C.sub.34H.sub.30O.sub.9 582.606 ##STR00080## Kuwanon P;
2- Deoxy Morus macroura C.sub.34H.sub.30O.sub.8 ##STR00081##
Kuwanon W Morus lhou C.sub.45H.sub.42O.sub.11 758.820 ##STR00082##
Kuwanon X Morus lhou C.sub.34H.sub.30O.sub.9 582.606 ##STR00083##
Kuwanon X; 3''-Epimer Morus alba (white mulberry)
C.sub.34H.sub.30O.sub.9 582.606 ##STR00084## Kuwanon Z Morus alba
(white mulberry) C.sub.34H.sub.26O.sub.10 594.573 ##STR00085##
Mongolicin C Morus mongolica C.sub.34H.sub.26O.sub.9 578.574
##STR00086## Moracenin C Morus sp. C.sub.45H.sub.44O.sub.11 760.836
##STR00087## Mulberrofuran C Morus bombycis (Moraceae) ##STR00088##
Mulberrofuran E Morus alba (white mulberry) (Moraceae)
C.sub.39H.sub.36O.sub.8 632.709 ##STR00089## Mulberrofuran I Morus
bombycis C.sub.34H.sub.24O.sub.8 560.559 ##STR00090## Mulberrofuran
J Morus lhou C.sub.34H.sub.28O.sub.9 580.590 ##STR00091##
Mulberrofuran J, 2-Epimer Morus bombycis ##STR00092## Mulberrofuran
O Morus alba 646.692 ##STR00093## Mulberrofuran P Morus alba (white
mulberry) C.sub.34H.sub.22O.sub.9 574.542 ##STR00094##
Mulberrofuran Q Morus alba (white mulberry)
C.sub.34H.sub.24O.sub.10 592.558 ##STR00095## Mulberrofuran S Morus
alba (white mulberry) C.sub.34H.sub.24O.sub.9 576.558 ##STR00096##
Mulberrofuran T Morus alba (white mulberry) C.sub.44H.sub.44O.sub.9
716.826 ##STR00097## Mulberrofuran U Morus insignis
C.sub.39H.sub.36O.sub.9 648.708 ##STR00098## Multicaulisin Morus
multicaulis C.sub.40H.sub.36O.sub.11 692.718 ##STR00099## Sanggenol
G Morus cathayana C.sub.30H.sub.34O.sub.7 694.734 ##STR00100##
Sanggenol J Morus cathayana C.sub.45H.sub.44O.sub.12 776.835
##STR00101## Sanggenol M Morus mongolica C.sub.44H.sub.44O.sub.11
748.825 ##STR00102## Sanggenon B Morus C.sub.33H.sub.30O.sub.9
570.595 ##STR00103## Sanggenon B; 7-O-(2,4- Dihydroxy benzoyl)
(Sanggenon S) Morus sp C.sub.40H.sub.34O.sub.12 706.701
##STR00104## Sanggenon D Morus cathayana C.sub.40H.sub.36O.sub.12
708.717 ##STR00105## Sanggenon E Morus Spp.
C.sub.45H.sub.44O.sub.12 776.835 ##STR00106## Sanggenon G Morus
alba C.sub.40H.sub.38O.sub.11 694.734 ##STR00107## Sanggenon G;
14,15-Dihydro, 15-hydroxy Morus sp. C.sub.40H.sub.40O.sub.12
712.749 ##STR00108## Sanggenon Q Morus mongolica
C.sub.40H.sub.36O.sub.12 708.717 ##STR00109## Sanggenon D;
3'-Epimer Morus cathayana C.sub.40H.sub.36O.sub.12 708.717
##STR00110## Sanggenon D; 2,3,3'- Triepimer Morus cathayana
C.sub.40H.sub.36O.sub.12 708.717
##STR00111## Sorocein B Sorocea bonplandii C.sub.40H.sub.34O.sub.9
658.703 ##STR00112## Sorocein H Sorocea bonplandii (Moraceae) and
Morus spp. C.sub.45H.sub.44O.sub.12 776.835 ##STR00113##
Wittiorumin B Morus wittiorum C.sub.40H.sub.36O.sub.12 708.717
##STR00114## Wittiorumin B; 1''-Epimer, 2'- deoxy Morus wittiorum
C.sub.40H.sub.36O.sub.11 692.718 ##STR00115## Wittiorumin E Morus
wittiorum C.sub.40H.sub.38O.sub.10 678.734 ##STR00116## Wittiorumin
F Morus wittiorum C.sub.39H.sub.36O.sub.9 648.708 ##STR00117##
Wittiorumin G Morus wittiorum C.sub.40H.sub.38O.sub.10 678.734
##STR00118## Yunanensin A Morus yunnanensis C.sub.39H.sub.28O.sub.8
624.645
[0103] Compounds in Table A can be extracted, isolated or purified
from the indicated plant species or certain plant parts (e.g., from
the bark, trunk, trunk bark, stem bark, root, root bark, bark
surface, leaves, fruits, flowers, other plant parts, or any
combination thereof) or can be prepared synthetically or
semi-synthetically as described in more detail below. In certain
embodiments, one or more compounds of Table A are enriched for or
are the major active ingredients in an extract of the indicated
plant species, wherein the enriched extract is obtained from a
whole plant or certain plant parts, such as leaves, bark, trunk,
trunk bark, stem, stem bark, twigs, tubers, root, root bark, bark
surface, young shoots, rhizomes, seed, fruit, androecium,
gynoecium, calyx, stamen, petal, sepal, carpel (pistil), flower, or
any combination thereof.
[0104] It is understood that any embodiment of the compounds of
structure (I) or (II), as set forth above, and any specific
substituent set forth herein for the compounds of structure (I) or
(II), as set forth above, may be independently combined with other
embodiments or substituents of compounds of structure (I) or (II)
to form embodiments of this disclosure not specifically set forth
above. In addition, in the event that a list of substituents is
listed for any particular R group in a particular embodiment or
claim, it is understood that each individual substituent may be
deleted from the particular embodiment or claim and that the
remaining list of substituents will be considered to be within the
scope of this disclosure.
[0105] For the purposes of administration, the compounds of the
present invention may be administered as a raw chemical or may be
formulated as pharmaceutical or nutraceutical compositions.
Pharmaceutical or nutraceutical compositions of the present
invention comprise a compound of structure (I) or (II) and a
pharmaceutically or nutraceutically acceptable carrier, diluent or
excipient. The compound of structure (I) or (II) is present in the
composition in an amount which is effective to treat a particular
disease or condition of interest--that is, in an amount sufficient
promote weight loss or any of the other associated indications
described herein, and generally with acceptable toxicity to a
patient. Weight loss and other activity of compounds of structure
(I) or (II) can be determined by one skilled in the art, for
example, as described in the Examples below. Appropriate
concentrations and dosages can be readily determined by one skilled
in the art.
[0106] Administration of the compounds of this disclosure, or their
pharmaceutically or nutraceutically acceptable salts, in pure form
or in an appropriate pharmaceutical or nutraceutical composition,
can be carried out via any of the accepted modes of administration
of agents for serving similar utilities. The pharmaceutical or
nutraceutical compositions of this disclosure can be prepared by
combining a compound of this disclosure with an appropriate
pharmaceutically or nutraceutically acceptable carrier, diluent or
excipient, and may be formulated into preparations in solid,
semi-solid, liquid or gaseous forms, such as tablets, capsules,
powders, granules, ointments, solutions, suppositories, injections,
inhalants, gels, microspheres, and aerosols. Typical routes of
administering such pharmaceutical or nutraceutical compositions
include oral, topical, transdermal, inhalation, parenteral,
sublingual, buccal, rectal, vaginal, or intranasal. The term
parenteral as used herein includes subcutaneous injections,
intravenous, intramuscular, intrasternal injection or infusion
techniques. Pharmaceutical or nutraceutical compositions of this
disclosure are formulated so as to allow the active ingredients
contained therein to be bioavailable upon administration of the
composition to a patient. Compositions that will be administered to
a subject or patient take the form of one or more dosage units,
where for example, a tablet may be a single dosage unit, and a
container of a compound of this disclosure in aerosol form may hold
a plurality of dosage units. Actual methods of preparing such
dosage forms are known, or will be apparent, to those skilled in
this art; for example, see Remington: The Science and Practice of
Pharmacy, 20th Edition (Philadelphia College of Pharmacy and
Science, 2000). The composition to be administered will, in any
event, contain a therapeutically effective amount of a compound of
this disclosure, or a pharmaceutically or nutraceutically
acceptable salt thereof, for treatment of a disease or condition of
interest in accordance with the teachings of this invention.
[0107] A pharmaceutical or nutraceutical composition of this
disclosure may be in the form of a solid or liquid. In one aspect,
the carrier(s) are particulate, so that the compositions are, for
example, in tablet or powder form. The carrier(s) may be liquid,
with the compositions being, for example, oral syrup, injectable
liquid or an aerosol, which is useful in, for example, inhalatory
administration.
[0108] When intended for oral administration, the pharmaceutical or
nutraceutical composition is in either solid or liquid form, where
semi-solid, semi-liquid, suspension and gel forms are included
within the forms considered herein as either solid or liquid.
[0109] As a solid composition for oral administration, the
pharmaceutical or nutraceutical composition may be formulated into
a powder, granule, compressed tablet, pill, capsule, chewing gum,
wafer, bar, or like form. Such a solid composition will typically
contain one or more inert diluents or edible carriers. In addition,
one or more of the following may be present; binders such as
carboxymethylcellulose, ethyl cellulose, cyclodextrin,
microcrystalline cellulose, gum tragacanth or gelatin; excipients
such as starch, lactose or dextrins, disintegrating agents such as
alginic acid, sodium alginate. Primogel, corn starch and the like;
lubricants such as magnesium stearate or Sterotex; glidants such as
colloidal silicon dioxide; sweetening agents such as sucrose or
saccharin; a flavoring agent such as peppermint, methyl salicylate
or orange flavoring; and a coloring agent.
[0110] When the pharmaceutical or nutraceutical composition is in
the form of a capsule, for example, a gelatin capsule, it may
contain, in addition to materials of the above type, a liquid
carrier such as polyethylene glycol or oil.
[0111] The pharmaceutical or nutraceutical composition may be in
the form of a liquid, for example, an elixir, syrup, solution,
emulsion or suspension. The liquid may be for oral administration
or for delivery by injection, as two examples. When intended for
oral administration, a useful composition contains, in addition to
the present compounds, one or more of a sweetening agent,
preservatives, dye/colorant and flavor enhancer, in a composition
intended to be administered by injection, one or more of a
surfactant, preservative, wetting agent, dispersing agent,
suspending agent, buffer, stabilizer and isotonic agent may be
included.
[0112] The liquid pharmaceutical or nutraceutical compositions of
this disclosure, whether they be solutions, suspensions or other
like form, may include one or more of the following adjuvants:
sterile diluents such as water for injection, saline solution, such
as physiological saline, Ringer's solution, isotonic sodium
chloride, fixed oils such as synthetic mono or diglycerides which
may serve as the solvent or suspending medium, polyethylene
glycols, glycerin, propylene glycol or other solvents;
antibacterial agents such as benzyl alcohol or methyl paraben;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. The parenteral
preparation can be enclosed in ampoules, disposable syringes or
multiple dose vials made of glass or plastic. Physiological saline
is a generally useful adjuvant. An injectable pharmaceutical or
nutraceutical composition is sterile.
[0113] A liquid pharmaceutical or nutraceutical composition of this
disclosure intended for either parenteral or oral administration
should contain an amount of a compound of this disclosure such that
a suitable dosage will be obtained.
[0114] The pharmaceutical or nutraceutical composition of this
disclosure may be intended for topical administration, in which
case the carrier may suitably comprise a solution, emulsion, cream,
lotion, ointment, or gel base. The base, for example, may comprise
one or more of the following; petrolatum, lanolin, polyethylene
glycols, bee wax, mineral oil, diluents such as water and alcohol,
and emulsifiers and stabilizers. Thickening agents may be present
in a pharmaceutical or nutraceutical composition for topical
administration. If intended for transdermal administration, the
composition may include a transdermal patch or iontophoresis
device.
[0115] The pharmaceutical or nutraceutical composition of this
disclosure may be intended for rectal administration, in the form,
for example, of a suppository, which will melt in the rectum and
release the drug. The composition for rectal administration may
contain an oleaginous base as a suitable nonirritating excipient.
Such bases include lanolin, cocoa butter and polyethylene
glycol.
[0116] The pharmaceutical or nutraceutical composition of this
disclosure may include various materials, which modify the physical
form of a solid or liquid dosage unit. For example, the composition
may include materials that form a coating shell around the active
ingredients. The materials that form the coating shell are
typically inert, and may be selected from, for example, sugar,
shellac, and other enteric coating agents. Alternatively, the
active ingredients may be encased in a gelatin capsule.
[0117] The pharmaceutical or nutraceutical composition of this
disclosure in solid or liquid form may include an agent that binds
to the compound of this disclosure and thereby assists in the
delivery of the compound. Suitable agents that may act in this
capacity include a monoclonal or polyclonal antibody, a protein or
a liposome.
[0118] The pharmaceutical or nutraceutical composition of this
disclosure in solid or liquid form may include reducing the size of
a particle to, for example, improve bioavailability. The size of a
powder, granule, particle, microsphere, or the like in a
composition, with or without an excipient, can be macro (e.g.,
visible to the eye or at least 100 .mu.m in size), micro (e.g., may
range from about 100 .mu.m to about 100 nm in size), nano (e.g.,
may no more than 100 nm in size), and any size in between or any
combination thereof to improve size and bulk density.
[0119] The pharmaceutical or nutraceutical composition of this
disclosure may consist of dosage units that can be administered as
an aerosol. The term aerosol is used to denote a variety of systems
ranging from those of colloidal nature to systems consisting of
pressurized packages. Delivery may be by a liquefied or compressed
gas or by a suitable pump system that dispenses the active
ingredients. Aerosols of compounds of this disclosure may be
delivered in single phase, bi-phasic, or tri-phasic systems in
order to deliver the active ingredient(s). Delivery of the aerosol
includes the necessary container, activators, valves,
subcontainers, and the like, which together may form a kit. One
skilled in the art, without undue experimentation, may determine
the most appropriate aerosol(s).
[0120] The pharmaceutical or nutraceutical compositions of this
disclosure may be prepared by methodology well known in the
pharmaceutical or nutraceutical art. For example, a pharmaceutical
or nutraceutical composition intended to be administered by
injection can be prepared by combining a compound of this
disclosure with sterile, distilled water so as to form a solution.
A surfactant may be added to facilitate the formation of a
homogeneous solution or suspension. Surfactants are compounds that
non-covalently interact with the compound of this disclosure so as
to facilitate dissolution or homogeneous suspension of the compound
in the aqueous delivery system.
[0121] The compounds of this disclosure, or their pharmaceutically
or nutraceutically acceptable salts, are administered in a
therapeutically effective amount, which will vary depending upon a
variety of factors including the activity of the specific compound
employed; the metabolic stability and length of action of the
compound; the age, body weight, general health, sex, and diet of
the patient; the mode and time of administration, the rate of
excretion; the drug combination; the severity of the particular
disorder or condition; and the subject undergoing therapy.
[0122] Compounds of this disclosure, or pharmaceutically or
nutraceutically acceptable derivatives thereof, may also be
administered simultaneously with, prior to, or after administration
of one or more other therapeutic agents. Such combination therapy
includes administration of a single pharmaceutical or nutraceutical
dosage formulation which contains a compound of this disclosure and
one or more additional active agents, as well as administration of
the compound of this disclosure and each active agent in its own
separate pharmaceutical or nutraceutical dosage formulation. For
example, a compound of this disclosure and another active agent can
be administered to the patient together in a single oral dosage
composition, such as a tablet or capsule, or each agent can be
administered in separate oral dosage formulations. Where separate
dosage formulations are used, the compounds of this disclosure and
one or more additional active agents can be administered at
essentially the same time, i.e., concurrently, or at separately
staggered times, i.e., sequentially; combination therapy is
understood to include all these regimens.
[0123] It is understood that in the present description,
combinations of substituents or variables of the depicted formulae
are permissible only if such contributions result in stable
compounds.
[0124] It will also be appreciated by those skilled in the art that
in the process described herein the functional groups of
intermediate compounds may need to be protected by suitable
protecting groups. Such functional groups include hydroxy, amino,
mercapto and carboxylic acid. Suitable protecting groups for
hydroxy include trialkylsilyl or diarylalkylsilyl (for example,
t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl),
tetrahydropyranyl, benzyl, and the like. Suitable protecting groups
for amino, amidino and guanidino include t-butoxycarbonyl,
benzyloxycarbonyl, and the like. Suitable protecting groups for
mercapto include --C(O)--R'' (where R'' is alkyl, aryl or
arylalkyl), p-methoxybenzyl, trityl and the like. Suitable
protecting groups for carboxylic acid include alkyl, aryl or
arylalkyl esters. Protecting groups may be added or removed in
accordance with standard techniques, which are known to one skilled
in the art and as described herein. The use of protecting groups is
described in detail in Green, T. W. and P. G. M. Wutz, Protective
Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As one of skill
in the art would appreciate, the protecting group may also be a
polymer resin such as a Wang resin, Rink resin or a
2-chlorotrityl-chloride resin.
[0125] It will also be appreciated by those skilled in the art,
although such protected derivatives of compounds of this invention
may not possess pharmacological activity as such, they may be
administered to a mammal and thereafter metabolized in the body to
form compounds of this disclosure which are pharmacologically
active. Such derivatives may therefore be described as "prodrugs".
All prodrugs of compounds of this invention are included within the
scope of this disclosure.
[0126] Furthermore, all compounds of this disclosure which exist in
free base or acid form can be converted to their pharmaceutically
or nutraceutically acceptable salts by treatment with the
appropriate inorganic or organic base or acid by methods known to
one skilled in the art. Salts of the compounds of this disclosure
can be converted to their free base or acid form by standard
techniques.
[0127] In some embodiments, compounds of the present disclosure can
be isolated from plant sources, for example, from those plants
included in Table A and elsewhere throughout the present
application. Suitable plant parts for isolation of the compounds
include leaves, bark, trunk, trunk bark, stem, stem bark, twigs,
tuber %, root, root bark, bark surface, young shoots, rhizomes,
seed, fruit, androecium, gynoecium, calyx, stamen, petal, sepal,
carpel (pistil), flower, or any combination thereof. In some
related embodiments, the compounds are isolated from plant sources
and synthetically modified to contain any of the recited
substituents. In this regard, synthetic modification of the
compound isolated from plants can be accomplished using any number
of techniques which are known in the art and are well within the
knowledge of one of ordinary skill in the art.
[0128] As noted above, the compounds of the present invention are
Diels-Alder adducts of a chalcone and prenylphenyl moiety. While
not wishing to be bound by theory, it is believed that the
Diels-Alder reaction occurs as part of the biosynthetic pathway in
various plants, and thus certain embodiments of this disclosure
include isolating the compounds from plants and using as is or
performing various synthetic modifications. However, in other
embodiments the compounds are prepared synthetically. For example,
the following Reaction Scheme illustrates a method of making
compounds of this invention. i.e., compound of structure (I) of
(II) using synthetic techniques:
##STR00119##
[0129] Representative compounds of structure (I) or (II) (e.g.,
(Ib) or (IIb)) can be prepared synthetically according to the
general procedure illustrated in Reaction Scheme 1. Chalcone A and
isoprenylphneyl B may be purchased from commercial sources,
isolated from plant sources or prepared according to procedures
known in the art. Reaction of chalcone A with isoprenylphneyl B
under appropriate Diels-Alder conditions (which are known in the
art) results in compounds of structure (I) or (II). Compounds of
structure (I) and (II) are regioisomers as a result of the
asymmetry of chalcone A. The various substituents (i.e., the "R"
groups) can be installed prior to Diels-Alder cyclization as
depicted in Reaction Scheme 1 or installed after cyclization.
Methods for modification of the above method and for adding various
R groups are well known in the art and within the grasp of one of
ordinary skill in the art.
[0130] It is understood that one skilled in the art may be able to
make these compounds by similar methods or by combining other
methods known to one skilled in the art. It is also understood that
one skilled in the art would be able to make, in a similar manner
as described below, other compounds of structure (I) or (II) not
specifically illustrated below by using the appropriate starting
components and modifying the parameters of the synthesis as needed.
In general, starting components may be obtained from sources such
as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix
Scientific, TCI, and Fluorochem USA, etc. or synthesized according
to sources known to those skilled in the art (see, for example.
Advanced Organic Chemistry: Reactions, Mechanisms, and Structure,
5th edition (Wiley, December 2000)) or prepared as described in
this invention.
[0131] In further embodiments, at least one Diels-Alder adduct of a
chalcone and prenylphenyl moiety of the present disclosure may be
combined with one or more weight management agents. A "weight
management agent," as used herein, refers to a biologically active
compound, molecule, or composition that allows a subject to manage
their weight, which may involve maintaining a particular weight
level, reducing weight gain or reducing weight. The biological
activity of the weight management agents may include reducing or
suppressing appetite, altering metabolic levels, altering lipid
metabolism, decreasing caloric intake, or the like. Exemplary
weight management agents include anorectic agents, lipase
inhibitors, cannabinoid receptor modulators, psychotropic agents,
insulin sensitizers, stimulants, satiety agents, or combinations
thereof.
[0132] In certain embodiments, Diels-Alder adducts of a chalcone
and prenylphenyl moiety of the present disclosure are used with at
least one other weight management agent, such as an anorectic
agent, a lipase inhibitor, a cannabinoid receptor modulator, a
psychotropic agent, an insulin sensitizer, a stimulant, or a
satiety agent. The weight management agents and the Diels-Alder
adducts of a chalcone and prenylphenyl moiety of the present
disclosure may be formulated together or separately. In addition,
the Diels-Alder adducts of a chalcone and prenylphenyl moiety of
the present disclosure may be administered or taken by a subject
simultaneously with, prior to, or after administration of the at
least one other weight management agent.
[0133] In certain embodiments, the anorectic agent is sibutramine,
diethylpropion, benzphetamine, phendimetrazine, or catecholamine.
In further embodiments, the lipase inhibitor is a Mutamba extract,
a Rosemary extract, carnosic acid, carnosol, lipostatin,
tetrahydrolipostatin, Punica granatum pericarp extract, Marchantia
polymorpha whole plant extract, Panax japonicas extract or
Platycodi radix extract.
[0134] In still further embodiments, the cannabinoid receptor
modulator is a cannabinoid receptor agonist, antagonist, or inverse
agonist. In yet further embodiments, the cannabinoid receptor
modulator is specific for cannabinoid receptor 1 (CB1), cannabinoid
receptor 2 (CB2), or both CB1 and CB2, such as a CB1 antagonist or
inverse agonist. Exemplary cannabinoid receptor modulators include
rimonabant,
N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-py-
razole-3-carboxamide (AM 251),
1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyraz-
ole-3-carboxamide (AM281),
4-[6-methoxy-2-(4-methoxypheyl)1-benzofuran-3-carbonyl]benzonitrile
(LY 320135), Magnolia extract, magnolol, honokiol, magnolol and
honokiol, purinol, or Piper Longum seed extract. In certain
embodiments, Diels-Alder adducts of a chalcone and prenylphenyl
moiety of the present disclosure may be used with a second, third,
fourth, or fifth Diels-Alder adduct of a chalcone and a
prenylphenyl moiety. In certain embodiments, the weight management
agents includes two, three, four, five, six, seven, eight, nine, or
ten Diels-Alder adducts of a chalcone and a prenylphenyl
moiety.
[0135] For example, certain embodiments Diels-Alder adducts of a
chalcone and prenylphenyl moiety of the present disclosure may be
used with an anorectic agent and a lipase inhibitor, or an
anorectic agent and a cannabinoid receptor modulator, or an
anorectic agent and a psychotropic agent, an anorectic agent and an
insulin sensitizer, or an anorectic agent and a stimulant, or an
anorectic agent and a satiety agent. In still further embodiments,
Diels-Alder adducts of a chalcone and prenylphenyl moiety of the
present disclosure may be used with a lipase inhibitor and a
stimulant, or a cannabinoid receptor modulator and a stimulant, or
a cannabinoid receptor modulator and a lipase inhibitor. Any of the
aforementioned compositions may further comprise a satiety agent or
a psychotropic agent or an insulin sensitizer.
[0136] In certain embodiments, Diels-Alder adducts of a chalcone
and prenylphenyl moieties used alone, in combination, or with
another weight management agent have a prenylphenyl moiety that is
a prenylated phenol, an isoprenylated flavonoid, a prenylated
flavonoid, a prenylated flavoinoid dimer, or a combination thereof.
In certain embodiments, the Diels-Alder adducts of a chalcone and a
prenylphenyl moiety have a prenylphenyl moiety that is an
isoprenylated flavonoid such as an isoprenylated flavone, flavonol,
flavanone, chalcone, isoflavone, isoflavanone, aurone, or stilbene.
In certain embodiments, Diels-Alder adducts of a chalcone and a
prenylphenyl moiety have a prenylated flavonoid that is a
prenylated flavone, flavonol, flavanone, chalcone, isoflavone,
isoflavanone, aurone, or stilbene. In further embodiments,
Diels-Alder adducts of a chalcone and a prenylphenyl moiety used
alone, in combination, or with one or more other weight management
agents purified from, isolated from, enriched for, or contained in
a Moru extract or a Milicia excelsa extract, such as Albanin G,
Kuwanon G, Kuwanon M, Cathayanon A, Morusin, Morusinol, Sanggenon
C, Sanggenon D, Sanggenon O, or any combination thereof.
[0137] In further embodiments, the weight management agent used
with Diels-Alder adducts of a chalcone and a prenylphenyl moiety is
a psychotropic agent such as a mood stabilizer, anti-depressant, or
anti-convulsant. In still further embodiments, the weight
management agent used with Diels-Alder adducts of a chalcone and a
prenylphenyl moiety is a stimulant such as caffeine,
dicaffeoylquinic acid or dextroamphetamine, or one or more
stimulant purified from, isolated from, enriched for, or contained
in a Yerba Mate extract, green tea extract, green coffee bean
extract, Cola nut extract, Citrus aurantium fruit extract, Gacinia
extract, Areca catechu fruit/seed extract or dextroamphetamine. In
even further embodiments, the weight management agent used with
Diels-Alder adducts of a chalcone and a prenylphenyl moiety is an
insulin sensitizer such as thiazolidinediones (e.g., rosiglitazone,
pioglitazone), oxazolidinediones, isoxazolidinediones, biguanides
(e.g., metformin), selective mTOT (mitochondrial Target of
Thiazolidinediones) modulators, cinnamon extract, banaba extract,
chromium, fish oil, acetic acid, D-chiro-inositol, or
.alpha.-lipoic acid. In yet further embodiments, the weight
management agent used with Diels-Alder adducts of a chalcone and a
prenylphenyl moiety is a satiety agent such as dodecanoic acid,
glyceryl dodecanoate, glyceryl 1,3-didodecanoate, glyceryl
tridodecanoate, and derivatives or mixtures thereof, or one or more
satiety agent purified from, isolated from, enriched for, or
contained in a hoodia extract, a pine nut extract, or a fiber
supplement.
[0138] As noted herein, compounds of a Diels-Alder adduct of a
chalcone and a prenylphenyl moiety may be obtained by chemical
synthesis or from a plant extract, such as a Morus or Milicia
extract. For example, Morus is a genus of flowering trees in the
family Moraceae, which comprises more than 30 species (known as
mulberries) that grow wild or under cultivation in many countries.
Exemplary Morus species include Morus alba L., Morus australis
Poir, Morus celtidifolia Kunth, Morus insignis, Morus mesozygia
Stapf, Morus microphylla, Morus nigra L., Morus rubra L., Morus
atropurpurea, Morus bombycis, Morus cathayana, Morus indica, Morus
lhou, Morus japonica, Morus kagavamae, Morus laevigata, Morus
latifolia, Morus liboensis, Morus macroura, Morus mongolica, Morus
multicaulis, Morus notabilis, Morus rotundiloba, Morus serrate,
Morus heterophyllus, Mortis tillaefblia, Morus trilobata, Morus
yunnanensis, and Morus wittiorum. In certain embodiments, a Morus
extract is from Morus alba, or a Morus extract is a mixture of
extracts from one, two, three, four, or five different Morus
species. A mixture of extracts may include extracts from two or
more Morus species listed in Table A.
[0139] Magnolia includes about 210 flowering plant species in the
subfamily Magnolioideae of the family Magnoliaceae. Magnolia, known
as Hou Pu, can grow wild or under cultivation in many countries.
Exemplary Magnolia species includes Magnolia obovata Thunhb.,
Magnolia officinalis Rehd. & Wilson, Magnolia rostrata K. W.
Smith, Magnolia tripelata, Magnolia globosa Hook. f & Thoms,
Magnolia sieboldii K. Kuoch, Magnolia wilsonii (Finet. &
Gagnep.) Rehd., Magnolia fraseri Walt. Magnolia macrophylla Michx,
Magnolia decidua (Q. Y. Zheng) V. S. Kumar, Magnolia dolichogyna
(Dandy ex Noot.) Figlar & Noot., Magnolia duclouxii Finet &
Gagnep., Magnolia figlarii V S. Kumar, Magnolia fordiana, Magnolia
emarginata Urb. & Ekman, Magnolia hamorii Howard, Magnolia
pallescens Urb. & Ekmran, Magnolia mahechae (Lozano) Govaerts,
Magnolia ptaritepuiana Stevermark, Magnolia striatifolia Little,
Magnolia mexicana DC., Magnolia minor (Urb. Govaerts, Magnolia
morii (Lozano) Govaerts, Magnolia narinensis (Lozano) Govaerts,
Magnolia neillii (Lozano) Govaerts, Magnolia ovata (A.St.-Hil.)
Spreng, Magnolia polyhypsophilla (Lozano) Govaerts, Magnolia
quetzal, Magnolia rimachii (Lozano) Govaerts, Magnolia sambuensis,
Magnolia delavayi Franchet, Magnolia fistulosa (Finet &
Gagnep.) Dandy, Magnolia henryi Dunn, Magnolia nana Dandy, Magnolia
odoratissima, Magnolia grandiflora L, and Magnolia guatemalensis.
In certain embodiments, a Magnolia extract is from Magnolia
officinalis, or a Magnolia extract is a mixture of extracts from
one, two, three, four, or five different Magnolia species.
[0140] Rosmarinus officinalis, commonly known as Rosemary, is a
woody, perennial herb with fragrant, evergreen, needle-like leaves
and white, pink, purple, or blue flowers, native to the
Mediterranean region. Rosemary is a member of the mint family
Lamiaceae, which includes many other herbs. Other exemplary
Rosemary species include Rosmarinus tomentosus, Rosmarinus
eriocalyx, and Rosmarinus palaui. In certain embodiments, a
Rosemary extract is from Rosmarinus officinalis, or a Rosemary
extract is a mixture of extracts from one, two, or three Rosemary
species.
[0141] The plant origin for most commonly utilized Yerba Mate is
Ilex paraguariensis, which is a species of holly (family
Aquifoliaceae) well known as a source of the beverage called mate.
Yerba Mate is native to subtropical South America in northeastern
Argentina, Bolivia, southern Brazil, Uruguay and Paraguay. The
genus is distributed throughout the world's different climates.
Exemplary Ilex species includes American Holly (Itex opaca),
Carolina Holly (Ilex ambigua), Chinese Holly (Ilex cornuta), Common
Winterberry (Ilex verticillata), Dahoon (Ilex cassine), Deciduous
Holly (Ilex decidua), English Holly (Ilex aquifolium), Australia
Holly (Ilex arnhemensis), Inkberry (Ilex glabra), Japanese Holly
(Ilex crenata), Large Gallberry (Ilex coriacea), Smooth Winterberry
(Ilex laevigata), Yaupon (Ilex vomitoria), Africa species (Ilex
mitis), and Ilex canariensis Alacaronesia, Ilex aquifolium). Ilex
mucronata, formerly the type species of Nemopanthus, is native to
eastern North America. Nemopanthus was treated as a monotypic genus
with eight species of the family Aquifoliaceae, now transferred to
Ilex based on molecular data (closely related to Ilex amelanchier).
In certain embodiments, a Yerba Mate extract is from Ilex
paraguariensis, or a Yerba Mate extract is a mixture of extracts
from one, two, three, four, or five Ilex species.
[0142] Guazuma, a genus of flowering trees in the family Malvaceae,
is widely found in the Caribbean, South American, Central America
and Mexico. Mutamba is a common name of Guazuma plant with various
medicinal values in traditional herbal medicine. Exemplary
condensed tannins of this disclosure maybe extracted from different
species of Guazuma plant, including Guazuma commersoniopsis,
Guazuma euguazuma, Guazuma gynophoricola, Guazuma blumei, Guazuma
bubroma, Guazuma burbroma, Guazuma corlacea, Guazuma crinita,
Guazuma grandiflora, Guazuma guazuma, Guazuma invira, Guazuma
iuvira, Guazuma longipedicellata, Guazuma parvifolia, Guazuma
polybotra, Guazuma polybotrya, Guazuma rosea, Guazuma tomentosa,
Guazuma ulmifolia, and Guazuma utilis. In certain embodiments, a
Mutamba extract is from Guazuma ulmifolia, or a Mutamba extract is
a mixture of extracts from one, two, three, four, or five Guazuma
species.
[0143] In certain embodiments, a composition comprising a
Diels-Alder adduct of a chalcone and a prenylphenyl moiety
comprises a Morus extract containing or enriched for at least one
Diels-Alder adduct of a chalcone and a prenylphenyl moiety, a
Magnolia extract, and a Yerba Mate extract, which extracts can be
mixed or used together in a 2:1:5 weight ratio to a 2:1:10 weight
ratio, respectively. In further embodiments, a composition
comprising a Diels-Alder adduct of a chalcone and a prenylphenyl
moiety and a weight management agent comprises a Morus extract
containing or enriched for at least one Diels-Alder adduct of a
chalcone and a prenylphenyl moiety, a Magnolia extract, and a Yerba
Mate extract, which extracts can be mixed or used together in a
2:1:5 weight ratio to a 2:1:10 weight ratio, respectively. In any
of the aforementioned embodiments, a Morus extract is a Morus alba
extract or extract enriched for Kuwanon G, Albanin G, Morusin, or
any combination thereof; a Magnolia extract is a Magnolia
officinalis extract or extract enriched for magnolol, honokiol,
both, or a high purity mixture of both; and a Yerba Mate extract is
an Ilex paraguayensis extract is enriched for caffeine,
dicaffeoylquinic acid, or both. In related embodiments, a weight
management agent is an anorectic agent, lipase inhibitor,
cannabinoid receptor modulator, psychotropic agent, insulin
sensitizer, stimulant, satiety agent, or any combination thereof.
In any of these embodiments, a weight management agent is an
anorectic agent. In any of these embodiments, a weight management
agent is a lipase inhibitor. In any of these embodiments, a weight
management agent is a cannabinoid receptor modulator. In any of
these embodiments, a weight management agent is a psychotropic
agent. In any of these embodiments, a weight management agent is an
insulin sensitizer. In any of these embodiments, a weight
management agent is a stimulant. In any of these embodiments, a
weight management agent is a satiety agent.
[0144] In certain embodiments, a composition comprising a
Diels-Alder adduct of a chalcone and a prenylphenyl moiety
comprises a Diels-Alder adduct of a chalcone and a prenylphenyl
moiety, Purinol (a high purity, such as at least 90% purity, or at
least 91%, 92%, 93%, 94%, or 95% purity, magnolol and honokiol
mixture isolated from Magnolia plant extract), and Yerba Mate
extract, wherein the Diels-Alder adduct of a chalcone and a
prenylphenyl moiety is Albanin G, Kuwanon G, Kuwanon M, Cathayanon
A, Morusin, Morusinol, Sanggenon C. Sanggenon D, Sanggenon O or any
combination thereof. In further embodiments, a composition
comprising a Diels-Alder adduct of a chalcone and a prenylphenyl
moiety and a weight management agent comprises a Diels-Alder adduct
of a chalcone and a prenylphenyl moiety, Purinol (a high purity,
such as at least 90% purity, or at least 91%, 92%, 93%, 94%, or 95%
purity) magnolol and honokiol mixture isolated from Magnolia plant
extract), and Yerba Mate extract, wherein the Diels-Alder adduct of
a chalcone and a prenylphenyl moiety is Albanin G, Kuwanon G,
Kuwanon M, Cathayanon A, Morusin, Morusinol, Sanggenon C, Sanggenon
D, Sanggenon O, or any combination thereof, and the Yerba Mate
extract is an Ilex paraguayensis extract enriched for caffeine,
dicaffeoylquinic acid, or both.
[0145] In certain embodiments, a composition comprising a
Diels-Alder adduct of a chalcone and a prenylphenyl moiety
comprises a Morus extract containing or enriched for at least one
Diels-Alder adduct of a chalcone and a prenylphenyl moiety, a
Magnolia extract, and a Mutamba extract, which can be mixed or used
together in a 2:1:5 weight ratio to a 2:1:10 weight ratio,
respectively. In further embodiments, a composition comprising a
Diets-Alder adduct of a chalcone and a prenylphenyl moiety and a
weight management agent comprises a Morus extract containing or
enriched for at least one Diels-Alder adduct of a chalcone and a
prenylphenyl moiety, a Magnolia extract, and a Mutamba extract,
which extracts can be mixed or used together in a 2:1:5 weight
ratio to a 2:1:10 weight ratio, respectively. In any of the
aforementioned embodiments, a Morus extract is a Morus alba extract
or extract enriched for Kuwanon G, Albanin G, Morusin, or any
combination thereof, and a Magnolia extract is a Magnolia
officinalis extract or extract enriched for magnolol, honokiol,
both, or a high purity mixture of both. In related embodiments, a
weight management agent is an anorectic agent, lipase inhibitor,
cannabinoid receptor modulator, psychotropic agent, insulin
sensitizer, stimulant, satiety agent, or any combination thereof.
In any of these embodiments, a weight management agent is an
anorectic agent. In any of these embodiments, a weight management
agent is a lipase inhibitor. In any of these embodiments, a weight
management agent is a cannabinoid receptor modulator. In any of
these embodiments, a weight management agent is a psychotropic
agent. In any of these embodiments, a weight management agent is an
insulin sensitizer. In any of these embodiments, a weight
management agent is a stimulant. In any of these embodiments, a
weight management agent is a satiety agent.
[0146] In certain embodiments, a composition comprising a
Diels-Alder adduct of a chalcone and a prenylphenyl moiety
comprises a Diels-Alder adduct of a chalcone and a prenylphenyl
moiety, purinol, and Mutamba extract, wherein the Diels-Alder
adduct of a chalcone and a prenylphenyl moiety is Albanin G,
Kuwanon G, Kuwanon M, Cathayanon A, Morusin, Morusinol, Sanggenon
C, Sanggenon D, Sanggenon O, or any combination thereof. In further
embodiments, a composition comprising a Diels-Alder adduct of a
chalcone and a prenylphenyl moiety and a weight management agent
comprises a Diels-Alder adduct of a chalcone and a prenylphenyl
moiety, purinol, and Mutamba extract, wherein the Diels-Alder
adduct of a chalcone and a prenylphenyl moiety is Albanin G,
Kuwanon G, Kuwanon M, Cathayanon A, Morusin, Morusinol, Sanggenon
C. Sanggenon D, Sanggenon O. or any combination thereof.
[0147] In certain embodiments, a composition comprising a
Diels-Alder adduct of a chalcone and a prenylphenyl moiety
comprises a Morus extract containing or enriched for at least one
Diels-Alder adduct of a chalcone and a prenylphenyl moiety, a
Rosemary extract, and a Yerba Mate extract, which can be mixed or
used together in a 2:5:5 weight ratio to a 2:5:10 weight ratio,
respectively. In further embodiments, a composition comprising a
Die s-Alder adduct of a chalcone and a prenylphenyl moiety and a
weight management agent comprises a Morus extract containing or
enriched for at least one Diels-Alder adduct of a chalcone and a
prenylphenyl moiety, a Rosemary extract, and a Yerba Mate extract,
which extracts can be mixed or used together in a 2:5:5 weight
ratio to a 2:5:10 weight ratio, respectively. In any of the
aforementioned embodiments, a Morus extract is a Morus alba extract
or extract enriched for Kuwanon G, Albanin G, Morusin, or any
combination thereof. In related embodiments, a weight management
agent is an anorectic agent, lipase inhibitor, cannabinoid receptor
modulator, psychotropic agent, insulin sensitizer, stimulant,
satiety agent, or any combination thereof. In any of these
embodiments, a weight management agent is an anorectic agent. In
any of these embodiments, a weight management agent is a lipase
inhibitor. In any of these embodiments, a weight management agent
is a cannabinoid receptor modulator. In any of these embodiments, a
weight management agent is a psychotropic agent. In any of these
embodiments, a weight management agent is an insulin sensitizer. In
any of these embodiments, a weight management agent is a stimulant.
In any of these embodiments, a weight management agent is a satiety
agent.
[0148] In certain embodiments, a composition comprising a
Diels-Alder adduct of a chalcone and a prenylphenyl moiety
comprises (1) a Diels-Alder adduct of a chalcone and a prenylphenyl
moiety, carnosol, and Yerba Mate extract or (2) a Diels-Alder
adduct of a chalcone and a prenylphenyl moiety, carnosic acid, and
Yerba Mate extract; wherein the Diels-Alder adduct of a chalcone
and a prenylphenyl moiety is Albanin G, Kuwanon G, Kuwanon M,
Cathayanon A.
[0149] Morusin. Morusinol, Sanggenon C, Sanggenon D, Sanggenon O or
any combination thereof, and the Yerba Mate extract is an Ilex
paraguayensis extract is enriched for caffeine, dicaffeoylquinic
acid, or both. In further embodiments, a composition comprising a
Diels-Alder adduct of a chalcone and a prenylphenyl moiety and a
weight management agent comprises (1) a Diels-Alder adduct of a
chalcone and a prenylphenyl moiety, carnosol, and Yerba Mate
extract or (2) a Diels-Alder adduct of a chalcone and a
prenylphenyl moiety, carnosic acid, and Yerba Mate extract; wherein
the Diels-Alder adduct of a chalcone and a prenylphenyl moiety is
Albanin G, Kuwanon G, Kuwanon M, Cathayanon A, Morusin, Morusinol,
Sanggenon C, Sanggenon D, Sanggenon O or any combination thereof,
and the Yerba Mate extract is an Ilex paraguayensis extract is
enriched for caffeine, dicaffeoylquinic acid, or both.
[0150] In certain embodiments, a composition comprising a
Diels-Alder adduct of a chalcone and a prenylphenyl moiety
comprises a Morus extract containing or enriched for at least one
Diels-Alder adduct of a chalcone and a prenylphenyl moiety, a
Rosemary extract, and a mutamba extract, which can be mixed or used
together in a 2:5:5 weight ratio to a 2:5:10 weight ratio,
respectively. In further embodiments, a composition comprising a
Diets-Alder adduct of a chalcone and a prenylphenyl moiety and a
weight management agent comprises a Morus extract containing or
enriched for at least one Diels-Alder adduct of a chalcone and a
prenylphenyl moiety, a Rosemary extract, and a mutamba extract,
which extracts can be mixed or used together in a 2:5:5 weight
ratio to a 2:5;10 weight ratio, respectively. In any of the
aforementioned embodiments, a Morus extract is a Morus alba extract
or extract enriched for Kuwanon G, Albanin G, Morusin, or any
combination thereof. In related embodiments, a weight management
agent is an anorectic agent, lipase inhibitor, cannabinoid receptor
modulator, psychotropic agent, insulin sensitizer, stimulant,
satiety agent, or any combination thereof. In any of these
embodiments, a weight management agent is an anorectic agent. In
any of these embodiments, a weight management agent is a lipase
inhibitor. In any of these embodiments, a weight management agent
is a cannabinoid receptor modulator. In any of these embodiments, a
weight management agent is a psychotropic agent. In any of these
embodiments, a weight management agent is an insulin sensitizer. In
any of these embodiments, a weight management agent is a stimulant.
In any of these embodiments, a weight management agent is a satiety
agent.
[0151] In certain embodiments, a composition comprising a
Diels-Alder adduct of a chalcone and a prenylphenyl moiety
comprises a (1) a Diels-Alder adduct of a chalcone and a
prenylphenyl moiety, carnosol, and mutamba extract or (2) a
Diels-Alder adduct of a chalcone and a prenylphenyl moiety,
carnosic acid, and mutamba extract: wherein the Diels-Alder adduct
of a chalcone and a prenylphenyl moiety is Albanin G, Kuwanon G,
Kuwanon M, Cathayanon A, Morusin, Morusinol, Sanggenon C, Sanggenon
D, Sanggenon O or any combination thereof. In further embodiments,
a composition comprising a Diels-Alder adduct of a chalcone and a
prenylphenyl moiety and a weight management agent comprises a (1) a
Diels-Alder adduct of a chalcone and a prenylphenyl moiety,
carnosol, and mutamba extract or (2) a Diels-Alder adduct of a
chalcone and a prenylphenyl moiety, carnosic acid, and mutamba
extract; wherein the Diels-Alder adduct of a chalcone and a
prenylphenyl moiety is Albanin G, Kuwanon G, Kuwanon M, Cathayanon
A, Morusin, Morusinol, Sanggenon C, Sanggenon D, Sanggenon O or any
combination thereof.
[0152] In certain embodiments, a composition comprising a
Diels-Alder adduct of a chalcone and a prenylphenyl moiety
comprises a Morus extract containing or enriched for at least one
Diels-Alder adduct of a chalcone and a prenylphenyl moiety, a
Rosemary extract, and an Areca extract, which can be mixed or used
together in a 2:5:5 weight ratio to a 2:5:10 weight ratio,
respectively. In further embodiments, a composition comprising a
Diels-Alder adduct of a chalcone and a prenylphenyl moiety and a
weight management agent comprises a Morus extract containing or
enriched for at least one Diels-Alder adduct of a chalcone and a
prenylphenyl moiety, a Rosemary extract, and an Areca extract,
which extracts can be mixed or used together in a 2:5:5 weight
ratio to a 2:5:10 weight ratio, respectively. In any of the
aforementioned embodiments, a Morus extract is a Morus alba extract
or extract enriched for Kuwanon G, Albanin G, Morusin, or any
combination thereof. In related embodiments, a weight management
agent is an anorectic agent, lipase inhibitor, cannabinoid receptor
modulator, psychotropic agent, insulin sensitizer, stimulant,
satiety agent, or any combination thereof. In any of these
embodiments, a weight management agent is an anorectic agent. In
any of these embodiments, a weight management agent is a lipase
inhibitor. In any of these embodiments, a weight management agent
is a cannabinoid receptor modulator. In any of these embodiments, a
weight management agent is a psychotropic agent. In any of these
embodiments, a weight management agent is an insulin sensitizer. In
any of these embodiments, a weight management agent is a stimulant.
In any of these embodiments, a weight management agent is a satiety
agent.
[0153] In certain embodiments, a composition comprising a
Diels-Alder adduct of a chalcone and a prenylphenyl moiety
comprises a (1) a Diels-Alder adduct of a chalcone and a
prenylphenyl moiety, carnosol, and Areca extract or (2) a
Diels-Alder adduct of a chalcone and a prenylphenyl moiety,
carnosic acid, and Areca extract; wherein the Diels-Alder adduct of
a chalcone and a prenylphenyl moiety is Albanin G, Kuwanon G,
Kuwanon M, Cathayanon A, Morusin, Morusinol, Sanggenon C. Sanggenon
D, Sanggenon O or any combination thereof. In further embodiments,
a composition comprising a Diels-Alder adduct of a chalcone and a
prenylphenyl moiety and a weight management agent comprises a (1) a
Diels-Alder adduct of a chalcone and a prenylphenyl moiety,
carnosol, and Areca extract or (2) a Diels-Alder adduct of a
chalcone and a prenylphenyl moiety, carnosic acid, and Areca
extract; wherein the Diels-Alder adduct of a chalcone and a
prenylphenyl moiety is Albanin G, Kuwanon G, Kuwanon M, Cathayanon
A. Morusin, Morusinol, Sanggenon C, Sanggenon D, Sanggenon O or any
combination thereof.
[0154] In any of the aforementioned embodiments, a composition
comprising an extract mixture (optionally including a weight
management agent) can be formulated with a pharmaceutically or
nutraceutically acceptable carrier, diluent or excipient. In
further embodiments, the pharmaceutical or nutraceutical
formulation comprises from about 0.5 weight percent (wt %) to about
90 wt %, about 0.5 wt % to about 80 wt %, about 0.5 wt % to about
75 wt %, about 0.5 wt % to about 70 wt %, about 0.5 wt % to about
50 wt %, about 1.0 wt % to about 40 wt %, about 1.0 wt % to about
20 wt %, about 1.0 wt % to about 10 wt %, about 3.0 wt % to about
9.0 wt %, about 5.0 wt % to about 10 wt %, about 3.0 wt % to about
6.5 wt % of the major active ingredients in an extract mixture, or
the like. In any of these embodiments, the pharmaceutical or
nutraceutical composition is formulated as a tablet, a capsule, a
powder, or granule. In related embodiments, a weight management
agent is an anorectic agent, lipase inhibitor, cannabinoid receptor
modulator, psychotropic agent, insulin sensitizer, stimulant,
satiety agent, or any combination thereof. In any of these
embodiments, a weight management agent is an anorectic agent. In
any of these embodiments, a weight management agent is a lipase
inhibitor. In any of these embodiments, a weight management agent
is a cannabinoid receptor modulator. In any of these embodiments, a
weight management agent is a psychotropic agent. In any of these
embodiments, a weight management agent is an insulin sensitizer. In
any of these embodiments, a weight management agent is a stimulant.
In any of these embodiments, a weight management agent is a satiety
agent.
[0155] In any of the aforementioned embodiments, a composition
comprising an extract mixture (optionally including a weight
management agent) wherein one of the extracts is a Morus extract
enriched for one or more major active ingredients, such as Kuwanon
G, Albanin G, Morusin, or any combination thereof. In certain
embodiments, a Morus extract is a Morus alba extract enriched for
Kuwanon G, Albanin G, Morusin, or any combination thereof. In
further embodiments, a pharmaceutical or nutraceutical formulation
comprising an extract mixture as described herein comprises from
about 0.5 weight percent (wt %) to about 5.0 wt % of the major
active ingredients, such as Kuwanon G, Albanin G, Morusin, or any
combination thereof, in a Morus extract, such as a Morus alba
extract or extract enriched for Kuwanon G, Albanin G, Morusin, or
any combination thereof.
[0156] In any of the aforementioned embodiments, a composition
comprising an extract mixture (optionally including a weight
management agent) wherein one of the extracts is a Guazuma
ulmifolia (Mutamba) extract enriched for one or more major active
ingredients. In certain embodiments, a Guazuma ulmifolia (Mutamba)
extract is enriched for a procyanidin, procyanidin B2, procyanidin
B5, procyanidin C1, procyanidin dimers, procyanidin trimers,
procyanidin tetramers, procyanidin pentamers, procyanidin hexamers,
condensed tannins, an oligomer of catechin or epicatechin,
epicatechin, or any combination thereof. In further embodiments, a
pharmaceutical or nutraceutical formulation comprising an extract
mixture as described herein comprises from about 0.5 weight percent
(wt %) to about 50 wt % of the major active ingredients in a
Guazuma ulmifolia (Mutamba) extract, such as a procyanidin,
procyanidin B2, procyanidin B5, procyanidin C1, procyanidin dimers,
procyanidin trimers, procyanidin tetramers, procyanidin pentamers,
procyanidin hexamers, condensed tannins, an oligomer of catechin or
epicatechin, epicatechin, or any combination thereof.
[0157] In any of the aforementioned embodiments, a composition
comprising an extract mixture (optionally including a weight
management agent) wherein one of the extracts is a Magnolia extract
enriched for one or more major active ingredients, such as
magnolol, honokiol, or both, or a high purity magnolol, honokiol,
or mixture thereof. In certain embodiments, the Magnolia extract is
a Magnolia officinalis extract enriched for magnolol, honokiol, or
both, or a high purity magnolol, honokiol, or mixture thereof
isolated from Magnolia plant extract that has at least about 90%,
91%, 92%, 93%, 94%, or 95% purity. In certain embodiments, a
pharmaceutical or nutraceutical formulation comprising an extract
mixture as described herein comprises from about 5.0 weight percent
(wt %) to about 10 wt % of the major active ingredients in a
Magnolia officinalis (Magnolia) extract, such as magnolol,
honokiol, both, or a high purity mixture of both.
[0158] In any of the aforementioned embodiments, a composition
comprising an extract mixture (optionally including a weight
management agent) wherein one of the extracts is a Yerba Mate
extract enriched for one or more major active ingredients. In
certain embodiments, a Yerba Mate extract is an Ilex paraguayensis
extract is enriched for caffeine, dicaffeoylquinic acid, or both.
In further embodiments, a pharmaceutical or nutraceutical
formulation comprising an extract mixture as described herein
comprises from about 0.5 weight percent (wt %) to about 5.0 wt % of
the major active ingredients in an Ilex paraguayensis (Yerba Mate)
extract, such as caffeine, dicaffeoylquinic acid, or both.
[0159] In any of the aforementioned embodiments, a composition
comprising an extract mixture (optionally including a weight
management agent) wherein one of the extracts is a Rosmarinus
officinalis (Rosemary) extract enriched for one or more major
active ingredients. In certain embodiments, a Rosmarinus
officinalis (Rosemary) extract is enriched for a carnosol,
carnosoic acid, ursolic acid, or any combination thereof. In
further embodiments, a pharmaceutical or nutraceutical formulation
comprising an extract mixture as described herein comprises from
about 1.0 weight percent (wt %) to about 10 wt % of the major
active ingredients in a Rosmarinus officinalis (Rosemary) extract,
such as carnosol, carnosoic acid, ursolic acid, or any combination
thereof.
[0160] In any of the aforementioned embodiments, a pharmaceutical
or nutraceutical formulation comprising an extract mixture
(optionally including a weight management agent) of a Morus
extract, a Magnolia extract, and an Ilex paraguayensis (Yerba Mate)
extract, will include from about 0.5 weight percent (wt %) to about
5.0 wt % of the major active ingredients in a Morus extract, from
about 5.0 wt % to about 10 wt % of the major active ingredients in
a Magnolia extract, and from about 0.5 wt % to about 5.0 wt % of
the major active ingredients in Yerba Mate extract. In certain
embodiments, a pharmaceutical or nutraceutical formulation
comprises about 1% of the major active ingredients in a Morus
extract (such as Kuwanon G, Albanin G, Morusin, or any combination
thereof), about 7% of the major active ingredients in a Magnolia
extract (such as magnolol, honokiol, both, or a high purity mixture
of both), and about 1% of the major active ingredients in a Yerba
Mate extract (such as caffeine, dicaffeoylquinic acid, or both). In
any of the aforementioned embodiments, a Morus extract is a Morus
alba extract or extract enriched for Kuwanon G, Albanin G, Morusin,
or any combination thereof, and a Magnolia extract is a Magnolia
officinalis extract or extract enriched for magnolol, honokiol,
both, or a high purity mixture of both. In any of these
embodiments, the pharmaceutical or nutraceutical composition is
formulated as a tablet, a capsule, a powder, or granule.
[0161] In any of the aforementioned embodiments, a pharmaceutical
or nutraceutical formulation comprising an extract mixture
(optionally including a weight management agent) of a Morus
extract, a Magnolia extract, and a Guazuma ulmifolia (Mutamba)
extract, will include from about 0.5 weight percent (wt %) to about
5.0 wt % of the major active ingredients in a Morus extract, from
about 5.0 wt % to about 10 wt % of the major active ingredients in
a Magnolia extract, and from about 0.5 wt % to about 50 wt % of the
major active ingredients in a Mutamba extract. In certain
embodiments, a pharmaceutical or nutraceutical formulation
comprises about 1% of the major active ingredients in a Morus
extract (such as Kuwanon G, Albanin G, Morusin, or any combination
thereof), about 7% of the major active ingredients in a Magnolia
extract (such as magnolol, honokiol, both, or a high purity mixture
of both), and about 1% of the major active ingredients in a Mutamba
extract (such as a procyanidin, procyanidin B2, procyanidin B5,
procyanidin C1, procyanidin dimers, procyanidin trimers,
procyanidin tetramers, procyanidin pentamers, procyanidin hexamers,
condensed tannins, an oligomer of catechin or epicatechin,
epicatechin, or any combination thereof). In any of the
aforementioned embodiments, a Morus extract is a Morus alba extract
or extract enriched for Kuwanon G, Albanin G, Morusin, or any
combination thereof, and a Magnolia extract is a Magnolia
officinalis extract or extract enriched for magnolol, honokiol,
both, or a high purity mixture of both. In any of these
embodiments, the pharmaceutical or nutraceutical composition is
formulated as a tablet, a capsule, a powder, or granule. In related
embodiments, a weight management agent is an anorectic agent,
lipase inhibitor, cannabinoid receptor modulator, psychotropic
agent, insulin sensitizer, stimulant, satiety agent, or any
combination thereof. In any of these embodiments, a weight
management agent is an anorectic agent. In any of these
embodiments, a weight management agent is a lipase inhibitor. In
any of these embodiments, a weight management agent is a
cannabinoid receptor modulator. In any of these embodiments, a
weight management agent is a psychotropic agent. In any of these
embodiments, a weight management agent is an insulin sensitizer. In
any of these embodiments, a weight management agent is a stimulant.
In any of these embodiments, a weight management agent is a satiety
agent.
[0162] In any of the aforementioned embodiments, a pharmaceutical
or nutraceutical formulation comprising an extract mixture
(optionally including a weight management agent) of a Morus
extract, a Rosmarinus officinalis (Rosemary) extract, and an Ilex
paraguayensis (Yerba Mate) extract, will include from about 0.5
weight percent (wt %) to about 5.0 wt % of the major active
ingredients in a Morus extract, from about 1.0 wt % to about 10 wt
% of the major active ingredients in a Rosemary extract, and from
about 0.5 wt % to about 5.0 wt % of the major active ingredients in
a Yerba Mate extract. In certain embodiments, a pharmaceutical or
nutraceutical formulation comprises about 1% of the major active
ingredients in a Morus extract (such as Kuwanon G, Albanin G,
Morusin, or any combination thereof), about 4.5% of the major
active ingredients in a Rosemary extract (such as carnosol,
carnosoic acid, ursolic acid, or any combination thereof), and
about 1% of the major active ingredients in a Yerba Mate extract
(such as caffeine, dicaffeoylquinic acid, or both). In any of the
aforementioned embodiments, a Morus extract is a Moru alba extract
or enriched extract. In any of these embodiments, the
pharmaceutical or nutraceutical composition is formulated as a
tablet, a capsule, a powder, or granule. In related embodiments, a
weight management agent is an anorectic agent, lipase inhibitor,
cannabinoid receptor modulator, psychotropic agent, insulin
sensitizer, stimulant, satiety agent, or any combination thereof.
In any of these embodiments, a weight management agent is an
anorectic agent. In any of these embodiments, a weight management
agent is a lipase inhibitor. In any of these embodiments, a weight
management agent is a cannabinoid receptor modulator. In any of
these embodiments, a weight management agent is a psychotropic
agent. In any of these embodiments, a weight management agent is an
insulin sensitizer. In any of these embodiments, a weight
management agent is a stimulant. In any of these embodiments, a
weight management agent is a satiety agent.
[0163] In further embodiments, a composition comprising a
Diels-Alder adduct of a chalcone and a prenylphenyl moiety as
provided herein (optionally including a weight management agent)
may be used in a method for treating or preventing weight gain or
obesity, promoting weight loss, appetite suppression, modifying
satiety, modifying fat uptake, increasing metabolism to promote
weight loss or prevent weight gain, maintaining body weight,
reducing body fat or fatty tissues, increasing muscle or lean body
mass, reducing hepatosteatosis, improving fatty liver, improving
one or more liver NASH scores, enhancing fatty acid metabolism in
liver, promoting a healthy lipid profile (by, e.g., lowering LDL
cholesterol, lowering total cholesterol, lowering triglyceride, or
increasing HDL), promoting glucose metabolism, reducing fasting
glucose levels, maintaining healthy glucose levels, reducing
caloric intake, improving caloric efficiency, reducing food intake,
reducing visceral fat, reducing waist circumference, reducing
body-to-mass index (BMI), increasing energy, increasing stamina,
maintaining energy level while dieting, promoting thermogenesis
reducing excess fluids, reducing water retention while maintaining
normal hydration, increasing muscle mass, improving fat-to-muscle
mass ratio, optimizing or improving body composition, optimizing or
improving hormonal balance for appitite control, maintaining normal
insulin, leptin, ghrelin, PYY, GIP or enterostatin levels or
functions, optimizing, managing or improving hormonal balance to
control satiety, maintaining or managing healthy CCK peptide,
GLP-1, bombesin, or somatostatin levels or functions, maintaining
healthy flora of intestinal tract, optimizing, improving or
managing digestion, inducing lipolysis, reducing intracellular
triglyceride accumulation, reducing fat accumulation in adipose
tissue or an adipocyte, maintaining healthy adiponectin levels,
managing or reducing lipogenesis or weight gain associated with
metabolism of fructose, glucose or both, reducing or controlling
oxidative stress associated with an overweight or obese mammal
(e.g., by reducing reactive oxygen species or oxidative free
radicals; improving ORAC (Oxygen Radical Absorption Capacity)
values; maintaining a healthy level of glutathione, superoxide
dismutase, catalase, peroxidase or endogenous antioxidants;
maintaining healthy oxidative homeostasis), controlling or managing
systemic inflammation associated with an overweight or obese mammal
(e.g., by promoting normal metabolism of arachidonic acid,
maintaining a normal level of pro-inflammatory cytokines), managing
mood stress or other mental disorders associated with an overweight
or obese mammal, or any combination thereof.
[0164] In related embodiments, a weight management agent for use
with the compositions to treat or improve the various
weight-related conditions is an anorectic agent, lipase inhibitor,
cannabinoid receptor modulator, psychotropic agent, insulin
sensitizer, stimulant, satiety agent, or any combination thereof.
In any of these embodiments, a weight management agent is an
anorectic agent. In any of these embodiments, a weight management
agent is a lipase inhibitor. In any of these embodiments, a weight
management agent is a cannabinoid receptor modulator. In any of
these embodiments, a weight management agent is a psychotropic
agent. In any of these embodiments, a weight management agent is an
insulin sensitizer. In any of these embodiments, a weight
management agent is a stimulant. In any of these embodiments, a
weight management agent is a satiety agent.
[0165] In certain embodiments, provided herein is an isolated
oligomer or a composition (e.g., for weight management or weight
loss) comprising a pharmaceutically acceptable excipient and an
oligomer, wherein the oligomer comprises from two to thirty
subunits, wherein the subunits have, at each occurrence,
independently the following structure (III):
##STR00120##
or a pharmaceutically acceptable salt, stereoisomer or tautomer
thereof, wherein R.sup.1a and R.sup.1b are, at each occurrence,
independently H, hydroxyl, halo, a gallic acid ester, a glycoside,
sulfhydryl, amino, aldehyde, C.sub.1-12 alkyl, C.sub.1-12 alkoxy,
C.sub.1-12 alkthio, C.sub.1-12 alkyamino, aryl, heteroaryl,
aralkyl, alkyl carbonyl, aralkylcarbonyl, or a direct bond to an
adjacent subunit; R.sup.2 is, at each occurrence, independently H
or an ether bond to an adjacent subunit; R.sup.3 is, at each
occurrence, independently H or a direct bond to an adjacent
subunit: R.sup.4 is, at each occurrence, OH or an ether bond to an
adjacent subunit; and R.sup.5a, R.sup.5b, R.sup.5c, R.sup.5d and
R.sup.5e are, at each occurrence, independently H, hydroxyl, halo,
a gallic acid ester, a glycoside, sulfhydryl, amino, aldehyde,
C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.1-12 alkthio, C.sub.1-12
alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl or
aralkylcarbonyl, wherein at least one of R.sup.1a, R.sup.1b,
R.sup.2, R.sup.3 or R.sup.4 is a direct bond or ether bond to an
adjacent subunit in certain embodiments, oligomers of flavan-3-ol,
with subunits as set forth in structure (III) and wherein at least
one of R.sup.1a, R.sup.1b, R.sup.2, R.sup.3 or R.sup.4 is a direct
bond or ether bond to an adjacent subunit, are dimers, trimers,
tetramers, pentamers, hexamers, heptamers, octamers, nonamers,
decamers, or up to 30 flavan-3-ol subunits (such a 30-mer would
have a total molecular weight of about 10.sup.3 Da).
[0166] In further embodiments, two adjacent subunits may have one
of the following structures (IIIa), (IIIb) or (IIIc):
##STR00121##
[0167] In any of the aforementioned embodiments, each of R.sup.1a
and R.sup.1b is, at each occurrence, H or a direct bond to an
adjacent subunit. In a further embodiment, R.sup.5a is, at each
occurrence, H and R.sup.5b is, at each occurrence, hydroxyl; or
R.sup.5a is, at each occurrence, hydroxyl and R.sup.5b is, at each
occurrence, H. In still further embodiments, each of R.sup.5c,
R.sup.5d and R.sup.5e is, at each occurrence, H. In certain
embodiments, oligomers of flavan-3-ol, with subunits as set forth
in structure (IIIa), (IIIb), or (IIIc) are dimers, trimers,
tetramers, pentamers, hexamers, heptamers, octamers, nonamers,
decamers, or up to 30 flavan-3-ol subunits (such a 30-mer would
have a total molecular weight of about 10.sup.3 Da).
[0168] In certain embodiments, provided herein is an isolated
oligomer or a composition (e.g., for weight management or weight
loss) comprising a pharmaceutically acceptable excipient and an
oligomer, wherein the oligomer comprises from two to thirty
subunits, wherein the subunits have, at each occurrence,
independently the following structure (IIId):
##STR00122##
or a pharmaceutically acceptable salt, stereoisomer or tautomer
thereof, wherein R.sup.1a and R.sup.1b are, at each occurrence,
independently H, hydroxyl, halo, a gallic acid ester, a glycoside,
sulfhydryl, amino, aldehyde, C.sub.1-12 alkyl, C.sub.1-12 alkoxy,
C.sub.1-12 alkthio, C.sub.1-12 alkyamino, aryl, heteroaryl,
aralkyl, alkyl carbonyl, aralkylcarbonyl, or a direct bond to an
adjacent subunit; and R.sup.5a, R.sup.5b, R.sup.5c, R.sup.5d and
R.sup.5e are, at each occurrence, independently H, hydroxyl, halo,
a gallic acid ester, a glycoside, sulfhydryl, amino, aldehyde,
C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.1-12 alkthio, C.sub.1-12
alkyamino, aryl, heteroaryl, aralkyl, alkyl carbonyl or
aralkylcarbonyl, wherein at least one of R.sup.1a, R.sup.1b,
position 2, or position 4 is a direct bond or ether bond to an
adjacent subunit. In certain embodiments, oligomers of flavan-3-ol,
with subunits as set forth in structure (IIId) and wherein at least
one of R.sup.1a, R.sup.1b, position 2, or position 4 is a direct
bond or ether bond to an adjacent subunit, are dimers, trimers,
tetramers, pentamers, hexamers, heptamers, octamers, nonamers,
decamers, or up to 30 flavan-3-ol subunits (such a 30-mer would
have a total molecular weight of about 10.sup.3 Da).
[0169] In further embodiments, two adjacent subunits may have one
of the following structures (IIIe), (IIIf) or (IIIg):
##STR00123##
Structures (IIIe) and (IIIf) are exemplary B-type linkages and
Structure (IIIg) is an example of an A-type linkage. More
specifically, Structure (IIIe) is a 4.fwdarw.8 linkage, wherein
R.sup.1a, R.sup.3 or both are available as a direct bond or ether
bond to one or more adjacent subunits up to total of 30 subunits,
and Structure (IIIe) is a 4.fwdarw.6 linkage, wherein R.sup.3,
R.sup.4 or both are available as a direct bond or ether bond to one
or more adjacent subunits up to total of 30 subunits.
[0170] In certain embodiments, the present disclosure provides a
composition comprising any of the aforementioned oligomers of
flavan-3-ol and at least one other weight management agent, such as
an anorectic agent, lipase inhibitor, cannabinoid receptor
modulator, psychotropic agent, insulin sensitizer, stimulant, or
satiety agent. In further embodiments, the anorectic agent is
sibutramine, diethylpropion, benzphetamine, phendimetrazine, or
catecholamine. In still further embodiments, the lipase inhibitor
is a Rosemary extract, carnosic acid, carnosol, lipostatin,
tetrahydrolipostatin, Punica granatum pericarp extract, Marchantia
polymorpha whole plant extract, Panax japonicas extract or
Platycodi radix extract. In yet further embodiments, the
cannabinoid receptor modulator is a cannabinoid receptor agonist,
antagonist, or inverse agonist, which may be specific for
cannabinoid receptor 1 (CB1), cannabinoid receptor 2 (CB2), or both
CB1 and CB2 (e.g., a cannabinoid receptor 1 (CB1) antagonist or
inverse agonist). In further embodiments, the cannabinoid receptor
modulator is rimonabant,
N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-py-
razole-3-carboxamide (AM 251),
1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyraz-
ole-3-carboxamide (AM281), 4-[6-methoxy-2-(4-methoxypheyl)
1-benzofuran-3-carbonyl]benzonitrile (LY 320135), a Morus extract,
Diels-Alder adduct of a chalcone and a prenylphenyl moiety from
plants (e.g., isolated from or contained in a Morus alba extract, a
Milicia excelsa extract, or is Albanin G, Kuwanon G, Kuwanon M,
Cathayanon A, Morusin, Morusinol. Sanggenon C, Sanggenon D,
Sanggenon O or a combination thereof), Magnolia extract, magnolol,
honokiol, magnolol and honokiol, purinol, or Piper Longum seed
extract. In still further embodiments, the psychotropic agent is a
mood stabilizer, anti-depressant, or anti-convulsant. In even
further embodiments, the insulin sensitizer is a thiazolidinedione
(e.g., rosiglitazone, pioglitazone), a biguanide (e.g., metformin),
or a selective mTOT modulator. In yet further embodiments, the
stimulant is a Yerba Mate extract, caffeine, green tea extract,
green coffee bean extract, Cola nut extract. Citrus aurantium fruit
extract, Gacinia extract, Areca catechu fruit/seed extract or
dextroamphetamine. In further embodiments, the satiety agent is
dodecanoic acid, glyceryl dodecanoate, glyceryl 1,3-didodecanoate,
glyceryl tridodecanoate and derivatives and mixtures thereof, or
hoodia extract, pine nut extract, or a fiber supplement.
[0171] In certain embodiments, a composition comprises a Mutamba
extract containing or enriched for one or more oligomers of
flavan-3-ol as described herein, a Morus extract containing or
enriched for at least one Diels-Alder adduct of a chalcone and a
prenylphenyl moiety, and a Magnolia extract, which can be mixed or
used together in a 5:2:1 weight ratio to a 10:2:1 weight ratio,
respectively. In any of the aforementioned embodiments, a Morus
extract is a Morus alba extract or extract enriched for Kuwanon G,
Albanin G, Morusin, or any combination thereof, and a Magnolia
extract is a Magnolia officinalis extract or extract enriched for
magnolol, honokiol, both, or a high purity mixture of both.
[0172] In certain embodiments, a composition comprises a Mutamba
extract containing or enriched for one or more oligomers of
flavan-3-ol as described herein, a Rosemary extract, and a Yerba
Mate extract, which can be mixed or used together in a 2:1:2 weight
ratio to a 1:1:1 weight ratio, respectively.
[0173] In still further embodiments, one or more oligomers of
flavan-3-ol or compositions of such oligomers of flavan-3-ol
compounds as described herein, (optionally including a weight
management agent) may be used in a method for treating or
preventing weight gain or obesity, promoting weight loss, appetite
suppression, modifying satiety, modifying fat uptake, increasing
metabolism to promote weight loss or prevent weight gain,
maintaining body weight, reducing body fat or fatty tissues,
increasing muscle or lean body mass, reducing hepatosteatosis,
improving fatty liver, improving one or more liver NASH scores,
enhancing fatty acid metabolism in liver, promoting a healthy lipid
profile (by, e.g., lowering LDL cholesterol, lowering total
cholesterol, lowering triglyceride, or increasing HDL), promoting
glucose metabolism, reducing fasting glucose levels, maintaining
healthy glucose levels, reducing caloric intake, improving caloric
efficiency, reducing food intake, reducing visceral fat, reducing
waist circumference, reducing body-to-mass index (BMI), increasing
energy, increasing stamina, maintaining energy level while dieting,
promoting thermogenesis reducing excess fluids, reducing water
retention while maintaining normal hydration, increasing muscle
mass, improving fat-to-muscle mass ratio, optimizing or improving
body composition, optimizing or improving hormonal balance for
appitite control, maintaining normal insulin, leptin, ghrelin, PYY,
GIP or enterostatin levels or functions, optimizing, managing or
improving hormonal balance to control satiety, maintaining or
managing healthy CCK peptide, GLP-1, bombesin, or somatostatin
levels or functions, maintaining healthy flora of intestinal tract,
optimizing, improving or managing digestion, inducing lipolysis,
reducing intracellular triglyceride accumulation, reducing fat
accumulation in adipose tissue or an adipocyte, maintaining healthy
adiponectin levels, managing or reducing lipogenesis or weight gain
associated with metabolism of fructose, glucose or both, reducing
or controlling oxidative stress associated with an overweight or
obese mammal (e.g., by reducing reactive oxygen species or
oxidative free radicals; improving ORAC (Oxygen Radical Absorption
Capacity) values; maintaining a healthy level of glutathione,
superoxide dismutase, catalase, peroxidase or endogenous
antioxidants; maintaining healthy oxidative homeostasis),
controlling or managing systemic inflammation associated with an
overweight or obese mammal (e.g., by promoting normal metabolism of
arachidonic acid, maintaining a normal level of pro-inflammatory
cytokines), managing mood stress or other mental disorders
associated with an overweight or obese mammal, or any combination
thereof.
[0174] In related embodiments, a weight management agent for use
with the compositions to treat or improve the various
weight-related conditions is an anorectic agent, lipase inhibitor,
cannabinoid receptor modulator, psychotropic agent, insulin
sensitizer, stimulant, satiety agent, or any combination thereof.
In any of these embodiments, a weight management agent is an
anorectic agent. In any of these embodiments, a weight management
agent is a lipase inhibitor. In any of these embodiments, a weight
management agent is a cannabinoid receptor modulator. In any of
these embodiments, a weight management agent is a psychotropic
agent. In any of these embodiments, a weight management agent is an
insulin sensitizer. In any of these embodiments, a weight
management agent is a stimulant. In any of these embodiments, a
weight management agent is a satiety agent.
[0175] In any of the aforementioned method of use embodiments, a
composition comprising an extract mixture (optionally including a
weight management agent) can be formulated with a pharmaceutically
or nutraceutically acceptable carrier, diluent or excipient. In
further embodiments, the pharmaceutical or nutraceutical
formulation comprises from about 0.5 weight percent (wt %) to about
90 wt %, about 0.5 wt % to about 80 wt %, about 0.5 wt % to about
75 wt %, about 0.5 wt % to about 70 wt %, about 0.5 wt % to about
50 wt %, about 1.0 wt % to about 40 wt %, about 1.0 wt % to about
20 wt %, about 1.0 wt % to about 10 wt %, about 3.0 wt % to about
9.0 wt %, about 5.0 wt % to about 10 wt %, about 3.0 wt % to about
6.5 wt % of the major active ingredients in an extract mixture, or
the like. In any of these embodiments, the pharmaceutical or
nutraceutical composition is formulated as a tablet, a capsule, a
powder, or granule.
[0176] In any of the aforementioned method of use embodiments, a
composition comprising an extract mixture (optionally including a
weight management agent) wherein one of the extracts is a Morus
extract enriched for one or more major active ingredients. In
further embodiments, a pharmaceutical or nutraceutical formulation
comprising an extract mixture as described herein comprises from
about 0.5 weight percent (wt %) to about 5.0 wt % of the major
active ingredients in a Morus extract enriched for, for example,
Kuwanon G, Albanin G, Morusin, or any combination thereof. In
certain of these embodiments, a Morus extract is a Morus alba
extract enriched for Kuwanon G, Albanin G, Morusin, or any
combination thereof.
[0177] In any of the aforementioned method of use embodiments, a
composition comprising an extract mixture (optionally including a
weight management agent) wherein one of the extracts is a Guazuma
ulmifolia (Mutamba) extract enriched for one or more major active
ingredients. In certain embodiments, a Guazuma ulmifolia (Mutamba)
extract is enriched for a procyanidin procyanidin B2, procyanidin
B5, procyanidin C1, procyanidin dimers, procyanidin trimers,
procyanidin tetramers, procyanidin pentamers, procyanidin hexamers,
condensed tannins, an oligomer of catechin or epicatechin,
epicatechin, or any combination thereof. In further embodiments, a
pharmaceutical or nutraceutical formulation comprising an extract
mixture as described herein comprises from about 0.5 weight percent
(wt %) to about 50 wt % of the major active ingredients in a
Guazuma ulmifolia (Mutamba) extract, such as a procyanidin
procyanidin B2, procyanidin B5, procyanidin C1, procyanidin dimers,
procyanidin trimers, procyanidin tetramers, procyanidin pentamers,
procyanidin hexamers, condensed tannins, an oligomer of catechin or
epicatechin, epicatechin, or any combination thereof.
[0178] In any of the aforementioned method of use embodiments, a
composition comprising an extract mixture (optionally including a
weight management agent) wherein one of the extracts is a Magnolia
extract enriched for one or more major active ingredients. In
further embodiments, a pharmaceutical or nutraceutical formulation
comprising an extract mixture as described herein comprises from
about 5.0 weight percent (wt %) to about 10 wt % of the major
active ingredients in a Magnolia extract, such as magnolol,
honokiol, both, or a high purity mixture of both. In certain of
these embodiments, a Magnolia extract is a Magnolia officinalis
extract enriched for magnolol, honokiol, or both, or a high purity
magnolol, honokiol, or mixture thereof isolated from a Magnolia
officinalis plant extract having at least 90%, 91%, 92%, 93%, 94%,
or 95% purity.
[0179] In any of the aforementioned method of use embodiments, a
composition comprising an extract mixture (optionally including a
weight management agent) wherein one of the extracts is a Ilex
paraguayensis (Yerba Mate) extract enriched for one or more major
active ingredients. In certain embodiments, an Ilex paraguayensis
(Yerba Mate) extract is enriched for caffeine, dicaffeoylquinic
acid, or both. In further embodiments, a pharmaceutical or
nutraceutical formulation comprising an extract mixture as
described herein comprises from about 0.5 weight percent (wt %) to
about 5.0 wt % of the major active ingredients in an Ilex
paraguayensis (Yerba Mate) extract, such as caffeine,
dicaffeoylquinic acid, or both.
[0180] In any of the aforementioned method of use embodiments, a
composition comprising an extract mixture (optionally including a
weight management agent) wherein one of the extracts is a
Rosmarinus officinalis (Rosemary) extract enriched for one or more
major active ingredients. In certain embodiments, a Rosmarinus
officinalis (Rosemary) extract is enriched for a carnosol,
carnosoic acid, ursolic acid, or any combination thereof. In
further embodiments, a pharmaceutical or nutraceutical formulation
comprising an extract mixture as described herein comprises from
about 1.0 weight percent (wt %) to about 10 wt % of the major
active ingredients in a Rosmarinus officinalis (Rosemary) extract,
such as carnosol, carnosoic acid, ursolic acid, or any combination
thereof.
[0181] In any of the aforementioned method of use embodiments, a
pharmaceutical or nutraceutical formulation comprising an extract
mixture (optionally including a weight management agent) of a Morus
extract, a Magnolia extract, and a Ilex paraguayensis (Yerba Mate)
extract, will include from about 0.5 weight percent (wt %) to about
5.0 wt % of the major active ingredients in a Morus extract, from
about 5.0 wt % to about 10 wt % of the major active ingredients in
a Magnolia extract, and from about 0.5 wt % to about 5.0 wt % of
the major active ingredients in Yerba Mate extract. In certain
embodiments, a pharmaceutical or nutraceutical formulation
comprises about 1% of the major active ingredients in a Morus
extract (such as Kuwanon G, Albanin G, Morusin, or any combination
thereof), about 7% of the major active ingredients in a Magnolia
extract (such as magnolol, honokiol, both, or a high purity mixture
of both), and about 1% of the major active ingredients in a Yerba
Mate extract (such as caffeine, dicaffeoylquinic acid, or both). In
any of the aforementioned embodiments, a Morus extract is a Morus
alba extract or extract enriched for Kuwanon G, Albanin G, Morusin,
or any combination thereof, and a Magnolia extract is a Magnolia
officinalis extract or extract enriched for magnolol, honokiol,
both, or a high purity mixture of both. In any of these
embodiments, the pharmaceutical or nutraceutical composition is
formulated as a tablet, a capsule, a powder, or granule.
[0182] In any of the aforementioned method of use embodiments, a
pharmaceutical or nutraceutical formulation comprising an extract
mixture (optionally including a weight management agent) of a Morus
extract, a Magnolia extract, and a Guazuma ulmifolia (Mutamba)
extract, will include from about 0.5 weight percent (wt %) to about
5.0 wt % of the major active ingredients in a Morus extract, from
about 5.0 wt % to about 10 wt % of the major active ingredients in
a Magnolia extract, and from about 0.5 wt % to about 50 wt % of the
major active ingredients in a Mutamba extract. In certain
embodiments, a pharmaceutical or nutraceutical formulation
comprises about 1% of the major active ingredients in a Morus
extract (such as Kuwanon G, Albanin G, Morusin, or any combination
thereof), about 7% of the major active ingredients in a Magnolia
extract (such as magnolol, honokiol, both, or a high purity mixture
of both), and about 1% of the major active ingredients in a Mutamba
extract (such as a procyanidin procyanidin B2, procyanidin B5,
procyanidin C1, procyanidin dimers, procyanidin trimers,
procyanidin tetramers, procyanidin pentamers, procyanidin hexamers,
condensed tannins, an oligomer of catechin or epicatechin,
epicatechin, or any combination thereof), In any of the
aforementioned embodiments, a Morus extract is a Morus alba extract
or extract enriched for Kuwanon G, Albanin G, Morusin, or any
combination thereof, and a Magnolia extract is a Magnolia
officinalis extract or extract enriched for magnolol, honokiol,
both, or a high purity mixture of both. In any of these
embodiments, the pharmaceutical or nutraceutical composition is
formulated as a tablet, a capsule, a powder, or granule.
[0183] In any of the aforementioned method of use embodiments, a
pharmaceutical or nutraceutical formulation comprising an extract
mixture (optionally including a weight management agent) of a Morus
extract, a Rosmarinus officinalis (Rosemary) extract, and a Ilex
paraguayensis (Yerba Mate) extract, will include from about 0.5
weight percent (wt %) to about 5.0 wt % of the major active
ingredients in a Morus extract, from about 1.0 wt % to about 10 wt
% of the major active ingredients in a Rosemary extract, and from
about 0.5 wt % to about 5.0 wt % of the major active ingredients in
a Yerba Mate extract. In certain embodiments, a pharmaceutical or
nutraceutical formulation comprises about 1% of the major active
ingredients in a Morus extract (such as Kuwanon G, Albanin G,
Morusin, or any combination thereof), about 4.5% of the major
active ingredients in a Rosemary extract (such as carnosol,
carnosoic acid, ursolic acid, or any combination thereof), and
about 1% of the major active ingredients in a Yerba Mate extract
(such as caffeine, dicaffeoylquinic acid, or both). In any of the
aforementioned embodiments, a Morus extract is a Morus alba extract
or extract enriched for Kuwanon G, Albanin G, Morusin, or any
combination thereof, and a Magnolia extract is a Magnolia
officinalis extract or extract enriched for magnolol, honokiol,
both, or a high purity mixture of both. In any of these
embodiments, the pharmaceutical or nutraceutical composition is
formulated as a tablet, a capsule, a powder, or granule.
EXAMPLES
Example 1
Preparation of Organic and Aqueous Extracts from Morus alba
[0184] Plant material from Morus alba L. root barks was ground to a
particle size of no larger than two millimeters (mm). Dried ground
plant material (60 grams (g)) was then transferred to an Erlenmeyer
flask and Methanol:Dichloromethane (1:1 volume ratio) (600
milliliters (mL)) was added. The mixture was shaken for one hour,
filtered and the biomass was extracted again with
Methanol:Dichloromethane (1:1 volume ratio) (600 mL). These organic
extracts were combined and evaporated under vacuum to provide 3.55
g of organic extract (OE). After organic extraction, the biomass
was air dried and extracted once with ultrapure water (600 mL). The
aqueous solution was filtered and freeze-dried to provide 4.44 g of
aqueous extract (AE).
[0185] Similar results were obtained using the same procedure, but
with the organic solvent being replaced with methanol or ethanol to
provide a methanol extract (ME) or ethanol extract (EE),
respectively. Other species and parts of plants and marine sample
were extracted using this same procedure.
Example 2
CB1 Binding Activity by Plant Extracts
[0186] Cannabinoid receptor binding assays were used as a primary
screening method to identify CB1 antagonist compounds. The assays
were performed using methods adapted from by Reggio et al., J. Med.
Chem. 41:5177, 1998 (CB1 receptor assay) and Munro et al., Nature
365:61, 1993 (CB2 receptor assay). Briefly, human cannabinoid
receptor protein bound to Chem-1 membrane was used in modified
HEPES (pH 7.4) buffer. A 10 microgram (.mu.g) aliquot of Chem-1
membrane, radioactively labeled [.sup.3H] CB1l-ligand SR141716A (2
nanomolar (nM)) (CB1 receptor antagonist) and test extracts or
positive control R (+)-WIN-55,212-2 (10 .mu.M) (CB1 receptor
agonist) were incubated in Incubation Buffer (50 mM HEPES (pH 7.4),
5 mM MgCl.sub.2, 1 mM CaCl2, 0.2% BSA) for 90 minutes at 37'C.
After incubation, the membranes were filtered and washed four times
and then the filters were counted to determine amount of [.sup.3H]
WIN-55,212-2 specifically bound to the CB1-membrane.
[0187] Table 1 presents the CB1 binding assay results from the OE
and AEs obtained from different plant parts of Morus alba: root
barks, fruits, leaves, stem barks and stem. The greatest inhibition
of CB1-ligand binding activity was found in OE obtained from root
barks with 91% inhibition at testing concentration of 100 .mu.g/mL
and 82% at testing concentration of 10 .mu.g/mL.
TABLE-US-00002 TABLE 1 Inhibition of CB1 ligand binding activity by
Extracts from Various Morus alba Plant Parts Active CB1 Plant
Source Tested Extract Extract Inhibition* Morus alba (root barks)
OE.sup..dagger., AE.sup..dagger-dbl. OE 91% (100 .mu.g/mL) Morus
alba (fruits) EE -- 82% (10 .mu.g/mL) Morus alba (leaves) EE.sup.#,
OE, AE, -- -- ME Morus alba (stem barks) AE -- -- Morus alba
(stems) ME -- -- *Results are expressed as a percent inhibition
relative to control radiolabeled SR141716A ligand. .sup..dagger.OE
means ground plant material extracted with Methanol:Dichloromethane
(1:1 volume ratio) as described in Example 1. .sup..dagger-dbl.AE
means OE extracted material that was air dried and extracted once
with ultrapure water, filtered and freeze-dried to provide aqueous
extract material as described in Example 1. .sup.#EE means that the
ground plant material was extracted with only ethanol. MB means
that the ground plant material was extracted with only
methanol.
[0188] These data show that organic extracts of Morus alba root
bark contain moderately high levels of a component that block the
binding of the labeled CB1-ligand to a human CB1 receptor protein.
In contrast, the other Morus alba plant tissues tested in this
experiment (fruits, leaves, stem bark, and stems) all had
undetectable levels of inhibition of CB1-ligand binding to CB1
receptor.
[0189] The CB1 Binding assay also detected inhibition activity from
other species of plants listed in Table 2. The common chemical
components in those Moraceae and Fabaceae plants are Diels-Alder
adducts of a chalcone and prenylphenyl moiety. The existence of
Diels-Alder adducts of a chalcone and prenylphenyl moiety is
detectable in different plant tissues, such as roots, stems,
rhizomes or whole plants.
TABLE-US-00003 TABLE 2 CB1 binding activity from plant extracts
containing prenylated flavonoids Family Latin Name CB1 Binding
activity Plant parts Moraceae Morus alba 82% at 10 .mu.g/ml Root
barks 91% at 100 .mu.g/ml Moraceae Milicia excelsa 29% at 10
.mu.g/ml Stem barks 113% at 100 .mu.g/ml Fabaceae Glycyrrhiza
glabara 48% at 10 .mu.g/ml Rhizome 67% at 100.mu.g/ml
Example 3
High Throughput Purification (HTP) of Active Plant Extracts
[0190] Organic extract material (400 mg) from the Morus alba root
bark extract obtained in Example 1 was loaded onto a prepacked (2
cm ID.times.8.2 cm, 10 g silica gel) column. The column was then
eluted using a Hitachi) High Throughput Purification (HTP) system
with a gradient mobile phase of (A) 50:50 volume ratio of
EtOAc:Hexane and (B) Methanol from 100% A to 100% B in 30 minutes
at a flow rate of 5 mL/min. The separation was monitored using a
broadband wavelength UV detector and the fractions were collected
in a 96-deep-well plate at 1.9 mL/well using a Gilson fraction
collector. The sample plate was dried under low vacuum and
centrifugation and then the samples were dissolved with 1.5 mL
dimethyl sulfoxide (DMSO) per well. A portion (100 .mu.L) was taken
and combined (based on UV trace) for the CB1 inhibition assay.
Column fractions having significant CB1 binding activity were
retained for further testing.
Example 4
Inhibition of CB1 Activity by Combined HTP Fractions from Morus
alba
[0191] Organic extract of Morus alba root bark from Example 3 was
further investigated by examining the combined HTP fractions for
inhibition of CB1 binding activity by labeled CB1-ligand. The
activity profile of combined HTP fractions indicates that more than
one component in the fractionated Morus alba organic extract might
contribute to the inhibition of CB1-ligand binding activity.
[0192] Table 3 presents the CB1 binding assay results from the
combined HTP fractions of Morus alba root bark extract.
TABLE-US-00004 TABLE 3 CB1-ligand binding inhibition of combined
Morus alba HTP fractions HTP Fraction Pools % CB1 Binding Activity
Inhibition Fraction 1 to 4 46% Fraction 5 to 36 88% Fraction 37 to
46 -55% Fraction 47 to 84 85% Fraction 85 to 95 78% Fraction 96
67%
Example 5
Isolation, Purification, and Identification of CB1-Binding
Inhibitors from Morus alba Extracts
[0193] An organic extract (11 g) from the root barks of Morus alba,
obtained as described in Example 1, was divided and loaded
separately onto two pre-packed flash columns (120 g silica,
particle size 32-60 .mu.m, 4 cm.times.19 cm), and then eluted with
Hexane, EtOAc and Methanol (as the mobile phase) at a flow rate of
20 mL/minutes. The gradients started with 95% Hexane/EtOAC for 5
minutes, then increased EtOAC from 5% to 100% over the duration of
25 minutes, and then held at 100% EtOAc for additional five
minutes, before increasing MeOH from 0% to 50% MeOH/EtOAC over a
next period of 15 minutes, finally changed the elution solution to
100% MeOH and eluted the column for another 16 minutes. The total
run time was 66 minutes and 88 fractions were generated for each
column. The fractions were analyzed by silica gel thin layer
chromatography (TLC) and pooled together to generate eight column
eluent pools. Each of the eight eluent pools was then tested using
the CB1 binding assay described in Example 2. The resulting CB1
binding assay data are shown in Table 4.
TABLE-US-00005 TABLE 4 CB1 activity by Silica Fraction Pools Eluent
Pool Number % CB1 Binding Activity Inhibition 1 -43% 2 -36% 3 -5% 4
64% 5 71% 6 29% 7 29% 8 35%
[0194] These data show that the highest level of inhibition of
CB1-ligand binding was in pool 4 (containing 1.4 g of material) and
pool 5 (1.7 g of material) when tested in the CB1 binding assay at
a concentration of 20 .mu.g/mL.
[0195] The resulting best active best pool (containing 300 mg of
material) was fractionated on a preparative C18 column (30
cm.times.250 cm) with a gradient mobile phase of water (A) and
methanol (B) over 60 minutes at a flow rate of 20 mL/minute to
generate 22 fraction pools. Mass Spectrometry (MS) analysis showed
that these pooled fractions of material contain three related
compounds, described in more detail below, all of which had
inhibitory activity in the CB1-binding assay described in Example
2.
[0196] Compound 1 (28.2 mg) was identified as a Diels-Alder adduct
of a chalcone and prenylphenyl moiety called Kuwanon G, also known
as Moracenin B or Albanin F, by High Resolution Electron Spray
Ionization Mass Spectroscopy (HRESIMS) (m/z) [M+H].sup.-=693.2329;
UV .lamda..sub.max (MeOH): 265, 320 nm; .sup.1H NMR (600 MHz,
DMSO-d.sub.6, 100.degree. C.) .delta. ppm 1.44 (s, 3H) 1.52 (br.
s., 3H) 1.58 (s, 3H) 1.92 (m, 2H) 3.08 (d, 3H) 3.56 (m, 2H) 4.29
(d, J=10.02 Hz, 1H) 4.48 (m, 1H) 5.07 (m, 1H) 5.14 (br. s, 1H) 5.93
(s, 2H) 5.96 (dd, J=8.35, 2.23 Hz, 1H) 6.02 (br s, 1H) 6.11 (d,
J=2.23 Hz, 1H) 6.41 (dd, J=8.35, 2.23 Hz, 1H) 6.51 (s, 1H) 6.60 (m,
1H) 7.13 (d, J=8.35 Hz, 1H) 7.28 (br s, 1H); .sup.13C NMR (126 MHz,
METHANOL-d.sub.4) .delta. ppm 16.35 (1C) 21.78 (1C) 23.35 (1C)
24.53 (1C) 37.72 (1C) 97.14 (1C) 101.57 (1C) 102.22 (1C) 102.33
(1C) 104.28 (1C) 106.55 (2C) 107.00 (1C) 107.21 (1C) 112.37 (1C)
114.47 (1C) 120.27 (1C) 121.62 (2C) 123.27 (1C) 131.05 (1C) 131.35
(2C) 132.62 (1C) 132.99 (1C) 155.16 (1C) 155.56 (1C) 156.38 (1C)
159.66 (1C) 160.39 (2C) 161.13 (1C) 161.88 (1C) 164.51 (1C) 164.63
(1C) 182.46 (1C) 208.68 (1C).
##STR00124##
[0197] Compound 2 (10.5 mg) was identified as Albanin G, also known
as Kuwanon H or Moracenin A, another Diels-Alder adduct of a
chalcone and prenylphenyl moiety by HRESIMS (m/z) [M-H].sup.-=759;
UV .lamda..sub.max (MeOH): 265, 320 nm; .sup.13C NMR (126 MHz,
METHANOL-d.sub.4) ppm 16.35 (1C) 16.47 (1C) 20.96 (1C) 21.79 (1C)
23.32 (1C) 24.51 (1C) 24.53 (1C) 33.74 (1C) 35.61 (1C) 36.81 (1C)
37.77 (1C) 97.19 (1C) 102.27 (1C) 102.33 (1C) 104.24 (1C) 106.07
(1C) 106.53 (2C) 107.34 (1C) 112.37 (1C) 113.94 (1C) 114.35 (1C)
120.17 (1C) 121.60 (2C) 122.31 (2C) 123.25 (1C) 130.21 (2C) 131.33
(2C) 132.96 (1C) 156.37 (3C) 157.07 (1C) 159.59 (1C) 160.37 (1C)
161.23 (1C) 161.77 (1C) 161.96 (1C) 162.21 (1C) 182.45 (1C) 208.82
(1C).
##STR00125##
Example 6
Inhibition of CB1, CB2 and .alpha.-Glucosidase by Kuwanon G and
Albanin G Purified from Morus alba
[0198] The CB1 binding assay, described in Example 2, was used to
test the Kuwanon G and Albanin G compounds isolated and identified
in Example 5. The Kuwanon G Albanin G compounds were tested at
concentrations ranging between 0.04 .mu.g/mL and 20 .mu.g/mL, to
obtain a dose-response curve for each compound. The sample
concentration was plotted against the percent inhibition and the
IC.sub.50 (defined as the concentration at which 50% inhibition of
binding activity is achieved in relation to the control) was
determined. CB1 assay data for each compound are shown in Table
5.
[0199] Inhibition of CB2 receptor-ligand binding activity of the
purified Kuwanon G and Albanin G compounds was also examined using
methods similar to those described in Example 2 for the CB1
receptor, with some modifications. Briefly, human cannabinoid CB2
receptor protein expressed in CHO-K1 cells were used in modified
HEPES buffer (pH 7.0). A 30 .mu.g aliquot of CB2-membrane was mixed
with tritium labeled nonspecific CB1 agonist [.sup.3H] WIN-55,212-2
(2.4 nM) and test samples of Kuwanon G and Albanin G compounds, or
just the nonspecific ligand R (+)-WIN-55,212-2 (10 .mu.M) (positive
control) were incubated in incubation buffer (20 mM HEPES (pH 7.0),
0.5 mg/ml BSA) for 90 minutes at 37'C. After incubation, the
membranes were filtered and washed; the filters were then counted
to determine the amount of radiolabeled [.sup.3H] WIN-55,212-2 that
was specifically bound to the CB2-membrane. The CB2 assay data for
each compound are shown in Table 5.
[0200] Inhibition of rice ea-glucosidase activity by each of the
purified Kuwanon G and Albanin G compounds was measured as follows.
Rice .alpha.-Glucosidase inhibition assay: the test compound or
vehicle was pre-incubated with 77 mU/ml enzyme Rice
.alpha.-glucosidase in MES buffer pH 6.3 for 15 minutes at
25.degree. C. The reaction was initiated with addition of 2 mM
p-nitrophenyl .alpha.-D-glucopyanoside, incubated for another 90
minute at 70.degree. C., and then terminated by adding of 1 M
sodium carbonate. The end-product p-nitrophenol was measured by
spectrophotometer. The rice .alpha.-glucosidase assay data for each
Morus alba compound is shown in Table 5.
TABLE-US-00006 TABLE 5 Inhibition of CB1, CB2 and
.alpha.-glucosidase by Kuwanon G and Albanin G % Inhibition Rice %
Inhibition .alpha.-glucosidase CB1-Binding CB1 IC.sub.50 CB2
IC.sub.50 Compound (100 .mu.g/ml) (20 .mu.g/ml) (.mu.M) (.mu.M)
Kuwanon G -22% 92% 10.1 28.9 Albanin G 66% 96% 0.1 6.6
[0201] These data demonstrate that the two major active
compounds--Kuwanon G and Albanin G--are CB1 selective inhibitors
and that the extract is free from any significant
.alpha.-glucosidase inhibition.
Example 7
CB1 and CB2 Binding Inhibition by Compounds Purified from Milicia
excelsa (African Teak)
[0202] The organic extract (8 g) from the stem barks of Milicia
excelsa, obtained using the methods described in Example 1, was
divided and loaded separately onto two pre-packed flash columns
(120 g silica, particle size 32-60 .mu.m, 4 cm.times.19 cm), then
the column was eluted with the gradient as described in Example 5.
A Diels-Alder adduct of a chalcone and prenylphenyl moiety was
isolated from one of the active fractions and identified as
Sanggenon C/D/O. The structure and spectroscopy data were as
follows: ESIMS (m/z) [M-2H].sup.- 706; UV .lamda..sub.max (MeOH):
265, 320 nm; .sup.1H NMR (500 MHz, METHANOL-d.sub.4) ppm 1.55 (s,
CH.sub.3, 3H) 1.58 (s, CH.sub.3, 3H) 1.82 (m, CH.sub.3, 3H) 2.28
(dd, J=18.65, 5.09 Hz, 1H) 2.39 (dd, J=17.80, 5.09 Hz, 1H) 2.69 (m,
1H) 2.94 (m, 1H) 3.87 (d, J=6.78 Hz, CH, 1H) 4.16 (br. s., CH, 1H)
4.49 (br. s., CH, 1H) 5.19 (br. s., 1H) 5.45 (br. s., 1H) 5.64 (a,
1H) 6.11 (d, J=2.26 Hz, 1H) 6.17 (dd, J=8.48, 2.26 Hz, 1H)
6.23-6.34 (m, 3H) 6.42 (dd, J=8.20, 1.70 Hz, 1H) 6.86 (d, J=8.19
Hz, 1H) 7.21 (d, J=8.48 Hz, 1H) 8.08 (d, J=8.76 Hz, 1H).
##STR00126##
[0203] Sanggenon was tested in the CB1 binding inhibition activity
assay as described in Example 2. The activity data are set forth in
Table 6.
TABLE-US-00007 TABLE 6 Inhibition of CB1 and CB2 by Sanggenon
isolated from Milicia excelsa CB1 CB1 IC.sub.50 CB2 IC.sub.50
Compound (20 .mu.g/ml) (.mu.M) (.mu.M) Sanggenon 96% 3.5 24
[0204] These data show that Sanggenon is a potent inhibitor of CB1
ligand binding with CB1 selectivity over the CB2 receptor
protein.
Example 8
Preparation of Various Milicia excelsa Extracts
[0205] Milicia EtOAc extract 8 was produced as follows: 5 kg of
dried Milicia excelsa stem barks were cut, crushed, and extracted
with approximately 4-fold volume (20 L) of ethyl alcohol (Food
grade. Korea Ethanol Supplies Company, Korea) in water (v/v). The
extraction solvent was treated at 80.degree. C., for 4 hrs and the
resulting extraction was filtered to obtain a supernatant that was
concentrated with evaporator at 40.degree. C. The above-described
extraction procedure was repeated two times. The resulting
extraction solutions were combined together and concentrated until
the volume become 1/25 of the original volumes. The concentrated
solution was then dried by vacuum freeze-drying to obtain 200 g of
crude Milicia excelsa EtOH extract powder.
[0206] 196 g of crude Milicia excelsa EtOH extract powder prepared
in the above procedure was suspended in 2 L of distilled water and
the suspension was vigorously mixed with 2 L of n-hexane to obtain
an n-hexane soluble fraction and water-soluble fraction. The
n-hexane soluble fraction was collected and the residual solution
was subjected to a second n-hexane extraction. The above-described
procedure was repeated four times and the resulting n-hexane
soluble fractions were combined and evaporated under vacuum to
obtain 74.8 g of n-hexane soluble extract 8-1 of Milicia excelsa
stem bark.
[0207] The water-soluble fraction of Milicia excelsa stem bark
prepared in above procedure was vigorously mixed with an equivalent
volume of ethyl acetate to obtain an ethyl acetate soluble fraction
and a water-soluble fraction. The ethyl acetate soluble fraction
was collected and the residual solution was subjected to the ethyl
acetate extraction again. This procedure was repeated four times.
The ethyl acetate soluble fractions and water-soluble fractions
were respectively evaporated under vacuum to obtain 63.9 g of ethyl
acetate soluble extract 8 and 35.34 g of water-soluble extract 8-2
of Milicia excelsa stem bark.
Example 9
Test of .alpha.-Glucosidase and CB1 Activity in Morus alba
Extracts
[0208] The Morus alba 70% EtOH extract and its enriched EtOAc
fraction generated from water/EtOAc partition were evaluated for
the activity against .alpha.-glucosidase in the assay described in
Example 6. These data are displayed in Table 7.
TABLE-US-00008 TABLE 7 CB1 and .alpha.-Glucosidase Inhibition
Activity of Morus alba Extracts .alpha.-Glucoside Inhibition Sample
(100 .mu.g/ml) CB1 Inhibition 70% EtOH 99% 64% (20 .mu.g/ml) EtOAc
Fraction -18% 58% (5 .mu.g/ml)
[0209] These data show that after EtOAc enrichment of Morus alba
ethanol extract, the ar-glucosidase inhibition activity of the
ethanol extract was eliminated, while the binding inhibition
activity of the extract (i.e., prenylated flavonoids) in the CB1
receptor binding assay was retained.
Example 10
In Vitro Study of CB1 Functional Activity of Compounds Isolated
from Morus alba and Other Plants
[0210] In vitro efficacy and CB1 binding specificity of compounds
isolated from Morus alba and Milicia excelsa were tested in the
human CB1 receptor binding assay, and the agonist or antagonist
function of the compounds was determined by measuring the effect on
cAMP or agonist-induced cAMP modulation. Assays were performed by
Ricerca Biosciences LLC, (Concord, Ohio) on plant extract samples
essentially as described by Breivogel et al., J. Biol. Chem.
273:16865, 1998 and Gonsiorek et al., Mol. Pharmacol. 57:1045,
2000.
CB1 Agonist Effect GPCR Functional Assay
[0211] Briefly, CHO-K1 cells were suspended in HBSS buffer
complemented with HEPES 20 mM (pH 7.4), then distributed in
microplates at a density of 10.sup.4 cells/well in the presence of
one of the following: HBSS (basal control), a reference agonist at
100 nM (stimulated control), a reference agonist (EC.sub.50
determination) or a plant extract test compound. Thereafter, the
adenylyl cyclase activator NKH 477 is added at a final
concentration of 3 AM. Following a 10 minute incubation at
37.degree. C., the cells are lysed and the fluorescence acceptor
(D2-labeled cAMP) and fluorescence donor (anti-cAMP antibody
labeled with europium cryptate) were added. After 60 min at room
temperature, the fluorescence transfer is measured at .lamda.ex=337
nm and .lamda.em=620 nm and 665 nm using a microplate reader
(Rubystar, BMG). The cAMP concentration is determined by dividing
the signal measured at 665 nm by that measured at 620 nm (ratio).
The results are expressed as a percent of the control response to
100 nM CP 55940 (full CB1 agonist). The standard reference agonist
is CP 55940, which is tested in each experiment at several
concentrations to generate a concentration-response curve from
which its EC.sub.50 value is calculated.
CB1 Antagonist Effect GPCR Functional Assay
[0212] The CHO-K1 cells are suspended in HBSS buffer complemented
with HEPES 20 mM (pH 7.4), then distributed in microplates at a
density of 10 cells/well and preincubated for five min at room
temperature in the presence of one of the following: the reference
antagonist, AM 281, at 3 .mu.M (basal control), HBSS (stimulated
control), the reference antagonist, for ICs determination, or the
test plant extract compounds. The reference agonist CP 55940 is
also present at a final concentration of 10 nM or is omitted from
the reaction mixture for the basal control. Thereafter, the
adenylyl cyclase activator NKH 477 is added at final concentration
of 3 .mu.M. Following 10 min incubation at 37.degree. C., the cells
are lysed and the fluorescence acceptor (D2-labeled cAMP) and
fluorescence donor (anti-cAMP antibody labeled with europium
cryptate) are added. After 60 min at room temperature, the
fluorescence transfer is measured at .lamda..sub.ex=337 nm and
.lamda..sub.em=620 nm and 665 nm using a microplate reader
(Rubystar, BMG). The cAMP concentration is determined by dividing
the signal measured at 665 nm by that measured at 620 nm (ratio).
The results are expressed as a percent inhibition of the control
response to nM CP 55940. The standard reference antagonist is AM
281, which is tested in each experiment at several concentrations
to generate a concentration-response curve from which its IC.sub.50
value is calculated. The compounds were tested at six different
concentrations (0.3, 1, 3, 10, 30, and 150 .mu.M) to generate a
dose curve. The results are set forth in Table 8 below.
TABLE-US-00009 TABLE 8 CB1 Agonist and Antagonist Inhibition CB1
Antagonist Inhibition Compound CB1 Agonist EC.sub.50 (.mu.M) (at
100 .mu.M) Kuwanon G 85 -1% Albanin G ND 17% Sanggenon 31 -14%
[0213] The data presented in Table 7, suggest that the Kuwanon G
and Sanggenon compounds are affecting the CB1 receptor activity
with agonist-like behavior instead of antagonist. In contrast,
Albanin G appears to have a modest antagonist-like activity with
the CB1 receptor.
Example 11
HPLC Quantification of Active Extracts from Morus alba
[0214] Extracts for raw materials were produced as follows: 20
grams of plant powder mixed with Diatomaceous earth was put into a
100 mL extraction cell. It was extracted with solvent (100% EtOH or
MeOH/CH.sub.2Cl.sub.2 at 1:1) using ASE 350 (Extraction condition:
Heat=5 minutes, Static=5 minutes, Flush=80 volume, Purge=900
seconds, Cycles=3, Pressure=1500 psi, Temperature=80.degree. C.,
Solvent C=100% Ethanol). After extraction, the solution was
filtered and collected, then concentrated with evaporator at
50.degree. C. to produce a solid extract.
[0215] Agilent HPLC/PDA system was used for the detection and
quantification of the Diels-Alder adducts of a chalcone and
prenylphenyl moiety Kuwanon G and Albanin in the Morus extracts.
The C18 reversed-phase column (Phenomenex, USA) was utilized as
Luna Sum, 50 mm long and 4.6 mm in diameter. A binary Purified
water (mobile phase A) and acetonitrile (mobile phase B) gradient
was used for elution. The flow rate was set to 1 ml/min passing
through the Luna C18 column with a column temperature of 40.degree.
C. The UV detector was set to read absorbance at 270 nm.
TABLE-US-00010 TABLE 9 Gradient Table of HPLC Analytical Method
Time (min) Mobile phase A Mobile phase B 0.0 50 50 5.0 50 50 30.0
20 80 30.5 50 50 35.0 50 50
[0216] Pure Kuwanon G, pure Albanin G, and Reference Standard
Material (RSM, Morus EtOAc fraction 17) were utilized as
quantification standards. All extract samples were prepared in a
concentration range from 3 mg/ml to 1 mg/ml after sonicating for
approximately 15 minutes. The sample solution was cooled in a flask
to room temperature and filtered through a 0.45 um nylon syringe
filter and 10 .mu.l of the sample was injected into HPLC.
[0217] Morus plants were collected from China and South Korea from
different geological locations in both countries. The HPLC
quantification of Kuwanon G and Albanin G content in different
species, different plant parts, collected from different locations,
and at different age of plants, are listed in Table 10 through
Table 14.
TABLE-US-00011 TABLE 10 Kuwanon G and Albanin G Content in Two
Species of Morus Plants % of Extract Extraction Sample Kuwanon G
AlbaninG Yield (%) Morus alba 5.13 3.98 15.8 4.6 3.09 14.1 Morus
catayana 0.4 ND 9.7
TABLE-US-00012 TABLE 11 Kuwanon G and Albanin G Contents in
Different Parts of Morus Plants The content(%) of extract
Extraction Morus alba Part Kuwanon G AlbaninG Yield (%) 1 Leaf 0.06
N.D 7.9 2 Fruit N.D N.D 23.1 3 Branch 0.20 0.25 7.4 4 Leaf, Branch
N.D N.D 7.3 Root bark 3.90 2.51 17.8 Root wood 0.15 N.D 5.4 Fine
root 3.52 2.98 15.4 Stem bark 0.97 0.61 10.2 Stem wood N.D N.D 3.2
5 Bark 3.48 1.26 24.6 Periderm 0.09 ND 21.5
TABLE-US-00013 TABLE 12 Kuwanon G and Albanin G Content in Morus
alba Root Barks from Different Geological Locations in Korea Active
content(%) Extraction Vendor Name Kuwanon G Albanin G Yield (%)
Kyoungdong 5.13 3.98 15.8 Kyoundong 1.23 0.82 17.4 Asan 3.11 1.81
19.2 Seon-il mulsan 1.99 0.59 10.2 Kyoungdong 0.80 N.D 11.05
TABLE-US-00014 TABLE 13 Kuwanon G and Albanin G Content in Morus
alba Root Barks purchased from Different Geological Locations in
China. The content(%)/Extract Extraction Vendor Name Kuwanon G
Albanin G Yield (%) Sichuan 3.64 2.82 14.1 Hubei 0.64 0.13 6 Hunan
0.14 0.18 5.65 Guizhou 4.60 3.09 14.1 Yunnan 0.67 0.33 8
Sichuan/Xichang 0.85 N.D 12.1 Sichuan/Mian Yang 4.66 2.74 11
Sichuan 3.41 2.73 14.5 Sichuan 3.44 2.67 15.1 Hunan 4.60 2.30
11.05
TABLE-US-00015 TABLE 14 Kuwanon G and Albanin G Content in
Different Age of Morus alba Root Barks. Content % Extract
Extraction Yield Location Years old Kuwanon G AlbaninG (%) A 2 8.85
6.12 12 3 3.49 2.40 8.9 B 2 2.49 1.00 8.6
Example 12
Preparation of Morus alba 70% EtOH Extract 12
[0218] Dried Morus alba roots and root barks (93.3 kg) were cut,
crushed, and then extracted with approximately seven-fold volume
(700 L) of 70% ethyl alcohol in water (v/v); the extraction was
carried out at 100.degree. C. for 4 hrs. The ethanol solution was
filtered to obtain the supernatant, which was then concentrated
with an evaporator under vacuum at 40.degree. C. This extraction
and concentration procedure was repeated two times. The extraction
solutions were then combined together and concentrated until the
volume become 1/25 of the original volume. The concentrated
solution was dried by vacuum freeze-drying to obtain 18.3 kg of
Morus alba 70% EtOH extract powder 12. The extraction yield was
about 19.6% (w/w). The major active component content is listed in
Table 16 of Example 20.
Example 13
Preparation of Morus alba EtOAc Fraction 13
[0219] Morus alba EtOH extract 12 from Example 12 (15 kg) was
extracted with approximately two-fold volume (300 L) of ethyl
acetate (EP grade, Ducksan Chemical, Korea), Extraction was
performed by homogenization of the extraction solution at 15,000
rpm for five minutes with homogenizer (IKA T25D, Germany). The well
homogenized extraction solution was then separated by centrifuge
(Beckman J-20XP, Germany) at 3,000 rpm (rotor# JLA 8.1000) for five
minutes. The upper layer (EtOAc soluble layer) was filtered by
filter paper (Hyundai Micro, No. 20, Korea) and the EtOAc solution
was collected. The residue (precipitate material) collected from
the centrifugation was re-extracted with two-fold volume (300 L) of
ethyl acetate (EP grade, Ducksan Chemical, Korea). The re-extracted
solution was agitated at 150 rpm for 2 hours. The resulting mixture
was then filtered (Hyundai Micro, No. 20, Korea) to obtain an
additional EtOAc extract solution. The above-described procedure
was repeated two times. The resulting three EtOAc extract solutions
were then combined and concentrated by evaporator at 40.degree. C.
to obtain the final EtOAc extract 13. The yield was 3.04 kg from 15
kg of 70% EtOH extract. The major active component content is
listed in Table 16 of Example 20.
Example 14
Preparation of Morus alba EtOAc Fraction 14
[0220] Morus alba EtOAc fraction 14 was produced from EtOH extract
using the extraction methods described in Example 12. 4.5 kg of
dried Morus alba roots and roots barks yielded 715 g of crude Morus
alba EtOH extract powder. Corresponding n-hexane soluble and water
soluble extracts were prepared from 691.4 g of the crude Morus alba
EtOH powder after solvent partition. This resulted in production of
95.9 g of the n-hexane soluble extract and 263.8 g of the
water-soluble extract of Morus alba. The water soluble extract was
further partitioned with an equivalent amount of ethyl acetate
(EtOAc) to produce an EtOAc extract. The final amount of Morus alba
EtOAc fraction 14 obtained from this process was 331.8 g. The major
active component content is listed in Table 16 of Example 20.
Example 15
Preparation of Morus alba EtOAC Fraction 15
[0221] Morus EtOAc fraction 15 was produced using the extraction
methods described in Examples 12 and 14. Dried Morus alba roots and
root barks (2.0 kg) yielded 283.5 g of crude Morus alba EtOH
extract powder. Corresponding n-hexane soluble and water soluble
extracts were prepared from 100 g of the crude Morus alba EtOH
powder according to the method of Example 14. This resulted in
production of 13.7 g of the n-hexane soluble extract and 38 g of
the water-soluble extract of Morus alba. After solvent partition
with EtOAc, the final amount of Morus alba EtOAc fraction 15
obtained from this process was 47.6 g. The major active component
content is listed in Table 16 of Example 20.
Example 16
Preparation of Morus alba EtOAC Fraction 16
[0222] Morus EtOAc fraction 16 was produced using the extraction
methods described in Examples 12 and 14. 3 kg of dried Morus alba
roots and root bark yielded 428 g of crude Morus alba EtOH extract
powder, Corresponding n-hexane soluble and water soluble extracts
were prepared from 300 g of the crude Morus alba EtOH powder
according to the methods of Example 14. This resulted in production
of 40.8 g of the n-hexane soluble extract and 92.7 g of the
water-soluble extract of Morus alba. After solvent partition with
EtOAc, the final amount of Morus alba EtOAc fraction 16 obtained
from this process was 150.1 g. The major active component content
is listed in Table 16 of Example 20.
Example 17
Preparation of Morus alba EtOAC Fraction 17
[0223] Morus EtOAc fraction 17 was produced using the extraction
methods described in Examples 12 and 14. 4 kg of dried Morus alba
roots and root bark yielded 570 g of crude Morus alba EtOH extract
powder. Corresponding n-hexane soluble and water soluble extracts
were prepared from 570 g of the crude Morus alba EtOH powder
according to the methods of Example 14. This resulted in production
of 80.5 g of the n-hexane soluble extract and 156 g of the
water-soluble extract of Morus alba. After solvent partition with
EtOAc, the final amount of Morus alba EtOAc fraction 17, obtained
from this process was 327 g. The major active component content is
listed in Table 16 of Example 20.
Example 18
Preparation of Morus alba 70% EtOH Precipitate Extract 18
[0224] Morus alba EtOH precipitate extract 18 was produced by
follows; 634 kilograms (KG) of dried Morus alba roots and root
barks were cut, crushed and extracted with approximately 7 fold
volume (3600 liters (L) of 70% ethyl alcohol in water (v/v); the
extraction solvent was treated at 80.degree. C., for 4 hrs; the
residue was filtered to obtain the supernatant which was then
concentrated with an evaporator at 40.degree. C. The
above-described procedure was repeated three times. The extraction
solutions were then concentrated until the volume become about 1/30
the original starting volumes. Then the concentrated solutions were
combined to evaporate again in order to reduce volume of
concentrated solution until 1/90 volume of the original extraction
solution. The concentrated solution was rested at room temperature
for 24 hours (hr) to allow separation into two layers (supernatant
and precipitate-layer). The precipitate was filtered and dried by
vacuum freeze-drying to obtain M. alba 70% EtOH precipitate powder.
A total of 24 kg of the resulting product was obtained from 634 kg
of raw plant material. The extraction yield was about 3.79% (w/w).
The major active component content is listed in Table 16 of Example
20.
Example 19
Preparation of Morus alba 70% EtOH Precipitate (19-1), EtOH
Combination (19-2), and EtOH Supernatant (19-3) Extracts
[0225] Morus alba EtOH precipitate extract was produced as follows:
465 kg of dried Morus alba roots and root bark were cut, crushed,
and extracted with approximately 10-fold volume (4500 L) of 70%
ethyl alcohol in water (v/v); the extraction solvent was treated at
80.degree. C. for 4 hrs; the residue was filtered to obtain the
supernatant which was concentrated with an evaporator at 40.degree.
C. Above-described procedure was repeated three times. The
extraction solutions were concentrated until the volume become 1/30
the original volume. The concentrated solutions were then combined
and evaporated again to reduce the volume of the concentrated
solution until 1/90 volume of the original extraction solution was
achieved. The concentrated solution was left at room temperature
for 24 hr to allow separation into a supernatant and precipitate
layer. The precipitate layer was then dried by vacuum to obtain 12
kg of Morus alba 70% EtOH precipitate extract powder. The
extraction yield was about 2.6% (w/w). The supernatant layer was
dried by vacuum drying to obtain 24 kg Morus alba 70% EtOH
supernatant extract powder. The extraction yield for the
supernatant extract was about 5.2%.
[0226] Morus alba 70% EtOH combination extract 19-2 was obtained by
blending 2 kg of precipitate extract 19-1 and 4 kg of supernatant
extract 19-3. The major active component content in both Morus alba
EtOH precipitate 19-1 and combination extract 19-2 is listed in
Table 16 of Example 20.
Example 20
HPLC Quantification of Active Content in Different Morus alba
Extracts
[0227] The detailed HPLC quantification method for Kuwanon G and
Albanin G was described in Example 11. With the complexity of crude
extracts, different elution solvents, such as the following, could
also be utilized: a binary 0.1% phosphoric acid in purified water
(mobile phase A) and acetonitrile (mobile phase B) gradient was
used for elution (Table 15).
TABLE-US-00016 TABLE 15 Gradient table of HPLC Analytical Method
Time (min) Mobile phase A Mobile phase B 0 33 67 5 33 67 55 24 76
59 0 100 65 0 100 66 33 67 75 33 67
[0228] The flow rate 0.8 ml/min passing through the Luna C18 column
with column temperature 35.degree. C. The UV detector was set up at
275 nm. The reference standard material (RSM, Morus EtOAc fraction
17) was extracted with DMSO and utilized for calculation. Table 16
lists the major active components of Kuwanon G and Albanin G in the
pilot and production grade Morn alba root bark extracts and
fractions described in Examples 12-19,
TABLE-US-00017 TABLE 16 Kuwanon G and Albanin G in Different Morus
alba Root Bark Extracts and Fractions % Total Sample Extract %
Kuwanon G % Albanin G Active 12 2.88 1.64 4.51 13 8.80 5.80 14.60
14 13.53 8.32 21.85 15 10.51 6.95 17.46 16 10.93 7.38 18.31 17
(RSM) 9.95 6.65 16.60 18 5.30 4.16 9.46 19-1 6.21 4.33 10.54 19-2
3.30 1.81 5.11
Example 21
Preparation of Rosemary Steam Distillated EtOH Extract
[0229] Dried Rosemary leaf (2.6 kg) was distillated with
approximately 6 fold volume (15 L) of water to remove essential
oils and volatile components at 100.degree. C. for 5 hrs. After
steam distillation the retained residue was filtered to remove the
water distillated solution from the residue and then, the residue
was re-extracted with 6-fold volume (15 L) of ethyl alcohol (95%)
at 80.degree. C. for 5 hrs. The resulting extraction was filtered
to obtain a supernatant that was concentrated using an evaporator
at 40.degree. C. The above-described extraction procedure was
repeated twice. The resulting supernatants were combined together
and then concentrated with an evaporator at 40.degree. C. The
resulting concentrated solution was then dried by vacuum
freeze-drying to obtain 357.1 g of Rosemary steam distillated EtOH
extract powder 21. The extraction yield was about 13.7% (w/w).
Chemical analysis showed that Rosemary extract 21 contained the
following compounds (percent weight): Carnosol: 2.97%; Carnosic
Acid: 2.00% and Ursolic acid: 18.49%; total actives of these three
compounds 23.80%.
Example 22
Preparation of Rosemary EtOH Extracts 22 and 22-1
[0230] Rosemary EtOH extract 22 was produced using essentially the
methods described in Example 21. 29 kg of dried Rosemary leaf was
distillated with approximately 10-fold volume (300 L) of water to
remove essential oils and volatile components at 100.degree. C. for
2 hrs. The residue was next filtered to remove water distillated
solution from the residue and then the residue was re-extracted
with 10 fold volume (300 L) of 95% ethyl alcohol at room
temperature (20.degree. C.) for 2 hrs. The resulting residue was
filtered to obtain supernatant that was concentrated with an
evaporator at 40.degree. C. The resulting residue was extracted
again with a 10-fold volume (300 L) of 70% ethyl alcohol (v/v) at
room temperature (20.degree. C.) for 2 hrs. The resulting extract
residue was filtered and the supernatants retained and combined.
The resulting supernatant was then concentrated with an evaporator
at 40.degree. C. The resulting concentrated solution was finally
dried by vacuum freeze-drying to obtain 2.5 kg of Rosemary water
distillated EtOH extract powder 22. The extraction yield was about
8.6% (w/w). HPLC Actives profile for Carnosol, Carnosic acid and
Ursolic acid of this extract are provided in Example 25, Table
18.
[0231] Rosemary EtOH extract 22-1 was produced as follows: 58.68 kg
of dried Rosemary leaf was distillated with approximately 10 fold
volume (600 L) water to remove essential oils and volatile
components at 100.degree. C. for 2 hrs; the residue was removed
from the water distillated solution by filtration and then the
residue was re-extracted with 10 fold volume (600 L) 95% ethyl
alcohol at room temperature (20.degree. C.) for 2 hrs. The residue
was filtered to obtain supernatant. The residue was then extracted
again with 10 fold volume (600 L) 70% ethyl alcohol (v/v) at room
temperature (20.degree. C.) for 2 hrs. The doubly extracted residue
was filtered to obtain supernatant. Both supernatants were combined
and concentrated with an evaporator at 40.degree. C. Concentrated
supernatants were dried by vacuum freeze-drying to obtain Rosemary
water distillated EtOH extract powder 22-1. The yield was 5.0 kg
Rosemary extract powder 22-1 obtained from 58.68 kg of raw
material. The extraction yield was about 8.5% (w/w).
Example 23
Preparation of Rosemary Water Distillated EtOH Extract 23
[0232] Rosemary EtOH extract was produced essentially as described
in Example 22. 62 kg of dried Rosemary leaf yielded 5.9 kg water
distillated extract powder. The extraction yield was about 9.5%
(w/w). HPLC Actives profile for Carnosol, Carnosic acid and Ursolic
acid in this extract 23 are provided in Example 25, Table 10.
Example 24
Preparation of Rosemary Steam Distillated EtOH Extract 24
[0233] Rosemary steam distillated EtOH extract was produced
essentially as described in Example 21. 2 kg of dried Rosemary leaf
yielded 317.7 g of water distillated extract powder designated lot#
RN348-3201. The extraction yield was about 15.8% (w/w). HPLC
Actives profile for Carnosol, Carnosic acid and Ursolic acid in
this extract are provided in Example 22, Table 10.
Example 25
Analytical Results for Rosemary Leaf Extract
[0234] The following analytical method was used to determine the
amount of Carnosol, Carnosic acid and Ursolic acid in Rosemary
steam distillated ethanol extract 24 and water distillated ethanol
extracts 22 and 23. An Agilent HPLC/PDA system was used with a C18
reversed-phase column (Phenomenex, USA, Luna 5 um, 250 mm.times.4.6
mm), for detection and quantitation of Carnosol, Carnosic acid and
Ursolic acid. A binary 0.1% phosphoric acid in purified water
(mobile phase A) and acetonitrile (mobile phase B) gradient was
used for elution as set forth in Table 9. The column flow rate was
set at 1 ml/min passing through the Luna C18 column with a column
temperature of 40.degree. C. The UV detector was set up to read
absorbance at 210 nm.
TABLE-US-00018 TABLE 17 Rosemary Gradient Elution Scheme Time (min)
Mobile phase A Mobile phase B 0 40 60 5 20 80 30 0 100 34 0 100 35
40 60 40 40 60
[0235] Carnosol, Carnosic acid and Ursolic acid standards were
purchase from Sigma and dissolved in DMSO. Highest level control
concentration of carnosol and carnosic acid was made to 0.1 mg/ml.
The highest level control concentration of ursolic acid was made to
0.3 mg/ml and diluted to (0.065 mg/ml or 0.02 mg/ml) using
methanol. The test sample concentration were adjusted to about 1
mg/ml in methanol in a volumetric flask and sonicated until the
sample dissolved (approximately 20 minutes). The sample flask was
then cooled to room temperature, mixed well and filtered through a
0.45 um nylon syringe filter and then 10 ul of sample was injected
into HPLC. HPLC results showed the Table 10.
TABLE-US-00019 TABLE 18 HPLC Results of Rosemary Leaf Extracts
Sample Extract Carnosol % Carnosic acid % Ursolic acid % Total % 22
8.84 4.37 7.83 21.04 23 6.52 5.32 10.47 22.31 24 4.07 2.69 14.53
21.30
Example 26
Preparation of Yerba Mate Ethyl Alcohol Extract 26
[0236] Yerba Mate (Ilex paraguariensis) EtOH extract was produced
as follows: 1 kg of dried Ilex paraguariensis leaf, was cut,
crushed, and extracted with a 20-fold volume (20 L) of 95% ethyl
alcohol at 85.degree. C. for 4 hrs. The resulting residue was
filtered to obtain a supernatant that was concentrated with an
evaporator at 40.degree. C. The resulting residue was extracted a
second time with 20-fold volume (20 L) of 95% ethyl alcohol (v/v)
at 85.degree. C. for 4 hrs and filtered to obtain a second
supernatant which was concentrated with an evaporator at 40.degree.
C. The resulting concentrated cake was dried under vacuum to obtain
260 g of Yerba Mate EtOH extract powder. The extraction yield was
about 26% (w/w).
Example 27
Preparation of Yerba Mate Ethyl Alcohol Extract 27
[0237] Yerba Mate EtOH extract was produced as follows: 150 kg of
dried Yerba Mate (Ilex paraguayensis) leaf were cut, crushed, and
extracted with approximately 7-fold volume (1050 L) of 70% ethyl
alcohol in water (v/v) and the extraction solvent held at
100.degree. C. for 4 hrs. The residue was filtered to obtain a
supernatant that was concentrated with an evaporator at 40.degree.
C. The above-described procedure was repeated twice. The extraction
solutions were then combined together and concentrated until the
volume become 1/25 of the original volume. The concentrated
solution was dried by vacuum freeze-drying to obtain 26.7 kg of
Mate 70% EtOH extract powder. The extraction yield is about 17.8%
(w/w). HPLC analysis, as described in Example 29, determined that
the amount of Caffeine in Yerba Mate extract 27 was 2.44% by
weight.
Example 28
Quantification of Caffeine in Yerba Mate Leaf Extract
[0238] The following analytical method was used to determine the
amount of Caffeine in the Yerba Mate leaf extracts. The same
Agilent HPLC/PDA system, including the C18 reversed-phase column
(Phenomenex, USA) described in Example 25 was used for the
detection of Caffeine and minor components. A binary 0.1%
phosphoric acid in purified water (mobile phase A) and methanol
(mobile phase B) gradient was used for elution of Mate sample
components as described in Table 11. The flow rate was set to 1
ml/min passing through the Luna C18 column with a column
temperature of 35.degree. C. The UV detector was set to read
absorbance at 275 nm.
TABLE-US-00020 TABLE 19 Yerba Mate Gradient Elution Scheme Time
(min) Mobile phase A Mobile phase B 0 95 5 5 95 5 65 0 100 75 0 100
80 95 5 90 95 5
[0239] The quantification standard--Caffeine was purchased from
Sigma. Dicaffeoylquinic acid (DCYA) standards were purchased from
Chengdu Biopurify Phytohemicals Ltd., and dissolved in DMSO. The
highest concentration level of caffeine and 4,5-DCYA was 0.05 mg/ml
and diluted to L5 from L1 (0.0031 mg/ml) using methanol. The
highest concentration level of 3,4-DCYA was 0.02 mg/ml and diluted
to L5 from L1 (0.00125 mg/ml) using methanol. The highest
concentration level of 3,5-DCYA was 0.025 mg/ml and diluted to L5
from L1 (0.0016 mg/ml) using methanol. Concentration of Yerba Mate
leaf extract samples were adjusted to about 1 mg/ml in methanol in
a volumetric flask and sonicated until dissolved (approximately 20
minutes), and then cooled to room temperature, mixed well and
filtered through a 0.45 um nylon syringe filter. 10 .mu.l of sample
was examined by HPLC. HPLC actives quantification results for Yerba
Mate extract 27 and weight loss Compositions 1 and 3 (exemplified
in Examples 38 and 39, respectively) are provided in Table 20.
TABLE-US-00021 TABLE 20 HPLC Quantification of Yerba Mate Extract
and Weight Loss Compositions Sample Caffeine 3-4 DCYA 3-5 DCYA 4-5
DCYA Total % 27 2.44 0.77 1.24 2.09 6.55 Composition 1 1.80 0.58
0.94 1.56 4.88 Composition 3 1.29 0.41 0.69 1.43 3.82
Example 29
Preparation of Magnolia Extracts 29 and 29A
[0240] Magnolia (Magnolia officinalis) extract 29 was produced as
follows. 70 kg of dried stem barks of Magnolia officinalis was cut,
crushed, and extracted with 70% ethyl alcohol and the extraction
solvent treated at 80.degree. C. for 4 hrs. The resulting the
residue was filtered to obtain a supernatant that was concentrated
with an evaporator at 40.degree. C. The above-described procedure
was repeated two times. The extraction solutions were then combined
together and concentrated until the volume become 1/25 of the
original volume. Sodium hydroxide (NaOH) was added into the
concentrated solution to obtain a final concentration of 1% NaOH.
After saponification at 80.degree. C. for 30 min, the solution was
extracted with 2-fold volume of Hexane with agitator for 1 hour.
The resulting Hexane soluble fraction was then collected and
above-described procedure repeated three times. The n-hexane
soluble fractions were combined and evaporated under vacuum until
the volume become 1/6 of the original volume. High purity Magnolia
extract was obtained after re-crystallization vacuum drying. 70 kg
of dried stem barks of Magnolia officinalis yielded 652 g of high
purity Magnolia extract. The extraction yield is 0.93%. A second
batch of Magnolia extract (29A) was also produced according to the
same procedure, Quantification of the active content in both
extracts is provided in the following example.
Example 30
Analytical Method for Analysis of Magnolia Stem Bark Extracts
[0241] The following analytical method was used to determine the
amount of Magnolol and Honokiol in Magnolia stem bark extracts. The
same Agilent HPLC/PDA system, including the C18 reversed-phase
column (Phenomenex, USA) described in Example 25 was used for the
detection of Magnolol and Honokiol. A binary purified water (mobile
phase A) and acetonitrile (mobile phase B) gradient was used for
detection of Magnolol and Honokiol as described in Table 13. The
flow rate was set to 1 ml/min passing through the Luna C18 column
with a column temperature of 35.degree. C. and absorbance was read
at 290 nm.
TABLE-US-00022 TABLE 21 Magnolia Gradient Elution Scheme Time (min)
Mobile phase A Mobile phase B 0.0 23 77 18.0 23 77 18.1 0 100 25.0
0 100 25.1 23 77 30.0 23 77
[0242] Magnolol and Honokiol standards were purchased from
Guangzhou Honsea Sunshine Bio Science and Technology co., Ltd. and
extracted with methanol. The highest standard concentration of
Honokiol and Magnolol was 0.2 mg/ml and diluted to L3 from L1 (0.05
mg/ml) using methanol. The Magnolia stem bark extract sample
concentration was adjusted to about 0.2 mg/ml and combination
sample concentration was 2 mg/ml in methanol. A volumetric flask
was used in sample preparation and sonicated until dissolved
(approximately 10 minutes), flask was cooled to room temperature
and QS with extraction method, mixed well and filtered through a
0.45 um nylon syringe filter and 20 .mu.l sample was analyzed by
HPLC. The HPLC quantification results are provided in Table 22.
TABLE-US-00023 TABLE 22 HPLC Results of Magnolol and Honokiol in
Magnolia Stem Bark Extracts Total % Sample Honokiol % Magnolol %
(by weight) 29A 46.23 51.54 97.77 29 51.20 47.20 98.40
Example 31
Preparation of Areca catechu 70% EtOH Extract 31
[0243] Ground Areca catechu seed (7 kg) was divided into two
portions of 3 kg and 4 kg, crushed and place into two extraction
units and extracted with about an 8-fold volume (about 240 L and
320 L) of 70% ethyl alcohol in water (v/v) and the extraction
solvent held at 90.degree. C. for 4 hrs. After filtering the
extract solution, the filtrate was concentrated with an evaporator
at 50.degree. C. until only a water solution remained, which was
then collected and frozen at -70.degree. C. The remaining crushed
seed material was then extracted again as before. The 1.sup.st and
2.sup.nd frozen extract solutions were then dried in a freeze dryer
device. The dried extracts were combined and ground into a fine
powder to obtain 930 g. The final extraction yield was about 13.2%
(w/w).
Example 32
Preparation of Mutamba (Guazuma ulmifolia) Stem Bark EtOH Extract
32
[0244] Mutamba EtOH extract 32 was produced as follows: 500 g of
dried Mutamba (Guazuma ulmifolia) stem bark was cut, crushed and
extracted with 20-fold volume (10 L) of 95% ethyl alcohol at
85.degree. C. for 4 hrs. The residue was filtered to obtain a
supernatant. The residue was then extracted a second time as before
and the two EtOH supernatants combined together and concentrated
with evaporator at 40.degree. C. The concentrated cake was dried by
vacuum drying to obtain 70 g of Mutamba stem bark EtOH extract
powder. The extraction yield was about 14% (w/w). HPLC analysis
showed that this extract contained 1.31% Procyanidin B2 and 0.86%
Epicatechin.
Example 33
Preparation of Mutamba Extracts 33, 33-1, and 33-2
[0245] Mutamba EtOH extract 33 was produced as follows: two
different batches of 2.3 kg of dried Mutamba stem bark were cut,
crushed and then each batch was extracted with 15-fold volume (30
L) of 95% ethyl alcohol at 85.degree. C. for 4 hrs. The residue was
filtered to obtain two batch supernatants. The two supernatants
were combined and concentrated with an evaporator at 40.degree. C.
The resulting concentrated cake was then dried by vacuum to obtain
370 g of Mutamba EtOH extract powder 33. The extraction yield is
about 8.04% (w/w).
[0246] The two residues from the above-extraction were then
extracted again with about a 15-fold volume (30 L) of 95% ethyl
alcohol (v/v) at 85.degree. C. for 4 hrs. The residue was filtered
to obtain a supernatant that was concentrated with an evaporator at
40.degree. C. The resulting concentrated cake was dried by vacuum
drying to obtain 290 g of Mutamba EtOH extract powder 33-1 and the
extraction yield was about 6.3% (w/w).
[0247] The Mutamba EtOH extract 33-2 was produced as follows:
Mutamba EtOH powder extracts 33 and 33-1 were combined together and
pulverized resulting in 660 g of Mutamba EtOH extract 33-2. The
final extraction yield was about 14.34% (w/w). HPLC analysis showed
that this extract contained 0.96% Procyanidin B2 and 0.62%
Epicatechin.
Example 34
Preparation of Mutamba EtOH Extract Fractions 34, 34-1, and
34-2
[0248] Mutamba EtOH extract derived fractions 34, 34-1, and 34-2
were produced as follows: 500 g of Mutamba stem bark EtOH extract
33-2 from Example 33, was suspended in 5 L of distilled water and
the suspension was mixed with 5 L of ethylacetate vigorously to
divide into an ethylacetate soluble fraction and a water-soluble
fraction. The ethylacetate soluble fraction was collected and the
residual solution was subjected to a second ethylacetate
extraction. The above-described procedure was repeated three times.
The collected ethylacetate soluble fractions were pooled and then
evaporated in vacuo to give 117 g of ethylacetate soluble extract
of Mutamba stem bark 34. HPLC analysis showed that this extract
contained 2.57% Procyanidin B2 and 2.24% Epicatechin.
[0249] A water-soluble fraction of Mutamba stem bark EtOH extract
33-2 prepared in Example 33 was vigorously mixed with an equivalent
volume of butyl alcohol and water, and allowed to separate into a
butyl alcohol soluble fraction and water-soluble fraction. The
butyl alcohol soluble fraction was collected and the residual
solution was subjected to the butyl alcohol extraction again. This
procedure was repeated three times. The respective fractions were
pooled and evaporated in vacuo to obtain 260 g of butyl alcohol
soluble extract 34-1 and 130 g of water-soluble extract 34-2 of
Mutamba stem bark. HPLC analysis showed that Mutamba extract
fraction 34-1 contained: 1.31% Procyanidin B2 and 0.86%
Epicatechin. HPLC analysis was not performed on Mutamba extract
fraction 34-2.
Example 35
Preparation of Mutamba EtOH Extract 35
[0250] Mutamba EtOH extract 35 was produced by follows: 920 g of
dried Mutamba stem bark was cut, crushed, and extracted with
20-fold volume (20 L) of 95% ethyl alcohol at 85.degree. C. for 4
hrs. The residue was filtered to obtain a supernatant and the
retained residue was then again extracted with 20 L of 95% ethyl
alcohol (v/v) and filtered as before. The resulting supernatants
were combined together and concentrated with an evaporator at
40.degree. C. The resulting concentrated cake was dried under
vacuum to obtain 132 g of Mutamba EtOH extract powder 35. The
extraction yield was about 14.34% (w/w). HPLC analysis showed that
extract 35 contained 1.03% Procyanidin B2 and 0.60%
Epicatechin.
Example 36
Preparation of Mutamba EtOH Extract 36
[0251] Mutamba EtOH extract 36 was produced as follows: 85.7 kg of
dried Mutamba stem bark was cut, crushed, and extracted with
approximately 8-fold volume (720 L) of 70% ethyl alcohol in water
(v/v) after incubation in the extraction solvent at 100 C for 4
hrs. The residue was filtered to obtain a retained supernatant and
the residue was then re-extracted twice more using the same
extraction procedure. The retained supernatants were then were
combined together and concentrated until the volume was 1/25 of the
original starting volume. This concentrated solution was then dried
by vacuum freeze-drying to obtain 13.9 Kg of Mutamba stem bark 70%
EtOH extract powder 36. The extraction yield was about 16.2% (w/w).
The extraction yield was about 14.34% (w/w). HPLC analysis showed
that extract 36 contained 1.03% Procyanidin B2 and 0.60%
Epicatechin.
Example 37
HPLC Quantification of Various Mutamba Extracts
[0252] An Agilent HPLC/PDA system was used for the detection and
quantitation of procyanidin B2 and epicatechin compounds in Mutamba
plant extracts obtained from trees grown in different locations,
using different tree parts, different tree sexes and different tree
ages. A C18 reversed-phase column (Agilent, USA) was used (Zorbax
eclipse XDB-C18, 3.5 um, 150 mm.times.4.6 mm. A binary column
gradient was used for elution of material from the column. Mobile
Phase A: 0.01% trifluoroacetic acid in purified water, and Mobile
Phase B: acetonitrile gradient was used for elution (Table 23). The
flow rate was set to 0.8 ml/min passing through the Luna C18 column
with a column temperature of 35.degree. C. The UV detector was set
to record absorbance at 275 nm.
TABLE-US-00024 TABLE 23 Gradient table of HPLC Analytical Method
Time (min) Mobile phase A Mobile phase B 0 92 8 10 83 17 20 80.6
19.4 25 77.4 22.6 28 70.0 29.8 30 35 65 38 0 100 40 0 100 42 92 8
45 92 8
[0253] Pure epicatechin reference sample was purchased from Sigma.
Pure procyanidin B2 was purchased from Chengdu Biopurify
Phytohemicals, Ltd. Both reference samples were dissolved in DMSO.
Highest level concentration range of epicatechin was 0.05 mg/ml and
diluted to L5 from L1 (0.003 mg/ml) using 50% methanol in water.
Highest level concentration of procyanidin B2 was 0.05 mg/ml and
diluted to L5 from L1 (0.003 mg/ml) using 50% methanol in water.
Concentration of the Mutamba extract samples were adjusted to 2
mg/ml in 50% methanol in water in a volumetric flask and sonicated
until dissolved (approximately 20 minutes), and then cooled to room
temperature, mixed well and filtered through a 0.45 um nylon
syringe filter. HPLC analysis was performed by injecting a 10 .mu.l
sample into the HPLC.
1. Belize
[0254] All plant materials were cut, crushed, and extracted with
70% EtOH and the procyanidin B2 and epicatechin contents analyzed
by HPLC as described above. The HPLC results are presented in Table
24.
TABLE-US-00025 TABLE 24 Procyanidin B2 and Epicatechin Content in
Mutamba Trees from Belize Mutamba Content (%) of Extract Extraction
Tree Sample Ext ID Tree Part Procyanidin B2 Epicatechin Total Yield
(%) One year old B1 Stem 0.67 0.94 1.61 8.9 female tree B2 Bark
1.18 1.06 2.24 17.1 Mature B3 Stem 0.99 1.04 2.03 6.5 female tree
B4 Bark 1.92 1.54 3.45 12.4 Female tree B5 Bark 4.49 2.21 6.70 17.1
(3 m height) B6 Stem 1.02 1.03 2.05 7.5 Female tree B7 Bark 2.98
1.55 4.52 20.4 (3-4 m B8 Stem 1.01 1.03 2.03 9.3 height) Mature
male B9 Bark 3.97 2.00 5.97 15.1 tree B10 Stem 0.31 0.70 1.01
7.2
2. India
[0255] Five different Mutamba samples from different tree parts
were purchased from a vendor in India. The plant materials were
cut, crushed, and extracted with MeOH/CH.sub.2Cl.sub.2 (1:1 volume
ratio) and the procyanidin B2 and epicatechin contents analyzed by
HPLC as described above. The HPLC results are presented in Table
25.
TABLE-US-00026 TABLE 25 Procyanidin B2 and Epicatechin Content in
Mutamba Trees from India Extract Content(%) of Extract Extraction
No. PART Procyanidin B2 Epicatechin Total Yield (%) I1-1 Leaf 0 0 0
13.4 I1-2 Fruit 0 0 0 12.7 I1-3 Fine stem 0 0 0 8.2 I1-4 Stem bark
0 0 0 5.6 I1-5 Stem wood 0 0 0 2.2
3. Six Different Countries.
[0256] S Mutamba stem bark samples from six different countries
were purchased from a vendor. The plant materials were cut,
crushed, and extracted with 100% EtOH (E) or 70% EtOH (70E) or
MeOH/CH.sub.2Cl.sub.2 (1:1 volume ratio) (OE) and the procyanidin
B2 and epicatechin contents analyzed by HPLC as described herein.
The HPLC results are presented in Table 26.
TABLE-US-00027 TABLE 26 Procyanidin B2 and Epicatechin Content in
Mutamba Stem Bark from Different Countries % Content/Extract
Extraction Extract Procyanidin Yield No. Country B2 Epicatechin
Total (%) Solvent U1 Peru 1.14 0.62 1.76 28 70E M2 Mexico 0.98 0.36
1.33 3 70E B11 Belize 1.31 0.75 2.06 13 70E P99 Panama 1.31 0.86
2.17 E E10 England 0.31 0.05 0.36 8.1 70E I1-4 India 0 0 0 5.6
OE
Example 38
Preparation of Magnolia:Morus:Yerba Mate Composition 1
[0257] Three component (Magnolia:Morus:Yerba Mate) Composition 1
was produced by from the following three plant extract components:
1.02 kg of dried Magnolia extract powder 29, 2.01 kg of Morus alba
root bark extract powder 18, and 10.08 kg of Yerba Mate extract
powder 27 were blended with v-type blender (Seo-kang Engineering,
Korea) at 30 rpm for 1 hour. The final blending weight ratio was
Magnolia:Morus:Yerba Mate of 1:2:10 and resulted in production of
12.65 kg of weight loss combination Composition 1. The major active
compound profile in Composition 1 was determined by HPLC analysis
as described in Examples 11, 20, 25, 28 and 30. The quantification
results are shown in the Table 27.
TABLE-US-00028 TABLE 27 Summary of Active Components in Composition
1 Magnolia Morus Yerba Honokiol Magnolol Kuwanon Albanin Mate % % G
% G % Caffeine % Composition 1 3.88 3.91 1.09 0.77 1.80
Example 39
Preparation of Morus:Rosemary:Yerba Mate Composition 3
[0258] Three component (Morus:Rosemary:Yerba Mate) Composition 3
was produced as follows: 2.4 kg of dried Morus root bark extract
powder 18, 6.0 kg of dried Rosemary extract powder 23, and 12.04 kg
of dried Yerba Mate extract powder 27 were blended with v-type
blender (Seo-kang Engineering, Korea) at 30 rpm for 1 hour. The
final blending weight ratio was Morus:Rosemary:Mate at 2:5:10 and
resulted in 20.4 kg of Composition 3. The active contents in the
composition were quantified with the HPLC illustrated in Examples
11, 20, 25, 28 and 30. The quantification results are shown in the
Table 28.
TABLE-US-00029 TABLE 28 Summary of Active Content in Composition 9
Rosemary % Morus Carnosic % Ursolic % % Mate Sample % Carnosol acid
acid Kuwanon G Albanin G % Caffeine Composition 3 1.59 0.44 3.45
0.77 0.53 1.29
Example 40
Preparation of Morus:Acceleris:Loesyn:Bakutrol Composition 6
[0259] Four component (Morus:Acceleris:Loesyn:Bakutrol) composition
6 was produced as follows: 968.6 g of Morus alba root bark extract
20, 484.3 g of Panax ginseng extract (Acceleris), and 387.4 g of
standardized Aloe chromones in Aloe vera inner leaf gel powder
(Loesyn) were combined in a capped one-gallon jar, then shaken and
turned in the jar to produce a uniform powder (Powder 1). 96.9 g of
Psoralea seed extract (Bakutrol) was placed in a 2-liter beaker,
and gradually one-half of Powder 1 was added to the Bakutrol liquid
until a granule-like mixture was achieved. This mixture was then
added to the remaining half of Powder 1 and mixed thoroughly.
Finally, the complete mixture was put into a blender for
pulverizing. The final combination composition 6 had a blending
weight ratio of Morus:Acceleris:Loesyn:Bakutrol at 10:5:4:1. The
active contents of Composition 6 are shown in Table 29.
TABLE-US-00030 TABLE 29 Summary of Active Contents in Composition 6
Acceleris Morus alba root Total bark extract Ginsenoside Loesyn % %
Bakutrol (Rd, Rg3, % Kuwanon Albanin % Sample Rk1, Rg5) Aloesin G G
Bakuchiol Composition 6 10.02 0.80 1.57 0.88 3.34
Example 41
Preparation of Morus:Rosemary:Areca Composition 9
[0260] Three component (Morus:Rosemary:Areca) composition 9 was
produced as follows: 153.2 g of dry Morus alba root bark extract
18, 382.2 g of Rosemary extract powder 22-1, and 765.8 g of Areca
catechu fruit extract powder 31, were blended together with a
ribbon blender (Hankook P. M. EMG, Korea) at 30 rpm for 1 hour to
obtain 1.301 kg of UP609. The blending ratio of
Morus:Rosemary:Areca was 2:5:10 (weight ratio). The active contents
of Composition 9 are shown in Table 30.
TABLE-US-00031 TABLE 30 Summary of Active Content of Composition 3
Rosemary Morus Carnosol Carnosic Ursolic Kuwanon Albanin Areca
Sample % acid % acid % G % G % -- Composition 2.14 2.01 5.12 0.63
0.45 -- 9
Example 42
Preparation of Magnolia:Morus:Mutamba Composition 2
[0261] Three component (Magnolia:Morus:Mutamba) Composition 2 was
produced as follows: 1.02 Kg of dry Magnolia extract powder 29,
2.04 Kg of dry Morus alba root bark extract powder 18, and 10.05 Kg
of dry Mutamba stem bark extract powder 36, were blended together
with a v-type blender (Seo-kang Engineering, Korea) at 30 rpm for 1
hour to obtain 13.05 Kg of Composition 2. The blending ratio of
Magnolia:Morus:Mutamba was 1:2:10 (weight ratio).
TABLE-US-00032 TABLE 31 Summary of Active Ingredients for
Composition 2 Magnolia Morus Mutamba Honokiol Magnolol Kuwanon G
Albanin G Procyanidin Sample % % % % B2 Epicatechin Composition 2
4.06 4.19 1.01 0.71 1.10 0.81
Example 43
Preparation of Mutamba:Rosemary:Mate Composition 4
[0262] Three component (Mutamba:Rosemary:Mate) Composition 4 was
produced as follows: 352.3 g of Mutamba stem bark extract powder
36, 352.1 g of Rosemary extract powder 22, and 352.1 g of Mate
extract powder 27, were blended with a ribbon-style blender
(Seo-kang Engineering, Korea) at 30 rpm for 1 hour to obtain 1.04
Kg of combination Composition 4. The blending ratio of
Mutamba:Rosemary:Mate was 1:1:1 (weight ratio).
TABLE-US-00033 TABLE 32 Summary of Active ingredients for
Composition 4 Mutamba Rosemary Pro- Mate Carnosol Carnosic Ursolic
cyanidin Caffeine Sample % acid % acid % B2 Epicatechin % Compo-
2.11 1.79 2.30 0.47 0.32 0.71 sition 4
Example 44
Preparation of Mutamba:Rosemary:Mate Composition 8
[0263] Three component (Mutamba:Rosemary:Mate) Composition 8 was
produced as follows: 640.2 g of Mutamba stem bark extract powder
36, 320.1 g of Rosemary extract powder 22, and 640.2 g of Mate
extract powder 27, were blended with a ribbon-style blender
(Seo-kang Engineering, Korea) at 30 rpm for 1 hour to obtain 1.52
Kg of combination Composition 8. The blending ratio of
Mutamba:Rosemary:Mate was 2:1:2 (weight ratio).
TABLE-US-00034 TABLE 33 Summary of Active Ingredients for
Composition 8 Rosemary Mutamba Carnosic Ursolic Procyanidin Mate
Sample Carnosol % acid % acid % B2 Epicatechin Caffeine %
Composition 8 1.64 0.94 3.56 0.53 0.45 2.44
Example 45
Acute Food Intake in Sprague-Dawley Rats as a Measure of Appetite
Suppression
[0264] This example describes the acute food intake rat animal
model for evaluation of the effects of diet and test compounds on
amount and rate of food intake of rats after a fasting period.
[0265] Method:
[0266] Male Sprague-Dawley (SD) rats (Koatech, Korea), eight weeks
of age at the beginning of the experiment, were used in this study.
During acclimation periods the animals were maintained on a regular
rat chow diet (2018S, Harlan, USA). The rats were housed in a
climate-controlled room maintained on a 12 hr/12 hr reverse
light/dark cycle. Rats were administered 0.5% CMC (carboxymethyl
cellulose) aqueous solution as vehicle or in combination with a
test composition 30 minutes prior to the start of the dark-phase
feeding cycle. Experimental testing commenced at the onset of the
12 hr dark cycle. Prior to initiation of a test cycle the rats were
fasted overnight (less than about 16 hours) to enhance their
hunger. Otherwise the animals had unlimited access to 45% high fat
diet (Harlan, USA) and tap water. Food intake was measured at 0, 1,
2, 4, 6, 8, 10 and 24 hours from the start of the experiment to
determine the acute food intake of each animal in a study, and
total body weight was also measured at 2, 8, and 24 hours.
Example 46
Acute Food Intake Study of Morus alba Extract in Sprague-Dawley
(SD) Rats
[0267] This Example presents a 24-hour food intake test carried
according to the Example 45 to determine the effect of
administration of Morus alba plant extract 15 on rat food intake.
SD rats were administered Morus alba extract 15 produced according
to Example 15, in a solution of 0.5% CMC (carboxymethyl cellulose)
30 minutes prior to the start of dark-phase feeding cycle. The
Morus alba extract was administered at a dose of 250, 500 and 1000
mg/kg of animal weight, 7 animals per group.
[0268] Table 34 shows the food intake test results for rats treated
with a single dose of Morus alba extract 15 at three different
amounts compared to control animals.
TABLE-US-00035 TABLE 34 Cumulative Food Intake in Non-Obese Fasting
Rats Fed a High Fat Diet Dose Cumulative Food Intake (hour) Group
(mg/kg) 1 2 4 6 8 10 24 Control -- Mean 3.45 5.33 8.73 15.35 20.57
22.99 26.85 SD 1.20 1.00 1.12 1.92 1.47 1.49 2.20 Morus 250 Mean
1.43 2.94 5.86 9.77 13.56 16.68 24.16 alba SD 0.98 1.50 3.13 5.47
6.15 6.55 4.63 p value 0.0067 0.0098 0.0763 0.0544 0.0376 0.0648
0.2338 500 Mean 1.72 2.97 5.23 8.37 12.28 14.01 19.93 SD 0.96 0.80
1.21 2.71 2.98 3.35 2.72 p value 0.0315 0.0029 0.0033 0.0070 0.0071
0.0087 0.0065 1,000 Mean 0.74 1.26 3.12 6.06 9.95 15.11 19.48 SD
0.31 1.00 2.12 2.97 1.57 2.41 3.06 p value 0.0007 0.0005 0.0080
0.0035 0.0000 0.0031 0.0088
[0269] The data presented in Table 34 shows that all of the Morus
alba treatment groups exhibited a statistically significant
reduction in cumulative food intake. Further, a dose dependent
reduction in food intake was observed in the first hour of food
intake measurement through to completion of the study. These
results demonstrate that Morus alba extract has a statistically
significant effect on food intake in rats, which indicates that
Morus alba extract can be used as a body weight control composition
via inhibition of food intake. Also, the reduced food intake from a
single oral dose of Morus alba extract lasted more than 10 hours.
Thus, it is feasible to achieve a reduced appetite, enhanced
satiety, or reduced food or caloric intake by once or twice per day
oral administration of Morus alba extract.
Example 47
Acute Food Intake Study of Milicia excelsa Extract 8 in SD Rats
[0270] This 24-hour food intake study was conducted according to
the Example 45. SD rats were administered Milicia excelsa extract 8
produced according to Example 8, at a dosage of 1000 mg/kg of
animal weight, in 0.5% CMC (carboxymethyl cellulose solution)
solution 30 min prior to the start of the dark-phase feeding
cycle.
[0271] As shown the Table 35 and Table 36, body weight and body
weight gain were reduced significantly at the 8 hr and 24 hour
study time points. Body weight gain was determined by measurement
of the weight difference for each study group between each
successive sample time point of the study. Milicia excelsa extract
treatment groups also exhibited reduced food intake at the 1 hr, 4
hr, 6 hr and 8 hr time points (Table 37). Treatment SD rats fed the
Milicia excelsa extracts were also reduced significantly in
cumulative food intake for 24 hour (Table 38).
TABLE-US-00036 TABLE 35 Body Weight in Non-Obese Fasting Rats Fed a
High Fat Diet Dose Body weight (g) Group (mg/kg) 0 h 2 h 8 h 24 h
Control -- Mean 203.34 209.74 219.06 217.22 SD 5.27 4.26 6.79 5.26
Milicia 1,000 Mean 202.69 207.01 205.04 205.96 excelsa SD 8.15 7.34
7.03 12.49 p 0.8680 0.4610 0.0050 0.0309 value
TABLE-US-00037 TABLE 36 Change in Body Weight Gain in Non-Obese
Fasting Rats Fed High Fat Diet Dose Body weight gain (g) Group
(mg/kg) 2 h 8 h 24 h Control -- Mean 6.41 15.73 13.88 SD 1.69 4.36
1.92 Milicia 1,000 Mean 4.32 2.35 3.27 excelsa SD 2.46 6.44 11.00 p
value 0.1028 0.0001 0.0144
TABLE-US-00038 TABLE 37 Food Intake per Time Point in Non-Obese
Fasting Rats Fed a High Fat Diet Dose Food Intake (g) per Time
Point Group (mg/kg) 1 h 2 h 4 h 6 h 8 10 h 24 h Control -- Mean
3.09 2.07 2.76 4.19 3.51 1.33 6.34 SD 1.24 1.54 1.64 1.02 2.64 1.60
2.83 Milicia 1,000 Mean 0.80 0.94 0.35 0.70 0.98 1.05 6.44 excelsa
SD 0.41 0.75 0.50 1.07 1.81 1.78 5.09 p value 0.0002 0.0570 0.0011
0.0000 0.0236 0.7210 0.9562
TABLE-US-00039 TABLE 38 Cumulative Food Intake in Non-Obese Fasting
Rats Fed High Fat Diet Dose Cumulative food intake (g) Group
(mg/kg) 1 h 2 h 4 h 6 h 8 10 h 24 h Control -- Mean 3.09 5.15 7.91
12.10 15.61 16.93 23.27 SD 1.24 1.96 1.57 2.01 3.75 3.23 1.57
Milicia 1,000 Mean 0.80 1.74 2.09 2.79 3.77 4.82 11.26 excelsa SD
0.41 0.80 1.05 2.04 3.50 4.46 8.06 p value 0.0002 0.0003 0.0000
0.0000 0.0000 0.0000 0.0010
[0272] These data show that Milicia excelsa extracts were effective
in significantly reducing body weight and body weight gain. In
addition, SD rats fed a normal chow diet after fasting had reduced
intake of food. Therefore, the present result suggests that Milicia
excelsa extract can be used as a body weight controller via
inhibition of food intake.
Example 48
High Fat Diet Induced Obesity (DIO) Mouse Model
[0273] C57CL/6J mice aged 4-6 weeks (Korea Research Institute of
Bioscience & Biotechnology, Ohchang, Korea) were housed in
Polycarbonate cages (five mice per cage) in a room with a 12 hr:12
hr light-dark cycle and an ambient temperature of 24.degree. C. All
the mice were fed a commercial chow diet for 1 week after arrival
in the animal facility. Mice were then divided into normal and
obesity groups and fed with normal diet (ND) and high fat diets
(HFD) respectively. The HFD group was divided into multiple
treatment groups: a high fat diet vehicle group (HFD), an orlistat
(purchased as OTC drug Alli.RTM.) positive control treatment group
(ORI, 40 mg/kg of animal weight, 2 times/day) and optionally a
sibutramine positive control treatment group (10 mg/kg, 1
time/day). The HFD contained 340 g of fat/kg of HFD (310 g lard
plus 30 g soybean oil; Harlan Laboratories, USA). The HFD was
formulated to provide 60% of the total energy generated by the diet
from fat by replacing carbohydrate energy with lard and soybean
oil, whereas the normal diet (ND) group was fed a diet providing
only 18% of the total diet energy from fat (Harlan Laboratories,
USA).
[0274] Body weight was measured once each week and feed intake was
measured twice per week. At the end of the experimental period,
following a 12 hr. fasting period, the animals were anesthetized
with ether, and blood was drawn from the abdominal vein. Liver and
kidney and adipose tissues (epididymal, retroperitoneal, perirenal
adipose tissues) were removed from each animal, rinsed with
physiological saline, and weighed. Serum concentrations of glucose,
total cholesterol, triglycerides, and LDL-cholesterol were
determined using automatic analyzer (INTEGRA 400, Roche, Germany).
Statistical significance of test results was measured using the
Student's t-test.
Example 49
High Fat Diet Induced Obesity (DIO) Rat Model
[0275] Male Spraue-Dawley rats, age 4-6 weeks (OrientBio, Inc.;
Seongnam. Korea) were housed individually in Polycarbonate cages in
a room with a 12 hr:12 hr light-dark cycle and an ambient
temperature of 24.degree. C. All the rats were fed a commercial
chow diet for 1 week after arrival in the animal facility. Rats
were then divided into normal and obesity groups and fed with
normal diet (ND) and high fat diets (HFD) respectively. The HFD
group was divided into multiple treatment groups: a high fat diet
vehicle group (HED), an orlistat (purchased as OTC drug Alli.RTM.)
positive control treatment group (ORI, 80 mg/kg of animal weight, 2
times/day). In some examples, sibutramine was used as a positive
control (SIB, 3 mg/kg for rat studies and 10 mg/ml for mice
studies).
[0276] Body weight was measured once or twice per week and feed
intake was measured twice per week. Body weight gain was determined
for each study group by measurement of the body weight difference
for each study group between each successive week of the study. At
the end of the experimental period, following a 12-h fasting
period, the animals were anesthetized with ether, and blood was
drawn from the abdominal vein. Liver and kidney and adipose tissues
(epididymal, retroperitoneal, perirenal adipose tissues) were
removed from each animal, rinsed with physiological saline, and
weighted. Serum concentrations of glucose, total cholesterol and
LDL-cholesterol were determined using automatic analyzer (INTEGRA
400, Roche, Germany). Statistical significance of test results was
performed by Student's t-test.
Example 50
Effect of Morus alba Ethanol Extract 19-2 on DIO Mice
[0277] Morus alba 70% ethanol extract 19-2 produced according to
the Example 19 was orally administrated to DIO mice as described in
the Example 48. The Morus alba extract was administered at two dose
levels: the G1 group at 500 mg/kg of animal weight and the G2 group
at 1000 mg/kg of animal weight. Animals were given an oral dose by
gavage two times per day. Study results for measurement of animal
body weight are shown in Table 39.
TABLE-US-00040 TABLE 39 Effect of Morus alba extract 19-2 on Total
Body Weight in DIO mice Weeks Group 0 1 2 3 4 5 6 7 ND Mean 28.21
28.34 28.45 28.50 28.29 28.31 28.43 28.66 (Normal Diet) SD 2.032
2.006 2.277 2.417 2.414 2.449 2.425 2.413 p value 0.0000 0.0000
0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 HFD Mean 43.36 41.24
40.29 41.26 42.64 43.32 45.05 46.23 (High Fat SD 2.414 2.118 2.712
3.496 3.796 4.081 4.515 4.550 Diet) ORI Mean 42.76 38.73 35.09
35.66 37.28 38.57 40.03 40.91 (40 mg/kg) SD 3.226 3.286 3.330 3.542
3.896 4.508 4.617 4.127 p value 0.6889 0.0987 0.0054 0.0088 0.0185
0.0516 0.0530 0.0346 G1 Mean 42.86 40.58 38.76 39.55 40.82 42.44
44.10 45.22 (500 mg/kg) SD 2.921 2.492 3.219 3.662 4.658 5.306
5.949 6.166 p value 0.7200 0.5917 0.3548 0.3909 0.4375 0.7321
0.7385 0.7281 G2 Mean 43.07 40.05 38.02 36.98 37.56 38.29 39.18
39.66 (1 g/kg) SD 0.835 0.287 0.094 0.014 0.012 0.024 0.021 0.017 p
value 0.8353 0.2868 0.0942 0.0136 0.0124 0.0242 0.0214 0.0171 p
value: compare to HFD by t-test
[0278] The data in Table 39 shows that the 01 group (low dosage of
Morus alba extract 19-2) did not exhibit a significant difference
body weight compared to the HFD group. In contrast, the G2 group
(high dosage of Morus alba extract 19-2) exhibited statistically
significant reductions in body weight at weeks 3, 4, 5, 6 and 7
when compared with the HFD group. The positive control group (ORI,
treated with orlistat at 40 mg/kg of body weight) showed
statistically significant reductions in body weight at weeks 2, 3,
4, and 7 when compared with the HFD group.
[0279] Table 40 shows the effects on body weight gain of each
group.
TABLE-US-00041 TABLE 40 Effect of Morus alba on Body Weight Gain in
Mice Fed High Fat Diet Weeks Group 1 2 3 4 5 6 7 ND Mean 0.13 0.24
0.29 0.08 0.10 0.22 0.45 (Normal SD 0.387 0.567 0.736 0.810 0.959
0.839 0.929 Diet) p value 0.0000 0.0000 0.0002 0.1790 0.8322 0.1256
0.0302 HFD (High Mean -2.05 -3.37 -3.41 -2.14 -1.82 -0.22 1.24 Fat
Diet) SD 0.990 0.266 0.548 1.214 1.145 2.417 3.030 ORI Mean -4.00
-7.38 -6.70 -5.01 -3.81 -2.36 -1.42 40 mg/kg SD 0.571 0.839 1.227
1.409 1.609 1.175 1.259 p value 0.0004 0.0000 0.0001 0.0005 0.0032
0.0059 0.0041 G1 Mean -2.27 -4.28 -3.49 -2.22 -0.60 1.06 2.18 500
mg/kg SD 0.921 1.316 1.429 2.075 2.660 3.240 3.383 p value 0.7247
0.0621 0.0887 0.1522 0.6745 0.6957 0.6660 G2 Mean -3.02 -5.05 -6.09
-5.51 -4.78 -3.89 -3.41 1000 mg/kg SD 0.936 1.076 1.244 0.728 1.084
1.511 2.136 p value 0.0521 0.0018 0.0001 0.0000 0.0001 0.0001
0.0002 p value: compare to HFD by t-test
[0280] The data in Table 40 show that the change in weight gain for
the high dosage group (G2) and the positive control group (ORI)
both showed the greatest change in weight gain between week 2 and
week 3 with the rate of weight loss relative to the HFD group
dropping from week 4 to week 7.
[0281] Table 41 shows the effects of the Morus alba extract on body
weight gain, food intake, and the food efficiency ratio (FER),
which is the average body weight gain per day over the study
period, divided by the average food intake per day over the study
period.
TABLE-US-00042 TABLE 41 Effect of Morus alba on Mice Fed a High Fat
Diet Average Average Food Body Food Efficiency Weight Gain Intake
Ratio Group (g/day) (g/day) (FER) ND Mean 0.010 2.973 0.003 (Normal
Diet) SD 0.020 0.134 0.007 p value 0.0302 0.0000 0.0255 HFD Mean
0.027 2.390 0.011 (High Fat Diet) SD 0.066 0.580 0.028 ORI Mean
-0.031 2.711 -0.011 (40 mg/kg) SD 0.040 0.721 0.015 p value 0.0041
0.0293 0.0043 G1 Mean 0.047 2.332 0.017 (500 mg/kg) SD 0.074 0.611
0.030 p value 0.6660 0.6909 0.5321 G2 Mean -0.074 1.934 -0.038
(1000 mg/kg) SD 0.046 0.508 0.024 p value 0.0002 0.0013 0.0001 FER
(Feed efficacy ratio) = Body weight gain(g/day)/Food intake(g/day)
p value: compare to HFD by t-test
[0282] The data presented in Table 41 show that the high dose Morus
alba treatment group (G2) and the positive control treatment group
(ORI) both showed a statistically significant decrease in body
weight gain per day of the study compared to the HFD group. In
addition, both the G2 and ORI groups also showed a statistically
significant decrease in FER as compared to the HFD group.
Interestingly, the ORI and G2 groups also showed statistically
significant changes in food intake per day of the study, with the
ORI showing an increase in food intake compared to the HFD group,
while the G2 group showed a decrease in average food intake per day
of the study compared to the HFD group, suggesting that the
mechanism of action is different between the two compositions.
[0283] Taken together, the data presented in this example indicate
that the Morus alba 70% ethanol extract 19-2 when taken at a dose
of 1000 mg/kg of subject body weight, twice per day, is effective
in helping to control food intake and body weight for subjects on a
high fat diet.
Example 51
Effect of Morus alba Extract Precipitate 19-1 on DIO Mice
[0284] Morus alba precipitate 19-1, produced by precipitation from
a concentrated 70% ethanol extract of Example 189, was orally
administrated to DIO mice using the methods described in Example
48. The Morus alba extract precipitate was administered at two dose
levels: the G1 group at 250 mg/kg of animal weight and the G2 group
at 500 mg/kg of animal weight. Animals were given an oral dose by
gavage two times per day. Study results for measurement of animal
body weight are shown in Table 42.
TABLE-US-00043 TABLE 42 Effect of Morus alba extract precipitate
19-1 on Total Body Weight Weeks Group 0 1 2 3 4 5 6 7 ND Mean 28.21
28.34 28.45 28.50 28.29 28.31 28.43 28.66 SD 2.032 2.006 2.277
2.417 2.414 2.449 2.425 2.413 p value 0.0000 0.0000 0.0000 0.0000
0.0000 0.0000 0.0000 0.0000 HFD Mean 43.36 41.24 40.29 41.26 42.64
43.32 45.05 46.23 SD 2.414 2.118 2.712 3.496 3.796 4.081 4.515
4.550 ORI Mean 42.76 38.73 35.09 35.66 37.28 38.57 40.03 40.91 40
mg/kg SD 3.226 3.286 3.330 3.542 3.896 4.508 4.617 4.127 p value
0.6889 0.0987 0.0054 0.0088 0.0185 0.0516 0.0530 0.0346 G1 Mean
43.10 39.97 37.09 36.78 39.00 40.83 41.77 42.82 250 mg/kg SD 2.990
2.678 2.290 2.576 2.788 3.162 3.383 3.930 p value 0.8566 0.3250
0.0294 0.0153 0.0572 0.2155 0.1394 0.1473 G2 Mean 42.92 38.48 34.67
34.35 35.21 35.65 35.22 35.85 500 mg/kg SD 3.028 2.365 1.806 1.523
1.688 2.011 2.189 2.101 p value 0.7591 0.0330 0.0005 0.0003 0.0004
0.0006 0.0002 0.0001 p value: compared to HFD by t-test
[0285] As shown the Table 42, body weight was significantly
decreased in a dose dependent manner in the Morus alba treatment
groups. In particular, the G1 group (low dosage of Morus alba
extract precipitate 19-1) exhibited a significant difference in
body weight compared to the HFD group for week 2 and week 3.
Further the weight gain trend, as compared to the HFD group
suggests a lowered rate of weight gain compared to the HFD group
even if the numbers for weeks 4, 5, 6 and 7 failed to achieve
statistical significance. Whereas the G2 group (high dosage of
Morus alba extract precipitate 19-1) exhibited statistically
significant reductions in body weight at weeks 2 through week 7,
when compared with the HFD group. The positive control group, ORI,
(treated with orlistat) showed statistically significant reductions
in body weight at weeks 2, 3, 4, and 7 when compared with the HFD
group.
[0286] Table 43 shows the effects on body weight gain in each study
group.
TABLE-US-00044 TABLE 43 Effect of Morus alba Extract Precipitate
19-1 on Mice Fed a High Fat Diet Weeks Group 1 2 3 4 5 6 7 ND Mean
0.13 0.24 0.29 0.08 0.10 0.22 0.45 SD 0.387 0.567 0.736 0.810 0.959
0.839 0.929 p value 0.0000 0.0000 0.0002 0.1790 0.8322 0.1256
0.0302 HFD Mean -2.12 -3.08 -2.10 -0.73 -0.04 1.69 2.87 SD 0.689
0.880 1.365 1.583 1.897 2.339 2.504 ORI Mean -4.00 -7.38 -6.70
-5.01 -3.81 -2.36 -1.42 40 mg/kg SD 0.571 0.839 1.227 1.409 1.609
1.175 1.259 p value 0.0004 0.0000 0.0001 0.0005 0.0032 0.0059
0.0041 G1 Mean -3.13 -6.02 -6.33 -4.11 -2.27 -1.34 -0.28 250 mg/kg
SD 1.272 1.916 2.678 2.375 2.316 2.654 2.757 p value 0.0732 0.0018
0.0017 0.0059 0.0614 0.0352 0.0372 G2 Mean -4.44 -8.25 -8.57 -7.71
-7.27 -7.70 -7.07 500 mg/kg SD 1.399 1.600 1.665 1.594 1.315 1.414
1.582 p value 0.0011 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 p
value: compare to HFD by t-test
[0287] The data in Table 43 show that both of the Morus alba
treatment groups and the positive control ORI group exhibited
statistically significant decreases in body weight gains each week
after the first week of treatment of as compared to the HFD group.
In addition, the data also show the weight loss effect of the Morus
alba treatment groups is dose dependent.
[0288] Table 44 shows the effects of the Morus alba extract
precipitate 19-1 on body weight gain, food intake, and the food
efficiency ratio (FER), which is the average body weight gain per
day over the study period, divided by the average food intake per
day over the study period
TABLE-US-00045 TABLE 44 Effect of Morus alba extract precipitate
19-1 on Mice Fed a High Fat Diet Average Average Food Body Food
Efficiency Weight Gain Intake Ratio Group (g/day) (g/day) (FER) ND
Mean 0.010 2.973 0.003 (Normal Diet) SD 0.020 0.334 0.007 p value
0.0302 0.0000 0.0255 HFD Mean 0.062 2.390 0.026 (High Fat Diet) SD
0.054 0.580 0.023 ORI Mean -0.031 2.711 -0.011 (40 mg/kg) SD 0.040
0.721 0.015 p value 0.0041 0.0293 0.0043 G1 Mean -0.006 2.468
-0.002 (250 mg/kg) SD 0.060 0.682 0.024 p value 0.0372 0.5874
0.0351 G2 Mean -0.154 1.937 -0.079 (500 mg/kg) SD 0.034 0.542 0.018
p value 0.0000 0.0016 0.0000 Feed efficacy ratio (FER) = Body
weight gain (g/day)/Food intake(g/day) p value: compare to HFD by
t-test
[0289] The data presented in Table 44 show that both of the Morus
alba treatment groups (G1 and G2) and the positive control
treatment group (ORI) all showed statistically significant
decreases in body weight gain per day of the study. The ORI and G2
treatment groups also showed a statistically significant decrease
in the average amount of food intake per day as compared to the HFD
group. In addition, the G1, G2, and ORI treatment groups also
showed a statistically significant decrease in FER as compared to
the HFD group.
[0290] Table 45 shows the effects of the Morus alba extract
precipitate 19-1 on several specific tissues that are known to have
increased fat content in subjects on a high fat diet.
TABLE-US-00046 TABLE 45 Effects of Morus alba extract precipitate
19-1 on Organ Weight Epididymal Retroperitoneal Perirenal Total
Group Liver Fat Fat Fat Fat.sup.1) ND Mean 0.87 0.58 0.14 0.09 0.81
(Normal SD 0.102 0.189 0.070 0.032 0.280 Diet) p value 0.0051
0.0000 0.0000 0.0001 0.0000 HFD Mean 1.44 1.98 0.54 0.43 2.95 (High
Fat SD 0.406 0.464 0.085 0.134 0.478 Diet) ORI Mean 1.12 1.92 0.47
0.33 2.73 (40 mg/kg) SD 0.204 0.359 0.055 0.094 0.433 p value
0.0842 0.8036 0.0791 0.1376 0.3649 G1 Mean 1.26 2.24 0.57 0.36 3.17
(250 mg/kg) SD 0.247 0.237 0.075 0.121 0.252 p value 0.3216 0.2006
0.5206 0.3072 0.2958 G2 Mean 1.06 1.42 0.35 0.19 1.97 (500 mg/kg)
SD 0.115 0.062 0.043 0.036 0.117 p value 0.0347 0.0118 0.0001
0.0014 0.0005 p value: compare to HFD by t-test
[0291] The data in Table 45 show that there is a statistically
significant difference between liver weight, epididymal fat,
retroperitoneal fat, perirenal fat and total fat between the ND
control group (fed a normal nutritional diet having a moderate
caloric intact of fat) and the HFD group. Interestingly, the high
dosage Morus alba extract precipitate group (G2) showed
statistically significant decreases in all categories of fat
measurement as compared with the HFD group, whereas the ORI
treatment group showed no statistically significant changes in any
of the measured values.
[0292] Table 46 shows the effects of the Morus alba extract
precipitate 19-1 on fasting glucose (F-Glu), total cholesterol
(T-chol) and LDL-cholesterol (LDL-C) as measure in blood samples
obtained at the end of the study.
TABLE-US-00047 TABLE 46 Effect of Morus alba extract precipitate
19-1 on Biochemistry Parameters F-Glu T-Chol LDL-C Group (mg/dL)
(mg/dL) (mg/dL) ND Mean 125.40 107.00 5.00 SD 29.628 20.347 1.793 p
value 0.0016 0.0085 0.0060 HFD Mean 253.20 193.60 9.08 SD 53.867
52.003 1.689 ORI Mean 222.43 134.57 4.27 SD 51.006 31.627 0.896 p
value 0.3375 0.0338 0.0001 G1 Mean 280.14 173.57 5.59 SD 39.015
29.580 1.608 p value 0.3361 0.4134 0.0046 G2 Mean 188.29 136.14
4.24 SD 36.523 37.936 1.664 p value 0.0313 0.0503 0.0006 p value:
compare to HFD by t-test
[0293] The data in Table 46 show that low dosage Morus alba extract
precipitate group (G1) had statistically significant decrease in
LDL-cholesterol compared to the HFD group. The high dosage Morus
alba extract precipitate group (G2) had statistically significant
decreases in total glucose, cholesterol, and LDL-cholesterol as
compared to the HFD group.
[0294] Taken together, the data presented in this example indicate
that the Morus alba precipitate extract 19-1 when taken at a dose
of 500 mg/kg of subject body weight, twice per day, is effective in
helping to control food intake and body weight for subjects eating
a high fat diet. In addition, subjects taking the 500 mg/kg dose of
Morus alba precipitate extract 19-1 also exhibited statistically
significant improvements in blood chemistry end points and tissue
fat levels as compared to control subjects in the HFD group. These
results demonstrate that Morus alba extracts enriched in the
Diels-Alder adducts of a chalcone and prenylphenyl moiety Kuwanon
G, and Albanin G can be used to control body weight, lower food
intake, lower tissue fat content, lower blood glucose levels,
decrease total cholesterol and decrease LDL-cholesterol.
Example 52
Effect of Morus alba Ethyl Acetate Extract 16 on DIO Rats
[0295] Morus alba ethyl acetate extract 16 produced as described in
Example 16 was orally administrated to DIO rats using the methods
described in Example 40. Study time period was 42 days. The Morus
alba extract was administered to group G1 using a dose of 500 mg/kg
of animal weight. Study animals were given an oral dose by gavage
two times per day. Study results for measurement of total animal
body weight for all study groups are shown in Table 47. Table 48
shows the effects on body weight gain when calculated as the
difference between a subject animal weighed on day 0 of the study
compared to the body weight measured at the end of each time point
day of the study.
[0296] The data presented in Table 47 show that Morus alba ethyl
acetate extract 16 when dosed at 500 mg/kg of animal body weight,
produced a statistically significant decrease in body weight from
day 2 to day 14, day 21, and day 28 as compared to the HFD group.
The positive control SIB (Sibutramine dosed at 3 mg/kg) group,
produced a statistically significant reduction in body weight at
day 2 through the remainder of the study (day 42).
TABLE-US-00048 TABLE 47 Effect of Morus alba Extract 16 (500 mg/kg
of total subject weight) on Total Body Weight of DIO Rats Days
Group 0 1 2 3 4 7 10 14 ND Mean 325.08 332.10 336.67 339.58 340.88
353.66 360.07 371.43 (Normal SD 12.82 12.98 14.15 14.35 14.96 18.07
18.48 19.63 Diet) p 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
value HFD Mean 364.82 368.90 374.50 378.61 381.19 391.61 400.99
411.04 (High Fat SD 7.46 8.74 7.87 8.52 9.22 9.25 10.64 11.51 Diet)
SIB Mean 364.15 368.06 356.78 357.21 358.78 368.11 377.69 388.70
(Sibtramine SD 10.40 10.28 11.60 13.06 13.82 15.94 17.24 19.19 3
mg/kg) p 0.870 0.848 0.001 0.001 0.001 0.001 0.002 0.007 value G1
Mean 364.35 366.90 366.10 364.73 362.01 373.26 385.46 393.89 (500
g/Kg) SD 9.34 8.58 8.71 10.97 18.12 18.56 14.42 15.84 p 0.901 0.612
0.036 0.006 0.010 0.015 0.014 0.013 value Days Group 18 21 24 28 31
35 38 42 ND Mean 379.57 388.12 396.23 405.93 412.33 424.10 431.21
439.27 (Normal SD 20.17 20.99 22.32 22.64 22.42 21.69 22.59 21.27
Diet) p 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 value HFD
Mean 420.30 428.30 436.77 448.90 455.75 465.73 471.56 483.09 (High
Fat SD 12.00 14.51 15.62 16.93 17.48 16.38 16.59 16.34 Diet) SIB
Mean 395.38 407.06 414.91 425.99 432.32 442.55 447.66 454.96
(Sibtramine SD 19.05 21.34 20.48 20.94 22.49 23.56 25.53 25.35 3
mg/kg) p 0.003 0.019 0.016 0.015 0.019 0.021 0.025 0.010 value G1
Mean 407.12 413.32 423.76 430.11 442.06 450.60 460.19 466.34 (500
g/Kg) SD 16.21 16.56 22.10 21.32 21.65 22.69 25.42 23.37 p 0.055
0.045 0.148 0.043 0.138 0.106 0.254 0.082 value p value: Compared
to HFD group
TABLE-US-00049 TABLE 48 Effect of Morus alba Extract 16 (500 mg/kg
of total subject weight) on Weight Gain in DIO Rats Days Group 1 2
3 4 7 10 14 18 ND Mean 7.02 11.58 14.50 15.80 28.57 34.99 46.34
54.49 (Normal Diet) SD 3.08 3.32 3.42 3.78 7.31 8.42 9.79 11.09 p
value 0.021 0.163 0.650 0.720 0.502 0.712 0.974 0.817 HFD Mean 4.07
9.67 13.78 16.37 26.79 36.16 46.21 55.48 (High Fat SD 1.93 2.48
3.55 3.16 3.65 5.10 7.36 7.20 Diet) SIB Mean 3.91 -7.37 -6.94 -5.37
3.96 13.54 24.55 31.23 (Sibtramine SD 1.89 3.49 6.76 6.91 9.81
10.77 12.83 12.49 3 mg/kg) p value 0.855 0.000 0.000 0.000 0.000
0.000 0.000 0.000 G1 Mean 2.55 1.76 0.38 -2.34 8.92 21.11 29.54
42.77 (500 g/Kg)) SD 3.17 5.89 9.54 17.57 18.29 13.92 16.59 16.33 p
value 0.214 0.002 0.001 0.008 0.013 0.008 0.013 0.043 Days Group 21
24 28 31 35 38 42 ND Mean 63.04 71.15 80.84 87.25 99.02 106.13
114.19 (Normal Diet) SD 11.64 13.71 14.31 14.17 13.46 14.72 14.88 p
value 0.930 0.889 0.604 0.559 0.757 0.925 0.531 HFD Mean 63.48
71.95 84.07 90.93 100.90 106.73 118.26 (High Fat SD 10.13 11.40
13.03 13.50 13.32 13.55 13.58 Diet) SIB Mean 42.91 50.75 61.84
68.17 78.40 83.51 90.81 (Sibtramine SD 14.03 13.21 13.27 14.71
15.22 17.02 16.74 3 mg/kg) p value 0.002 0.001 0.001 0.002 0.003
0.004 0.001 G1 Mean 48.97 59.41 65.76 77.71 86.25 95.84 101.99 (500
g/Kg)) SD 18.04 22.91 22.14 22.76 23.59 25.92 23.90 p value 0.044
0.145 0.040 0.136 0.109 0.259 0.082 p value: Compared to HFD
group
[0297] The data in Table 48 show that the G1 Morus alba ethyl
acetate extract 16 treatment group exhibited statistically
significant decreases in body weight gains from day 2 to day 21,
and then day 28 of the study as compared to the HFD group. The
positive control SIB (Sibutramine dosed at 3 mg/kg) showed
statistically significant decreases in body weight gains from day 2
to completion of the study on day 42 as compared to the HFD
group.
[0298] Table 49 shows the effects of Morus alba extract Ethyl
Acetate Extract 16 on DIO Rats for the following end points:
average body weight gain per day of the study, average food intake
per day of the study, and the food efficiency ratio (FER) which is
calculated as the average body weight gain per day over the study
period, divided by the average food intake per day over the study
period.
TABLE-US-00050 TABLE 49 Effect of Morus alba ethyl acetate extract
16 on Rats Fed a High Fat Diet Food Food Efficiency Weight Gain
Intake Ratio Group (g/day) (g/day) (FER) ND Mean 2.720 16.460 0.165
(Normal Diet) SD 0.350 0.650 0.019 p value 0.531 0.041 0.059 HFD
Mean 2.820 15.420 0.183 (High Fat Diet) SD 0.320 0.700 0.021 SIB
Mean 2.160 13.880 0.155 (Sibtramine SD 0.400 0.660 0.024 3 mg/kg) p
value 0.001 0.007 0.013 G1 Mean 2.430 16.300 0.149 (500 g/Kg)) SD
0.570 0.660 0.033 p value 0.082 0.077 0.015 Food Efficiency Ratio
(FER) = (body weight gain (g/day)/food intake (g/day) p value:
Compared to HFD group
[0299] The data in Table 49 show that the ethyl acetate extract of
Morus alba as administered to group G1 showed a statistically
significantly lower Food Efficiency Ratio as compared to the HFD
group. The SIB positive control treatment group showed statically
lower values than the HFD group for Weight Gain per day, Food
Intake per day, and Food Efficiency Ratio.
TABLE-US-00051 TABLE 50 Effects of Morus alba extract 16 on Fat
Deposit Weight in Rats Fed a High Fat Diet Epididymal
Retroperitoneal Perirenal Total Group Fat Fat Fat Fat ND Mean 5.556
2.397 1.468 9.420 (Normal SD 1.300 0.593 0.389 2.207 Diet) p value
0.001 0.001 0.007 0.001 HFD Mean 8.636 4.637 2.370 15.643 (High Fat
SD 1.986 1.463 0.799 4.179 Diet) SIB Mean 6.698 3.190 1.562 11.450
(Sibtramine SD 1.064 0.701 0.399 1.999 3 mg/kg) p value 0.017 0.015
0.013 0.013 G1 Mean 5.911 2.648 1.405 9.964 (500 g/Kg)) SD 0.404
0.624 0.242 1.183 p value 0.002 0.002 0.004 0.002 p value: Compared
to HFD group
[0300] The data in Table 50 show that there is a statistically
significant difference between Epididymal Fat, Retroperitoneal Fat,
Perirenal Fat and Total Fat between the ND control group, fed a
normal diet having a moderate caloric intact of fat, and the HFD
group. Morus alba extract treated group (G1) and the positive
control treatment group (SIB) showed statistically significant
decreases in all categories of fat measurement as compared with the
HFD group, demonstrating that the Morus alba extract 16 is
effective in reducing the amount of fat present in DIO rats.
[0301] Table 51 shows the effects of the Morus alba extract 16 on
fasting glucose (F-Glu), total cholesterol (T-chol) and
LDL-cholesterol (LDL-C) as measure in blood samples obtained at the
end of the study.
TABLE-US-00052 TABLE 51 Effect of Morus alba extract 16 on
Biochemistry Parameters in DIO Rats T-Chol Triglyceride Fasting
Glucose Group (mg/dL) (mg/dL) (mg/dL) ND Mean 98.67 40.89 112.67
(Normal Diet) SD 10.89 17.61 10.57 p value 0.027 0.753 0.001 HFD
Mean 113.40 44.50 135.70 (High Fat Diet) SD 15.36 30.37 13.85 SIB
Mean 106.60 41.10 136.00 (Sibtramine SD 10.83 23.50 12.01 3 mg/kg)
p value 0.269 0.783 0.959 G1 Mean 90.40 32.20 113.80 (500 g/Kg)) SD
9.29 14.28 11.33 p value 0.001 0.268 0.001 p value: Compared to HFD
group
[0302] The data in Table 51 show that Morus alba extract 16
treatment group had statistically significant decrease in
Total-Cholesterol and Fasting Glucose as compared to the HFD group.
The SIB treatment group showed no statistically significant changes
compared to the HFD group for any of the measured endpoints.
[0303] These data show that the Morus alba extract 16 when taken at
a dose of 500 mg/kg of body weight, twice per day, is effective in
lowering the rate of body weight gain in rats fed a high fat diet.
In addition, subjects taking the 500 mg/kg dose of extract 16 also
exhibited statistically significant improvements in blood chemistry
end points (Table 51) and tissue fat levels (Table 50) as compared
to control subjects in the HFD group. These results demonstrate
that Morus alba extracts enriched in Kuwanon G and Albanin G can be
used to control body weight gain, lower tissue fat content, lower
fasting blood glucose levels, and decrease total cholesterol
levels.
Example 53
Effect of Mutamba Ethanol Extract 32 on DIO Mice
[0304] Mutamba ethanol extract 32, produced according to the
Example 32, was orally administrated to DIO mouse model as
illustrated in the Example 48. The Mutamba extract was administered
to treatment group G1 at 1000 mg/kg of animal weight per day. G1
treatment group animals were given an oral dose of 500 mg/kg by
gavage two times per day. Study results for measurement of animal
body weight are shown in Table 52.
TABLE-US-00053 TABLE 52 Effect of Mutamba Extract 32 on Total Body
Weight in DIO Mice Weeks Group 0 1 2 3 4 5 6 7 8 ND Mean 27.98
28.04 28.12 28.56 27.99 28.05 28.37 28.67 29.39 (Normal SD 1.657
1.561 1.602 1.982 1.467 1.517 1.493 1.513 1.977 Diet) p value
0.0000 0.0028 0.0014 0.000 0.000 0.0005 0.0003 0.000 0.000 HFD Mean
39.72 38.80 39.56 40.78 41.80 43.21 44.69 46.10 46.74 (High SD
3.576 4.005 3.672 3.301 3.178 3.776 3.612 3.085 3.144 Fat Diet) SIB
Mean 39.70 34.24 33.96 36.05 36.64 37.42 38.54 38.74 39.95 10 mg/kg
SD 3.575 1.812 1.948 1.579 3.568 4.384 5.499 5.556 5.766 p value
0.990 0.049 0.029 0.0348 0.0552 0.070 0.082 0.039 0.058 ORI Mean
39.96 37.37 37.05 37.41 37.60 38.12 40.04 41.77 42.06 40 mg/kg SD
3.489 4.190 4.401 4.659 4.860 5.563 6.028 5.998 6.246 p value 0.920
0.596 0.357 0.223 0.145 0.129 0.1769 0.1889 0.1730 G1 Mean 39.79
34.93 36.51 35.78 36.06 37.75 37.41 39.70 41.53 1000 mg/kg SD 3.771
3.548 4.453 2.564 2.706 3.213 3.465 4.039 4.074 p value 0.978
0.1448 0.271 0.0281 0.0152 0.0393 0.0116 0.0226 0.0535 p value:
compare to HFD by t-test
[0305] The data in Table 52 show that the Mutamba ethanol extract
32, 1000 mg/kg/day treatment group showed statistically significant
decreases in body weight from week 3 through to week 7 of the study
as compared to the HFD group. The positive control SIB (sibutramine
dosed at 10 mg/kg) showed statistically significant decreases in
total body weight from week 1, 2, 3 and 7 of the study as compared
to the HFD group.
[0306] Study results for measurement of animal body weight gain are
shown in Table 53.
TABLE-US-00054 TABLE 53 Effect of Mutamba extract 32 on Body Weight
Gain in DIO Mice Weeks Group 1 2 3 4 5 6 7 8 ND Mean 0.07 0.14 0.58
0.01 0.08 0.39 0.69 1.41 (Normal SD 0.393 0.545 1.306 0.757 0.654
0.606 0.779 1.018 Diet) p value 0.1193 0.4309 0.4576 0.0009 0.0000
0.0015 0.0011 0.0000 HFD Mean -0.93 -0.16 1.06 2.07 3.49 4.97 6.38
7.01 (High Fat SD 1.121 0.941 0.606 1.097 1.142 1.483 1.739 1.863
Diet) SIB Mean -5.46 -4.84 -2.75 -2.17 -1.38 -0.27 -0.06 1.15 10
mg/kg SD 3.088 2.260 1.968 1.997 2.462 3.360 3.216 3.563 p value
0.0150 0.0038 0.0262 0.0047 0.0054 0.0159 0.0062 0.0149 ORI Mean
-2.59 -2.90 -2.55 -2.35 -1.84 0.08 1.81 2.10 40 mg/kg SD 0.792
1.346 1.714 1.792 2.536 3.102 3.188 3.124 p value 0.0268 0.0058
0.0022 0.0015 0.0027 0.0130 0.0228 0.0166 G1 Mean -4.86 -3.28 -4.01
-3.73 -2.04 -2.38 -0.09 1.74 1000 mg/kg SD 3.302 2.576 2.049 2.271
2.083 2.423 1.974 1.540 p value 0.0357 0.0345 0.0038 0.0009 0.0008
0.0004 0.0006 0.0012 p value: compare to HFD by t-test
[0307] The data in Table 53 show that the Mutamba ethanol extract
32, 1000 mg/kg/day treatment group and the positive control
treatment group SIB and ORI, all showed statistically significant
decreases in body weight gain across all weeks of the study.
[0308] Study results for effects of treatments on body weight gain,
food intake, and the food efficiency ratio (FER) are shown in Table
54.
TABLE-US-00055 TABLE 54 Effect of Mutamba Extract 32 on DIO Mice
Average Average Food Body Food Efficiency Weight Gain Intake Ratio
Group (g/day) (g/day) (FER) ND Mean 0.025 2.836 0.009 (Normal Diet)
SD 1.018 0.253 0.018 p value 0.0013 0.0001 0.0013 HFD Mean 0.125
2.536 0.049 (High Fat Diet) SD 1.863 0.213 0.033 SIB Mean 0.021
2.724 0.008 (10 mg/kg) SD 3.563 0.676 0.064 p value 0.0149 0.2868
0.0111 ORI Mean 0.038 2.826 0.013 (40 mg/kg) SD 3.124 0.410 0.056 p
value 0.0166 0.0162 0.0093 G1 Mean 0.031 2.631 0.012 (1000 mg/kg)
SD 1.540 0.667 0.027 p value 0.0012 0.5819 0.0010 Feed efficacy
ratio (FER) = Body weight gain (g/day)/Food intake(g/day) p value:
compared to HFD by t-test
[0309] The data presented in Table 54 show that average body weight
gain per day and food efficiency ratio (FER) endpoints were
significantly lowered in the Mutamba and SIB treatment groups, when
compared to the high fat diet group. The ORI treatment resulted in
statistically significant decreases in average body weight gain per
day, average food intake per day and FER.
[0310] Study results for effects of treatments on blood
biochemistry parameters are shown in Table 55.
TABLE-US-00056 TABLE 55 Effect of Mutamba Extract 32 on
Biochemistry Parameters in DIO Mice ALT AST T-chol LDL-C HDL-C TG
Group (U/L) (U/L) (mg/dL) (mg/dL) (mg/dL) (mg/dL) ND Mean 13.78
45.56 120.40 8.10 60.08 33.00 (Normal Diet) SD 1.171 1.137 10.431
1.140 4.609 12.062 p value 0.0571 0.0687 0.0059 0.0421 0.0560
0.0968 HFD Mean 78.62 87.84 192.00 11.42 70.78 48.40 (High Fat
Diet) SD 54.749 38.225 32.458 2.853 9.671 13.777 SIB Mean 37.00
65.95 155.75 6.18 68.73 49.75 (10 mg/kg) SD 34.273 19.436 53.786
2.636 15.400 16.091 p value 0.2290 0.3357 0.2483 0.0254 0.8127
0.8958 ORI Mean 40.42 67.96 184.40 6.78 78.52 143.60 (40 mg/kg) SD
23.183 14.479 22.030 0.976 5.498 102.878 p value 0.1887 0.3085
0.6763 0.0088 0.1584 0.1072 G1 Mean 34.34 55.60 182.60 5.42 108.30
34.40 (1000 mg/kg) SD 11.243 6.784 8.905 0.672 51.059 14.029 p
value 0.1456 0.1326 0.5620 0.0079 0.1770 0.1500 p value: compare to
HFD by t-test
[0311] The data in Table 55 show that the Mutamba, ORI and SIB
treatment groups, all exhibited statistically significant decrease
in LDL-cholesterol compared to the HFD group.
[0312] Table 56 shows the effects of the Mutamba ethanol extract 32
treatment on measurements in DIO mice on several histopathological
measures of fatty liver and the resulting calculated Non-Alcoholic
Staetohepatitis (NASH) score of the liver.
TABLE-US-00057 TABLE 56 Effect of Mutamba Extract 32 on Liver
Pathology in DIO Mice Pathology Indications Lobular Hepatocellular
Steatosis Inflammation Ballooning NASH Group (0-3) (0-3) (0-2)
(sum) ND Mean 0.00 0.13 0.00 0.13 (Normal SD 0.000 0.354 0.000
0.354 Diet) p value 0.0000 0.0004 0.0000 0.0054 HFD Mean 2.50 1.50
1.00 5.00 (High Fat SD 1.000 0.577 0.000 1.414 Diet) SIB Mean 0.50
0.50 0.25 1.25 (10 mg/kg) SD 1.000 0.577 0.500 1.893 p value 0.0300
0.0498 0.0240 0.0192 ORI Mean 1.20 1.60 0.60 3.40 (40 mg/kg) SD
0.837 0.894 0.548 2.074 p value 0.0708 0.8529 0.1930 0.2315 G1 Mean
0.80 1.20 0.00 2.00 (1000 SD 0.447 0.447 0.000 0.707 mg/kg) p value
0.0108 0.4071 0.0000 0.0041 p value: compare to HFD by t-test
[0313] The data in Table 56 show that there are statistically
significant differences in the amount of Steatosis. Lobular
inflammation, Hepatocellular ballooning and the NASH score between
the ND control group, fed a normal diet having a moderate caloric
intact of fat, and the HFD group. Treatment group G1 showed
statistically significant decreases in Steatosis, Hepatocellular
ballooning and NASH score as compared with the HFD group. The SIB
treatment group showed statistically significant decreases in
Steatosis, Lobular inflammation, Hepatocellular ballooning and NASH
score as compared with the HFD group. The ORI treatment group did
not show any statistically significant changes compared to the HFD
group. These data demonstrate that the Mutamba extract 32 was
effective in reducing the amount of liver damage present in mice
fed a high fat diet.
[0314] Overall, the data presented in this example show that
Mutamba ethanol extract 32 was effective in lowering body weight
LDL blood cholesterol and fatty liver in mice fed a high fat
diet.
Example 54
Effect of Mutamba Ethanol Extract 33-2 on DIO Mice
[0315] Mutamba ethanol extract 33-2, produced according to the
Example 33, was orally administrated to DIO mouse model as
illustrated in the Example 48. The Mutamba extract was administered
to treatment group G1 at 1000 mg/kg of animal weight per day. G1
treatment group animals were given an oral dose of Mutamba 33-2 at
1000 mg/kg by gavage two times per day. G2 treatment group animals
were given an oral dose of Mutamba ethanol extract 33-2 at 500
mg/kg by gavage two times per day. Study results for measurement of
animal body weight gain are shown in Table 57.
TABLE-US-00058 TABLE 57 Effect of Mutamba Extract 33-2 on Body
Weight Gain in DIO Mice Weeks Group 1 2 3 4 5 6 7 8 ND Mean 0.15
0.30 0.08 -0.13 -0.07 0.45 -0.10 0.14 (Normal SD 0.529 0.405 0.747
0.952 0.952 0.906 0.747 0.968 Diet) p value 0.0590 0.1707 0.0035
0.0001 0.0000 0.0001 0.0000 0.0000 HFD Mean 0.85 0.81 2.12 4.11
5.47 6.79 7.55 8.21 (High Fat SD 0.374 0.596 0.642 0.457 0.349
0.177 1.046 1.493 Diet) SIB Mean -2.72 -2.39 -0.39 1.47 2.54 3.99
5.15 6.27 (10 mg/kg) SD 1.090 1.286 1.719 1.615 1.705 1.931 2.051
1.890 p value 0.0004 0.0026 0.0289 0.0166 0.0167 0.0312 0.0729
0.1399 ORI Mean -3.05 -2.81 -2.63 -1.28 0.43 3.52 3.72 5.13 (40
mg/kg) SD 1.312 1.370 1.786 1.859 1.706 1.384 1.445 1.461 p value
0.0007 0.0018 0.0016 0.0021 0.0021 0.0057 0.0031 0.0172 G1 Mean
0.39 1.02 2.19 3.13 3.99 4.29 5.19 4.78 (1000 mg/kg) SD 0.397 0.465
0.833 1.239 1.187 1.616 2.104 1.768 p value 0.1143 0.5666 0.8935
0.1815 0.0486 0.0253 0.0819 0.0177 G2 Mean -1.65 -1.63 0.08 2.02
3.63 5.65 6.30 7.82 (500 mg/kg) SD 0.843 1.730 2.212 2.686 3.249
3.895 4.567 4.408 p value 0.0009 0.0321 0.1221 0.1568 0.2756 0.5510
0.5829 0.8747 p value: compare to HFD by t-test.
[0316] The data in Table 57 show that the Mutamba ethanol extract
33-2, 1000 mg/kg/day treatment group showed statistically
significant decreases in body weight gain at weeks 5, 6 and 8 of
the study. The G2, 500 mg/kg/day treatment group showed
statistically significant decreases in body weight gain at weeks 1,
and 2 of the study.
[0317] Study results for effects of treatments on body weight gain,
food intake, and the food efficiency ratio (FER) are shown in Table
58.
TABLE-US-00059 TABLE 58 Effect of Mutamba extract 33-2 on DIO Mice
Body Weight Food Gain Food Intake Efficiency Ratio Group (g/day)
(g/day) (FER) ND Mean 0.003 2.395 0.001 (Normal SD 0.018 0.206
0.007 Diet) p value 0.0000 0.4282 0.0000 HFD Mean 0.149 2.341 0.064
(High Fat SD 0.027 0.179 0.012 Diet) SIB Mean 0.114 2.624 0.043 (10
mg/kg) SD 0.034 0.372 0.013 p value 0.1399 0.0118 0.0458 ORI Mean
0.093 2.769 0.034 (40 mg/kg) SD 0.027 0.436 0.010 p value 0.0172
0.0016 0.0037 G1 Mean 0.087 2.337 0.037 (1000 mg/kg) SD 0.032 0.249
0.014 p value 0.0177 0.9588 0.0180 G2 Mean 0.142 2.503 0.057 (500
mg/kg) SD 0.080 0.642 0.032 p value 0.8747 0.3421 0.6966 Feed
efficacy ratio (FER) = Body weight gain (g/day)/Food intake (g/day)
p value: compare to HFD by t-test
[0318] The data presented in Table 58 show that average body weight
gain per day and food efficiency ratio (FER) endpoints were
statistically significantly lowered in the Mutamba ethanol extract
33-2, 1000 mg/kg/day treatment group compared to the high fat diet
group.
[0319] Study results for effects of treatments on blood
biochemistry parameters are shown in Table 59.
TABLE-US-00060 TABLE 59 Effect of Mutamba extract 33-2 on
Biochemistry Parameters in DIO Mice T-chol LDL-C TG Group (mg/dL)
(mg/dL) (mg/dL) ND Mean 89.60 7.04 22.00 (Normal Diet) SD 9.659
2.304 7.071 p value 0.0000 0.1018 0.0097 HFD Mean 213.25 10.13
37.00 (High Fat Diet) SD 21.639 2.617 9.487 SIB Mean 158.60 5.26
46.60 (10 mg/kg) SD 5.030 1.389 15.126 p value 0.0130 0.0086 0.2631
ORI Mean 182.40 7.42 41.20 (40 mg/kg) SD 12.482 1.593 16.514 p
value 0.0306 0.0955 0.4616 G1 Mean 168.00 6.14 22.60 (1000 mg/kg)
SD 35.763 1.191 5.079 p value 0.0628 0.0181 0.5066 p value: compare
to HFD by t-test
[0320] The data in Table 59 show that Mutamba extract 33-2, 1000
mg/kg/day treatment group decreased LDL-cholesterol in a
statistically significant fashion as compared to the HFD group.
[0321] The data presented in this example show that Mutamba extract
33-2 when administered at 1000 mg/kg of subject body weight showed
significantly decreased body weight gain, Food Efficiency Ratio,
and LDL-cholesterol. Therefore, the present results indicate that
Mutamba extract 33-2 can be used as a body weight and blood
cholesterol controller.
Example 55
Efficacy Study of Mutamba Fractions 34, 34-1, and 34-2 on a DIO
Mouse Model
[0322] Mutamba fractions 34, 34-1, and 34-2 produced according to
the example 34 was orally administrated to DIO mice as described in
Example 48. Three Mutamba fraction treatment groups were: G1 at 250
mg/kg of EtOA fraction 34; G2 at 250 mg/kg of BuOH fraction 34-1;
and G3 at 250 mg/kg of Water fraction 34-2. The treatment article
was given orally by gavaging 2 times per day.
[0323] The EtOA fraction of Mutamba treatment group (G1) didn't
show any effect on body weight. However, the BuOH fraction
treatment group (G2) showed significantly decreased body weight at
week 4, 6, 7, and 8. The water fraction treatment group (G3) showed
significantly decreased body weight at week 2 when compared to the
HFD group (Table 60).
TABLE-US-00061 TABLE 60 Effect of Mutamba Fractions 34, 34-1, and
34-2 on Body Weight in Mice Fed a High Fat Diet Weeks Group 0 1 2 3
4 5 6 7 8 ND Mean 27.34 27.49 27.64 27.42 27.21 27.27 27.79 27.24
27.48 SD 1.283 1.271 1.242 1.444 1.831 1.700 1.601 1.465 1.381 p
value 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000
0.0000 HFD Mean 41.07 41.92 41.88 43.18 45.18 46.54 47.85 48.62
49.27 SD 1.955 2.295 2.486 2.132 2.295 2.110 1.834 1.353 0.933 SIB
Mean 38.41 35.69 36.03 38.02 39.89 40.95 42.40 43.57 44.69 SD 2.522
3.066 3.200 3.812 3.817 4.185 4.412 4.534 4.322 p value 0.1290
0.0121 0.0202 0.0472 0.0458 0.0466 0.0556 0.0711 0.0788 ORI Mean
39.76 36.71 36.95 37.14 38.49 40.19 43.28 43.49 44.90 SD 3.037
1.792 1.674 2.110 2.927 2.871 2.544 2.674 2.635 p value 0.4846
0.0064 0.0092 0.0038 0.0074 0.0079 0.0198 0.0105 0.0167 G1 Mean
37.39 36.44 37.29 39.04 40.69 42.40 44.37 45.27 46.55 SD 4.593
5.544 5.215 5.225 4.695 4.013 3.926 3.721 3.519 p value 0.1823
0.1092 0.1531 0.1840 0.1263 0.1067 0.1487 0.1343 0.1807 G2 Mean
38.00 38.93 38.73 38.17 38.32 40.16 40.92 41.91 42.36 SD 2.719
4.010 4.241 5.007 5.136 5.224 3.599 3.378 3.001 p value 0.1013
0.2294 0.2337 0.1067 0.0438 0.0572 0.0103 0.0076 0.0032 G3 Mean
37.97 37.32 36.97 38.90 40.53 42.15 43.60 44.40 45.38 SD 2.741
4.074 3.009 3.738 4.141 4.756 4.610 4.646 4.815 p value 0.1000
0.0854 0.0346 0.0827 0.0862 0.1334 0.1293 0.1264 0.1461 p value:
compare to HFD by t-test.
[0324] The BuOH fraction 34-1 treatment group (G2) body weight gain
was significantly decreased at weeks 6, 7, and 8. Otherwise, the
EtOA fraction 34 treatment group (G1) showed significantly
decreased body weight at week 1 and the water fraction 34-2
treatment group (G3) at week 2 when compared to HFD group (Table
61).
TABLE-US-00062 TABLE 61 Effect of Mutamba fractions 34, 34-1, and
34-2on Body Weight Gain in Mice Fed a High Fat Diet Weeks Group 1 2
3 4 5 6 7 8 ND Mean 0.15 0.30 0.08 -0.13 -0.07 0.45 -0.10 0.14 SD
0.529 0.405 0.747 0.952 0.952 0.906 0.747 0.968 p value 0.0590
0.1707 0.0035 0.0001 0.0000 0.0001 0.0000 0.0000 HFD Mean 0.85 0.81
2.12 4.11 5.47 6.79 7.55 8.21 SD 0.374 0.596 0.642 0.457 0.349
0.177 1.046 1.493 SIB Mean -2.72 -2.39 -0.39 1.47 2.54 3.99 5.15
6.27 SD 1.090 1.286 1.719 1.615 1.705 1.931 2.051 1.890 p value
0.0004 0.0026 0.0289 0.0166 0.0167 0.0312 0.0729 0.1399 ORI Mean
-3.05 -2.81 -2.63 -1.28 0.43 3.52 3.72 5.13 SD 1.312 1.370 1.786
1.859 1.706 1.384 1.445 1.461 p value 0.0007 0.0018 0.0016 0.0021
0.0021 0.0057 0.0031 0.0172 G1 Mean -0.95 -0.10 1.65 3.30 5.01 6.98
7.88 9.16 SD 1.149 0.723 0.744 0.895 1.356 1.657 1.765 1.639 p
value 0.0204 0.0831 0.3539 0.1464 0.4988 0.8108 0.7554 0.3978 G2
Mean 0.93 0.73 0.17 0.32 2.16 2.92 3.90 4.36 SD 1.467 1.768 2.894
3.226 2.843 1.143 1.323 1.868 p value 0.9170 0.9341 0.2109 0.0575
0.0589 0.0014 0.0029 0.0124 G3 Mean -0.65 -1.00 0.93 2.56 4.18 5.63
6.43 7.41 SD 1.584 1.407 1.207 1.661 2.288 2.369 2.560 2.947 p
value 0.1004 0.0484 0.1225 0.1161 0.2782 0.3374 0.4428 0.6410 p
value: compare to HFD by t-test.
[0325] There were no significant changes on FER when mice were
treated with the EtOA fraction 34 and water fraction 34-1, but the
BuOH fraction 34-1 treatment group showed borderline changes (Table
62).
TABLE-US-00063 TABLE 62 Effect of Mutamba fractions 34, 34-1, and
34-2 on Mice Fed a High Fat Diet FER Body weight Food (Food gain
intake efficiency Group (g/day) (g/day) ratio) ND Mean 0.003 2.395
0.001 SD 0.018 0.206 0.007 p value 0.0000 0.4282 0.0000 HFD Mean
0.149 2.341 0.064 SD 0.027 0.179 0.012 SIB Mean 0.114 2.624 0.043
SD 0.034 0.372 0.013 p value 0.1399 0.0118 0.0458 ORI Mean 0.093
2.769 0.034 SD 0.027 0.436 0.010 p value 0.0172 0.0016 0.0037 G1
Mean 0.167 2.532 0.066 SD 0.030 0.206 0.012 p value 0.3978 0.0086
0.8046 G2 Mean 0.079 1.978 0.040 SD 0.034 0.158 0.017 p value
0.0124 0.0000 0.0513 G3 Mean 0.135 2.244 0.060 SD 0.054 0.251 0.024
p value 0.6410 0.2182 0.7871 FER (Feed efficacy ratio) = Body
weight gain (g/day)/Food intake (g/day) p value: compare to HFD by
t-test
[0326] In the BuOH fraction 34-1 of Mutamba treatment group (G2),
ALT, HDL-C and TG showed significant changes (Table 63).
TABLE-US-00064 TABLE 64 Effect of Mutmba fractions 34, 34-1, and
34-2 on Liver Pathology in Mice Fed a High Fat Diet Indications
Lobular Hepatocellular Steatosis Inflammation ballooning NSAH Group
(0-3) (0-3) (0-2) (sum) ND Mean 0.00 1.00 0.00 1.00 SD 0.000 0.000
0.000 0.000 p value 0.0000 0.0006 0.0001 0.0000 HFD Mean 2.75 2.50
1.75 7.00 SD 0.500 0.577 0.500 0.816 SIB Mean 1.20 1.60 0.40 3.20
SD 0.837 0.548 0.548 1.304 p value 0.0142 0.0479 0.0066 0.0015 ORI
Mean 1.00 1.60 0.80 3.40 SD 0.707 0.548 0.837 1.949 p value 0.0042
0.0479 0.0871 0.0111 G2 Mean 1.40 1.40 0.80 3.60 SD 0.548 0.548
0.837 1.871 p value 0.0066 0.0222 0.0871 0.0108 G3 Mean 1.60 1.60
0.80 4.00 SD 0.548 0.548 0.837 1.871 p value 0.0141 0.0479 0.0871
0.0212 p value: compare to HFD by t-test
[0327] Both the BuOH fraction 34-1 (G2) and the water fraction 34-2
(G3) treatment groups showed a significantly decreased NASH score
when compared with the high fat diet (Table 64).
TABLE-US-00065 TABLE 64 Effect of Mutmba fractions 34, 34-1, and
34-2 on Liver Pathology in Mice Fed a High Fat Diet Indications
Lobular Hepatocellular Steatosis Inflammation ballooning NSAH Group
(0-3) (0-3) (0-2) (sum) ND Mean 0.00 1.00 0.00 1.00 SD 0.000 0.000
0.000 0.000 p value 0.0000 0.0006 0.0001 0.0000 HFD Mean 2.75 2.50
1.75 7.00 SD 0.500 0.577 0.500 0.816 SIB Mean 1.20 1.60 0.40 3.20
SD 0.837 0.548 0.548 1.304 p value 0.0142 0.0479 0.0066 0.0015 ORI
Mean 1.00 1.60 0.80 3.40 SD 0.707 0.548 0.837 1.949 p value 0.0042
0.0479 0.0871 0.0111 G2 Mean 1.40 1.40 0.80 3.60 SD 0.548 0.548
0.837 1.871 p value 0.0066 0.0222 0.0871 0.0108 G3 Mean 1.60 1.60
0.80 4.00 SD 0.548 0.548 0.837 1.871 p value 0.0141 0.0479 0.0871
0.0212 p value: compare to HFD by t-test
[0328] As is evident from the results, the 250 mg/kg BuOH fraction
34-1 treatment group showed significantly decreased body weight
gain, FER, TG and NASH score. Therefore, the Mutamba BuOH extract
34-1 can be used as a body weight and blood cholesterol and fatty
liver controller.
Example 56
Efficacy Study of Mutamba EtOH Extract 32 (in DIO Rats
[0329] Mutamba ethanol extract 32 produced according to the example
32 was orally administrated by gavage to rats in a DIO model (as
described in Example 49) at a dosage of 500 mg/kg twice a day.
[0330] In the Mutamba treatment group (G3), body weight was
significantly decreased at days 87, 91, 94 and 98, when compared
with the high fat diet group (Table 65).
TABLE-US-00066 TABLE 65 Effect of Mutamba Ethanol Extract 32 on
Body Weight in Rats Fed a High Fat Diet Days Group 0 3 7 10 14 17
21 24 ND Mean 320.66 335.39 351.46 362.05 368.52 377.64 390.40
394.90 SD 21.51 13.89 16.55 17.24 16.75 19.96 20.15 20.59 p value
0.026 0.009 0.011 0.009 0.008 0.009 0.009 0.010 HFD Mean 343.82
361.82 381.42 393.40 403.22 413.10 426.37 431.33 SD 16.89 15.61
18.18 17.87 20.45 19.91 20.62 21.41 G3 Mean 342.70 357.67 370.89
379.63 386.71 395.49 407.00 413.14 SD 15.73 17.54 18.61 24.35 28.12
32.59 32.30 34.30 p value 0.890 0.661 0.326 0.266 0.248 0.260 0.220
0.271 Days Group 28 31 35 38 42 45 49 ND Mean 401.13 408.06 415.74
418.68 429.37 431.83 433.91 SD 21.63 22.46 23.46 23.78 24.65 25.65
26.05 p value 0.005 0.006 0.005 0.003 0.005 0.004 0.003 HFD Mean
442.03 451.15 461.94 468.41 478.74 484.72 487.24 SD 20.68 22.52
23.43 23.53 25.85 25.99 23.49 G3 Mean 417.65 429.28 434.91 447.75
451.41 458.17 461.74 SD 41.27 38.57 45.77 37.53 36.07 32.24 30.48 p
value 0.202 0.234 0.204 0.255 0.142 0.129 0.117 Days Group 52 56 60
64 67 70 74 ND Mean 436.97 444.94 450.37 455.47 459.98 462.51
469.00 SD 25.29 26.04 25.19 25.07 24.89 25.54 27.48 p value 0.003
0.002 0.003 0.003 0.003 0.003 0.003 HFD Mean 489.77 499.73 505.17
507.01 509.35 512.15 520.72 SD 23.99 22.08 25.59 24.33 21.21 22.39
22.28 G3 Mean 465.06 470.63 480.31 481.18 482.58 488.84 494.84 SD
37.71 32.38 30.66 31.51 32.54 34.38 34.29 p value 0.139 0.083 0.139
0.124 0.104 0.172 0.132 Days Group 77 80 84 87 91 94 98 ND Mean
473.26 474.59 481.74 483.20 488.38 490.31 493.26 SD 26.55 28.19
27.62 30.18 30.58 30.68 31.12 p value 0.004 0.003 0.005 0.004 0.004
0.003 0.002 HFD Mean 523.55 524.71 528.65 532.17 539.97 545.50
553.65 SD 24.23 19.80 19.32 16.34 19.53 21.76 22.44 G3 Mean 497.11
499.54 497.76 499.07 504.37 509.43 512.23 SD 36.46 31.10 35.05
34.15 35.65 30.19 25.98 p value 0.149 0.107 0.074 0.049 0.047 0.030
0.010 p value: Compared to HFD group
[0331] In the Mutamba treatment group, the body weight gain was
decreased when compared with the high fat diet group (Table
66).
TABLE-US-00067 TABLE 66 Effect of Mutamba Ethanol Extract 32 on
Body Weight Gain in Rats Fed a High Fat Diet Days Group 3 7 10 14
17 21 24 ND Mean 11.51 27.57 38.17 44.64 53.76 66.51 71.02 SD 2.76
5.38 7.10 6.59 10.20 11.04 11.93 p value 0.130 0.053 0.036 0.006
0.028 0.034 0.041 HFD Mean 13.94 33.54 45.53 55.34 65.23 78.49
83.45 SD 2.59 4.54 2.95 4.64 5.13 6.03 6.70 G3 Mean 12.40 25.62
34.35 41.43 50.22 61.73 67.87 SD 5.57 7.68 9.81 14.62 18.93 18.90
20.62 p value 0.529 0.044 0.023 0.047 0.084 0.060 0.099 Days Group
28 31 35 38 42 45 49 ND Mean 77.25 84.18 91.86 94.80 105.49 107.95
110.03 SD 13.25 14.26 14.55 16.36 16.70 18.23 18.63 p value 0.016
0.015 0.009 0.005 0.007 0.005 0.005 HFD Mean 94.16 103.28 114.06
120.54 130.87 136.85 139.36 SD 7.11 9.02 10.20 8.38 10.01 10.78
8.84 G3 Mean 72.37 84.01 89.63 102.47 106.14 112.89 116.47 SD 27.18
24.16 32.13 23.10 22.37 18.58 17.03 p value 0.081 0.087 0.097 0.091
0.028 0.016 0.012 Days Group 52 56 60 64 67 70 74 ND Mean 113.09
121.05 126.49 131.59 136.10 138.63 145.12 SD 17.60 17.50 17.72
17.83 17.86 18.66 20.23 p value 0.004 0.003 0.003 0.006 0.009 0.011
0.010 HFD Mean 141.90 151.86 157.29 159.14 161.47 164.27 172.84 SD
8.67 6.76 10.82 9.54 9.06 8.89 7.86 G3 Mean 119.78 125.36 135.03
135.91 137.31 143.56 149.56 SD 17.66 18.97 18.57 18.51 18.91 20.95
20.69 p value 0.017 0.009 0.023 0.017 0.015 0.043 0.025 Days Group
77 80 84 87 91 94 98 ND Mean 149.38 150.71 157.86 159.32 164.50
166.42 169.37 SD 19.36 20.70 19.62 22.22 22.23 22.93 25.00 p value
0.011 0.015 0.021 0.025 0.016 0.011 0.007 HFD Mean 175.67 176.84
180.77 184.29 192.10 197.62 205.77 SD 7.93 4.16 5.99 4.07 4.99 7.00
8.87 G3 Mean 151.83 154.27 152.48 153.80 159.09 164.15 166.95 SD
22.32 17.01 20.58 19.22 20.56 15.80 12.99 p value 0.031 0.012 0.010
0.005 0.005 0.001 0.000
[0332] Weight gain (g/day) and Food efficiency ratio (FER) were
significantly lowered in the Mutmaba treatment group as compared to
the high fat diet group (Table 67).
TABLE-US-00068 TABLE 67 Effect of Mutamba Ethanol Extract 32 on DIO
Rats Weight Food FER gain intake (Food efficiency Group (g/day)
(g/day) ratio) ND Mean 1.728 16.265 0.107 SD 0.255 0.868 0.018 p
value 0.007 0.669 0.010 HFD Mean 2.100 15.953 0.132 SD 0.090 0.787
0.005 G3 Mean 1.704 15.693 0.109 SD 0.133 0.709 0.006 p value 0.000
0.693 0.000 FER (Food efficiency ratio) = (body weight gain
(g/day)/food intake (g/day) p value: Compared to HFD group
[0333] In the Mutamba treatment group of absolute organ weight,
Perirenal, Retroperitoneal and Total fat pad were significantly
decrease, when compared with the high fat diet group (Table
68).
TABLE-US-00069 TABLE 68 Effects of Mutamba Ethanol Extract 32 on
Absolute Organ Weights in Rats Fed a High Fat Diet Epididymal
Retroperitoneal Perirenal Group Fat Fat Fat Total Fat ND Mean 0.014
0.008 0.005 0.027 SD 0.004 0.002 0.001 0.006 p value 0.007 0.001
0.004 0.002 HFD Mean 0.020 0.013 0.007 0.040 SD 0.003 0.002 0.001
0.005 G3 Mean 0.017 0.009 0.005 0.030 SD 0.003 0.002 0.001 0.006 p
value 0.054 0.006 0.010 0.008 p value: Compared to HFD group
[0334] These results show that both body weight and body weight
gain were significantly decreased in the Mutamba treatment groups.
At treatment group, FER (food efficiency ratio) and visceral fat
weights were also significantly decreased. Therefore, the present
results suggest that Mutamba extract can be used as a body weight
controller.
Example 57
Effect of Morus alba Ethyl Acetate Extract 15 Combined with
Magnolia Extract 29 on DIO Mice
[0335] Morus alba ethyl acetate extract 15 produced as described in
Example 15 and Magnolia extract 29 produced according to Example 29
were combined and blended to a ratio of 2:1 by weight. The
combination composition was orally administrated to DIO mice as
described in Example 48 at a dosage of 300 mg/kg of animal weight
(200 mg/kg Morus and 100 mg/kg Magnolia). The study time period was
seven weeks. Table 69 shows the shows the effects of the
combination composition on body weight gain, that is, the change in
body weight in each study group as measured at the beginning of the
study compared to the weight measured on the day of each time point
of the study.
TABLE-US-00070 TABLE 69 Effect of Morus alba extract 15 Combined
with Magnolia extract 29 on Body Weight Gain in Mice Fed a High Fat
Diet Weeks Group 1 2 3 4 5 6 7 ND Mean -0.09 -0.25 0.09 0.35 0.13
0.56 0.79 SD 0.202 0.513 0.418 0.408 0.500 0.528 0.543 p value
0.1250 0.4003 0.2140 0.1935 0.0758 0.0351 0.0243 HFD Mean -0.55
0.16 1.45 1.95 3.29 4.40 5.77 SD 0.891 0.665 1.324 1.455 1.643
1.373 1.476 ORI Mean -3.20 -4.88 -3.34 -2.61 -2.04 -1.65 -0.07 (40
mg/kg) SD 0.611 1.463 1.502 1.859 1.640 1.526 1.437 p value 0.0011
0.0028 0.0072 0.0174 0.0081 0.0029 0.0033 G1 Mean -1.87 -2.78 -2.15
-1.62 -1.56 -1.48 -0.56 (300 mg/kg) SD 1.126 1.369 1.533 1.546
1.976 2.316 2.240 p value 0.1001 0.0144 0.0152 0.0180 0.0121 0.0077
0.0051 p value: compared to HFD by t-test
[0336] The data in Table 69 show that the animals treated with a
composition comprising Morus alba 15 combined with Magnolia extract
29 (treatment group G1) exhibited statistically significant
decreases in body weight gains from week 2 through to week 7 of the
study as compared to the HFD group. The positive control ORI
(Orilistat) showed statistically significant decreases in body
weight gains from week 1 through to week 7 of the study as compared
to the HFD group.
[0337] Table 70 shows the effects of Morus alba extract 15 and
Magnolia extract 29 combination composition on DOI mice for the
following end points: average body weight gain per day of the
study, average food intake per day of the study, and the food
efficiency ratio (FER) which is calculated as the average body
weight gain per day over the study period, divided by the average
food intake per day over the study period.
TABLE-US-00071 TABLE 70 Effect of Morus alba extract 15 Combined
with Magnolia Extract 29 on DIO Mice Body Weight Food Efficiency
Gain Food Intake Ratio Group (g/day) (g/day) (FER) ND Mean 0.018
3.558 0.005 SD 0.012 0.560 0.003 p value 0.0243 0.3067 0.0242 HFD
Mean 0.128 3.342 0.038 SD 0.033 0.807 0.010 ORI Mean -0.002 3.961
0.000 (40 mg/kg) SD 0.032 2.330 0.007 p value 0.0033 0.2661 0.0006
G1 Mean -0.012 2.261 -0.006 (300 mg/kg) SD 0.050 0.858 0.022 p
value 0.0051 0.0010 0.0189 Feed Efficacy Ratio (FER) = Body weight
gain (g/day)/Food intake (g/day) p value: compare to HFD by
t-test
[0338] The data in Table 70 show that the composition having Morus
alba extract 15 combined with Magnolia extract 29 (treatment group
G1) showed a statistically significant effect on lowering body
weight gain, food intake and lowering the Food Efficiency Ratio as
compared to the HFD group. The ORI positive control treatment group
showed statistically lower values than the HFD group for Body
Weight Gain per day and Food Efficiency Ratio.
[0339] Table 71 shows the effects of Morus alba extract 15 and
Magnolia extract 29 combination composition on DIO mice for total
liver organ weight, the weights of three fatty deposits: epididymal
fat, retroperitoneal fat, perirenal fat, and total fat (the sum of
the previous three fatty tissues).
TABLE-US-00072 TABLE 71 Effect of Morus alba extract 15 Combined
with Magnolia Extract 29 on Absolute Liver Weight and Weight of
Fatty Deposits in DIO Mice Epi- Retro- didymal peritoneal Perirenal
Total Group Liver Fat Fat Fat Fat* ND Mean 1.00 0.53 0.15 0.08 0.76
SD 0.145 0.150 0.064 0.017 0.222 p value 0.0410 0.0002 0.0000
0.0117 0.0001 HFD Mean 1.22 2.60 0.69 0.31 3.60 SD 0.215 0.424
0.051 0.118 0.521 ORI Mean 1.05 1.92 0.55 0.21 2.68 40 SD 0.137
0.230 0.039 0.055 0.310 mg/kg p value 0.2282 0.0236 0.0034 0.1729
0.0177 G1 Mean 1.23 1.73 0.52 0.20 2.45 300 SD 0.274 0.466 0.076
0.110 0.643 mg/kg p value 0.9286 0.0148 0.0036 0.1649 0.0145 *Total
fat is sum of the three fat deposits (epididymal, retroperitoneal
and perirenal fat) p value: compare to HFD by t-test
[0340] The data in Table 71 show that there is a statistically
significant difference in the weight of liver, epididymal fat,
retroperitoneal fat, perirenal fat and total fat between the ND
control group, fed a normal diet having a moderate caloric intact
of fat, and the HFD group. In addition, treatment group G1 getting
the composition of Morus alba extract 15 combined with Magnolia
extract 29, along with the positive control treatment group (ORI),
showed statistically significant decreases in two fat deposits
(Epididymal Fat and Retroperitoneal Fat) and Total Fat as compared
with the HFD group. These data, demonstrate the composition of
Morus alba extract combined with Magnolia extract is effective at
reducing the amount of fat present in DIO mice.
[0341] Table 72 shows the effects of the composition comprising
Morus alba 15 combined with Magnolia extract 29 on measurements in
DIO mice of fasting glucose (F-Glu), total cholesterol (T-chol) and
LDL-cholesterol (LDL-C) as measure in blood samples obtained at the
end of the study.
TABLE-US-00073 TABLE 72 Effect of Morus alba Extract 15 Combined
with Magnolia Extract 29 on Biochemistry Parameters F-Glu T-Chol
Triglyceride LDL-C Group (mg/dL) (mg/dL) (mg/dL) (mg/dL) ND Mean
178.67 105.67 30.56 4.54 (Normal Diet) SD 56.934 17.349 11.886
1.705 p value 0.0004 0.0000 0.7005 0.0807 HFD Mean 334.50 176.75
28.00 6.25 (High Fat Diet) SD 30.957 10.996 6.976 0.480 ORI Mean
225.50 134.00 62.50 3.30 (40 mg/kg) SD 52.208 30.299 64.717 1.134 p
value 0.0115 0.0379 0.3653 0.0030 G1 Mean 251.20 145.60 24.00 2.28
(300 mg/kg) SD 51.237 16.652 7.071 0.965 p value 0.0250 0.0150
0.4244 0.0001
[0342] The data in Table 46 show the combination composition
treatment group G1 and the positive control treatment group (ORI)
showed statistically significant decreases in total cholesterol,
LDL-cholesterol, and fasting glucose. These data, demonstrate the
Morus alba and Magnolia combinations composition when administered
at 300 mg/kg of body weight is effective in reducing cholesterol
and fasting glucose levels in DIO mice.
Example 58
Effect of Morus alba Ethyl Acetate Extract 15 Combined With Yerba
Mate Extract 26 on DIO Mice
[0343] Morus alba ethyl acetate extract 15 produced as described in
Example 15 and Yerba Mate extract 26 produced according to Example
26 were combined and blended to a ratio of 1:5 by weight. The
combination composition was orally administrated to DIO mice as
described in Example 48 at a dosage of 1200 mg/kg of animal weight
(200 mg/Kg Morus and 1000 mg/Kg Yerba Mate). The study time period
was seven weeks. Table 73 shows the effects of the combination
composition on total body weight and Table 74 shows the effects of
the combination composition on body weight gain.
TABLE-US-00074 TABLE 73 Effect of Morus alba extract 15 Combined
with Yerba Mate extract 26 on Total Weight in DIO Mice Weeks Group
0 1 2 3 4 5 6 7 ND Mean 28.60 28.51 28.34 28.69 28.94 28.73 29.16
29.39 (Normal SD 2.249 2.244 2.341 2.491 2.094 2.107 2.087 2.095
Diet) p value 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000
0.0000 HFD Mean 37.98 38.18 38.72 40.01 40.51 41.85 42.96 44.32
(High Fat SD 2.3816 1.9830 2.2782 1.5612 1.9172 1.7894 1.9183
2.0291 Diet) ORI Mean 38.40 35.20 33.58 35.12 35.84 36.41 36.80
38.38 (40 mg/kg) SD 2.895 3.211 2.730 2.568 2.320 2.416 2.534 2.257
p value 0.8094 0.1510 0.0465 0.0343 0.0371 0.0226 0.0174 0.0157 G1
Mean 39.14 37.04 35.59 34.76 35.15 35.85 36.40 38.06 (1200 mg/kg)
SD 2.985 2.430 2.268 2.564 2.496 2.590 2.669 2.696 p value 0.5169
0.4739 0.1085 0.0197 0.0195 0.0130 0.0104 0.0138 p value: compare
to HFD by t-test
TABLE-US-00075 TABLE 74 Effect of Morus alba extract 15 Combined
with Yerba Mate extract 26 on Weight Gain in DIO Mice Weeks Group 1
2 3 4 5 6 7 ND Mean -0.09 -0.25 0.09 0.35 0.13 0.56 0.79 (Normal SD
0.202 0.513 0.418 0.408 0.500 0.528 0.543 Diet) p value 0.1250
0.4003 0.2140 0.1935 0.0758 0.0351 0.0243 HFD Mean -0.55 0.16 1.45
1.95 3.29 4.40 5.77 (High Fat SD 0.891 0.665 1.324 1.455 1.643
1.373 1.476 Diet) ORI Mean -3.20 -4.88 -3.34 -2.61 -2.04 -1.65
-0.07 (40 mg/kg) SD 0.611 1.463 1.502 1.859 1.640 1.526 1.437 p
value 0.0011 0.0028 0.0072 0.0174 0.0081 0.0029 0.0033 G1 Mean
-2.10 -3.55 -4.38 -3.99 -3.29 -2.74 -1.08 (1200 mg/kg) SD 0.655
1.671 2.413 2.464 2.567 2.878 2.484 p value 0.0195 0.0116 0.0092
0.0097 0.0079 0.0076 0.0053 p value: compare to HFD by t-test
[0344] The data in Table 73 show that the animals in the
combination composition of Morus alba extract 15 and Yerba Mate
extract 26 treatment group (G1) exhibited statistically significant
decreases in total body weight from week 3 through to week 7 of the
study as compared to the HFD group. The positive control ORI
(orilistat dosed at 40 mg/kg) showed statistically significant
decreases in total body weight from week 2 through to week 7 of the
study as compared to the HFD group.
[0345] The data in Table 74 show that the animals in the
combination composition of Morus alba extract 15 and Yerba Mate
extract 26 treatment group (G1) and the positive control ORI group
of animals both exhibited statistically significant decreases in
body weight gains from week 1 through to week 7 of the study as
compared to the HFD group.
[0346] Table 75 shows the effects of Morus alba extract 15 and
Yerba Mate extract 26 combination composition on DIO mice for the
following end points: average body weight gain per day of the
study, average food intake per day of the study, and the food
efficiency ratio (FER) which is calculated as the average body
weight gain per day over the study period, divided by the average
food intake per day over the study period.
TABLE-US-00076 TABLE 75 Effect of Morus alba extract 15 Combined
with Yerba Mate extract 26 on DIO Mice Food Efficiency Weight Gain
Food Intake Ratio Group (g/day) (g/day) (FER) ND Mean 0.018 3.558
0.005 (Normal SD 0.012 0.560 0.003 Diet) p value 0.0243 0.3067
0.0242 HFD Mean 0.128 3.342 0.038 (High SD 0.033 0.807 0.010 Fat
Diet) ORI Mean -0.002 3.961 0.000 (40 mg/kg) SD 0.032 2.330 0.007 p
value 0.0033 0.2661 0.0006 G1 Mean -0.024 2.435 -0.010 (1200 mg/kg)
SD 0.055 0.475 0.023 p value 0.0053 0.0020 0.0143 FER(Feed efficacy
ratio) = Body weight gain(g/day)/Food intake(g/day) p value:
compare to HFD by t-test
[0347] The data in Table 75 show that the Morus alba extract 15 and
Yerba Mate extract 26 combination composition treatment group G1
showed a statistically significant effect on lowering body weight
gain, food intake and lower Food Efficiency Ratio as compared to
the HFD group. The ORI positive control treatment group showed
statistically lower values than the HFD group for Body Weight Gain
per day and Food Efficiency Ratio.
[0348] Table 76 shows the effects of Morus alba extract 15 and
Yerba Mate extract 26 combination composition on DOI mice for the
weight of Retroperitoneal Fat.
TABLE-US-00077 TABLE 76 Effect of Morus alba and Yerba Mate
Combination Composition on Retroperitoneal Fat in DIO Mice Group
Retroperitoneal Fat ND Mean 0.15 (Normal SD 0.064 Diet) p value
0.0000 HFD Mean 0.69 (High SD 0.051 Fat Diet) ORI Mean 0.55 (40
mg/kg) SD 0.039 p value 0.0034 G1 Mean 0.57 (1200 mg/kg) SD 0.080 p
value 0.0281 p value: compare to HFD by t-test
[0349] The data in Table 76 show that there is a statistically
significant difference in the weight of retroperitoneal fat,
between the ND control group, fed a normal diet having a moderate
caloric intact of fat, and the HFD group. In addition, the Morus
alba extract 15 and Yerba Mate extract 26 combination composition
treatment group G1 and the positive control treatment group (ORI)
showed statistically significant decreases in Retroperitoneal Fat
as compared with the HFD group. These data, demonstrate the Morus
alba and Yerba Mate combinations composition is effective in
reducing the amount of retroperitoneal fat present in DIO mice.
[0350] Table 77 shows the effects of the Morus alba 15 and Yerba
Mate extract 26 combination composition on measurements in DIO mice
of fasting glucose (F-Glu), total cholesterol (T-chol),
triglyceride and LDL-cholesterol (LDL-C) as measure in blood
samples obtained at the end of the study.
TABLE-US-00078 TABLE 77 Effect of Morus alba and Yerba Mate
Combination Composition on Biochemistry Parameters in DIO Mice
F-Glu T-Chol Triglyceride LDL-C Group (mg/dL) (mg/dL) (mg/dL)
(mg/dL) ND Mean 178.67 105.67 30.56 4.54 (Normal SD 56.934 17.349
11.886 1.705 Diet) p value 0.0004 0.0000 0.7005 0.0807 HFD Mean
334.50 176.75 28.00 6.25 (High SD 30.957 10.996 6.976 0.480 Fat
Diet) ORI Mean 225.50 134.00 62.50 3.30 (40 mg/kg) SD 52.208 30.299
64.717 1.134 p value 0.0115 0.0379 0.3653 0.0030 G1 Mean 279.80
146.60 10.60 4.50 (1200 mg/kg) SD 42.275 14.170 3.209 0.667 p value
0.0681 0.0102 0.0015 0.0032 p value: compare to HFD by t-test
[0351] The data in Table 77 show the combination composition
treatment group G1 and the positive control treatment group (ORI)
showed statistically significant decreases in total cholesterol,
LDL-cholesterol, and fasting glucose.
[0352] Overall, the data presented in this example show that the
combination composition composed of Morus alba ethyl acetate
extract 15 (200 mg/Kg) and Yerba Mate extract 26 (1000 mg/Kg) was
effective in lowering total body weight and the rate of body weight
gain in mice fed a high fat diet.
Example 59
Effect of Morus Alba Ethyl Acetate Extract 14 Combined with
Rosemary Extract 21 on DIO Mice
[0353] Morus alba ethyl acetate extract 14 produced as described in
Example 14 and Rosemary extract 21 produced according to Example 21
were combined and blended to a ratio of 2:5 by weight. The
combination composition was orally administrated to DIO mice as
described in Example 48 at a dosage of 700 mg/kg of animal weight
(200 mg/Kg Morus and 500 mg/Kg Rosemary). The study time period was
seven weeks. Table 78 shows the effects of the combination
composition on total body weight gain and Table 79 shows the
effects of the combination composition on body weight gain.
TABLE-US-00079 TABLE 78 Effect of Morus alba extract 14 Combined
with Rosemary extract 21 on Weight Weeks Group 0 1 2 3 4 5 6 7 ND
Mean 29.01 29.30 29.42 29.27 29.25 29.73 29.82 30.11 (Normal SD
2.824 2.881 2.523 2.315 2.368 2.739 2.942 3.113 Diet) p value
0.0002 0.0002 0.0001 0.0000 0.0000 0.0000 0.0000 0.0000 HFD Mean
41.86 41.60 42.53 43.65 44.78 46.09 46.64 47.86 (High Fat SD 3.812
3.776 3.617 3.425 3.293 2.957 2.694 2.219 Diet) ORI Mean 42.39
38.83 36.64 36.48 38.57 40.07 40.95 42.99 (40 mg/kg) SD 3.489 4.433
4.681 3.948 4.221 4.690 4.775 4.910 p value 0.8079 0.2718 0.0349
0.0073 0.0174 0.0238 0.0294 0.0514 G1 Mean 43.03 37.59 36.41 37.43
39.04 40.40 40.60 41.89 (700 mg/kg) SD 2.702 1.930 2.597 3.630
4.103 4.381 4.527 4.505 p value 0.5340 0.0312 0.0046 0.0091 0.0189
0.0209 0.0157 0.0156 p value: compare to HFD by t-test
TABLE-US-00080 TABLE 79 Effect of combination with Morus alba
extract 14 Combined with Rosemary extract 21 on Body Weight Fain in
DIO Mice Weeks Group 1 2 3 4 5 6 7 ND Mean 0.29 0.41 0.26 0.23 0.71
0.81 1.10 SD 0.530 0.704 0.665 0.875 0.724 0.723 0.834 p value
0.0779 0.5236 0.0056 0.0007 0.0002 0.0003 0.0005 HFD Mean -0.27
0.67 1.79 2.92 4.23 4.77 5.99 SD 0.248 0.525 0.732 0.866 1.080
1.378 1.933 ORI Mean -3.56 -5.75 -5.91 -3.82 -2.33 -1.44 0.60 40
mg/kg SD 1.391 1.935 1.852 1.463 1.675 1.512 1.550 p value 0.0002
0.0000 0.0000 0.0000 0.0000 0.0000 0.0003 G1 Mean -5.44 -6.62 -5.60
-3.99 -2.63 -2.43 -0.67 700 mg/kg SD 1.377 1.585 1.900 2.081 2.057
2.200 2.382 p value 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000
0.0003 p value: compare to HFD by t-test
[0354] The data in Table 78 show that the animals treated with
Morus alba extract 1.4 combined with Rosemary extract 21 (treatment
group G1) exhibited statistically significant decreases in total
body weight from week 1 through to week 7 of the study as compared
to the HFD group. The positive control ORI (Orilistat dosed at 40
mg/kg) showed statistically significant decreases in total body
weight from week 2 through to week 6, but not weeks 1 and 7 of the
study as compared to the HFD group.
[0355] The data in Table 79 show that the animals in treatment
group (G1) who received Morus alba extract 14 combined with
Rosemary extract 21 and the positive control ORI group both
exhibited statistically significant decreases in body weight gain
from week 1 through to week 7 of the study as compared to the HFD
group.
[0356] Table 80 shows the effects of Morus alba extract 14 combined
with Rosemary extract 21 combination composition on DIO mice for
the following end points: average body weight gain per day of the
study, average food intake per day of the study, and the food
efficiency ratio (FER) which is calculated as the average body
weight gain per day over the study period, divided by the average
food intake per day over the study period.
TABLE-US-00081 TABLE 80 Effect of Morus alba Extract 14 Combined
with Rosemary Extract 21 on DIO Mice Food Body Efficiency Weight
Gain Food Intake Ratio Group (g/day) (g/day) (FER) ND Mean 0.023
3.046 0.007 SD 0.017 0.188 0.006 p value 0.0005 0.0000 0.0003 HFD
Mean 0.125 2.376 0.053 SD 0.040 0.400 0.017 ORI Mean 0.012 2.711
0.005 40 mg/kg SD 0.032 0.456 0.012 p value 0.0003 0.0069 0.0002 G1
Mean -0.014 2.180 -0.006 700 mg/kg SD 0.050 0.474 0.023 p value
0.0003 0.1126 0.0005 Feed Efficacy Ratio (FER) = Body Weight
Gain(g/day)/Food Intake(g/day) p value: compare to HFD by
t-test
[0357] The data in Table 80 show that the Morus alba extract 14
combined with Rosemary extract 21 combination composition treatment
group G1 showed a statistically significant effect on lowering
average weight gain per day and lower Food Efficiency Ratio as
compared to the HFD group. The ORI positive control treatment group
showed statically lower values than the HFD group for average
weight gain per day, average Food Intake per day and Food
Efficiency Ratio.
[0358] Table 81 shows the effects of Morus alba extract 14 combined
with Rosemary extract 21 combination composition on DIO mice for
the weight of Perirenal Fat.
TABLE-US-00082 TABLE 81 Effect of Morus alba 14 combined with
Rosemary extract 21 on Perirenal Fat in DIO mice Group Perirenal
Fat ND Mean 0.08 (Normal SD 0.026 Diet) p value 0.0001 HFD Mean
0.58 (High SD 0.127 Fat Diet) ORI Mean 0.43 (40 mg/kg) SD 0.186 p
value 0.1369 G1 Mean 0.40 (700 mg/kg) SD 0.152 p value 0.0479 p
value: compare to HFD by t-test
[0359] The data in Table 81 show that there is a statistically
significant difference in the weight of perirenal fat, between the
ND control group, fed a normal diet having a moderate caloric
intact of fat, and the HFD group. In addition, the Morus alba
extract 14 combined with Rosemary extract 21 composition treatment
group G1 and the positive control treatment group (ORI) showed
statistically significant decreases in Perirenal Fat as compared
with the HFD group. These data demonstrate the Morus alba and
Rosemary combination compositions are effective in reducing the
amount of perirenal fat present in DIO mice.
[0360] Overall, the data presented in this example show that the
combination composition composed of Morus alba ethyl acetate
extract 14 (200 mg/Kg) and Rosemary extract 21 (500 mg/Kg) was
effective in lowering total body weight and the rate of body weight
gain in mice fed a high fat diet.
Example 60
Efficacy Study of Mutamba Ethanol Extract 35 Combined with Morus
alba EtOAC Fraction of Ethanol Extract 15 in DIO Mice
[0361] Mutamba ethanol extract 35 produced according to the example
35 and Morus alba EtOAc fraction of ethanol extract 15 produced
according to example 15 was blended in a ratio of 5:1. The combined
composition was orally administrated to DIO mice as described in
the Example 48 at a dosage of 1200 mg/kg (G1) twice a day by
gavage.
[0362] The treatment group (G1) shoed significantly decreased
weight gain was at weeks 4, 5, 6 and 7 as compared to the high fat
diet group (Table 82).
TABLE-US-00083 TABLE 82 Effect of Mutamba Extract 35 Combined with
Morus alba Extract 15 on Total Body Weight in DIO Mice Weeks Group
0 1 2 3 4 5 6 7 ND Mean 28.60 28.51 28.34 28.69 28.94 28.73 29.16
29.39 SD 2.249 2.244 2.341 2.491 2.094 2.107 2.087 2.095 p value
0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 HFD Mean
37.98 38.18 38.72 40.01 40.51 41.85 42.96 44.32 SD 2.3816 1.9830
2.2782 1.5612 1.9172 1.7894 1.9183 2.0291 ORI Mean 38.40 35.20
33.58 35.12 35.84 36.41 36.80 38.38 SD 2.895 3.211 2.730 2.568
2.320 2.416 2.534 2.257 p value 0.8094 0.1510 0.0465 0.0343 0.0371
0.0226 0.0174 0.0157 G1 Mean 37.22 35.36 34.49 35.24 35.09 34.12
34.70 35.24 SD 2.177 1.465 0.928 1.161 1.309 1.269 1.695 1.570 p
value 0.9374 0.5169 0.4739 0.1085 0.0197 0.0195 0.0130 0.0104 p
value: compare to HFD by t-test
[0363] The treatment group (G1), the body weight gain was
significantly decreased after the second week treatment of the
experiment (Table 83) and such effect was lasted to the end of the
treatment.
TABLE-US-00084 TABLE 83 Effect of Mutamba Extract 35 Combined with
Morus alba Extract 15 on Weight Gain in DIO Mice Weeks Group 1 2 3
4 5 6 7 ND Mean -0.09 -0.25 0.09 0.35 0.13 0.56 0.79 SD 0.202 0.513
0.418 0.408 0.500 0.528 0.543 p value 0.1250 0.4003 0.2140 0.1935
0.0758 0.0351 0.0243 HFD Mean -0.55 0.16 1.45 1.95 3.29 4.40 5.77
SD 0.891 0.665 1.324 1.455 1.643 1.373 1.476 ORI Mean -3.20 -4.88
-3.34 -2.61 -2.04 -1.65 -0.07 SD 0.611 1.463 1.502 1.859 1.640
1.526 1.437 p value 0.0011 0.0028 0.0072 0.0174 0.0081 0.0029
0.0033 G1 Mean -1.86 -2.74 -1.98 -2.13 -3.10 -2.53 -1.98 SD 0.973
1.483 1.258 0.901 2.036 1.246 1.068 p value 0.1080 0.0378 0.0067
0.0011 0.0051 0.0005 0.0002 p value: compare to HFD by t-test
[0364] The treatment group (G1) of FER was significantly decreased,
when compared with the high fat diet group (Table 84)
TABLE-US-00085 TABLE 84 Effect of Mutamba Extract 35 Combined with
Morus alba Extract 15 on DIO Mice Weight Gain Food intake FER Group
(g/day) (g/day) (Food efficiency ratio) ND Mean 0.018 3.558 0.005
SD 0.012 0.560 0.003 p value 0.0243 0.3067 0.0242 HFD Mean 0.128
3.342 0.038 SD 0.033 0.807 0.010 ORI Mean -0.002 3.961 0.000 SD
0.032 2.330 0.007 p value 0.0033 0.2661 0.0006 G1 Mean -0.044 2.652
-0.017 SD 0.024 0.273 0.009 p value 0.0002 0.0004 0.0004 FER(Feed
efficacy ratio) = Body weight gain(g/day)/Food intake(g/day) p
value: compare to HFD by t-test
[0365] The treatment group (G1) of glucose, total cholesterol and
LDL-C were significantly decreased as compared to the high fat diet
group (Table 85).
TABLE-US-00086 TABLE 85 Effect of Mutamba Extract 35 Combined with
Morus alba Extract 15 on Biochemistry Parameters Glu T-chol TG
LDL-C Group (mg/dL) (mg/dL) (mg/dL) (mg/dL) HD Mean 178.67 105.67
30.56 4.54 SD 56.934 17.349 11.886 1.705 p value 0.0004 0.0000
0.7005 0.0807 HFD Mean 334.50 176.75 28.00 6.25 SD 30.957 10.996
6.976 0.480 ORI Mean 225.50 134.00 62.50 3.30 SD 52.208 30.299
64.717 1.134 p value 0.0115 0.0379 0.3653 0.0030 G1 Mean 194.80
116.20 14.60 2.76 SD 33.596 33.732 8.173 0.948 p value 0.0004
0.0114 0.0354 0.0003
[0366] Absolute weights of epididymal fat pads, retroperitoneal
fat, perirenal fat and total fat pads were significantly decreased
in the treatment group (G1) as compared to the high fat diet group
(Table 86).
TABLE-US-00087 TABLE 85 Effect of Mutamba Extract 35 Combined with
Morus alba Extract 15 on Biochemistry Parameters Glu T-chol TG
LDL-C Group (mg/dL) (mg/dL) (mg/dL) (mg/dL) ND Mean 178.67 105.67
30.56 4.54 SD 56.934 17.349 11.886 1.705 p value 0.0004 0.0000
0.7005 0.0807 HFD Mean 334.50 176.75 28.00 6.25 SD 30.957 10.996
6.976 0.480 ORI Mean 225.50 134.00 62.50 3.30 SD 52.208 30.299
64.717 1.134 p value 0.0115 0.0379 0.3653 0.0030 G1 Mean 194.80
116.20 14.60 2.76 SD 33.596 33.752 8.173 0.948 p value 0.0004
0.0114 0.0354 0.0003
[0367] Overall, the data show that body weight and body weight gain
were significantly decreased in DIO mice treated with Mutamba
extract 35 combined with Morus alba extract 15. FER (food
efficiency ratio) and fat weights were also significantly
decreased. Furthermore, fasting glucose, total cholesterol and
LDL-cholesterol levels were significantly decreased by sample
treatment. Therefore, the present example indicates that the
combination of Mutamba extract with Morus alba extract can be used
as a body weight, glucose level, and cholesterol level
controller.
Example 61
Efficacy Study of Mutamba Ethanol Extract 35 Combined with Yerba
Mate Extract 26 in DIO Mice
[0368] Mutamba ethanol extract 35 produced according to the example
35 and Mate extract 26 produced according to example 26 were
blended in a ratio of 1:1. The dual combination composition was
orally administrated to DIO mice as described in the example 48 at
a dosage of 2000 mg/kg (G1) twice a day.
[0369] Weight gain was significantly decreased in the treatment
group (G1) after the third week of treatment (Table 87) and that
effect lasted to the end of the treatment.
TABLE-US-00088 TABLE 87 Effect of Mutamba Combined with Yerba Mate
on Weight Gain in DIO Mice Weeks Group 1 2 3 4 5 6 7 ND Mean -0.09
-0.25 0.09 0.35 0.13 0.56 0.79 SD 0.202 0.513 0.418 0.408 0.500
0.528 0.543 p value 0.1250 0.4003 0.2140 0.1935 0.0758 0.351 0.0243
HFD Mean -0.55 0.16 1.45 1.95 3.29 4.40 5.77 SD 0.891 0.665 1.324
1.455 1.643 1.373 1.476 SIB Mean -3.63 -5.37 -4.69 -4.82 -4.25
-3.90 -2.27 SD 1.504 2.317 2.250 2.227 1.862 2.276 2.960 p value
0.00885 0.0078 0.00553 0.00358 0.00119 0.00135 0.00511 ORI Mean
-3.20 -4.88 -3.34 -2.61 -2.04 -1.65 -.0.07 SD 0.611 1.463 1.502
1.859 1.640 1.526 1.437 p value 0.0011 0.0028 0.0072 0.0174 0.0081
0.0029 0.0033 G1 Mean -0.11 -1.28 -1.43 -2.25 -1.84 -2.03 -0.45 SD
0.546 1.290 1.361 1.794 2.060 2.241 1.849 p value 0.3970 0.0709
0.0373 0.0141 0.0104 0.0020 0.0031 p value: compare to HFD by
t-test
[0370] FER was significantly decreased in the treatment group (G1)
as compared with the high fat diet group (Table 87).
TABLE-US-00089 TABLE 87 Effect of Mutamba Combined with Yerba Mate
on DIO Mice Weight Gain Food Intake FER Group (g/day) (g/day) (Food
efficiency ratio) ND Mean 0.018 3.558 0.005 SD 0.012 0.560 0.003 p
value 0.0243 0.3067 0.0242 HFD Mean 0.128 3.342 0.038 SD 0.033
0.807 0.010 SIB Mean -0.051 3.564 -0.014 SD 0.066 1.671 0.018 p
value 0.0051 0.5332 0.0042 ORI Mean -0.002 3.961 0.000 SD 0.032
2.330 0.007 p value 0.0033 0.2661 0.0006 G1 Mean -0.010 2.212
-0.005 SD 0.041 0.334 0.018 p value 0.0031 0.0000 0.0038 FER(Feed
efficacy ratio) = body weight gain(g/day)/Food intake(g/day) p
value: compare to HFD by t-test
[0371] These data show that weight gain and FER were significantly
decreased in the treatment group (G1). Therefore, the present
example indicates that the combination of Mutamba extract and Yerba
Mate extract can be used as a body weight controller.
Example 62
Efficacy Study of Mutamba Ethanol Extract 35 Combined with Magnolia
Extract 29 in DIO Mice
[0372] Mutamba ethanol extract 35 produced according to the example
35 and Magnolia extract 29 produced according to example 29 was
blended in a ratio of 10:1. The dual combination composition was
orally administrated to DIO mice as described in the example 48 at
a dosage of 1100 mg/kg (G1) twice a day.
[0373] Weight gain was significantly decreased in the treatment
group (G1) after the third week of treatment (Table 88) and such
effect lasted until the end of the treatment.
TABLE-US-00090 TABLE 88 Effect of Mutamba Extract Combined with
Magnolia Extract on Weight Gain in DIO Mice Weeks Group 1 2 3 4 5 6
7 ND Mean -0.09 -0.25 0.09 0.35 0.13 0.56 0.79 SD 0.202 0.513 0.418
0.408 0.500 0.528 0.543 p value 0.1250 0.4003 0.2140 0.1935 0.0758
0.0351 0.0243 HFD Mean -0.55 0.16 1.45 1.95 3.29 4.40 5.77 SD 0.891
0.665 1.324 1.455 1.643 1.373 1.476 SIB Mean -3.63 -5.37 -4.69
-4.82 -4.25 -3.90 -2.27 SD 1.504 2.317 2.250 2.227 1.862 2.276
2.960 p value 0.00885 0.0078 0.00553 0.00358 0.00119 0.00135
0.00511 OR1 Mean -3.20 -4.88 -3.34 -2.61 -2.04 -1.65 -0.07 SD 0.611
1.463 1.502 1.859 1.640 1.526 1.437 p value 0.0011 0.0028 0.0072
0.0174 0.0081 0.0029 0.0033 G1 Mean -1.18 -2.51 -2.77 -3.99 -3.80
-3.41 -1.65 SD 0.537 0.550 1.229 2.247 2.458 3.214 3.681 p value
0.2326 0.0008 0.0038 0.0068 0.0047 0.0079 0.0175 p value: compare
to HFD by t-test
[0374] FER was changed in the treatment group (G1) as compared with
the high fat diet group (Table 89).
TABLE-US-00091 TABLE 89 Effect of Mutamba Extract Combined with
Magnolia Extract on DIO Mice Weight Gain Food Intake FER Group
(g/day) (g/day) (Food efficiency ratio) ND Mean 0.018 3.558 0.005
SD 0.012 0.560 0.003 p value 0.0243 0.3067 0.0242 HFD Mean 0.128
3.342 0.038 SD 0.033 0.807 0.010 ORI Mean -0.002 3.961 0.000 SD
0.032 2.330 0.007 p value 0.0033 0.2661 0.0006 G1 Mean -0.037 2.265
-0.016 SD 0.082 0.306 0.036 p value 0.0175 0.0000 0.0475 FER(Feed
efficacy ratio) = Body weight gain(g/day)/Food intake(g/day) p
value: compare to HFD by t-test
[0375] TG was significantly decreased in the treatment group (G1)
as compared with the high fat diet group (Table 90).
TABLE-US-00092 TABLE 90 Effect of Mutamba Extract Combined with
Magnolia Extract on Biochemistry Parameter Glu T-chol TG Group
(mg/dL) (mg/dL) (mg/dL) ND Mean 178.67 105.67 30.56 SD 56.934
17.349 11.886 p value 0.0004 0.0000 0.7005 HFD Mean 334.50 176.75
28.00 SD 30.957 10.996 6.976 ORI Mean 225.50 134.00 62.50 SD 52.208
30.299 64.717 p value 0.0115 0.0379 0.3653 G1 Mean 280.50 164.25
15.75 SD 34.933 13.200 3.202 p value 0.0600 0.1959 0.0188
[0376] The NASH score was significantly decreased in the treatment
group (G1) as compared with the high fat diet group (Table 91).
TABLE-US-00093 TABLE 91 Effect of the treatment group (G1) of Liver
Pathology in DIO Mice Indications Lobular Hepatocellular Steatosis
Inflammation ballooning NASH Group (0-3) (0-3) (0-2) (sum) ND Mean
0.00 1.33 0.00 1.20 SD 0.000 0.707 0.000 0.789 p value 0.0000
0.8816 0.0000 0.0000 HFD Mean 1.86 1.29 1.29 4.43 SD 0.900 0.488
0.488 1.272 ORI Mean 1.00 1.40 1.00 3.40 SD 0.000 0.548 0.000 0.548
p value 0.0620 0.7114 0.2258 0.1236 G1 Mean 1.00 1.00 0.80 2.80 SD
0.000 0.000 0.837 0.837 p value 0.0620 0.2258 0.2309 0.0322 p
value: compare to HFD by t-test
[0377] These data show that body weight gain and FER were
significantly decreased in the treatment groups (G1). Furthermore,
TG and NASH score were also significantly decreased in the
treatment groups. Therefore, the present example shows that Mutamba
extract combined with Magnolia extract can be used as a body
weight, cholesterol level, and fatty liver controller.
Example 63
Efficacy Study of Morus alba Ethyl Acetate Extract 17 Combined with
Magnolia Extract 29 and Yerba Mate Extract 26 on DIO Mice
[0378] Morus alba ethyl acetate fraction 17 produced according to
Example 17, Magnolia extract 29 produced according to Example 29,
Yerba Mate extract 26 produced according to Example 26, and
Rosemary extract 21 produced according to the Example 21 were
tested individually or combined on DIO mouse model as described in
Example 48. The combination of Morus, Magnolia, and Yerba Mate
extracts were blended to a blend ratio of 2:1:5 by weight to make
combination Composition 1A.
[0379] DIO mice were divided to eight treatment groups; HFD (high
fat diet group), ORI (orlistat, 40 mg/kg/day, twice daily), the
combination composition treatment group G1 (Morus, Magnolia, Mate
Composition 1A at 800 mg/kg/day), treatment group G2 (Magnolia
extract 29, 100 mg/kg/day), treatment group G3 (Yerba Mate extract
26, 500 mg/kg/day), treatment group G4 (Morus extract, 200
mg/kg/day) and two Rosemary extract 21 treatment groups (05, 500
mg/kg/day and G6, 1000 mg/kg/day). All test samples were orally
administrated to DIO mice as described in the Example 48. The daily
dosage for each treatment group was divided in half and
administrated twice daily. The study time period was eight weeks.
The effects of these treatments in DIO mice are shown in Table 92
for total body weight and Table 93 for weight gain.
TABLE-US-00094 TABLE 92 Effect of Various Individual or Combined
Extracts on Total Body Weight in DIO Mice Weeks Group 0 1 2 3 4 5 6
7 8 ND Mean 29.24 29.11 29.03 29.47 29.36 29.51 29.75 29.76 30.14
SD 1.020 0.967 1.201 1.166 1.428 1.309 1.509 1.270 1.321 p value
0.0001 0.0001 0.000 0.000 0.000 0.000 0.000 0.000 0.000 HFD Mean
41.31 41.73 42.12 43.64 44.94 46.25 47.78 48.43 49.29 SD 2.932
2.771 2.856 2.884 2.870 3.172 3.247 3.224 2.783 ORI Mean 40.22
37.98 34.08 35.32 36.38 37.65 38.75 39.96 39.99 SD 2.816 2.547
1.277 1.186 1.867 2.284 2.931 3.235 3.633 p value 0.528 0.035
0.0001 0.0001 0.0001 0.0003 0.0005 0.0011 0.0006 G1 Mean 39.33
36.75 32.50 33.96 35.06 35.42 34.61 36.44 37.50 (Combo) SD 1.591
1.623 1.554 1.035 1.145 1.736 1.180 1.271 1.474 p value 0.2118
0.0064 0.0001 0.0001 0.0001 0.0001 0.0000 0.0000 0.000 G2 Mean
40.13 39.50 38.12 39.05 39.92 40.72 41.43 42.76 43.63 Magnolia SD
2.687 2.362 2.324 1.832 1.428 1.612 1.907 2.253 2.869 p value
0.4852 0.1641 0.0239 0.0081 0.0033 0.0035 0.0020 0.0054 0.006 G3
Mean 40.78 41.32 42.33 43.36 43.83 44.38 45.48 46.16 47.34 Yerba SD
2.806 2.856 3.702 3.909 4.013 4.355 4.578 4.112 4.200 Mate p value
0.755 0.807 0.915 0.887 0.592 0.415 0.340 0.311 0.366 G4 Mean 40.57
40.23 38.41 39.82 40.84 42.59 44.28 45.78 47.87 Moras SD 2.591
2.264 2.989 2.904 2.703 2.277 2.693 2.945 2.799 p value 0.6524
0.3307 0.0526 0.0450 0.0288 0.0448 0.0698 0.1672 0.3989
TABLE-US-00095 TABLE 93 Effect of Various Individual or Combined
Extracts on Weight Gain in DIO Mice Weeks Group 1 2 3 4 5 6 7 8 ND
Mean -0.13 -0.22 0.22 0.11 0.27 0.50 0.52 0.89 SD 0.367 0.377 0.238
0.463 0.390 0.635 0.447 0.562 p value 0.0057 0.0011 0.0080 0.0010
0.0005 0.0004 0.0002 0.000 HFD Mean 0.42 0.81 2.34 3.64 4.94 6.47
7.13 7.98 SD 0.238 0.636 1.229 1.343 1.526 1.873 1.815 1.904 ORI
Mean -2.24 -6.14 -4.91 -3.84 -2.57 -1.47 -0.26 -0.23 SD 1.123 2.286
1.286 1.228 1.355 1.507 1.728 2.055 p value 0.0018 0.0005 0.0000
0.0000 0.0000 0.0000 0.0001 0.0000 G1 Mean -2.58 -6.83 -5.37 -4.27
-3.91 -4.72 -2.89 -1.83 (Combo) SD 1.466 0.942 0.729 2.160 2.740
1.320 1.880 2.089 p value 0.0096 0.0000 0.0000 0.0000 0.0001 0.0000
0.0000 0.0000 G2 Mean -0.63 -2.01 -1.08 -0.22 0.59 1.30 2.63 3.50
Magnolia SD 0.394 1.150 1.555 1.999 2.263 3.241 3.197 4.010 p value
0.0005 0.0008 0.0018 0.0029 0.0030 0.0070 0.0134 0.0329 G3 Mean
0.54 1.56 2.58 3.05 3.60 4.71 5.38 6.56 Yerba SD 0.345 1.020 1.627
1.825 2.189 2.356 2.120 2.236 Mate p value 0.4874 0.1617 0.7760
0.5424 0.2474 0.1822 0.1570 0.2647 G4 Mean -0.33 -2.16 -0.75 0.27
2.03 3.71 5.21 7.30 Morus SD 0.430 2.722 2.368 1.877 1.476 1.358
1.976 2.020 p value 0.0038 0.0437 0.0178 0.0051 0.0072 0.0154
0.1112 0.5628 G5 Mean 0.19 -1.11 0.14 0.86 1.68 3.00 4.37 5.31
Rosemary SD 0.314 1.009 1.435 1.960 2.165 2.330 2.011 2.276 (low) p
value 0.1801 0.0028 0.0171 0.0169 0.0131 0.0175 0.0318 0.0519 G6
Mean 0.77 -1.13 -1.36 -3.30 -2.41 -1.90 -1.05 0.58 Rosemary SD
0.713 1.428 1.598 1.861 0.961 2.206 3.400 4.129 (high) p value
0.4048 0.0177 0.0032 0.0001 0.0000 0.0002 0.0010 0.0046 p value:
compare to HFD by t-test
[0380] Table 92 shows that mice treated with Morus-Magnolia-Mate
Composition 1A (treatment group 01) and the positive control ORI
all exhibited statistically significant decreases in total weight
from week 2 through to week 8 as compared to the HFD group. The
Magnolia treatment group (G2) exhibited statistically significant
decreases in weight from week 3 through to week 8 as compared to
the HFD group. The Morus treatment group (G4) exhibited
statistically significant decreases in weight from week 4 through
to week 5 as compared to the HFD group. The high dose Rosemary
(1000 mg/Kg/day) treatment group (G6) exhibited statistically
significant decreases in weight from week 4 through to week 8 as
compared to the HFD group. The Yerba Mate treatment group (G3) and
the low dose Rosemary (500 mg/Kg/day) treatment group (G5) did not
exhibit any statistically significant decreases in weight during
the study as compared to the HFD group.
[0381] Table 93 shows that the animals in the Morus-Magnolia-Mate
combination composition 1A (treatment group G1), Magnolia treatment
group (G2), Rosemary (1000 mg/Kg/day) treatment group G6, and the
positive control ORI group all exhibited statistically significant
decreases in weight gain from week 1 through to week 8 as compared
to the HFD group. The Morus treatment group (G4) exhibited
statistically significant decreases in weight gain from week 1
through week 6 as compared to the HFD group. The low dose Rosemary
(500 mg/Kg/day) treatment group (G5) exhibited statistically
significant decreases in weight gain from week 2 through to week 7
as compared to the HFD group. The Yerba Mate treatment group (G3)
did not exhibit any statistically significant decreases in weight
gain during the study as compared to the HFD group.
[0382] Table 94 shows that the individual extracts as well as the
combination animals appear to induce more of a reduced food intake
at the beginning of treatment.
TABLE-US-00096 TABLE 94 Effect of various Individual or Combined
Extracts on Feed Intake Change (g/day) in DIO Mice Days Group 4 7
11 14 21 25 28 32 ND Mean 3.81 3.34 3.58 3.55 2.88 2.54 3.58 3.27
SD 0.062 0.020 0.171 0.424 0.742 1.336 0.034 0.342 p value 0.0145
0.0820 0.0157 0.1165 0.9279 0.9527 0.0086 0.1326 HFD Mean 2.71 2.93
2.57 2.73 2.82 2.60 2.92 2.65 SD 0.179 0.177 0.061 0.085 0.165
0.068 0.080 0.090 ORI Mean 2.23 2.56 3.01 3.18 3.27 3.27 3.44 3.27
SD 0.035 0.009 0.245 0.226 0.365 0.189 0.229 0.024 p value 0.0649
0.0987 0.1312 0.1205 0.2570 0.0418 0.0930 0.0111 G1 Mean 1.35 2.06
2.48 2.91 2.68 2.48 2.15 3.23 SD 0.139 0.054 0.231 0.022 0.594
0.349 0.052 0.614 p value 0.0137 0.0220 0.6476 0.1019 0.7735 0.6762
0.0976 0.3187 G2 Mean 2.33 2.69 2.50 2.44 2.68 2.90 2.65 2.67 SD
0.127 0.379 0.309 0.245 0.514 0.099 0.031 0.002 p value 0.1362
0.4975 0.7879 0.2547 0.7489 0.0697 0.0486 0.8840 G3 Mean 2.32 2.73
2.66 2.42 2.26 2.42 2.37 2.47 SD 0.351 0.252 0.276 0.082 0.002
0.118 0.971 0.299 p value 0.2983 0.4618 0.6914 0.0639 0.1311 0.2054
0.5107 0.4838 G4 Mean 1.78 2.47 2.32 2.69 3.03 3.06 3.07 2.99 SD
0.436 0.087 0.222 0.007 0.075 0.151 0.012 0.134 p value 0.1090
0.0808 0.2639 0.5327 0.2382 0.0580 0.1179 0.0975 G5 Mean 2.22 1.90
2.19 2.39 2.33 2.47 2.65 2.65 SD 0.349 0.288 0.144 0.054 0.311
0.365 0.028 0.240 p value 0.2234 0.0499 0.0758 0.0408 0.1899 0.6774
0.0472 0.9740 G6 Mean 3.22 2.07 3.12 2.59 1.61 1.51 1.85 2.95 SD
0.686 0.759 0.469 0.066 0.702 0.945 1.414 0.104 p value 0.4173
0.2583 0.2389 0.2000 0.1411 0.2463 0.3996 0.0940 Days Group 35 39
42 46 49 53 ND Mean 3.18 3.36 1.36 3.45 3.64 3.03 SD 0.663 0.115
2.964 0.151 0.382 0.141 p value 0.4491 0.0170 0.5610 0.0830 0.1256
0.3868 HFD Mean 2.70 2.49 2.81 2.89 2.92 2.80 SD 0.280 0.113 0.125
0.191 0.106 0.257 ORI Mean 3.35 3.17 3.43 3.18 3.54 3.17 SD 0.127
0.057 0.222 0.255 0.078 0.019 p value 0.0959 0.0169 0.0759 0.3243
0.0222 0.1757 G1 Mean 3.30 2.88 3.23 3.12 3.01 2.92 SD 0.563 0.659
0.431 0.589 0.185 0.227 p value 0.3099 0.5001 0.3228 0.6409 0.6021
0.6557 G2 Mean 2.98 2.32 2.85 2.36 2.93 2.85 SD 0.325 0.500 0.054
0.907 0.570 0.276 p value 0.4516 0.6851 0.7622 0.5085 0.9799 0.8771
G3 Mean 2.69 2.52 2.82 2.43 2.65 2.59 SD 0.057 0.139 0.090 0.123
0.040 0.104 p value 0.9709 0.8800 0.9459 0.1064 0.0780 0.3990 G4
Mean 3.03 2.85 2.99 3.02 3.03 2.87 SD 0.177 0.179 0.276 0.118 0.042
0.212 p value 0.2945 0.1382 0.5031 0.4844 0.3010 0.7991 G5 Mean
2.74 2.46 1.73 2.25 2.61 2.32 SD 0.245 0.151 1.445 0.031 0.337
0.104 p value 0.8976 0.8429 0.4015 0.0432 0.3326 0.1332 G6 Mean
2.62 2.65 6.70 2.81 3.00 2.64 SD 0.179 0.236 5.610 0.302 0.191
0.054 p value 0.7709 0.4780 0.5060 0.7859 0.6817 0.4876 p value:
compare to HFD by t-test
[0383] Table 95 shows that the animals in the Morus-Magnolia-Mate
combination composition 1A (treatment group G1), Magnolia treatment
group (G2), high dose Rosemary (1000 mg/Kg/day) treatment group G6,
and the positive control ORI group of animals, all exhibited
statistically significant decreases in average weight gain and FER
as compared to the HFD group. The Yerba Mate treatment group (G3)
and the low dose of Rosemary (500 mg/Kg/day) treatment group (G5)
exhibited statistically significant decreases in food intake as
compared to the HFD group. The Morus treatment group (G4) did not
exhibit any statistically significant decreases in average daily
weight gain, FER, or food intake as compared to the HFD group.
TABLE-US-00097 TABLE 95 Effect of Various Individual or Mixed
Extracts on Average Weight Gain Food Efficiency Weight Gain Food
Intake Ratio Group (g/day) (g/day) (FER) ND Mean 0.017 3.181 0.005
(Normal SD 0.011 0.906 0.003 Diet) p value 0.0002 0.0202 0.0002 HFD
Mean 0.153 2.752 0.056 (High SD 0.037 0.178 0.013 Fat Diet) ORI
Mean -0.004 3.147 -0.001 (40 mg/Kg/day) SD 0.040 0.371 0.013 p
value 0.0000 0.0000 0.0000 G1 Mean -0.035 2.700 -0.013 Composition
1A SD 0.040 0.615 0.015 (800 mg/Kg/day) p value 0.0000 0.6676
0.0000 G2 Mean 0.067 2.653 0.025 Magnolia SD 0.077 0.363 0.029 (100
mg/Kg/day) p value 0.0329 0.2023 0.0422 G3 Mean 0.126 2.525 0.050
Mate SD 0.043 0.279 0.017 (500 mg/Kg/day) p value 0.2647 0.0007
0.5321 G4 Mean 0.140 2.800 0.050 Morus SD 0.039 0.392 0.014 (200
mg/Kg/day) p value 0.5628 0.5614 0.4879 G5 Mean 0.102 2.350 0.043
Rosemary SD 0.044 0.428 0.019 (500 mg/Kg/day) p value 0.0519 0.0001
0.2149 G6 Mean 0.011 2.810 0.004 Rosemary SD 0.079 1.686 0.028
(1000 mg/Kg/day) p value 0.0046 0.8592 0.0042 FER(Feed efficacy
ratio) = Body weight gain(g/day)/Food intake(g/day) p value:
compare to HFD by t-test
[0384] Table 96 shows that absolute weights of epididymal fat pads,
retroperitoneal fat, and perirenal fat were significantly decreased
in the Morus treatment group (G4) and Magnolia treatment group (G2)
as compared to the high fat diet group. Absolute weights of
perirenal fat were also significantly reduced in the
Morus-Magnolia-Mate Composition 1A treatment group (G1) and the low
dose Rosemary (500 mg/Kg/day) treatment group (G5) as compared to
the high fat diet group. Absolute liver weights of were
significantly reduced in the Morus-Magnolia-Mate Composition 1A
treatment group (G1), Magnolia treatment group (G2), and the
positive control ORI group as compared to the high fat diet
group.
TABLE-US-00098 TABLE 96 Effect of Various Individual or Combined
Extracts on Absolute Organ Weight in DIO Mice Retro- Epi- perito-
Peri- didymal neal Renal Total Group Liver Fat Fat Fat Fat* ND Mean
1.07 0.46 0.11 0.06 0.63 (Normal SD 0.062 0.126 0.042 0.017 0.181
Diet) p 0.0009 0.0001 0.0000 0.0001 0.0000 value HFD Mean 2.11 2.10
0.57 0.68 3.56 (High SD 0.368 0.400 0.090 0.141 0.454 Fat Diet) ORI
Mean 1.19 2.03 0.50 0.29 2.82 (40 SD 0.093 0.831 0.191 0.129 1.137
mg/Kg/ p 0.0013 0.4854 0.4229 0.0005 0.1700 day) value G1 Mean 1.38
2.12 0.56 0.25 2.92 Com- SD 0.026 0.411 0.147 0.063 0.603 position
p 0.0046 0.4.730 0.7973 0.0001 0.0771 1A value (800 mg/Kg/ day G2
Mean 1.36 2.77 0.69 0.43 3.88 Mag- SD 0.238 0.320 0.067 0.111 0.420
nolia p 0.0018 0.0496 0.0328 0.0057 0.2287 (100 value mg/Kg/ day)
G3 Mean 1.80 2.44 0.60 0.58 3.63 Mate SD 0.487 0.302 0.071 0.132
0.233 (500 p 0.2194 0.5014 0.5729 0.2404 0.7381 mg/Kg/ value day)
G4 Mean 1.82 2.86 0.69 0.49 4.04 Morus SD 0.375 0.322 0.080 0.090
0.339 (200 p 0.2112 0.0242 0.0384 0.0201 0.0628 mg/Kg/ value day)
G5 Mean 1.81 2.80 0.64 0.50 3.94 Rose- SD 0.617 0.305 0.107 0.132
0.279 mary p 0.1293 0.0359 0.2955 0.0436 0.1127 (500 value mg/Kg/
day) G6 Mean 1.59 2.49 0.62 0.47 3.59 Rose- SD 0.409 0.744 0.137
0.317 1.120 mary p 0.0682 0.6075 0.5254 0.1826 0.9570 (1000 value
mg/Kg/ day) *Total fat is sum of the three fat pads (epididymal
retroperitoneal and perirenal fat) p value: compare to HFD by
t-test
[0385] Table 97 shows that relative organ weights show similar
results to those seen for absolute organ weights (shown in Table
96).
TABLE-US-00099 TABLE 97 Results of Relative Organ Weight Change in
DIO Mice Epididymal Retroperitoneal PeriRenal Total Group Liver Fat
Fat Fat Fat* ND Mean 0.04 0.02 0.00 0.00 0.002 (Normal SD 0.002
0.004 0.001 0.0001 0.006 Diet) p value 0.0691 0.0004 0.0000 0.0001
0.0000 HFD Mean 0.04 0.05 0.01 0.01 0.08 (High Fat SD 0.006 0.011
0.003 0.003 0.013 Diet) ORI Mean 0.03 0.05 0.01 0.01 0.07 (40
mg/Kg/ SD 0.003 0.018 0.004 0.003 0.024 day) p value 0.0007 0.7407
0.7513 0.0011 0.7628 G1 Mean 0.04 0.06 0.01 0.01 0.08 Composition
SD 0.002 0.009 0.003 0.001 0.013 1A p value 0.0449 0.2021 0.1606
0.0002 0.6631 (800 mg/Kg/ day) G2 Mean 0.03 0.06 0.02 0.01 0.09
Magnolia SD 0.004 0.006 0.002 0.002 0.007 (100 mg/Kg/ p value
0.0014 0.0108 0.0132 0.0069 0.0279 day) G3 Mean 0.04 0.05 0.01 0.01
0.08 Mate SD 0.008 0.009 0.003 0.003 0.011 (500 mg/Kg/ p value
0.2066 0.4308 0.4915 0.3310 0.5468 day) G4 Mean 0.04 0.06 0.02 0.01
0.09 Morus SD 0.006 0.009 0.002 0.001 0.010 (200 mg/Kg/ p value
0.1561 0.0470 0.0818 0.0098 0.0973 day) G5 Mean 0.04 0.06 0.01 0.01
0.09 Rosemary SD 0.009 0.011 0.004 0.002 0.013 (500 mg/Kg/ p value
0.3783 0.0422 0.2069 0.0364 0.0834 day) G6 Mean 0.04 0.06 0.02 0.01
0.09 Rosemary SD 0.005 0.014 0.003 0.006 0.019 (1000 mg/Kg p value
0.1763 0.1587 0.1027 0.2684 0.2608 day)
Example 64
Effect of Morus alba Ethyl Acetate Extract 18 Combined with
Magnolia Extract 29A and Yerba Mate Extract 27 on DIO Mice
[0386] Morus alba precipitate from ethanol extract 18, produced as
described in Example 18, Magnolia extract 29A, produced as
described in Example 29, and Yerba Mate extract 27 produced
according to Example 27, were combined and blended to a ratio of
2:1:10 by weight to make combination Composition 1 as described in
Example 38. Composition 1 was orally administrated twice per day to
DIO mice as described in Example 48 at two different dosages.
Treatment group G1 was administered 650 mg/kg of animal weight (100
mg/Kg Morus, 50 mg/Kg Magnolia, and 500 mg/Kg Mate) and treatment
group G2 was administered 1300 mg/kg of animal weight (200 mg/Kg
Morus, 100 mg/Kg Magnolia, and 1000 mg/Kg Mate). The study time
period was seven weeks. Table 98 shows the effects of the
Composition 1 on weight gain.
TABLE-US-00100 TABLE 98 Effect of Composition 1 (Morus, Magnolia,
and Mate) on Weight Gain in DIO Mice Weeks Group 1 2 3 4 5 6 7 ND
Mean -0.25 -0.39 -0.31 -0.60 -0.28 0.01 0.74 (Normal SD 0.698 0.670
0.418 0.543 0.448 0.702 0.768 Diet) p value 0.0585 0.0268 0.9565
0.0270 0.0063 0.0008 0.0009 HFD Mean -1.01 -1.59 -0.35 1.25 2.88
4.11 4.68 (High Fat SD 0.531 0.246 0.775 1.344 1.564 1.569 1.556
Diet) ORI Mean -5.43 -6.69 -5.27 -3.47 -2.34 -1.08 0.02 (40 mg/kg)
SD 1.106 1.822 1.249 1.010 1.450 1.721 1.806 p value 0.0000 0.0000
0.0000 0.0001 0.0005 0.0004 0.0010 G1 Mean -2.31 -3.72 -2.95 -1.44
-0.46 0.25 1.05 (650 mg/kg) SD 0.740 0.826 1.680 1.571 2.452 2.853
3.303 p value 0.0068 0.0021 0.0087 0.0143 0.0233 0.0122 0.0234 G2
Mean -1.85 -3.32 -2.63 -2.17 -0.67 1.03 1.39 (1300 mg/kg) SD 0.831
1.646 1.664 1.752 2.050 2.700 3.089 p value 0.0709 0.0314 0.0179
0.0038 0.0126 0.0345 0.0320 p value: compare to HFD by t-test
[0387] The data in Table 98 show that the animals in the
Composition 1 (Morus. Magnolia, and Mate) treatment group (G1) and
the positive control ORI group both exhibited statistically
significant decreases in body weight gain from week 1 through to
week 7 as compared to the HFD group. Treatment group (G2) exhibited
statistically significant decreases in body weight gain from week 2
through to week 7 as compared to the HFD group.
[0388] Table 99 shows the effects of the Composition 1 (Morus,
Magnolia, and Mate) on DIO mice for the following end points:
average body weight gain per day of the study, average food intake
per day of the study, and the food efficiency ratio (FER).
TABLE-US-00101 TABLE 99 Effect of Composition 1 (Morus, Magnolia,
and Mate) on DIO Mice Food Efficiency Weight Gain Food Intake Ratio
Group (g/day) (g/day) (FER) ND Mean 0.016 3.401 0.005 (Normal SD
0.016 0.556 0.005 Diet) p value 0.0009 0.0000 0.0007 HFD Mean 0.100
2.592 0.038 (High SD 0.033 0.521 0.013 Fat Diet) ORI Mean 0.000
3.195 0.000 (40 mg/kg) SD 0.038 0.752 0.012 p value 0.0010 0.0000
0.0007 G1 Mean 0.022 2.617 0.009 (650 mg/kg) SD 0.070 0.754 0.027 p
value 0.0234 0.9052 0.0225 G2 Mean 0.030 2.315 0.013 (1300 mg/kg)
SD 0.066 0.618 0.028 p value 0.0320 0.0004 0.0512 Feed Efficacy
Ratio (FER) = Body weight gain(g/day)/Food intake(g/day) p value:
compare to HFD by t-test
[0389] The data in Table 99 show that the Composition 1 (Morus,
Magnolia, and Mate) treatment group G1 showed a statistically
significant effect on lowering average weight gain per day and a
lower Food Efficiency Ratio as compared to the HFD group. Treatment
group G2 showed a statistically significant effect on lowering of
the average weight gain per day and Food Intake as compared to the
HFD group. But, the effect on Food Efficiency Ratio was not
statistically significant. The ORI positive control treatment group
showed statically lower values than the HFD group for average
weight gain per day, average Food Intake per day, and Food
Efficiency Ratio.
[0390] Table 100 shows the effects of the Composition 1 (Morus,
Magnolia, and Mate) on measurements in DIO mice of Alanine
Transaminase (ALT), Aspartate transaminase (AST), triglyceride
(TG), total cholesterol (T-chol), and LDL-cholesterol (LDL-C) as
measured in blood samples obtained at the end of the study.
TABLE-US-00102 TABLE 100 Effect of Composition 1 (Morus, Magnolia,
and Mate) on Biochemistry Parameters in DIO Mice ALT AST T-chol TG
LDL-C Group (U/L) (U/L) (mg/dL) (mg/dL) (mg/dL) ND Mean 19.51 48.56
116.78 10.00 4.00 (Normal SD 1.561 6.396 6.418 2.872 0.682 Diet) p
0.0277 0.0088 0.0016 0.0001 0.0126 value HFD Mean 51.32 87.77
181.70 33.10 8.23 (High SD 38.335 37.269 46.466 10.816 4.300 Fat
Diet) ORI Mean 21.83 65.65 137.33 27.83 4.03 (40 SD 5.707 17.307
28.261 6.555 0.301 mg/kg) p 0.0389 0.1970 0.0543 0.3020 0.0130
value G1 Mean 20.69 59.44 150.14 12.67 5.00 (650 SD 6.722 12.564
39.418 5.007 1.243 mg/kg) p 0.0334 0.0462 0.1642 0.0007 0.0473
value G2 Mean 23.61 52.74 170.00 8.43 6.13 (1300 SD 6.923 4.977
39.383 1.988 1.652 mg/kg) p 0.0500 0.0158 0.5955 0.0000 0.1848
value p value: compare to HFD by t-test
[0391] The data in Table 100 shows that the Composition 1 (Morus,
Magnolia, and Mate) treatment group G1 exhibited a statistically
significant decreases in ALT, AST, triglyceride, and
LDL-cholesterol as compared to the HFD group. Treatment group G2
showed statistically significant decreases in ALT, AST,
triglyceride as compared to the HFD group. Whereas the ORI
treatment group showed a statistically significant decreases in ALT
and LDL-cholesterol as compared to the HFD group.
[0392] Table 101 shows the effects of the Composition 1 (Morus,
Magnolia, and Mate) on measurements in DIO mice on several
histopathological measures of fatty liver and the resulting
calculated Non-Alcoholic Staetohepatitis (NASH) score of the
liver.
TABLE-US-00103 TABLE 101 Effects of Composition 1 (Morus, Magnolia,
and Mate) on Liver Pathology Pathology Indications Lobular
Hepatocellular Steatosis Inflammation ballooning NSAH Group (0-3)
(0-3) (0-2) (sum) ND Mean 0.00 1.33 0.00 1.33 (Normal SD 0.000
0.500 0.000 0.500 Diet) p value 0.0000 0.1226 0.0000 0.0000 HFD
Mean 2.20 1.70 1.50 5.40 (High SD 0.919 0.483 0.527 1.647 Fat Diet)
ORI Mean 0.67 1.33 0.67 2.67 (40 mg/kg) SD 0.516 0.516 0.516 1.366
p value 0.0023 0.1736 0.0081 0.0042 G1 Mean 1.14 1.71 0.86 3.71
(650 mg/kg) SD 0.690 0.488 0.690 1.113 p value 0.0214 0.9531 0.0454
0.0330 G2 Mean 1.57 1.43 0.86 3.86 (1300 mg/kg) SD 0.976 0.535
0.690 1.864 p value 0.1958 0.2920 0.0454 0.0916 p value: compare to
HFD by t-test
[0393] The data in Table 101 show that there is a statistically
significant difference in the amount of Steatosis, Hepatocellular
ballooning and NASH score between the ND control group, fed a
normal diet having a moderate caloric intact of fat, and the HFD
group. Treatment group G1 and the ORI treatment group both showed
statistically significant decreases in Steatosis, Hepatocellular
ballooning and NASH score as compared with the HFD group. Treatment
group G2 showed statistically significant decrease in
Hepatocellular ballooning as compared with the HFD group. These
data, demonstrate that Composition 1 (Morus, Magnolia, and Mate)
was effective in reducing the amount of liver damage present in
mice fed a high fat diet.
[0394] Overall, the data presented in this Example 48 show that the
Morus-Magnolia-Mate combination, composition was effective in
lowering body weight and the rate of body weight gain in mice fed a
high fat diet.
Example 65
Effect of Morus alba Ethyl Acetate Extract 18 Combined with
Magnolia Extract 29A and Yerba Mate Extract 27 on DIO Rats
[0395] Morus alba precipitate from ethanol extract 18, produced as
described in Example 18, Magnolia extract 29A, produced as
described in Example 29, and Yerba Mate extract 27 produced
according to Example 27, were combined and blended to a ratio of
2:1:10 by weight to make combination Composition 1 as described in
Example 38. Composition 1 was orally administrated twice per day to
DIO rats as described in Example 49 at two different dosages.
Treatment group G1 was administered at 650 mg/kg of animal weight
(100 mg/Kg Morus, 50 mg/Kg Magnolia, and 500 mg/Kg Mate) and
treatment group G2 was administered 1300 mg/kg of animal weight
(200 mg/Kg Morus, 100 mg/Kg Magnolia, and 1000 mg/Kg Mate). The
study time period was 55 days. Table 102 shows the effects of the
Morus-Magnolia-Mate Composition 1 on weight gain.
TABLE-US-00104 TABLE 102 Effect of Composition 1 (Morus, Magnolia,
and Mate) on Weight Gain in DIO Rats Days Group 3 7 10 14 17 21 24
28 31 35 ND Mean 4.83 11.86 18.42 23.43 23.61 32.45 39.57 44.19
51.02 52.69 SD 5.54 7.84 7.31 8.03 13.36 13.98 13.08 13.85 16.00
14.55 p value 0.018 0.064 0.028 0.008 0.010 0.009 0.013 0.004 0.006
0.001 HFD Mean 13.22 22.38 32.85 49.08 52.91 63.73 72.81 81.45
89.85 98.23 SD 6.81 12.32 14.21 19.87 23.30 24.20 28.42 25.48 28.13
26.72 ORI Mean 2.80 1.04 0.82 3.40 3.34 15.89 19.89 28.19 33.33
38.43 80 SD 13.85 15.80 17.11 18.18 20.72 21.08 20.72 22.69 24.86
25.56 mg/kg p value 0.085 0.010 0.001 0.000 0.001 0.001 0.001 0.001
0.001 0.000 G1 Mean 5.41 10.66 16.37 23.46 27.10 35.47 39.45 50.43
55.77 59.14 650 SD 7.91 15.65 17.89 21.15 23.91 26.95 26.12 23.36
24.86 24.97 mg/kg p value 0.053 0.119 0.062 0.026 0.046 0.045 0.028
0.024 0.023 0.009 G2 Mean 3.34 11.73 13.71 23.51 27.28 30.82 32.74
39.96 44.32 50.17 1300 SD 7.13 12.66 16.49 22.14 28.51 31.25 30.99
30.99 36.09 34.43 mg/kg p value 0.013 0.110 0.026 0.029 0.070 0.035
0.018 0.011 0.014 0.008 Days Group 37 42 45 49 52 55 ND Mean 55.89
62.78 66.06 75.05 80.44 78.60 SD 12.98 14.33 19.09 18.66 20.38
19.52 p value 0.002 0.001 0.001 0.002 0.001 0.001 HFD Mean 103.01
113.20 124.09 130.99 141.23 143.39 SD 29.41 29.26 31.14 33.04 32.99
34.29 ORI Mean 43.95 49.06 59.88 63.68 71.12 68.11 80 SD 28.85
30.54 29.53 30.43 29.27 29.33 mg/kg p value 0.001 0.001 0.001 0.001
0.001 0.000 G1 Mean 66.27 76.79 84.95 91.78 102.42 101.21 650 SD
23.92 23.19 24.56 22.05 21.64 18.93 mg/kg p value 0.016 0.016 0.015
0.016 0.016 0.011 G2 Mean 56.75 60.92 70.72 76.03 83.73 83.84 1300
SD 32.51 32.25 33.38 32.29 30.68 30.62 mg/kg p value 0.010 0.004
0.005 0.005 0.003 0.003 p value: Compared to HFD group
[0396] The data in Table 102 show that the animals in the
Composition 1 (Morus, Magnolia, and Mate) treatment group (G1)
exhibited statistically significant decreases in body weight gain
from day 14 through to day 55 of the study as compared to the HFD
group. Treatment group (G2) exhibited statistically significant
decreases in body weight gain on day 3, day 10, day 14 and day 21
through to day 55 of the study as compared to the HFD group.
Treatment group (ORI) exhibited statistically significant decreases
in body weight gain on day 7 through to day 55 of the study as
compared to the HFD group.
[0397] Table 103 shows the effect of Composition 1 (Morus.
Magnolia, and Mate) on DOI rats for the following end points:
average body weight gain per day of the study, average food intake
per day of the study, and the food efficiency ratio (FER).
TABLE-US-00105 TABLE 103 Effect of Composition 1 (Morus, Magnolia,
and Mate) on DIO Rats Food Body Efficiency Weight Gain Food Intake
Ratio Group (g/day) (g/day) (FER) ND Mean 1.40 24.26 0.06 (Normal
SD 0.35 0.72 0.01 Diet) p value 0.001 0.073 0.000 HFD Mean 2.56
21.14 0.12 (High SD 0.61 2.37 0.02 Fat Diet) ORI Mean 1.22 25.71
0.05 (80 mg/kg) SD 0.52 1.09 0.02 p value 0.000 0.023 0.000 G1 Mean
1.81 19.62 0.09 (650 mg/kg) SD 0.34 1.00 0.02 p value 0.011 0.303
0.009 G2 Mean 1.50 19.57 0.08 (1300 mg/kg) SD 0.55 1.31 0.03 p
value 0.003 0.302 0.002 Feed Efficacy Ratio (FER) = Body weight
gain(g/day)/Food intake(g/day) p value: Compared to HFD group
[0398] The data in Table 103 show that Composition 1 (Morus,
Magnolia, and Mate) treatment groups G1 and G2 both showed a
statistically significant effect on lowering average Body Weight
Gain per day and a lower Food Efficiency Ratio as compared to the
HFD rat group. The ORI positive control treatment group showed
statistically lower values than the HFD group for average Body
Weight Gain per day, average Food Intake per day and Food
Efficiency Ratio.
[0399] Table 104 shows the effects of Composition 1 (Morus,
Magnolia, and Mate) on several specific fatty tissues that are
known to increase in fat content in rats subjected to a high fat
diet.
TABLE-US-00106 TABLE 104 Effect of Composition 1 (Morus, Magnolia,
and Mate) on Fatty Tissue Weights in DIO Rats Retro- Total Group
Epididymal peritoneal Perirenal Fat ND Mean 11.14 12.41 3.70 27.25
(Normal Diet) SD 2.32 3.04 0.65 5.47 p value 0.000 0.000 0.000
0.000 HFD Mean 22.84 28.27 8.90 60.01 (High Fat Diet) SD 2.82 6.58
1.93 7.98 ORI Mean 17.87 19.21 6.11 43.19 (80 mg/kg) SD 5.44 3.00
1.69 9.56 p value 0.044 0.005 0.008 0.002 G1 Mean 18.40 21.73 6.71
46.83 (650 mg/kg) SD 3.05 4.37 1.66 7.93 p value 0.009 0.037 0.029
0.005 G2 Mean 15.01 18.44 5.15 38.60 (1300 mg/kg) SD 1.11 5.58 1.07
8.78 p value 0.000 0.006 0.001 0.000 p value: Compared to HFD
group
[0400] The data in Table 104 show that there is a statistically
significant difference between Epididymal Fat, Retroperitoneal Fat,
Perirenal Fat and Total Fat between the ND control group, fed a
normal nutritional diet having a moderate caloric intact of fat,
and the HFD group. Treatment groups ORI, G1 and G2, all showed
statistically significant decreases in all categories of fat
measurement as compared with the HFD group, showing that
Composition 1 (Morus, Magnolia, and Mate) had a statistically
significant effect on lowering the accumulation of fat deposits in
rats fed a high fat diet.
[0401] Table 105 shows the effect of Composition 1 (Morus,
Magnolia, and Mate) on DOI rats on several histopathological
measures of fatty liver and the resulting calculated Non-Alcoholic
Staetohepatitis (NASH) score of the liver.
TABLE-US-00107 TABLE 105 Effects of Composition 1 (Morus, Magnolia,
and Mate) on Liver Pathology Pathological Indications Lobular
Inflam- Hepatocellular Steatosis mation ballooning NASH Group (0-3)
(0-3) (0-2) (sum) ND Mean 0.000 1.857 0.000 1.857 (Normal Diet) SD
0.000 0.378 0.000 0.378 p value 0.000 0.180 0.000 0.000 HFD Mean
1.875 2.125 1.625 5.625 (High Fat SD 1.126 0.354 0.744 1.923 Diet)
ORI Mean 0.750 1.500 0.675 2.875 (80 mg/kg) SD 0.707 0.535 0.744
1.727 p value 0.031 0.015 0.018 0.009 G1 Mean 0.625 1.625 0.250
2.500 (650 mg/kg) SD 0.518 0.518 0.463 1.069 p value 0.013 0.041
0.001 0.001 G2 Mean 0.500 1.500 0.500 2.500 (1300 mg/kg) SD 0.535
0.535 0.535 0.926 p value 0.008 0.015 0.004 0.001 p value: Compared
to HFD group
[0402] The data in Table 105 show Composition 1 (Morus, Magnolia,
and Mate) treatment groups G1 and G2, as well as the positive
control treatment group (ORI), all showed statistically significant
decreases in Steatosis, Lobular Inflammation, Hepatocellular
ballooning and NASH score as compared with the HFD group. These
data, demonstrate the Composition 1 (Morus, Magnolia, and Mate) was
effective in reducing the amount of liver damage present in rats
fed a high fat diet.
[0403] Table 106 shows the effects of Composition 1 (Morus,
Magnolia, and Mate) on measurements in DIG mice of Alanine
Transaminase (ALT), Aspartate transaminase (AST), and triglyceride
(TG), as measured in blood samples obtained at the end of the
study.
TABLE-US-00108 TABLE 106 Effect of Composition 1 (Morus, Magnolia,
and Mate) on Biochemistry Parameters in DIO Rats ALT AST TG Group
(U/L) (U/L) (mg/dL) ND Mean 41.13 152.69 105.75 (Normal Diet) SD
9.27 27.97 64.38 p value 0.204 0.416 0.259 HFD Mean 54.75 168.00
137.13 (High Fat Diet) SD 26.46 43.12 40.50 ORI Mean 35.65 146.39
216.38 (80 mg/kg) SD 7.54 24.18 100.04 p value 0.085 0.242 0.067 G1
Mean 34.00 127.29 83.63 (650 mg/kg) SD 9.10 25.14 28.79 p value
0.067 0.041 0.010 G2 Mean 39.35 114.89 97.00 (1300 mg/kg) SD 6.19
17.66 30.45 p value 0.149 0.010 0.043 p value: compare to HFD by
t-test
[0404] The data in Table 106 shows that Composition 1 (Morus,
Magnolia, and Mate) treatment groups 01 and G2, exhibited a
statistically significant decreases in AST and total glucose as
compared to the HFD group. Whereas the ORI treatment group showed
no statistically significant decreases in any of the measured blood
components compared to the HFD group.
[0405] Overall, the data presented in this example show that
Composition 1 (Morus, Magnolia, and Mate) was effective in lowering
body weight and the rate of body weight gain in rats fed a high fat
diet, and in lowered a number of measures of the effects of a high
fat diet on rat physiology.
Example 66
Effect of Morus alba Ethyl Acetate Extract Precipitate 18 Combined
with Rosemary Extract 24 and Yerba Mate Extract 27 on DIO Mice
[0406] Morus alba precipitate from ethanol extract 18, produced as
described in Example 18, Rosemary extract 24, produced as described
in Example 24, and Yerba Mate extract 27 produced according to
Example 27, were combined and blended to a ratio of 2:5:10 by
weight to make combination Composition 3 as described in Example
39. The Morus-Rosemary-Mate combination Composition 3 was orally
administrated twice per day to DOI mice as described in Example 48.
Treatment group G1 was administered 1700 mg/kg of animal weight
(200 mg/Kg Morus, 500 mg/Kg Rosemary, and 1000 mg/Kg Mate) of the
combination composition each day, divided between the two doses.
The study time period was seven weeks. Table 107 shows the effects
of Composition 3 on body weight gain.
TABLE-US-00109 TABLE 107 Effect of Composition 3 on Weight Gain in
DIO Mice Weeks Group 1 2 3 4 5 6 7 ND Mean -0.25 -0.39 -0.31 -0.60
-0.28 0.01 0.74 SD 0.698 0.670 0.418 0.543 0.448 0.702 0.768 p
value 0.0585 0.0268 0.9565 0.0270 0.0063 0.0008 0.0009 HFD Mean
-1.01 -1.59 -0.35 1.25 2.88 4/11 4.68 SD 0.531 0.246 0.775 1.344
1.564 1.569 1.556 ORI Mean -5.43 -6.69 -5.27 -3.47 -2.34 -1.08 0.02
40 SD 1.106 1.822 1.249 1.010 1.450 1.721 1.806 mg/kg p value
0.0000 0.0000 0.0000 0.0001 0.0005 0.0004 0.0010 G1 Mean -2.34
-3.69 -3.34 -2.45 -0.72 0.17 0.32 1700 SD 0.628 0.551 0.709 1.148
1.639 1.969 1.959 mg/kg P value 0.0043 0.0007 0.0004 0.0010 0.0086
0.0048 0.0021 p value: compare to HFD by t-test
[0407] The data in Table 107 show that the animals in the
Composition 3 treatment group (G1) and the positive control
treatment group (ORI) all exhibited statistically significant
decreases in body weight gain from week 1 through to week 7 as
compared to the HFD group.
[0408] Table 108 shows the effects of Composition 3 on DIO mice for
the following end points: average body weight gain per day of the
study, average food intake per day of the study, and the food
efficiency ratio (FER) which is calculated as the average body
weight gain per day over the study period, divided by the average
food intake per day over the study period.
TABLE-US-00110 TABLE 108 Effect of Composition 3 on DIO Mice Food
Efficiency Weight Gain Food Intake Ratio Group (g/day) (g/day)
(FER) ND Mean 0.016 3.401 0.005 (Normal Diet) SD 0.016 0.556 0.005
p value 0.0009 0.0000 0.0007 HFD Mean 0.100 2.592 0.038 (High Fat
SD 0.033 0.521 0.013 Diet) ORI Mean 0.000 3.195 0.000 (40 mg/kg) SD
0.038 0.752 0.012 p value 0.0010 0.0000 0.0007 G1 Mean 0.007 2.384
0.003 (1700 mg/kg) SD 0.042 0.603 0.017 p value 0.0021 0.0051
0.0026 Feed Efficacy Ratio (FER) = Body weight gain (g/day)/Food
intake (g/day) p value: compare to HFD by t-test
[0409] The data in Table 108 show that both the Composition 3 on
treatment group G1 and ORI treatment group showed a statistically
significant effect on lowering average Weight Gain per day, Food
Intake per day, and a lower Food Efficiency Ratio as compared to
the HFD group.
[0410] Table 109 shows the effects of Composition 3 treatment on
measurements in DIO mice of Alanine Transaminase (ALT), Aspartate
transaminase (AST), and triglyceride (TG) as measure in blood
samples obtained at the end of the study.
TABLE-US-00111 TABLE 109 Effect of Composition 3 on Biochemistry
Parameters in DIO Mice ALT AST TG Group (U/L) (U/L) (mg/dL) ND Mean
19.51 48.56 10.00 (Normal Diet) SD 1.561 6.396 2.872 p value 0.0277
0.0088 0.0001 HFD Mean 51.32 87.77 33.10 (High Fat Diet) SD 38.335
37.269 10.816 ORI Mean 21.83 65.65 27.83 (40 mg/kg) SD 5.707 17.307
6.555 p value 0.0389 0.1970 0.3020 G1 Mean 20.41 56.14 6.86 (1700
mg/kg) SD 3.291 15.588 2.545 p value 0.0314 0.0323 0.0000 p value:
compare to HFD by t-test
[0411] The data in Table 109 shows that the combination composition
treatment group G1, exhibited a statistically significant decreases
in ALT. AST, and total glucose as compared to the HFD group. In
contrast, the ORI treatment group showed a statistically
significant decrease in ALT as compared to the HFD group
[0412] Table 110 shows the effects of Composition 3 treatment on
measurements in DIO mice on several histopathological measures of
fatty liver and the resulting calculated Non-Alcoholic
Staetohepatitis (NASH) score of the liver.
TABLE-US-00112 TABLE 110 Effects of Composition 3 on Liver
Pathology Pathology Indications Lobular Hepatocellular Steatosis
Inflammation ballooning NASH Group (0-3) (0-3) (0-2) (sum) ND Mean
0.00 1.33 0.00 1.33 (Normal SD 0.000 0.500 0.000 0.500 Diet) p
value 0.0000 0.1226 0.0000 0.0000 HFD Mean 2.20 1.70 1.50 5.40
(High SD 0.919 0.483 0.527 1.647 Fat Diet) ORI Mean 0.67 1.33 0.67
2.67 (40 SD 0.516 0.516 0.516 1.366 mg/kg) p value 0.0023 0.1736
0.0081 0.0042 G1 Mean 0.57 1.43 0.57 2.57 (1700 SD 0.535 0.535
0.535 1.512 mg/kg) p value 0.0008 0.2920 0.0029 0.0026 p value:
compare to HFD by t-test
[0413] The data in Table 110 show that there is a statistically
significant difference in the weight of Steatosis, Hepatocellular
ballooning and NASH score between the ND control group, fed a
normal diet having a moderate caloric intact of fat, and the HFD
group. Treatment group G1 and the ORI treatment group both showed
statistically significant decreases in Steatosis, Hepatocellular
ballooning and NASH score as compared with the HFD group. These
data, demonstrate that Composition 3 was effective in reducing the
amount of liver damage present in mice fed a high fat diet.
[0414] Overall, the data presented in this example show that the
Morus-Rosemary-Mate combination Composition 3 was effective in
lowering body weight and the rate of body weight gain, and in
lowered a number of measures of the effects of a high fat diet on
mice physiology.
Example 67
Effect of Morus alba Ethyl Acetate Extract 18 Combined with
Rosemary Extract 24 and Yerba Mate Extract 27 on DIO Rats
[0415] Morus alba precipitate from ethanol extract 18, produced as
described in Example 18, Rosemary extract 24, produced as described
in Example 24, and Yerba Mate extract 27 produced according to
Example 27, were combined and blended to a ratio of 2:5:10 by
weight to make combination Composition 3, as further described in
Example 39. Composition 3 was orally administrated twice per day to
DIO mice as described in Example 40. Treatment group G1 was
administered at 850 mg/kg of animal weight (100 mg/Kg Morus, 250
mg/Kg Rosemary, and 500 mg/Kg Mate) of the combination composition
each day, divided between the two doses. Treatment group G2 was
administered 1700 mg/kg of animal weight (200 mg/Kg Morus, 500
m&/Kg Rosemary, and 1000 mg/Kg Mate) of Composition 3 each day,
divided between the two doses. The study time period was 55 days.
Table 111 shows the effects of Composition 3 on weight gain in DIO
rats.
TABLE-US-00113 TABLE 111 Effect of Composition 3 on Weight Gain in
DIO Rats Days Group 3 7 10 14 17 21 24 28 31 35 ND Mean 4.83 11.86
18.42 23.43 23.61 32.45 39.57 44.19 51.02 52.69 (Normal SD 5.54
7.84 7.31 8.03 13.36 13.98 13.08 13.85 16.00 14.55 Diet) p value
0.018 0.064 0.028 0.008 0.010 0.009 0.013 0.004 0.006 0.001 HFD
Mean 13.22 22.38 32.85 49.08 52.91 63.73 72.81 81.45 89.85 98.23
Diet) SD 6.81 12.32 14.21 19.87 23.30 24.20 28.42 25.48 28.13 26.72
ORI Mean 2.80 1.04 0.82 3.40 3.34 15.89 19.89 28.19 33.33 38.43 80
SD 13.85 15.80 17.11 18.18 20.72 21.08 20.72 22.69 24.86 25.56
mg/kg p value 0.085 0.010 0.001 0 0.001 0.001 0.001 0.001 0.001 0
G1 Mean 2.43 0.96 -2.63 9.60 9.73 19.30 24.89 34.45 38.94 49.14 850
SD 10.48 14.63 17.94 22.04 24.09 20.97 23.58 24.14 27.15 29.57
mg/kg p value 0.031 0.007 0.001 0.002 0.003 0.002 0.003 0.002 0.002
0.004 G2 Mean 1.15 10.32 9.91 18.64 19.17 23.79 26.36 33.51 36.98
40.18 1700 SD 4.15 8.83 11.76 15.28 17.88 17.51 19.39 19.17 16.28
18.19 mg/kg p value 0.001 0.043 0.004 0.004 0.006 0.002 0.002 0.001
0.001 0 Days Groups 37 42 45 49 52 55 ND Mean 55.89 62.78 66.06
75.05 80.44 78.60 (Normal SD 12.98 14.33 19.09 18.66 20.38 19.52
Diet) p value 0.002 0.001 0.001 0.002 0.001 0.001 HFD Mean 103.01
113.20 124.09 130.99 141.23 143.39 Diet) SD 29.41 29.26 31.14 33.04
32.99 34.29 ORI Mean 43.95 49.06 59.88 63.68 71.12 68.11 80 SD
28.85 30.54 29.53 30.43 29.27 29.33 mg/kg p value 0.001 0.001 0.001
0.001 0.001 0 G1 Mean 59.49 66.72 72.17 77.28 89.07 90.26 850 SD
29.43 30.02 32.84 31.98 32.63 31.29 mg/kg p value 0.010 0.007 0.006
0.005 0.007 0.006 G2 Mean 48.93 56.27 60.77 67.82 77.31 76.09 1700
SD 20.30 23.41 23.04 23.37 22.97 18.93 mg/kg p value 0.001 0.001 0
0.001 0.001 0.001 p value: Compared to HFD group
[0416] The data in Table 111 show that the animals in Composition 3
treatment groups (G1) and (G2) both exhibited, statistically
significant decreases in body weight gain from day 3 through to day
55 of the study as compared to the HFD group. The control treatment
group (ORI) exhibited statistically significant decreases in body
weight gain on day 7 through to day 55 of the study as compared to
the HFD group.
[0417] Table 112 shows the effects of Composition 3 on DOI rats for
the following end points: average body weight gain per day of the
study, average food intake per day of the study, and the food
efficiency ratio (FER) which is calculated as the average body
weight gain per day over the study period, divided by the average
food intake per day over the study period.
TABLE-US-00114 TABLE 112 Effect of Composition 3 on DIO Rats Body
Weight Food Efficiency Gain Food Intake Ratio Group (g/day) (g/day)
(FER) ND Mean 1.40 24.26 0.06 (Normal Diet) SD 0.35 0.72 0.01 p
value 0.001 0.073 0.000 HFD Mean 2.56 21.14 0.12 (High Fat Diet) SD
0.61 2.37 0.02 ORI Mean 1.22 25.71 0.05 (80 mg/kg) SD 0.52 1.09
0.02 p value 0.000 0.023 0.000 G1 Mean 1.61 19.65 0.08 (850 mg/kg)
SD 0.56 1.34 0.03 p value 0.006 0.327 0.006 G2 Mean 1.36 18.74 0.07
(1700 mg/kg) SD 0.34 0.62 0.02 p value 0.001 0.134 0.000 p value:
Compared to HFD group
[0418] The data in Table 12 show that Composition 3 treatment
groups G1 and G2 both showed a statistically significant effect on
lowering average weight gain per day and a lower Food Efficiency
Ratio as compared to the HFD rat group. The ORI positive control
treatment group showed statistically lower values than the HFD
group for average weight gain per day, average Food Intake per day
and Food Efficiency Ratio.
[0419] Table 113 shows the effects of the Morus-Rosemary-Mate
combination composition (Lot# IRM-1101) on several specific fatty
tissues that are known to increase in fat content in rats subjected
to a high fat diet.
TABLE-US-00115 TABLE 113 Effects of the Morus-Rosemary-Mate
combination composition (Lot# IRM-1101) on fatty tissue weights in
rats fed high fat diet Total Group Epididymal Retroperitoneal
Perirenal Fat* ND Mean 11.14 12.41 3.70 27.25 (Normal SD 2.32 3.04
0.65 5.47 Diet) p value 0.000 0.000 0.000 0.000 HFD Mean 22.84
28.27 8.90 60.01 (High Fat SD 2.82 6.58 1.93 7.98 Diet) ORI Mean
17.87 19.21 6.11 43.19 (80 SD 5.44 3.00 1.69 9.56 mg/kg) p value
0.044 0.005 0.008 0.002 G1 Mean 17.31 19.15 6.14 42.60 (850 SD 2.82
5.11 1.22 5.93 mg/kg) p value 0.002 0.008 0.005 0.000 G2 Mean 14.57
17.20 5.59 37.36 (1700 SD 3.67 4.11 1.60 9.03 mg/kg) p value 0.000
0.002 0.002 0.000 *Total fat is sum of the three fat deposits
(epididymal, retroperitoneal and perirenal fat) p value: compare to
HFD by t-test
[0420] The data in Table 113 show that there is a statistically
significant difference in the weight of Epididymal Fat,
Retroperitoneal Fat, Perirenal Fat and Total Fat between the ND
control group, fed a normal diet having a moderate caloric intact
of fat, and the HFD group. In addition, Composition 3 treatment
groups G1 and G2, as well as the positive control treatment group
(ORI), all showed statistically significant decreases in Epididymal
Fat, Retroperitoneal Fat, Perirenal Fat and Total Fat as compared
with the HFD group. These data, demonstrate Composition 3 was
effective in reducing the amount of fat present in DIO rats.
[0421] Table 114 shows the effects of Composition 3 on DIO rats on
several histopathological measures of fatty liver and the resulting
calculated Non-Alcoholic Staetohepatitis (NASH) score of the
liver.
TABLE-US-00116 TABLE 114 Effects of Composition 3 on Liver
Pathology in DIO Rats Pathology Indications Lobular Hepatocellular
Steatosis Inflammation ballooning NASH Group (0-3) (0-3) (0-2)
(sum) ND Mean 0.000 1.857 0.000 1.857 (Normal SD 0.000 0.378 0.000
0.378 Diet) p value 0.000 0.180 0.000 0.000 HFD Mean 1.875 2.125
1.625 5.625 (High SD 1.126 0.354 0.744 1.923 Fat Diet) ORI Mean
0.750 1.500 0.625 2.875 (80 SD 0.707 0.535 0.744 1.727 mg/kg) p
value 0.031 0.015 0.018 0.009 G1 Mean 1.500 1.625 1.125 4.250 (850
SD 0.535 0.518 0.641 1.389 mg/kg) p value 0.409 0.041 0.172 0.123
G2 Mean 0.875 1.625 0.500 3.000 (1700 SD 0.354 0.518 0.535 0.756
mg/kg) p value 0.031 0.041 0.004 0.003 p value: Compared to HFD
group
[0422] The data in Table 114 show that there is a statistically
significant difference in the amount of Steatosis, Hepatocellular
ballooning and NASH score between the ND control group, fed a
normal diet having a moderate caloric intact of fat, and the HFD
group. Composition 3 treatment group G1 showed a statistically
significant decrease in Lobular Inflammation as compared with the
HFD group. Composition 3 treatment group G2 and the ORI positive
control treatment group both showed statistically significant
decreases in Steatosis, Hepatocellular ballooning and NASH score as
compared with the HFD group. These data, demonstrate Composition 3
was effective in reducing the amount of liver damage present in
rats fed a high fat diet.
[0423] Table 115 shows the effects of Composition 3 treatment on
measurements in DIO rats of Alanine Transaminase (ALT). Aspartate
transaminase (AST), Alkaline Phospatase (ALP), total cholesterol
(T-chol), triglyceride (T-Glu), and LDL-cholesterol (LDL-C) as
measured in blood samples obtained at the end of the study.
TABLE-US-00117 TABLE 115 Effect of Composition 3 on Biochemistry
Parameters in DIO Rats ALT AST ALP T-chol TG LDL-C Group (U/L)
(U/L) (U/L) (mg/dL) (mg/dL) (mg/dL) ND Mean 41.13 152.69 361.23
100.00 105.25 10.05 (Nor- SD 9.27 27.97 89.16 12.34 64.38 2.30 mal
p 0.204 0.416 0.821 0.006 0.259 0.249 Diet) value HFD Mean 54.75
168.00 348.58 124.63 137.13 11.15 (High SD 26.46 43.12 126.40 17.44
40.50 1.10 Fat Diet) ORI Mean 35.65 146.39 480.16 110.13 216.38
10.63 (80 SD 7.54 24.18 288.95 21.16 100.04 1.36 mg/kg) p 0.085
0.242 0.266 0.158 0.06 0.411 value G1 Mean 34.36 106.55 218.34
83.00 77.25 6.46 (850 SD 16.47 38.38 48.70 28.42 69.24 2.49 mg/kg)
p 0.090 0.009 0.024 0.004 0.058 0.001 value G2 Mean 30.35 78.53
197.31 71.88 48.75 5.80 (1700 SD 5.36 21.66 77.04 16.16 15.29 1.39
mg/kg) p 0.035 0.000 0.014 0.000 0.000 0.000 value p value: compare
to HFD by t-test
[0424] The data in Table 115 shows that the combination composition
treatment group G1, exhibited a statistically significant decreases
in AST, ALP, total cholesterol, and LDL-cholesterol as compared to
the HFD group. Treatment group G2 showed statistically significant
decreases in AST, ALT, AST, ASP, total-cholesterol, triglyceride,
and LDL-cholesterol as compared to the HFD group. Whereas the ORI
treatment group did not show any statistically significant
decreases in any of the measured blood chemistry components as
compared to the HFD group.
[0425] Overall, the data presented in this example show that the
Morus-Rosemary-Mate combination. Composition 3 was effective in
lowering body weight and the rate of body weight gain, and in
lowered a number of different measures of the effects of a high fat
diet on rat physiology.
Example 68
Efficacy Study of Mutamba Ethanol Extract 35 Combined with Magnolia
Extract 29 and Yerba Mate Extract 26 in DIO Mice
[0426] Mutamba ethanol extract 35 produced according to Example 35
and Magnolia extract 29 produced according to Example 29 and Yerba
Mate extract 26 produced according to Example 26 was combined by
blending three components in a ratio of 5:1:5 to create Composition
10. The combined three ingredient composition was orally
administrated to DIO mice as described in the example 48 at a
dosage of 1,100 mg/kg (G1) twice a day.
[0427] The total body weight of mice was significantly decreased
after the third week in the group treated with
Mutamba:Magnolia:Yerba Mate Composition 10 (treatment group G1)
(Table 116).
TABLE-US-00118 TABLE 116 Effect of Mutamba:Magnolia:Yerba Mate
Composition 10 on Total Weight in DIO Mice Weeks Group 0 1 2 3 4 5
6 7 8 ND Mean 29.24 29.11 29.03 29.47 29.36 29.51 29.75 29.76 30.14
SD 1.020 0.967 1.201 1.166 1.428 1.309 1.509 1.270 1.321 p value
0.0001 0.0001 0.0001 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 HFD
Mean 41.31 41.73 1100.00 43.64 44.94 46.25 47.78 48.43 49.29 SD
2.932 2.771 2.856 2.884 2.870 3.172 3.247 3.224 2.783 ORI Mean
40.22 37.98 34.08 35.32 36.38 37.65 38.75 39.96 39.99 SD 2.816
2.547 1.277 1.186 1.867 2.284 2.931 3.235 3.633 p value 0.9795
0.5280 0.0351 0.0001 0.0001 0.0001 0.0003 0.0005 0.0011 G1 Mean
40.33 39.53 38.38 38.24 38.66 39.30 40.04 40.86 42.05 SD 2.619
2.629 2.333 3.240 3.631 3.511 3.866 3.839 4.445 p value 0.9605
0.5544 0.1886 0.0324 0.0122 0.0077 0.0049 0.0038 0.0041 P value:
compare to HFD by t-test
[0428] The weight gain in the Mutamba:Magnolia:Yerba Mate
Composition 10 treatment group (G1) was significantly decreased
after the first week of treatment (Table 117).
TABLE-US-00119 TABLE 117 Effect of Mutamba:Magnolia:Yerba Mate
Composition 10 on Weight Gain in DIO Mice Weeks Group 1 2 3 4 5 6 7
8 ND Mean -0.13 -0.22 0.22 0.11 0.27 0.50 0.52 0.89 SD 0.367 0.377
0.238 0.463 0.390 0.635 0.447 0.562 p value 0.0057 0.0011 0.0080
0.0010 0.0005 0.0004 0.0002 0.0002 HFD Mean 0.42 0.81 2.34 3.64
4.94 6.47 7.13 7.98 SD 0.2382 0.63623 1.229 1.3426 1.5261 1.8734
1.8149 1.9044 ORI Mean -2.24 -6.14 -4.91 -3.84 -2.57 -1.47 -0.26
-0.23 SD 1.123 2.286 1.286 1.228 1.355 1.507 1.728 2.055 p value
0.0018 0.0005 0.0000 0.0000 0.0000 0.0000 0.0001 0.0000 G1 Mean
-0.80 -1.94 -2.09 -1.67 -1.02 -0.29 0.53 1.73 SD 0.359 0.861 1.102
1.507 1.420 1.733 1.700 2.169 p value 0.0000 0.0001 0.0001 0.0001
0.0000 0.0001 0.0001 0.0003 p value: compare to HFD by t-test
[0429] Food efficiency ratio (FER) was significantly lowered in the
treatment group (G1) as compared to the high fat diet group (Table
118).
TABLE-US-00120 TABLE 118 Effect of Mutamba:Magnolia:Yerba Mate
Composition 10 on DIO Mice Body weight FER gain Food intake (Food
efficiency Group (g/day) (g/day) ratio) ND Mean 0.017 3.181 0.005
SD 0.011 0.906 0.003 p value 0.0002 0.0202 0.0002 HFD Mean 0.153
2.752 0.056 SD 0.037 0.178 0.013 ORI Mean -0.004 3.147 -0.001 SD
0.040 0.371 0.013 p value 0.0000 0.0000 0.0000 G1 Mean 0.033 2.397
0.014 SD 0.042 0.383 0.017 p value 0.0003 0.0001 0.0009 FER (Feed
efficacy ratio) = Body weight gain (g/day)/Food intake (g/day) p
value: compare to HFD by t-test
[0430] Plasma glucose and TG was significantly decreased in the
Mutamba:Magnolia:Yerba Mate Composition 10 treatment group (G1) as
compared to the high fat diet group (Table 119).
TABLE-US-00121 TABLE 119 Effect of Mutamba:Magnolia:Yerba Mate
Composition 10 on Biochemistry Parameters in DIO Mice ALT AST
T-chol TG TP LDL-C Group (U/L) (U/L) (mg/dL) (mg/dL) (g/dL) (mg/dL)
ND Mean 16.84 39.90 107.40 23.40 5.22 3.72 SD 0.899 0.781 6.107
3.715 0.164 0.683 p value 0.0718 0.3426 0.0100 0.0504 0.0924 0.0539
HFD Mean 88.83 72.99 229.75 38.50 5.65 10.13 SD 52.704 58.845
42.883 9.983 0.465 4.200 Mean 23.03 51.40 155.83 88.33 5.08 4.28
ORI SD 6.720 8.052 23.558 32.222 0.172 0.770 p value 0.0872 0.5174
0.0074 0.0185 0.0240 0.0673 Mean 26.82 57.90 198.40 20.20 4.98 8.74
G1 SD 5.287 12.930 15.307 9.680 0.130 1.016 p value 0.0995 0.5890
0.1677 0.0273 0.0596 0.5615
[0431] Absolute weight of liver and perirenal fat pads were
significantly decreased in the treatment group (G1) as compared
with the high fat diet group (Table 120).
TABLE-US-00122 TABLE 120 Effect of Mutamba:Magnolia:Yerba Mate
Composition 10 on Absolute Organ Weights in DIO Mice Epididymal
Retroperitoneal Group Liver Fat Fat PeriRenal Fat Total Fat* ND
Mean 1.07 0.46 0.11 0.06 0.63 SD 0.062 0.126 0.042 0.017 0.181 p
value 0.0000 0.0009 0.0001 0.0000 0.0001 HFD Mean 2.11 2.30 0.57
0.68 3.56 SD 0.368 0.400 0.090 0.141 0.454 ORI Mean 1.19 2.03 0.50
0.29 2.82 SD 0.093 0.831 0.191 0.129 1.137 p value 0.0005 0.0013
0.4854 0.4229 0.0005 G1 Mean 1.48 2.92 0.68 0.40 4.00 SD 0.306
0.218 0.037 0.107 0.289 p value 0.0036 0.4972 0.1737 0.004 0.9531
*Total fat is sum of the three fat pads(epididymal,
retroperitoneal, and perirenal fat) p value: compare to HFD by
t-test
[0432] The NASH score was significantly decreased in the treatment
group (G1) as compared with the high fat diet group (Table
121).
TABLE-US-00123 TABLE 121 Effect of Mutamba:Magnolia:Yerba Mate
Composition 10 on Liver Pathology in DIO mice Indications Lobular
Steatosis Inflammation Hepatocellular NSAH Group (0-3) (0-3)
ballooning (0-2) (sum) ND Mean 0.00 1.00 0.00 1.00 SD 0.000 0.000
0.000 0.000 p value 0.0000 0.0104 -- 0.0000 HFD Mean 2.83 1.50 2.00
6.33 SD 0.408 0.548 0.000 0.816 ORI Mean 0.33 1.50 0.33 2.17 SD
0.516 0.548 0.516 1.472 p value 0.0000 1.0000 0.0000 0.0001 G1 Mean
1.67 1.50 1.17 4.33 SD 0.816 0.548 0.408 1.506 p value 0.0107
1.0000 0.0005 0.0169 p value: compare to HFD by t-test
[0433] These data show that body weight, body weight gain. FER
(food efficiency ratio), visceral fat weights and NASH score in
liver were significantly decreased by Mutamba:Magnolia:Yerba Mate
Composition 10. Also, TG was decreased by treatment with this
triple combination. Therefore, this example shows that the
combination of Mutamba:Magnolia:Yerba Mate as provided in
Composition 10 is useful as a body weight reducer, as well as a
dyslipidemia and fatty liver reducer.
Example 69
Efficacy Study of Mutamba EtOH Extract 35 in Combination with
Magnolia Extract 29 and Morus alba Ethyl Acetate Fraction 15 in DIO
Mice
[0434] Mutamba ethanol extract 35 produced according to Example 35,
Magnolia extract 29 produced according to Example 29, and Morus
alba ethyl acetate fraction 15 produced according to Example 15
were combined by blending three components in a ratio of 5:1:2 to
generate Composition 2. The combined three ingredient Composition 2
was orally administrated to DIO mice as described in the example 48
at a dosage of 800 mg/kg (G1) twice a day.
[0435] The body weight was significantly decreased in the
Mutamba:Magnolia:Morus Composition 2 treatment group (G1) after the
second week treatment (Table 122).
TABLE-US-00124 TABLE 122 Effect of Mutamba:Magnolia:Morus
Composition 2 on Total Weight in DIO Mice Weeks Group 0 1 2 3 4 5 6
7 8 ND Mean 29.24 29.11 29.03 29.47 29.36 29.51 29.75 29.76 30.14
SD 1.020 0.967 1.201 1.166 1.428 1.309 1.509 1.270 1.321 p value
0.0001 0.0001 0.0001 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 HFD
Mean 41.31 41.73 42.12 43.64 44.94 46.25 47.78 48.43 49.29 SD 2.932
2.771 2.856 2.884 2.870 3.172 3.247 3.224 2.783 ORI Mean 40.22
37.98 34.08 35.32 36.38 37.65 38.75 39.96 39.99 SD 2.816 2.547
1.277 1.186 1.867 2.284 2.931 3.235 3.633 p value 0.9795 0.5280
0.0351 0.0001 0.0001 0.0001 0.0003 0.0005 0.0011 G1 Mean 39.43
35.89 32.37 34.48 35.19 34.97 34.87 35.43 35.84 SD 1.286 1.100
2.028 1.569 2.115 2.445 2.082 2.548 3.005 p value 0.4966 0.2684
0.0043 0.0004 0.0004 0.0004 0.0003 0.0001 0.0001 p value: compare
to HFD by t-test
[0436] The body weight gain was significantly decreased in the
Mutamba:Magnolia:Morus Composition 2 treatment group G1 after the
first week of treatment as compared to the high fat diet group
(Table 123).
TABLE-US-00125 TABLE 123 Effect of Mutamba:Magnolia:Morus
Composition 2 on Weight Gain in DIO Mice Weeks Group 1 2 3 4 5 6 7
8 ND Mean -0.13 -0.22 0.22 0.11 0.27 0.50 0.52 0.89 SD 0.367 0.377
0.238 0.463 0.390 0.635 0.447 0.562 p value 0.0057 0.0011 0.0080
0.0010 0.0005 0.0004 0.0002 0.0002 HFD Mean 0.42 0.81 2.34 3.64
4.94 6.47 7.13 7.98 SD 0.238 0.636 1.229 1.343 1.526 1.873 1.815
1.904 ORI Mean -2.24 -6.14 -4.91 -3.84 -2.57 -1.47 -0.26 -0.23 SD
1.123 2.286 1.286 1.228 1.355 1.507 1.728 2.055 p value 0.0018
0.0005 0.0000 0.0000 0.0000 0.0000 0.0001 0.0000 G1 Mean -3.54
-7.06 -4.95 -4.24 -4.46 -4.56 -4.00 -3.59 SD 2.241 3.041 1.944
1.735 1.894 1.115 1.365 1.982 p value 0.0001 0.0001 0.0001 0.0001
0.0000 0.0001 0.0001 0.0003 p value: compare to HFD by t-test
[0437] Food efficiency ratio (FER) was significantly lowered in the
Mutamba:Magnolia:Morus Composition 2 treatment group (G1) as
compared to the high fat diet group (Table 124).
TABLE-US-00126 TABLE 124 Effect of Mutamba:Magnolia:Morus
Composition 2 on DIO Mice Body weight FER gain Food intake (Food
efficiency Group (g/day) (g/day) ratio) ND Mean 0.017 3.181 0.005
SD 0.011 0.906 0.003 p value 0.0002 0.0202 0.0002 HFD Mean 0.153
2.752 0.056 SD 0.037 0.178 0.013 ORI Mean -0.004 3.147 -0.001 SD
0.040 0.371 0.013 p value 0.0000 0.0000 0.0000 G1 Mean -0.069 2.391
-0.029 SD 0.038 0.795 0.016 p value 0.0003 0.0107 0.0009 FER (Feed
efficacy ratio) = Body weight gain (g/day)/Food intake (g/day) p
value: compare to HFD by t-test
[0438] ALP and total cholesterol were significantly decreased in
the Mutamba:Magnolia:Morus Composition 2 treatment group (G1) as
compared to the high fat diet group (Table 125).
TABLE-US-00127 TABLE 125 Effect of Mutamba:Magnolia:Morus
Composition 2 on Biochemical Parameters in DIO Mice ALT AST ALP
T-chol TG TP LDL-C Group (U/L) (U/L) (U/L) (mg/dL) (mg/dL) (g/dL)
(mg/dL) ND Mean 16.84 39.90 195.74 107.40 23.40 5.22 3.72 p value
0.0718 0.0464 0.1655 0.0100 0.0504 0.0924 0.0539 HFD Mean 88.83
97.80 235.03 229.75 38.50 5.65 10.13 SD 52.704 35.293 31.025 42.883
9.983 0.465 4.200 ORI Mean 23.03 51.40 177.35 155.83 88.33 5.08
4.28 SD 6.720 8.052 13.486 23.558 32.222 0.172 0.770 p value 0.0872
0.0760 0.0034 0.0074 0.0185 0.0240 0.0673 G1 Mean 20.53 50.43
174.75 172.50 20.50 4.80 5.63 SD 5.437 6.001 24.604 10.661 11.733
0.000 0.903 p value 0.0802 0.0728 0.0227 0.0411 0.0581 0.0107
0.1195
[0439] Absolute weights of liver and perirenal fat pads were
significantly decreased in the treatment group (G1) as compared to
the high fat diet group (Table 126).
TABLE-US-00128 TABLE 126 Effect of Mutamba:Magnolia:Morus
Composition 2 on Absolute Organ Weights in DIO Mice Epididymal
Retroperitoneal Group Liver fat fat PeriRenal fat Total Fat* ND
Mean 1.07 0.46 0.11 0.06 0.63 SD 0.062 0.126 0.042 0.017 0.181 p
value 0.0000 0.0009 0.0001 0.0000 0.0001 HFD Mean 2.11 2.30 0.57
0.68 3.56 SD 0.368 0.400 0.090 0.141 0.454 ORI Mean 1.19 2.03 0.50
0.29 2.82 SD 0.093 0.831 0.191 0.129 1.137 p value 0.0005 0.0013
0.4854 0.4229 0.0005 G1 Mean 1.27 1.71 0.46 0.21 2.38 SD 0.091
0.464 0.152 0.048 0.657 p value 0.0036 0.4972 0.1737 0.0004 0.9531
*Total fat is sum of the three fat pads(epididymal, retroperitoneal
and perirenal fat) p value: compare to HFD by t-test
[0440] The NASH score was significantly decreased in the
Mutamba:Magnolia:Morus Composition 2 treatment group (G1) as
compared to the high fat diet (Table 127).
TABLE-US-00129 TABLE 127 Effect of Mutamba:Magnolia:Morus
Composition 2 on Liver Pathology in DIO Mice Indications Lobular
Steatosis Inflammation Hepatocellular NASH Group (0-3) (0-3)
ballooning (0-2) (sum) ND Mean 0.00 1.00 0.00 1.00 SD 0.000 0.000
0.000 0.000 p value 0.0000 0.0104 -- 0.0000 HFD Mean 2.83 1.50 2.00
6.33 SD 0.408 0.548 0.000 0.816 ORI Mean 0.33 1.50 0.33 2.17 SD
0.516 0.548 0.516 1.472 p value 0.0000 1.0000 0.0000 0.0001 G1 Mean
0.25 1.25 0.25 1.75 SD 0.500 0.500 0.500 0.957 p value 0.0000
0.4860 0.0000 0.0000 p value: compare to HFD by t-test
[0441] Overall these results show that total body weight, weight
gain. FER (food efficiency ratio), visceral fat weights and NASH
score in liver were significantly decreased by treatment with
Mutamba:Magnolia:Morus Composition 2. In addition, ALP and total
cholesterol were decreased by treatment of this combination.
Therefore, Mutamba:Magnolia:Morus Composition 2 can be used as a
body weight reducer, dyslipidemia and fatty liver reducer.
Example 70
Efficacy Study of Mutamba EtOH Extract 35 Combined with Yerba Mate
Extract 26 and Morus alba Ethyl Acetate Fraction 15 in DIO Mice
[0442] Mutamba ethanol extract 35 produced according to Example 35
and Yerba Mate extract 26 produced according to Example 26, and
Morus alba ethyl acetate fraction 15 produced according to Example
15 were combined by blending the three components in a ratio of
5:5:2, respectively. The three ingredient Mutamba:Yerba Mate:Morus
Composition 2 was orally administrated to DIO mice as described in
Example 48 at a dosage of 1,200 mg/kg (G1) twice a day.
[0443] The body weight was significantly decreased in the
Mutamba:Yerba Mate:Morus Composition 2 treatment group (G1) after
the second week treatment of the experiment (Table 128).
TABLE-US-00130 TABLE 128 Effect of Mutamba:Yerba Mate:Morus
Composition 2 on Total Body Weight in DIO Mice Weeks Group 0 1 2 3
4 5 6 7 8 ND Mean 29.24 29.11 29.03 29.47 29.36 29.51 29.75 29.76
30.14 SD 1.020 0.967 1.201 1.166 1.428 1.309 1.509 1.270 1.321 p
value 0.0001 0.0001 0.0001 0.0000 0.000 0.000 0.000 0.000 0.000 HFD
Mean 41.31 41.73 42.12 43.64 44.94 46.25 47.78 48.43 49.29 SD 2.932
2.771 2.856 2.884 2.870 3.172 3.247 3.224 2.783 ORI Mean 40.22
37.98 34.08 35.32 36.38 37.65 38.75 39.96 39.99 SD 2.816 2.547
1.277 1.186 1.867 2.284 2.931 3.235 3.633 p value 0.980 0.528 0.035
0.0001 0.0001 0.0001 0.0003 0.0005 0.0011 G1 Mean 40.34 38.20 34.45
36.47 38.27 39.49 40.66 41.34 42.12 SD 2.315 2.636 5.926 4.533
3.764 3.702 3.471 4.117 4.080 p value 0.933 0.540 0.048 0.017 0.008
0.006 0.007 0.004 0.008 p value: compare to HFD by t-test
[0444] Weight gain was significantly decreased after the first week
of treatment in mice of the Mutamba:Yerba Mate:Morus Composition 2
treatment group (G1) (Table 129).
TABLE-US-00131 TABLE 129 Effect of Mutamba:Yerba Mate:Morus
Composition 2 on Weight Gain in DIO Mice Weeks Group 1 2 3 4 5 6 7
8 ND Mean -0.13 -0.22 0.22 0.11 0.27 0.50 0.52 0.89 SD 0.367 0.377
0.238 0.463 0.390 0.635 0.447 0.562 p value 0.0057 0.0011 0.0080
0.0010 0.0005 0.0004 0.0002 0.0002 HFD Mean 0.42 0.81 2.34 3.64
4.94 6.47 7.13 7.98 SD 0.238 0.636 1.229 1.343 1.526 1.873 1.815
1.904 ORI Mean -2.24 -6.14 -4.91 -3.84 -2.57 -1.47 -0.26 -0.23 SD
1.123 2.286 1.286 1.228 1.355 1.507 1.728 2.055 p value 0.0018
0.0005 0.0000 0.0000 0.0000 0.0000 0.0001 0.0000 G1 Mean -2.14
-5.89 -3.87 -2.07 -0.85 0.32 1.00 1.78 SD 1.727 5.455 4.013 3.209
3.197 3.079 3.717 3.966 p value 0.0380 0.0126 0.0016 0.0000 0.0000
0.0000 0.0000 0.0000 p value: compare to HFD by t-test
[0445] The food efficiency ratio (FER) was significantly lowered in
the Mutamba:Yerba Mate:Morus Composition 2 treatment group (G1) as
compared to the high fat diet group (Table 130).
TABLE-US-00132 TABLE 130 Effect of Mutamba:Yerba Mate:Morus
Composition 2 on DIO Mice Weight Gain Food Intake FER Group (g/day)
(g/day) (Food Efficiency Ratio) ND Mean 0.017 3.181 0.005 SD 0.011
0.906 0.003 p value 0.0002 0.0202 0.0002 HFD Mean 0.033 2.752 0.014
SD 0.037 0.178 0.013 ORI Mean -0.004 3.147 -0.001 SD 0.040 0.371
0.013 p value 0.0000 0.0000 0.0000 G1 Mean 0.034 2.586 0.013 SD
0.076 0.509 0.029 p value 0.0000 0.1122 0.0000 FER (Feed efficacy
ratio) = Body weight gain (g/day)/Food intake (g/day) p value:
compare to HFD by t-test
[0446] TG was significantly decreased in the Mutamba:Yerba
Mate:Morus Composition 2 treatment group (G1) as compared to the
high fat diet group (Table 131).
TABLE-US-00133 TABLE 131 Effect of Mutamba:Yerba Mate:Morus
Composition 2 on Biochemistry Parameters ALT AST T-chol TG TP LDL-C
Group (U/L) (U/L) (mg/dL) (mg/dL) (g/dL) (mg/dL) ND Mean 16.84
39.90 107.40 23.40 5.22 3.72 SD 0.899 0.781 6.107 3.715 0.164 0.683
p value 0.0718 0.0464 0.0100 0.0504 0.0924 0.0539 HFD Mean 88.83
97.80 229.75 38.50 5.65 10.13 SD 52.704 35.293 42.883 9.983 0.465
4.200 ORI Mean 23.03 51.40 155.83 88.33 5.08 4.28 SD 6.720 8.052
23.558 32.222 0.172 0.770 p value 0.0872 0.0760 0.0074 0.0185
0.0240 0.0673 G1 Mean 27.87 55.83 194.00 8.50 5.15 8.47 SD 7.631
9.902 15.887 2.429 0.105 1.731 p value 0.1029 0.0949 0.0936 0.0078
0.1192 0.4036
[0447] Absolute weights of liver and perirenal fat pads were
significantly decreased in the Mutamba:Yerba Mate:Morus Composition
2 treatment group (G1) as compared to the high fat diet group
(Table 132).
TABLE-US-00134 TABLE 132 Effect of Mutamba:Yerba Mate:Morus
Composition 2 on Absolute Organ Weights in DIO Mice Epididymal
Retroperitoneal Group Liver Fat Fat PeriRenal Fat Total Fat* ND
Mean 1.07 0.46 0.11 0.06 0.63 SD 0.062 0.126 0.042 0.017 0.181 p
value 0.0000 0.0009 0.0001 0.0000 0.0001 HD Mean 2.11 2.30 0.57
0.68 3.56 SD 0.368 0.400 0.090 0.141 0.454 ORI Mean 1.19 2.03 0.50
0.29 2.82 SD 0.093 0.831 0.191 0.129 1.137 p value 0.0005 0.0013
0.4854 0.4229 0.0005 G1 Mean 1.47 2.54 0.64 0.35 3.54 SD 0.184
0.723 0.071 0.066 0.786 p value 0.0036 0.4972 0.1737 0.0004 0.9531
*Total fat is sum of the three fat pads(epididymal, retroperitoneal
and perirenal fat) p value: compare to HFD by t-test
[0448] The treatment group (G1), NASH score was significantly
decreased, when compared with the high fat diet (Table 133).
TABLE-US-00135 TABLE 133 Effect of Mutamba:Yerba Mate:Morus
Composition 2 on Liver Pathology in DIO Mice Indications Lobular
Steatosis Inflammation Hepatocellular NSAH Group (0-3) (0-3)
ballooning (0-2) (sum) ND Mean 0.00 1.00 0.00 1.00 SD 0.000 0.000
0.000 0.000 p value 0.0000 0.0104 -- 0.0000 HFD Mean 2.83 1.50 2.00
6.33 SD 0.408 0.548 0.000 0.816 ORI Mean 0.33 1.50 0.33 2.17 SD
0.516 0.548 0.516 1.472 p value 0.0000 1.0000 0.0000 0.0001 G1 Mean
1.50 1.50 1.33 4.33 SD 0.548 0.548 0.516 1.033 p value 0.0007
1.0000 0.0101 0.0040 p value: compare to HFD by t-test
[0449] Overall the data show that total body weight, weight gain,
FER (food efficiency ratio), visceral fat weights, and NASH score
in liver were significantly decreased in mice treated with
Mutamba:Yerba Mate:Morus Composition 2. Also, TG was decreased by
treatment with this triple combination. Therefore, the present
results indicate that a Mutamba:Yerba Mate:Morus combination, such
as Composition 2, can be used as a body weight reducer, as well as
a dyslipidemia and fatty liver reducer.
Example 71
Efficacy Study of Mutamba, Morus alba and Magnolia Combination
Composition 2 in DIO Mice
[0450] The combination of Mutamba ethanol extract 36 produced
according to Example 36, Morus alba precipitate from ethanol
extract 18 produced according to the Example 18, and Magnolia
extract 29A produced according to Example 29, was made by blending
the three components in a ratio of 10:1:2, respectively. The
combined three ingredient Composition 2 was orally administrated to
DIO mice as described in the example 48 at two different dosages of
650 mg/kg (G1 treatment group) and 1300 mg/kg (G2 treatment group),
twice a day.
[0451] Low dose treatment group (G1) didn't show any change in
total body weight, but the total body weight was significantly
decreased in the high dose treatment group (G2) after the third
week of treatment (Table 134).
TABLE-US-00136 TABLE 134 Effect of Mutamba:Morus:Magnolia
Composition 2 on Total Weight in DIO Mice Weeks Group 0 1 2 3 4 5 6
7 ND Mean 27.51 27.26 27.12 27.20 26.91 27.23 27.52 28.75 SD 1.489
1.753 1.689 1.525 1.683 1.648 1.735 1.700 p value 0.0000 0.0000
0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 HFD Mean 39.23 38.22
37.64 38.89 40.48 42.11 43.34 43.91 SD 2.805 3.052 3.574 3.957
4.400 4.784 4.590 4.382 ORI Mean 38.61 33.17 31.91 33.34 35.14
36.27 37.52 38.62 SD 2.587 2.687 2.795 2.752 2.835 3.260 3.074
3.275 p value 0.6475 0.0031 0.0030 0.0060 0.0130 0.0136 0.0108
0.0166 G1 Mean 39.10 36.66 35.75 36.22 37.73 39.35 40.43 41.88 SD
2.043 1.985 2.341 2.506 3.367 3.060 2.846 3.162 p value 0.9167
0.2556 0.2424 0.1378 0.1847 0.2016 0.1591 0.3121 G2 Mean 39.68
35.98 34.70 34.14 34.61 35.40 36.45 36.66 SD 2.809 1.532 0.872
2.124 1.763 2.598 2.913 3.279 p value 0.7507 0.0954 0.0668 0.0115
0.0023 0.0043 0.0033 0.0021 p value: compare to HFD by t-test
[0452] The body weight gain of low (01) and high (G2) dose
treatment groups showed a significant decrease during the
administration periods (Table 135).
TABLE-US-00137 TABLE 135 Effect of Mumbamba:Morus:Magnolia
Composition 2 on Weight Gain in DIO Mice Weeks Group 1 2 3 4 5 6 7
ND Mean -0.25 -0.39 -0.31 -0.60 -0.28 0.01 0.74 SD 0.698 0.670
0.418 0.543 0.448 0.702 0.768 p value 0.0585 0.0268 0.9565 0.0270
0.0063 0.0008 0.0009 HFD Mean -1.01 -1.59 -0.35 1.25 2.88 4.11 4.68
SD 0.531 0.246 0.775 1.344 1.564 1.569 1.556 ORI Mean -5.43 -6.69
-5.27 -3.47 -2.34 -1.08 0.02 SD 1.106 1.822 1.249 1.010 1.450 1.721
1.806 p value 0.0000 0.0000 0.0000 0.0001 0.0005 0.0004 0.0010 G1
Mean -2.44 -3.34 -2.88 -1.37 0.26 1.33 2.78 SD 0.864 1.314 1.625
2.138 2.097 1.473 1.987 p value 0.0053 0.0178 0.0096 0.0272 0.0550
0.0256 0.1278 G2 Mean -3.70 -4.97 -5.54 -5.06 -4.28 -3.23 -3.02 SD
1.717 1.300 1.387 1.749 2.042 2.627 2.357 p value 0.0007 0.0001
0.0000 0.0000 0.0000 0.0001 0.0000 p value: compare to HFD by
t-test
[0453] FER was significantly lower only in the high dose treatment
group (G2) as compared to the HFD group (Table 136). The low dose
treatment group (G1) showed only a significant change in food
intake (g/day).
TABLE-US-00138 TABLE 136 Effect of Mutamba:Morus:Magnolia
Composition 2 on DIO Mice Weight Gain Food Intake FER Group (g/day)
(g/day) (Food efficiency ratio) ND Mean 0.016 3.401 0.005 SD 0.016
0.556 0.005 p value 0.0009 0.0000 0.0007 HFD Mean 0.100 2.592 0.038
SD 0.033 0.521 0.013 ORI Mean 0.000 3.195 0.000 SD 0.038 0.752
0.012 p value 0.0010 0.0000 0.0007 G1 Mean 0.059 2.288 0.026 SD
0.042 0.615 0.018 p value 0.1278 0.0000 0.2300 G2 Mean -0.064 2.112
-0.030 SD 0.050 0.587 0.024 p value 0.0000 0.0000 0.0000 p value:
compare to HFD by t-test
[0454] AST and TG were significantly decreased in the low dose
treatment group (G1) as compared to the high fat diet group. For
the high dose treatment group (G2), ALT, AST, glucose. TG and
LDL-cholesterol were significantly decreased as compared to the
high fat diet group (Table 137).
TABLE-US-00139 TABLE 137 Effect of Mutamba:Morus:Magnolia
Composition 2 on Biohemistry Parameters ALT AST Glu T-chol TG LDL-C
HDL-C Group (U/L) (U/L) (mg/dL) (mg/dL) (mg/dL) (mg/dL) (mg/dL) ND
Mean 19.51 48.56 196.78 116.78 10.00 4.00 62.74 SD 1.561 6.396
31.104 6.418 2.872 0.682 3.076 p value 0.0277 0.0088 0.0153 0.0016
0.0001 0.0126 0.1600 HFD Mean 51.32 87.77 234.70 181.70 33.10 8.23
68.86 SD 38.335 37.269 30.159 46.466 10.816 4.300 10.217 ORI Mean
21.83 65.65 194.00 137.33 27.83 4.03 62.42 SD 5.707 17.307 47.147
28.261 6.555 0.301 9.746 p value 0.0389 0.1970 0.0521 0.0543 0.3020
0.0130 0.3121 G1 Mean 26.01 58.71 278.00 171.43 15.43 5.94 69.36 SD
5.616 4.985 36.699 14.351 7.091 1.350 3.455 p value 0.0681 0.0365
0.0217 0.5251 0.0018 0.1427 0.6755 G2 Mean 22.94 50.77 206.71
156.86 12.71 4.87 71.83 SD 4.301 5.880 19.129 8.726 3.450 0.948
2.622 p value 0.0445 0.0120 0.0475 0.1304 0.0001 0.0376 0.2659
[0455] The visceral fat pads weights of high dose treatment group
(G2) were significantly decreased as compared to the HFD group
(Table 138).
TABLE-US-00140 TABLE 138 Effect of Mutamba:Morus:Magnolia
Composition 2 on Absolute Organ Weights in DIO Mice \ Epididymal
Retroperitoneal Group Liver Fat Fat PeriRenal Fat Total Fat* ND
Mean 0.99 0.50 0.13 0.07 0.71 SD 0.062 0.099 0.041 0.020 0.145 p
value 0.0884 0.0000 0.0000 0.0001 0.0000 HFD Mean 1.27 2.41 0.61
0.43 3.45 SD 0.455 0.381 0.140 0.160 0.387 ORI Mean 0.99 2.08 0.57
0.28 2.94 SD 0.153 0.278 0.065 0.082 0.390 p value 0.0988 0.0718
0.4852 0.0436 0.0169 G1 Mean 1.34 2.54 0.66 0.40 3.60 SD 0.212
0.315 0.063 0.080 0.402 p value 0.7295 0.4823 0.4307 0.6849 0.4660
G2 Mean 1.18 1.71 0.45 0.21 2.38 SD 0.116 0.425 0.102 0.074 0.584 p
value 0.5600 0.0030 0.0180 0.0049 0.0004 *Total fat is sum of the
three fat pads(epididymal, retroperitoneal and perirenal fat) p
value: compare to HFD by t-test
[0456] Low dose treatment group (G1), NASH score didn't show any
change but high dose treatment group (G2) showed significantly
decreased (Table 139)
TABLE-US-00141 TABLE 139 Effect of Mutamba:Morus:Magnolia
Composition 2 on Liver Pathology in DIO Mice Indications Lobular
Steatosis Inflammation Hepatocellular NSAH Group (0-3) (0-3)
ballooning (0-2) (sum) ND Mean 0.00 1.33 0.00 1.33 SD 0.000 0.500
0.000 0.500 p value 0.0000 0.1226 0.0000 0.0000 HFD Mean 2.20 1.70
1.50 5.40 SD 0.919 0.483 0.527 1.647 ORI Mean 0.67 1.33 0.67 2.67
SD 0.516 0.516 0.516 1.366 p value 0.0023 0.1736 0.0081 0.0042 G1
Mean 1.43 1.57 1.57 4.57 SD 0.535 0.535 0.535 1.397 p value 0.0656
0.6124 0.7882 0.2957 G2 Mean 0.86 1.86 1.00 3.71 SD 0.378 0.378
0.577 0.756 p value 0.0025 0.4837 0.0837 0.0240 p value: compare to
HFD by t-test
[0457] Overall, these data show that body weight, body weight gain,
FER (food efficiency ratio), visceral fat weights and NASH score in
liver were significantly decreased in mice treated with a
Mutamba:Morus alba:Magnolia extract combination, such as
Composition 2. Also, ALT, AST, glucose, TG and LDL-cholesterol were
decreased by treatment with Mutamba:Morus alba:Magnolia Composition
2. Therefore, this example indicates that a Mutamba:Morus
alba:Magnolia combination can be used as a body weight,
dyslipidemia and fatty liver reducer.
Example 72
Efficacy Study of Mutamba, Morus alba and Magnolia Combined
Composition 2 in DIO Rats
[0458] The combination of Mutamba ethanol extract 36 produced
according to Example 36, Morus alba precipitate from ethanol
extract 18 produced according to the Example 18, and Magnolia
extract 29A produced according to Example 29, was made by blending
the three components in a ratio of 10:1:2, respectively. The
combined three ingredient Composition 2 was orally administrated to
DIO rats as described in the Example 49 at two different dosages of
650 mg/kg (S3) and 1,300 mg/kg (S4), twice a day.
[0459] Both treatment groups showed significantly decreased weight
gain as compared to the high fat diet group during the treatment
period (Table 140).
TABLE-US-00142 TABLE 140 Effect of Mutamba:Morus:Magnolia
Composition 2 on Weight Gain in DIO Rats Days Group 3 7 10 14 17 21
24 28 ND Mean 4.83 11.86 18.42 23.43 23.61 32.45 39.57 44.19 SD
5.54 7.84 7.31 8.03 13.36 13.98 13.08 13.85 p value 0.018 0.064
0.028 0.008 0.010 0.009 0.013 0.004 HFD Mean 13.22 22.38 32.85
49.08 52.91 63.73 72.81 81.45 SD 6.81 12.32 14.21 19.87 23.30 24.20
28.42 25.48 ORI Mean 2.80 1.04 0.82 3.40 3.34 15.89 19.89 28.19 SD
13.85 15.80 17.11 18.18 20.72 21.08 20.72 22.69 p value 0.085 0.010
0.001 0.000 0.001 0.001 0.001 0.001 S3 Mean 3.13 8.39 7.24 15.28
18.67 28.05 33.78 46.44 SD 6.37 11.46 18.77 25.03 25.18 24.62 27.20
29.85 p value 0.008 0.034 0.009 0.010 0.014 0.011 0.014 0.025 S4
Mean 0.91 3.77 4.26 11.73 10.38 19.06 21.24 32.23 SD 6.65 19.19
21.33 23.76 25.97 28.02 29.34 29.81 p value 0.003 0.040 0.008 0.004
0.004 0.004 0.003 0.003 Days Group 31 35 37 42 45 49 52 55 ND Mean
51.02 52.69 55.89 62.78 66.06 75.05 80.44 78.60 SD 16.00 14.55
12.98 14.33 19.09 18.66 20.38 19.52 p value 0.006 0.001 0.002 0.001
0.001 0.002 0.001 0.001 HFD Mean 89.85 98.23 103.01 113.20 124.09
130.99 141.23 143.39 SD 28.13 26.72 29.41 29.26 31.14 33.04 32.99
34.29 ORI Mean 33.33 38.43 43.95 49.06 59.88 63.68 71.12 68.11 SD
24.86 25.56 28.85 30.54 29.53 30.43 29.27 29.33 p value 0.001 0.000
0.001 0.001 0.001 0.001 0.001 0.000 S3 Mean 49.33 57.29 64.63 75.22
81.57 89.39 93.54 94.40 SD 32.53 33.53 36.23 36.50 40.84 41.18
41.99 42.09 p value 0.019 0.018 0.036 0.038 0.036 0.044 0.025 0.024
S4 Mean 36.71 48.98 56.15 66.03 74.80 82.92 87.16 87.63 SD 31.67
34.96 36.72 41.21 42.08 41.84 44.58 44.29 p value 0.003 0.007 0.014
0.021 0.020 0.024 0.016 0.014 p value: Compared to HFD group
[0460] Weight gain (g/day) and Food efficiency ratio (FER) were
significantly decreased in both the low dose S3 and high dose S4
treatment groups as compared to the high fat diet group (Table
141).
TABLE-US-00143 TABLE 141 Effect of Mutamba:Morus:Magnolia
Composition 2 on DIO Rats FER Weight gain Food Intake (Food
efficiency Group (g/day) (g/day) ratio) ND Mean 1.40 24.26 0.06 SD
0.35 0.72 0.01 p value 0.001 0.073 0.000 HFD Mean 2.56 21.14 0.12
SD 0.61 2.37 0.02 ORI Mean 1.22 25.71 0.05 SD 0.52 1.09 0.02 p
value 0.000 0.023 0.000 S3 Mean 1.69 19.55 0.08 SD 0.75 1.78 0.03 p
value 0.024 0.327 0.024 S4 Mean 1.56 19.47 0.08 SD 0.79 1.96 0.04 p
value 0.014 0.320 0.017 p value: Compared to HFD group
[0461] Absolute organ weight, as well as epididymal,
retroperitoneal, perirenal and total fat pads, were significantly
decreased in rats treated with low dose Composition 2 (S3) group of
as compared to the high fat diet group. Absolute organ weight, as
well as epididymal, perirenal and total fat pads, were
significantly decreased in mice treated with high dose Composition
2 (S4) as compared to the high fat diet group (Table 142).
TABLE-US-00144 TABLE 142 Effect of Mutamba:Morus:Magnolia
Composition 2 on Absolute Organ Weights in DIO rats Epididymal
Retroperitoneal Perirenal Total Group Fat Fat Fat Fat ND Mean 11.14
12.41 3.70 27.25 SD 2.32 3.04 0.65 5.47 p value 0.000 0.000 0.000
0.000 HFD Mean 22.84 28.27 8.90 60.01 SD 2.82 6.58 1.93 7.98 ORI
Mean 17.87 19.21 6.11 43.19 SD 5.44 3.00 1.69 9.56 p value 0.044
0.005 0.008 0.002 S3 Mean 15.42 20.67 6.26 42.35 SD 3.69 6.26 1.36
9.70 p value 0.001 0.033 0.008 0.001 S4 Mean 16.79 22.64 6.75 46.18
SD 3.45 5.47 1.70 9.35 p value 0.003 0.093 0.039 0.010 p value:
Compared to HFD group
[0462] Steatosis, Lobular inflammation, Hepatocellular ballooning
and NASH scores were decreased in both the low dose S3 and high
dose S4 treatment groups as compared to the high fat diet group
(Table 143).
TABLE-US-00145 TABLE 143 Effect of Mutamba:Morus:Magnolia
Composition 2 on Liver Pathology in DIO Rats Indications Lobular
Steatosis Inflammation Hepatocellular NSAH Group (0-3) (0-3)
Ballooning (0-2) (sum) ND Mean 0.000 1.857 0.000 1.857 SD 0.000
0.378 0.000 0.378 p value #DIV/0! 0.180 #DIV/0! 0.000 HFD Mean
1.875 2.125 1.625 5.625 SD 1.126 0.354 0.744 1.923 ORI Mean 0.750
1.500 0.625 2.875 SD 0.707 0.535 0.744 1.727 p value 0.031 0.015
0.018 0.009 S3 Mean 0.750 1.125 0.500 2.375 SD 0.463 0.354 0.535
0.744 p value 0.020 0.000 0.004 0.001 S4 Mean 0.571 1.571 0.286
2.429 SD 0.787 0.535 0.488 1.272 p value 0.024 0.032 0.001 0.003 p
value: Compared to HFD group
[0463] T-Cholesterol, triglycerides (TG) and LDL-Cholesterol were
decreased in both the low dose S3 and high dose S4 treatment groups
as compared to the high fat diet group (Table 144).
TABLE-US-00146 TABLE 144 Effect of Mutamba:Morus:Magnolia
Composition 2 on Biochemistry Parameters in DIO Rats T-chol TG
LDL-C Group (mg/dL) (mg/dL) (mg/dL) ND Mean 100.00 105.25 10.05 SD
12.34 64.38 2.30 p value 0.006 0.259 0.249 HFD Mean 124.63 137.13
11.15 SD 17.44 40.50 1.10 ORI Mean 110.13 216.38 10.63 SD 21.16
100.04 1.36 p value 0.158 0.067 0.411 S3 Mean 92.38 89.00 7.68 SD
14.93 40.29 1.79 p value 0.001 0.032 0.001 S4 Mean 78.86 74.57 6.50
SD 10.99 24.24 1.87 p value 0.000 0.003 0.000 p value: Compared to
HFD group
[0464] Overall, these data show that total body weight, weight
gain, FER (food efficiency ratio), visceral fat weight, and NASH
score in liver were significantly decreased in mice treated with a
mixture of Mutamba, Morus alba and Magnolia extracts. Also,
total-cholesterol, triglyceride (TG) and LDL-cholesterol were
decreased by treatment with the triple combination of Mutamba,
Morus alba and Magnolia. Therefore, this example indicates that
combinations such as Composition 2 can be used as a body weight
reducer, as well as dyslipidemia and fatty liver reducers.
Example 73
Efficacy of Mutamba Ethanol Extract 35, Magnolia Extract 29, Yerba
Mate Extract 26 and Morus EtOAC Fraction 15 Mixed Composition 11 in
DIO Mice
[0465] Mutamba ethanol extract 35 produced according to Example 35,
Magnolia extract 29 produced according to Example 29, Yerba Mate
extract 26 produced according to example 26 and Morus alba ethyl
acetate fraction 15 produced according to example 15, were combined
by blending the four components in a ratio of 5:1:5:2,
respectively. The combined four ingredient Composition 11 was
orally administrated to DIO mice as described in the example 48 at
a total dosage of 1,300 mg/kg (01) twice a day.
[0466] Total body weight was significantly decreased in mice of
treatment group (G1), which were given Mutamba:Magnolia:Yerba
Mate:Morus Composition 11, after the second week treatment during
the experiment (Table 145).
TABLE-US-00147 TABLE 145 Effect of Quadruple Composition 11on Total
Body Weight in DIO Mice Weeks Group 0 1 2 3 4 5 6 7 8 ND Mean 29.24
29.11 29.03 29.47 29.36 29.51 29.75 29.76 30.14 SD 1.020 0.967
1.201 1.166 1.428 1.309 1.509 1.270 1.321 p value 0.0001 0.0001
0.0001 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 HFD Mean 41.31
41.73 42.12 43.64 44.94 46.25 47.78 48.43 49.29 SD 2.932 2.771
2.856 2.884 2.870 3.172 3.247 3.224 2.783 ORI Mean 40.22 37.98
34.08 35.32 36.38 37.65 38.75 39.96 39.99 SD 2.816 2.547 1.277
1.186 1.867 2.284 2.931 3.235 3.633 p value 0.9795 0.5280 0.0351
0.0001 0.0001 0.0001 0.0003 0.0005 0.0011 G1 Mean 40.05 37.69 35.67
36.88 37.21 37.56 38.03 38.22 39.32 SD 2.838 2.191 2.473 2.830
2.280 2.356 2.496 3.072 3.470 p value 0.7625 0.4910 0.0270 0.0033
0.0036 0.0009 0.0007 0.0004 0.0005 p value: compare to HFD by
t-test
[0467] Weight gain was significantly decreased in the
Mutamba:Magnolia:Yerba Mate:Morus Composition 11 treatment group
(G1) after the first week treatment during the experiment (Table
146).
TABLE-US-00148 TABLE 146 Effect of Quadruple Composition 11 on
Weight Gain in DIO Mice Weeks Group 1 2 3 4 5 6 7 8 ND Mean -0.13
-0.22 0.22 0.11 0.27 0.50 0.52 0.89 SD 0.367 0.377 0.238 0.463
0.390 0.635 0.447 0.562 p value 0.0057 0.0011 0.0080 0.0010 0.0005
0.0004 0.0002 0.0002 HFD Mean 0.42 0.81 0.34 3.64 4.94 6.47 7.13
7.98 SD 0.238 0.636 1.229 1.343 1.526 1.873 1.815 1.904 ORI Mean
-2.24 -6.14 -4.91 -3.84 -2.57 -1.47 -0.26 -0.23 SD 1.123 2.286
1.286 1.228 1.355 1.507 1.728 2.055 p value 0.0018 0.0005 0.0000
0.0000 0.0000 0.0000 0.0001 0.0000 G1 Mean -2.36 -4.38 -3.17 -2.84
-2.49 -2.02 -1.83 -0.73 SD 2.030 2.834 2.907 2.555 1.915 1.985
2.015 1.864 p value 0.0370 0.0136 0.0022 0.0004 0.0001 0.0000
0.0000 0.0000 p value: compare to HFD by t-test
[0468] Food efficiency ratio (FER) was significantly lowered in the
treatment group (G1) as compared to the high fat diet group (Table
147).
TABLE-US-00149 TABLE 147 Effect of Mutamba:Magnolia:Yerba
Mate:Morus Composition on DIO Mice Body weight FER gain Food intake
(Food efficiency Group (g/day) (g/day) ratio) ND Mean 0.017 3.181
0.005 SD 0.11 0.906 0.003 p value 0.0002 0.0202 0.0002 HFD Mean
0.153 2.752 0.056 SD 0.037 0.178 0.013 ORI Mean -0.004 3.147 -0.001
SD 0.040 0.371 0.013 p value 0.0000 0.0000 0.0000 GI Mean -0.014
2.470 -0.006 SD 0.036 0.609 0.015 p value 0.0000 0.0253 0.0000
FER(Feed efficacy ratio) = Body weight gain(g/day)/Food
intake(g/day) p value: compared to HFD by t-test
[0469] Plasma glucose, total cholesterol and TG were significantly
decreased in treatment group G1 as compared to the high fat diet
group (Table 148).
TABLE-US-00150 TABLE 148 Effect of Mutamba:Magnolia:Yerba
Mate:Morus Composition 11 on Biochemistry Parameters in DIO Mice
ALT AST ALP Glu T-chol TG TP LDL-C HDL-C Group (U/L) (U/L) (U/L)
(mg/dL) (mg/mL) (mg/mL) (g/dL) (mg/dL) (mg/dL) ND Mean 16.84 39.90
195.74 157.60 107.40 23.40 5.22 3.72 61.66 SD 0.899 0.781 42.213
25.265 6.107 3.715 0.164 0.683 2.970 p value 0.0718 0.0464 0.1655
0.0001 0.0100 0.0504 0.0924 0.0539 0.0037 HFD Mean 88.83 97.80
235.03 347.00 229.75 38.50 5.65 10.13 75.05 SD 52.704 35.293 31.025
42.237 42.883 9.983 0.465 4.200 6.251 ORI Mean 23.03 51.40 177.35
269.17 155.83 88.33 5.08 4.28 75.57 SD 6.720 8.052 13.486 46.232
23.558 32.222 0.172 0.770 5.837 p value 0.0872 0.0760 0.0034 0.0274
0.0274 0.0185 0.0240 0.0673 0.8971 GI Mean 19.00 52.28 207.40
278.60 173.40 10.00 5.10 6.14 70.80 SD 6.552 9.987 24.104 32.238
27.355 3.674 0.200 1.383 10.836 p value 0.0761 0.0781 0.1750 0.0278
0.0468 0.0006 0.0468 0.0832 0.5113 p value: compare to HFD by
t-test
[0470] Absolute weights of liver and total fat pads were
significantly decreased in the Mutamba:Magnolia:Yerba Mate:Morus
Composition 11 treatment group G1 as compared to the high fat diet
group (Table 149).
TABLE-US-00151 TABLE 149 Effect of Mutamba:Magnolia:Yerba
Mate:Morus Composition 11 on Absolute Organ Weights in DIO Mice
Epididymal Retroperitoneal PeriRenal Total Group Liver Fat Fat Fat
Fat* ND Mean 1.07 0.46 0.11 0.06 0.63 SD 0.062 0.126 0.042 0.017
0.181 p value 0.0000 0.0009 0.0001 0.0000 0.0001 HFD Mean 2.11 2.30
0.57 0.68 3.56 SD 0.368 0.400 0.090 0.141 0.454 ORI Mean 1.19 2.03
0.50 0.29 2.82 SD 0.093 0.831 0.191 0.129 1.137 p value 0.0005
0.0013 0.4854 0.4229 0.0005 G1 Mean 1.38 2.12 0.56 0.25 2.92 SD
0.026 0.411 0.147 0.063 0.603 p value 0.0000 0.0046 0.4730 0.7973
0.0001 *Total fat is sum of the three fat pads(epididymal,
retroperitoneal and perirenal fat) p value: compare to HFD by
t-test
[0471] The NASH score was significantly decreased in the
Mutamba:Magnolia:Yerba Mate:Morus Composition 11 treatment group
(G1) as compared to the high fat diet mice (Table 150).
TABLE-US-00152 TABLE 150 Effect of Mutamba:Magnolia:Yerba
Mate:Morus Composition 11 on Liver Pathology in DIO Mice
Indications Lobular Steatosis Inflammation Hepatocellular NSAH
Group (0-3) (0-3) ballooning (0-2) (sum) ND Mean 0.00 1.00 0.00
1.00 SD 0.000 0.000 0.000 0.000 p value 0.0000 0.0104 -- 0.0000 HFD
Mean 2.83 1.50 2.00 6.33 SD 0.408 0.548 0.000 0.816 ORI Mean 0.33
1.50 0.33 2.17 SD 0.516 0.548 0.516 1.472 p value 0.0000 1.0000
0.0000 0.0001 G1 Mean 1.00 1.40 0.80 3.20 SD 1.225 0.548 0.837
2.490 p value 0.0258 0.7699 0.0062 0.0457 p value: compare to HFD
by t-test
[0472] Overall, these data show that total body weight, weight
gain, FER (food efficiency ratio), visceral fat weights and NASH
score in liver were significantly decreased in mice treated with a
mixture of Mutamba, Magnolia, Yerba Mate and Morus alba extracts.
Also, plasma glucose, total cholesterol and TG were decreased in
mice treated with this combination. Therefore, this example
indicates that a combination such as Composition 11 can be used as
a body weight reducer, as well as dyslipidemia and fatty liver
controllers.
Example 74
Triple Extract Combinations Show a Synergistic Effect on Reducing
Weight Gain
[0473] In this study, four different triple combination
compositions and one quadruple combination composition were tested
in diet induced obesity (DIO) mice, as described in Example 48, to
examine their effect on total body weight and weight gain. The five
compositions tested were as follows: [0474] (1) Composition 1A of
Example 63 (3 components)--Magnolia (100 mg/kg):Morus alba (200
mg/kg):Yerba Mate (500 mg/kg); [0475] (2) Composition 2A (3
components)--Magnolia (100 mg/kg):Morus alba (200 mg/kg):Mutamba
(500 mg/kg); [0476] (3) Composition 10 of Example 68 (3
components)--Magnolia (100 mg/kg):Yerba Mate (500 mg/kg):Mutamba
(500 mg/kg); [0477] (4) Composition 12 (3 components)--Morus alba
(200 mg/kg):Yerba Mate (500 mg/kg):Mutamba (500 mg/kg); and [0478]
(5) Composition 11 of Example 73 (4 components)--Magnolia (100
mg/kg):Morus alba (200 mg/kg):Yerba Mate (500 mg/kg):Mutamba (500
mg/kg).
Effect of Each Extract Tested Individually on Weight Loss
[0479] As shown in FIG. 1, a statistically significant difference
in total weight is seen in weeks 2-8 for Magnolia extract and in
weeks 3-5 for Morus alba extract as compared to the high fat diet
(HFD) group. The total weight difference for the other treatment
groups was not statistically significant as compared to the HFD
group. When examining reductions in weight gain, however, the
Magnolia, Morus alba and Rosemary treatment groups showed
statistically significant reductions at more time points as
compared to the HFD group. Yerba Mate and Mutamba treatment groups
showed no obvious reduction in weight gain when compared to the HFD
group, but a gradual trend of less weight gain was observed for
both groups (though not statistically significant) over the course
of the 8 week treatment,
Effect of Multicomponent Compositions on Mean Body Weight
[0480] As shown in FIG. 2, four of the five combinations showed
statistically significant weight differences as compared to the HFD
group within one week of oral treatment. The only group that did
not have a statistically significant weight loss at week one was
the group treated with Composition 10 (note this group does not
include the Morus alba extract), but statistically significant less
body weight when compared to the HFD group was observed for this
group and the rest of the treatment groups from week 2 through the
end of the experiment (FIG. 2). In addition, weight gain was
reduced in all combination groups beginning in week one after
treatment and this effect lasted through the end of the experiment
(see FIG. 2 and Table 151). Such a quick onset, together with the
long lasting, weight loss and reduced weight gain caused by each of
the combination compositions was unexpected.
[0481] Based on the data from DIO mice treated with the individual
components, a theoretical calculation of the additive weight gain
was made and these calculated values are provided in Table 151. All
five compositions showed an unexpected synergy because each
composition caused a greater weight loss than was predicted from
the additive effect of each individual component.
TABLE-US-00153 TABLE 151 Weight Gain in DIO Mice 2 Weeks after
Treatment with a Multicomponent Composition Weight Dosage Gain/Loss
Composition Group (mg/kg) (g) 1A 10 2A 12 11 Morus 200 -2.16 g x x
x x Magnolia 100 -2.01 g x x x x Yerba 500 1.55 g x x x x Mate
Mutamba 500 0.79 g x x x x HFD -- 0.81 g Weight Gain/Loss Expected
-2.62 g +0.33 g -3.38 g +0.18 g -1.83 g Observed -7.56 g -1.95 g
-6.34 g -5.89 g -4.74 g
[0482] Furthermore, by the end of treatment (8 weeks), mice treated
with Composition 1A, 2A, and 1 showed less mean body weights than
positive control Orlistat, while mice treated with Compositions 10
and 12 were essentially the same weight and slightly above the
Orlistat treated group (FIG. 2). At the end of the 8 week
treatment, mice treated with Composition 1A, 2A, and 11 showed a
weight reduction ranging from about 0.73 g to about 3.6 g, which
was better than the slight weight decrease (0.23 gram) seen in the
Orlistat group. Mice treated with Compositions 10 and 12 had
marginal body weight gains of 1.73 g and 1.78 g, respectively,
which is much less than the 3.5 g to 7.3 g weight gains observed in
mice treated with the components individually at the end of the 8
week treatment (see FIGS. 1 and 2).
[0483] Overall, these data show that the combinations of three or
four components containing Diels-Alder Adducts of chalcone and
prenylphenol showed unexpected quicker and long lasting reductions
in both body weight and weight gain as compared to mice fed a high
fat diet. Other unexpected benefits include a reduced food
efficiency ratio, reduced triglyceride and total cholesterol
levels, improved NASH scores, and reduced fat deposits in liver
(data not shown).
Example 75
Effect of Isolated Morus Extract Active Ingredients on Acute Food
Intake in Sprague-Dawley Rats
[0484] This Example presents a 24-hour food intake test carried
according to the Example 45 to determine the effect of food intake
by rats administered active ingredients (Kuwanon G and Albanin G)
isolated from EtOAc Morus alba root bark extract. S-D rats were
administered Morus alba Isolate Composition A containing 48.3%
Kuwanon G and 46.6% Albanin G with a combined Diels-Alder adduct of
chalcone and prenylphenol having about 94.9% purity, which was
produced using a method similar to that described in Example 5, in
a solution of 0.5% CMC (carboxymethyl cellulose) 30 minutes prior
to the start of dark-phase feeding cycle. The Morus alba Isolate
Composition A was administered at a dose of 92.5 and 185 mg/kg
(this dose is essentially the same active content as found in the
500 and 1000 mg/kg of Morus alba EtOAc extract 15 described in
Example 15) with 10 animals per group.
[0485] Table 152 shows the weight gain results for rats treated
with a single dose of Morus alba Isolate Composition A at two
different amounts compared to control animals.
TABLE-US-00154 TABLE 152 Weight Gain in Non-Obese Fasting Rats Fed
a High Fat Diet Weight Gain after Dose Treatment (hours) Group
(mg/kg) 2 8 24 Control -- Mean 6.71 18.30 12.31 S.D. 2.33 4.88 3.61
Isolate 92.5 Mean 3.11 11.26 12.16 Composition A S.D. 3.42 5.86
3.92 p value 0.0126 0.0062 0.9247 185 Mean 3.81 8.26 10.22 S.D.
3.26 8.21 8.64 p value 0.0321 0.0040 0.4871
[0486] The data of Table 152 show that Morus alba Isolate
Composition A (which includes 94.9% total Kuwanon G and Albanin G)
is capable of inducing a statistically significant reduction in
weight gain for at least 8 hours after treatment.
[0487] Table 153 shows the food intake test results for rats
treated with a single dose of Morus alba Isolate Composition A at
two different amounts compared to control animals.
TABLE-US-00155 TABLE 153 Cumulative Food Intake in Non-Obese
Fasting Rats Fed a High Fat Diet Dose Cumulative Food Intake (hour)
Group (mg/Kg) 1 2 4 6 8 10 24 Control -- Mean 2.12 5.97 10.75 13.78
20.36 22.66 27.35 SD 1.65 1.70 1.92 2.16 2.94 2.43 2.26 Isolate
92.5 Mean 0.89 3.47 7.18 9.81 14.33 17.12 25.71 SD 0.90 2.75 4.29
3.82 4.42 3.54 3.87 p value 0.0590 0.0273 0.0326 0.0123 0.0025
0.0009 0.2672 185 Mean 1.13 2.74 4.51 6.16 8.14 9.43 17.94 SD 0.84
1.36 2.74 4.48 6.44 7.54 9.07 p value 0.1137 0.0002 0.0000 0.0003
0.0001 0.0003 0.0096
[0488] These data together show that both Morus alba treatment
groups exhibited a statistically significant reduction in
cumulative food intake. Further, a dose dependent reduction in food
intake was observed in the second hour of food intake measurement
through to completion of study. These results demonstrate that
active ingredients Kuwanon G and Albanin G isolated from a Morus
alba extract has a statistically significant effect on food intake
in rats indicating that Kuwanon G and Albanin G from Morus alba
extracts can be used in body weight control compositions that
inhibit food intake. Also, the reduced food intake from a single
oral dose of the isolated Morus alba active ingredients lasted more
than 10 hours. Thus, it is feasible to achieve a reduced appetite,
enhanced satiety, or reduced food or caloric intake by once or
twice per day oral administration of a composition comprising
Diels-Alder Adducts of chalcone and prenylphenol Kuwanon G and
Albanin G isolated from Morus alba root bark extract.
Example 76
Effect of Various Compositions Having Morus Extract on Weight Gain,
Feed Intake, and Levels of Leptin, Ghrelin and CCK Peptide in DIO
Mice
[0489] Obese mice were treated with various three component
compositions to test the anti-obesity and appetite effects of such
compositions. Three component Composition 1 (Magnolia:Morus:Yerba
Mate) produced according to the Example 38, three component
Composition 2 (Magnolia:Morus:Mutamba) produced according to the
Example 42, three component Composition 3 (Morus:Rosemary:Yerba
Mate) produced according to the Example 39, and three component
Composition 9 (Morus:Rosemary:Areca) produced according to the
Example 41, were each separately administrated orally to DIO mice
as described in the Example 48. The high fat diet (HFD) mice were
divided into multiple treatment groups with a total of 10 animals
per group. The mice were treated with one of the following:
Composition 1 (1300 mg/kg/day), Composition 2 (1300 mg/kg/day),
Composition 3 (1700 mg/kg/day), or Composition 9 (1700 mg/kg/day).
Control groups included a normal diet group given vehicle only (ND,
negative control), a high fat diet group given vehicle only (HFD,
negative control), an orlistat group (ORI, 40 mg/kg, 2 times/day,
positive control), and a sibutramine group (10 mg/kg, 1 time/day,
positive control). Body weight and feed intake were measured daily
for 2 weeks after treatment and then twice a week until the end of
the 7 week study. At week 2 and week 7 after treatment, mice were
fasted for 16 hours and then five animals were examined to measure
plasma concentrations of leptin and ghrelin. Leptin and ghrelin
levels were measured by using commercial ELISA kits specific for
leptin or ghrelin (Millipore Co., Billerica, Mass.) according to
the manufacturer's instructions. Measurements were taken using a
microplate reader (Victor.TM. X3, PerkinElmer Inc., USA) and
analyzed with PerkinElmer 2030 workstation computer software.
[0490] After 2 weeks of treatment, all treatment groups showed a
tendency toward having decreased body weight gain. Mice treated
with Compositions 1, 2 and 9 showed significantly decreased body
weight gain by the end of the study (Table 154). Similarly, the
orlistat positive control showed significant reduction in body
weight gain at 7 weeks, but those treated with Sibutramine did
not.
TABLE-US-00156 TABLE 154 Effect of Different Morus Compositions on
Weight Gain Weight Gain Weight Gain Group 2-week T-test vs HFD
7-week T-test vs HFD ND -0.91 0.303 0.79 0.004 HFD -1.68 -- 5.73 --
Orlistat -5.07 0.031 0.56 0.013 Sibutramine -1.40 0.675 6.12 0.761
Composition 1 -2.61 0.457 1.89 0.047 Composition 2 -2.54 0.245
-0.77 0.001 Composition 3 -0.80 0.458 3.88 0.302 Composition 9
-2.45 0.428 -0.04 0.002
[0491] After 7 weeks of treatment, the feed intake of all treatment
groups except orlistat showed a tendency toward decreased feed
intake when compared to the HFD group. In particular, groups
treated with Sibutramine, Composition 1, and Composition 9 showed a
statistically significant reduction of average food intake after 7
weeks of treatment (Table 155).
TABLE-US-00157 TABLE 155 Effect of Different Morus Compositions on
Feed Intake Average Feed Intake Average Feed Intake Group 2-week
T-test vs HFD 7-week T-test vs HFD ND 3.63 0.002 3.85 0.014 HFD
2.62 -- 3.05 -- Orlistat 2.79 0.233 3.09 0.894 Sibutramine 2.87
0.292 2.67 0.037 Composition 1 2.64 0.930 2.65 0.096 Composition 2
2.19 0.455 1.99 0.001 Composition 3 2.43 0.483 2.73 0.281
Composition 9 2.13 0.132 1.95 0.010
[0492] Moreover, the food efficiency ratio (FER) was decreased in
all Composition treatment groups during the entire period of
dosing. The group treated with Composition 2 showed a significant
decrease both after 2 weeks and 7 weeks of treatment, while
Composition 1 showed significant changes after 2 weeks of treatment
and Composition 9 was significant only after 7 weeks of treatment.
Positive control orlistat significantly reduced FER, while the
changes observed in the sibutramine and Composition 3 treatment
group were not statistically significant (Table 156).
TABLE-US-00158 TABLE 156 Effect of Different Morus Compositions on
FER Food Efficiency Ratio (FER)* Group 2 weeks 7 weeks ND Mean
-0.023 0.004 SD 0.014 0.005 p value.sup..dagger. 0.0349 0.0098 HFD
Mean -0.059 0.050 SD 0.045 0.023 Orlistat Mean -0.223 0.005 SD
0.095 0.020 p value 0.0003 0.0106 Sibutramine Mean -0.099 0.054 SD
0.048 0.008 p value 0.0698 0.7289 Composition 1 Mean -0.127 0.019
SD 0.078 0.025 p value 0.0276 0.0713 Composition 2 Mean -0.164
-0.009 SD 0.062 0.014 p value 0.0495 0.0011 Composition 3 Mean
-0.049 0.036 SD 0.066 0.025 p value 0.6976 0.3792 Composition 9
Mean -0.089 0.000 SD 0.053 0.012 p value 0.1877 0.0024 *Feed
Efficiency Ratio (FER) = Weight Gain (g/day)/Feed Intake (g/day)
.sup..dagger.p value: Compared to HFD by t-test
[0493] Plasma leptin and active ghrelin levels in DIO mice after 2
weeks and 7 weeks of treatment with Compositions 1, 2, 3 and 9 are
listed in Table 157. HFD animals had a dramatically increased
leptin level compared to normal diet control (ND) animals at week 2
and at week 7. The active ghrelin level in the HFD animals was
dramatically reduced in comparison to ND mice, with an even more
dramatic reduction detectable after 7 weeks of treatment as
compared to the reduction after 2 weeks of treatment. The orlistat
positive control significantly reduced leptin level after 2 weeks
of treatment as compared to the HFD negative control group, but
showed similar levels at 7 weeks. Orlistat had no effect on active
ghrelin levels at either week 2 or week 7 as compared to the HFD
negative control group. The same time frame (2 weeks after
treatment) in which a noticeable change in leptin level was
observed after treatment with orlistat was also the same time frame
that the greatest reduction in weight gain was observed, while no
average feed intake reduction was observed throughout the treatment
period. The sibutramine positive control showed somewhat reduced
leptin level after 2 weeks of treatment as compared to the HFD
group, and showed no leptin level change at week 7. Similar to
orlistat, sibutramine had no effect on active ghrelin levels at
either week 2 or week 7 as compared to the HFD negative control
group.
TABLE-US-00159 TABLE 157 Effect of Different Morus Compositions on
Leptin and Ghrelin Levels Leptin (ng/ml) Active Ghrelin (pg/ml)
Group 2-week 7-week 2-week 7-week ND 1.7 .+-. 0.04 5.0 .+-. 0.08
1258.2 1203.2 HFD 20.1 .+-. 0.20 20.1 .+-. 0.16 975.3 578.8
Orlistat 11.6 .+-. 0.06 19.0 .+-. 0.24 1316.4 1353.7 Sibutramine
17.0 .+-. 0.43 20.4 .+-. 0.00 1001.3 600.1 Composition 1 15.7 .+-.
0.26 19.6 .+-. 0.05 1254.6 1141.0 Composition 2 11.4 .+-. 0.70 17.9
.+-. 0.05 1084.1 1354.5 Composition 3 17.6 .+-. 0.36 19.9 .+-. 0.39
682.6 831.0 Composition 9 16.0 .+-. 0.16 18.7 .+-. 0.04 1618.7
1234.2
[0494] Composition 2 showed a similar level of leptin reduction as
observed for orlistat, and had the most reduced leptin level at
week 7 as compared to the Composition 1, 3, and 9 treatment groups.
Furthermore, at the end of 7 weeks, the Composition 2 treated group
showed greatest reduction in (or least amount of) weight gain as
compared to the Composition 1, 3, and 9 treatment groups. The
reduction in leptin level from all four Composition treatment
groups paralleled the observed reduction in weight gain, feed
intake, and FERs, with Composition 2 showing the best efficacy
followed by Compositions 9, 1, and 3.
[0495] Cholecystokinin (CCK) is a peptide hormone that is a
physiological ligand for the gastrin/CCK-B receptor, while the
CCK-A receptor binds only sulfated CCK peptides. CCK peptides,
mainly produced in small intestinal endocrine I-cells, regulate
pancreatic enzyme secretion and growth, gallbladder contraction,
intestinal motility, satiety, and inhibit gastric acid secretion.
CCK peptides also stimulate digestion of fat and protein. Secretion
of CCK by the duodenal and intestinal mucosa is stimulated by fat-
or protein-rich chyme entering the duodenum. CCK then inhibits
gastric emptying, gastric acid secretion, and mediates digestion in
the duodenum. CCK stimulates the acinar cells of the pancreas to
release water and ions and stimulates the secretion of pancreatic
digestive enzymes that catalyze the digestion of fat, protein, and
carbohydrates.
[0496] Therefore, the effect on CCK peptide levels in mice from
treatment with Composition 2 (Magnolia:Morus:Mutamba), produced
according to the Example 42, or Composition 3 (Morus:Rosemary:Yerba
Mate), produced according to the Example 39, was examined to
determine the extent to which weight gain and/or appetite were
affected through this peptide hormone pathway. Briefly, CCK peptide
levels were measured after 7 weeks of administering Compositions 1
and 3 to mice on a high fat diet as described in Example 48.
Negative controls include mice on a normal diet (NC) and mice on a
high fat diet (HFD), each group administered vehicle only. The
positive controls were orlistat and sibutrimine. CCK levels were
detected using a CCK ELISA assay kit according to the
manufacturer's instructions (Abnova, Taipei City, Tiawan).
Measurements were performed using microplate reader Victor.TM. X3
(PerkinElmer Inc., Waltham, Mass.) and computer software on the
PerkinElmer 2030 workstation.
TABLE-US-00160 TABLE 158 Effect of Morus-Containing Compositions on
CCK Peptide Levels % CCK binding Group % .+-. SD P-value* NC 30.5
.+-. 3.8 0.033 HFD 7.7 .+-. 1.9 -- Orlistat 72.5 .+-. 3.4 0.017
Sibutramine 12.3 .+-. 1.6 0.121 Composition 2 12.8 .+-. 1.3 0.047
Composition 3 18.2 .+-. 2.9 0.036 *P < 0.05 versus the HFD
control group
[0497] As shown in Table 158, the CCK levels of the treated groups
were increased compared to those of the negative control (HFD)
group indicating that at least one of the components of Composition
2 and Composition 3 affect CCK modulation in mice.
Example 77
Effect of Morus-Containing Compositions and Rosemary Extract on
Lipid Accumulation
[0498] The ability to regulate the cell cycle and differentiation
of adipocytes are important in the development and physiology of
obesity. Adipocytes arise from multipotent mesenchymal precursor
cells that commit to become preadipocytes, which can either remain
dormant or differentiate into adipocytes. During terminal
differentiation, the fibroblast-like preadipocytes undergo a series
of morphological and biochemical changes to eventually accumulate
lipid droplets. These in vitro differentiated adipocytes share
similar morphology with adipocytes in vivo. Mouse embryo fibroblast
cell line, 3T3-L1, is a well-characterized cell line used to
examine insulin-induced glucose uptake and mechanisms of obesity
development (e.g., lipid accumulation).
[0499] Three component Composition 3 (Morus:Rosemary:Yerba Mate)
produced according to the Example 39, three component Composition 2
(Magnolia:Morus:Mutamba) produced according to the Example 42, and
Rosemary EtOH extract 22 produced according to the Example 22, were
examined for their effect on lipid accumulation in 3T3-L1
adipocytes. Briefly, 3T3 L1 cells (American Type Culture
Collection) were cultured in Dulbecco's modified Eagle's medium
(DMEM) (GIBCO) containing 10% bovine calf serum until confluent.
Two days post-confluence (DO), cells were stimulated to
differentiate with DMEM containing 10% fetal bovine serum (FBS), 5
.mu.g/ml insulin, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) and 1
.mu.M dexamethasone for two days (D2). Differentiated cells were
then maintained in 10% FBS/DMEM medium with 5 pig/ml insulin for
another two days (D4), followed by culturing with 10% FBS/DMEM
medium for four days (D8).
TABLE-US-00161 TABLE 159 Effect of Composition 3
(Morus:Rosemary:Yerba Mate) on Lipid Accumulation Samples
Concentration Inhibition (%) TNF-.alpha. 10 ng/ml 61.0 Composition
3 40 .mu.g/ml 0.5 80 .mu.g/ml 31.6 120 .mu.g/ml 62.4 160 .mu.g/ml
85.2
[0500] To examine lipid accumulation, four different concentrations
of Composition 3 (40, 80, 120 and 160 ug/ml) were tested with
medium from Days 0 to 8 of adipogenesis. The culture medium was
replaced every two days, and the cells were stained with Oil-Red O
on Day 8. The lipid staining showed that an 8 day incubation with
Composition 3 during the differentiation period significantly
inhibited 3T3-L1 adipogenesis in a dose-dependent manner (Table
159).
TABLE-US-00162 TABLE 160 Effect of Composition 2
(Magnolia:Morus:Mutamba) on Lipid Accumulation Samples
Concentration Inhibition (%) TNF-.alpha. 10 ng/ml 68.9 Composition
2 80 .mu.g/ml 15.3 120 .mu.g/ml 74.7 160 .mu.g/ml 88.4
[0501] Composition 2, used at 10, 80, 120 and 160 .mu.g/ml, also
showed high level efficacy of inhibiting lipid accumulation,
particularly at a concentration of about 120 .mu.g/ml or more
(Table 160).
TABLE-US-00163 TABLE 161 Effect of Rosemary EtOH extract 22 on
Lipid Accumulation Samples Concentration Inhibition (%) TNF-.alpha.
10 ng/ml 74.1 .+-. 6.06 Rosemary 20 .mu.g/ml 22.4 .+-. 5.76 40
.mu.g/ml 56.5 .+-. 0.86 80 .mu.g/ml 61.3 .+-. 5.93 160 .mu.g/ml
75.3 .+-. 1.02
[0502] Similarly, lipid accumulation in cells treated with Rosemary
EtOH extract 22 at 20, 40, 80 and 160 .mu.g/ml, which also showed
high level, dose-dependent efficacy on inhibiting lipid
accumulation (Table 161).
Example 78
Effect of Morus:Rosemary:Yerba Mate Composition on Triglyceride
Accumulation
[0503] Using the same adipocyte differentiation model as described
in Example 77, triglyceride levels were analyzed using an enzymatic
ELISA assay according to the manufacturer's instructions (Cayman
Chemical Co., Ann Arbor, Mich.). Briefly, 3T3-L1 cells were treated
with three component Composition 3 (Morus:Rosemary:Yerba Mate),
produced according to the Example 39, at concentrations of 80, 120
and 160 .mu.g/ml in 6 well plates during adipocyte differentiation
for 8 days. The cells were washed with PBS, scraped with
homogenizing solution, residual cell lysate was centrifuged at
3,000 g for 5 minutes to remove fat layers, and the supernatants
were assayed for triglyceride levels.
TABLE-US-00164 TABLE 162 Effect of Composition 3
(Morus:Rosemary:Yerba Mate) on Intracellular Triglyceride
Accumulation Samples Concentration Inhibition (%) TNF-.alpha. 10
ng/ml 19.4 Composition 3 80 .mu.g/ml 11.6 120 .mu.g/ml 35.7 160
.mu.g/ml 49.9
[0504] As adipocytes differentiate, their intracellular levels of
triglyceride increase continuously. This study shows that
Composition 3 effectively inhibited triglyceride accumulation in
differentiating 3T3-L1 preadipocytes, with concentrations of about
120 .mu.g/ml or more significantly decreasing triglyceride
accumulation (Table 162).
Example 79
Effect of Morus-Containing Compositions on Lipolysis
[0505] To determine the extent of lipolysis induced by
Morus-containing compositions, fully differentiated 3T3-L1
adipocytes (mature adipocytes) were treated with three component
Composition 1 (Magnolia:Morus:Yerba Mate) produced according to the
Example 38 or with three component Composition 3
(Morus:Rosemary:Yerba Mate) produced according to the Example 39,
for 24 hr and 48 hr with serum free DMEM. Isoproterenol (10 .mu.M)
was used as a positive control. The conditioned medium was
recovered and free glycerol released was assayed by using a
lipolysis assay kit (Sigma-Aldrich Inc., USA) following the
manufacturer's instructions.
TABLE-US-00165 TABLE 163 Effect of Morus-Containing Compositions on
Lipolysis % of Control Samples Concentration 24 hrs 48 hrs Control
-- 100 100 Isoproterenol 10 .mu.M 464.2 525.0 Composition 1 62.5
.mu.g/ml 74.6 104.5 125 .mu.g/ml 87.6 120.6 250 .mu.g/ml 178.0
128.8 Composition 3 62.5 .mu.g/ml 61.6 183.0 125 .mu.g/ml 88.3
211.6 250 .mu.g/ml 138.5 212.5
[0506] Composition 1 induced lipolysis in a dose dependent manner
and also may induce early phase lipolysis, while Composition 3
induced lipolysis in a dose and time dependent manner (Table
163).
Example 80
Anti-Oxidant Effect of Various Different Extracts and Various
Morus-Containing Compositions
[0507] Oxygen radicals, expected to be increased in obese subjects,
have an important role in the pathogenesis of many diseases.
Oxidative stress results when free radical formation is greatly
increased or protective antioxidant mechanisms are compromised.
Cells have developed an enzymatic anti-oxidant pathway against
reactive oxygen species (ROS) that are generated during oxidative
metabolism: first, the dismutation of superoxide anion
(O.sub.2.sup.-) to hydrogen peroxide (H.sub.2O.sub.2) catalyzed by
superoxide dismutase (SOD); and then, the conversion of
H.sub.2O.sub.2 to H.sub.2O by glutathione peroxidase (GPx) or
catalase (CAT) (Dalle-Donne et al., Clin. Chem. 52:601, 2006). The
activity of first- and second-step antioxidant enzymes must be
balanced to prevent oxidative damage in cells, which may contribute
to various pathological processes.
[0508] The DPPH (2,2-diphenyl-1-picrylhydrazyl) test is quick and
simple test for measuring anti-oxidation activity. The DPPH radical
has a violet color in solution, but changes to light yellow or
colorless when reduced to DPPH-H (2,2-diphenyl-1-picrylhydrazin)
after reaction with free radicals. The color change can be followed
spectrophotometrically at 517 nm to determine the anti-oxidative
potential of a composition. Briefly, a 0.2 mM solution of DPPH in
DMSO was mixed with each test sample at concentrations of 20, 40,
80 and 160 .mu.g/ml. Ascorbic acid was used as a positive control.
After a 30 minute incubation in the dark, the color change (i.e.,
decrease in absorbance) was measured at 517 nm in a
spectrophotometer. DPPH inhibitory activity is expressed as a
percent inhibition (Table 164).
[0509] The following samples were tested: (1) Rosemary EtOH extract
22 produced according to the Example 22, (2) Magnolia extract 29
produced according to the Example 29, (3) Mutamba extract 35
produced according to the Example 35, (4) Yerba Mate EtOH extract
was produced according to the Example 26, (5) Morus EtOH
precipitate extract 18 produced according to the Example 35, (6)
three component Composition 1 (Magnolia:Morus:Yerba Mate) produced
according to the Example 38, (7) three component Composition 2
(Magnolia:Morus:Mutamba) produced according to the Example 42, and
(8) three component Composition 3 (Morus:Rosemary:Yerba Mate)
produced according to the Example 39.
TABLE-US-00166 TABLE 164 Anti-Oxidative Activity of Various
Extracts and Composition Inhibition (%) Samples 20 .mu.g/ml 40
.mu.g/ml 80 .mu.g/ml 160 .mu.g/ml Ascorbic acid 50.8 .+-. 0.83 53.6
.+-. 1.72 64.2 .+-. 0.86 76.6 .+-. 0.30 Rosemary 32.8 .+-. 1.57
49.5 .+-. 1.28 51.6 .+-. 0.85 62.8 .+-. 1.00 extract Magnolia 33.0
.+-. 0.67 39.6 .+-. 0.64 41.3 .+-. 0.32 50.0 .+-. 0.42 extract
Mutamba 42.2 .+-. 0.52 41.8 .+-. 0.57 43.8 .+-. 0.89 46.3 .+-. 0.72
extract Mate extract 41.0 .+-. 0.26 42.7 .+-. 0.43 43.3 .+-. 0.25
45.7 .+-. 0.41 Morus extract 28.3 .+-. 1.68 42.0 .+-. 1.75 61.8
.+-. 2.00 87.1 .+-. 1.21 Composition 1 38.3 .+-. 0.77 43.4 .+-.
0.48 45.4 .+-. 0.88 46.4 .+-. 0.73 Composition 2 44.5 .+-. 1.16
44.4 .+-. 1.10 46.8 .+-. 0.87 50.8 .+-. 1.02 Composition 3 35.8
.+-. 1.24 45.1 .+-. 0.88 45.5 .+-. 0.15 50.9 .+-. 0.10
[0510] All test samples showed DPPH inhibitory effects (Table 164).
The DPPH scavenging activity of the test samples (in decreasing
order) was as follows: Morus extract>Rosemary
extract>Compositions 2, 3 and 1, and all other extracts of Mate,
Mutamba and Magnolia.
Example 81
Clinical Safety and Efficacy Dose-Escalation Evaluation of
Combination Compositions for Weight Loss in Humans
[0511] A safety and efficacy dose escalation study of Composition
1A (see Example 63), Composition 2A (see Example 74), and
Composition 3 (see Example 39) were each tested at three dose
levels in six subjects per study arm. Each composition was
formulated in capsule form, wherein each capsule contains 250 mg of
Composition 1A, 2A, or 3. Composition 1A is a combination of
Magnolia extract, Morus alba extract, and Yerba Mate extract at a
ratio of 1:2:5, respectively. Composition 2A is a combination of
Magnolia extract, Morus alba extract, and Mutamba extract at a
ratio of 1:2:5, respectively. Composition 3 is a combination of
Morus alba extract, Rosemary extract, and Yerba Mate extract at a
ratio of 2:5:10, respectively. Placebo capsules formulated for the
studies contain CMC (carboxymethyl cellulose) and are identical in
appearance to the capsules of Compositions 1A, 2A, and 3.
[0512] The test compositions (Composition 1A, Composition 2A, or
Composition 3) were taken with meals three times per day. Study
participants received instructions for dosing and storage
conditions of the test product. The study population included a
total of 54 subjects (male and female) between 18 and 50 years of
age, having a body mass index (BMI) ranging from about 30 to about
40, and in generally good health as determined by a medical
history. The test compositions were evaluated at 750 mg/day, 1,500
mg/day and 2,250 mgs/day. The study did not include a placebo
control except for stabilization, as explained further below. The
study was a double blind study for a period of six weeks, first two
weeks on placebo for stabilization and then crossing over to a low
dose active arm. After three individuals completed a study arm, all
safety parameters were evaluated and a decision was made whether to
continue enrolling for that dose level. If that lower dose level
was safe, then subjects for the next higher dose were enrolled. The
duration of the treatment with Composition 1A, Composition 2A, and
Composition 3 was for six weeks (including the two week placebo
stabilization period) for each participant. The total duration of
the study for each patient was about 8-10 weeks, which included
screening, randomization, and active treatment.
[0513] The primary objective of the study was to establish a
maximum tolerated dose (MTD), dose limiting toxicities, and/or
maximum feasible dose of the study product. The following
parameters were evaluated: CBC with Manual Differential, EKG, Blood
Pressure, Vitals, CMP (including Kidney and Liver function tests)
and Adverse Event Analysis.
[0514] A secondary objective was to evaluate the effect of various
doses on lipid profiles (TG, Chol, HDL, LDL), as well as fasting
glucose and insulin levels.
[0515] A tertiary objective was to evaluate the effect of multiple
doses on satiety and dietary intake.
[0516] A quaternary objective was to evaluate the efficacy of the
study product on anthropometric measurements, such as change in
weight, change in BMI, change in waist/hip ratio, arm
circumference, and thigh circumference, each as compared to
baseline.
[0517] The protocol was approved by the IRB prior to the initiation
of any study related procedures and all subjects signed the IRB
reviewed informed Consent.
Statistical Analysis
[0518] The study included three dose levels (Dose 1, Dose 2 and
Dose 3), and differences within time periods for each arm (within
groups tested), differences between two of three dose levels for
each time point (between groups tested), and differences among
three dose levels for each time point (among groups tested) were
analyzed for each endpoint.
[0519] For all endpoints in all types of variable, non-parametric
statistical methods were tested. The differences among dose levels
were tested for nominal significance using Kruskal-Wallis. The
differences between dose levels (i.e., Dose 1 versus Dose 2, Dose 1
versus Dose 3, and Dose 2 versus Dose 3) were tested for nominal
significance using the Wilcoxon Mann-Whitney test. The differences
within time periods for each dose levels were tested for nominal
significance using Wilcoxon Signed Ranks test or Sign test. For
categorical endpoints, the difference in the distribution, between
dose levels was tested using non-parametric Chi-square Test.
[0520] A Modified per Protocol (Mod PP) analysis was performed and
used to assess the efficacy variables of the study. The Mod PP
population involves analyzing together all subjects randomly
assigned to one of the treatments, excluding the subjects who
didn't take the study product. Subjects with at least one post-dose
visit completed were included in the analysis.
[0521] All safety endpoints were analyzed using non-parametric
statistical, methods. CBC with manual differential, EKG, Blood
Pressure, Vitals, CMP (including Kidney and Liver function tests)
were analyzed using Kruskal-Wallis for comparison among Dose
Levels, and Wilcoxon Mann Whitney for comparison between Dose
Levels. Comparison within time periods for each Dose Level was
assessed using Wilcoxon Signed Ranks test or Sign Test.
[0522] In obtaining comparable documentation on adverse events
(AEs), the investigator asked the subject the following open,
standardized, questions at each visit. Frequency and intensity of
AE's and serious AE's were recorded in detail, based on the
subject's interviews during each visit. Recorded AE's were grouped
by general type of event (body system). Differences in AE patterns
between product groups were assessed by Cochran-Q test. Moreover,
McNemar Change test were used to assess the differences in AE
patterns between product groups.
[0523] All efficacy endpoints were analyzed using non-parametric
methods. Lipid Panels, Change in fasting glucose and insulin,
Change in weight, BMI, waist/hip Ratio, Arm and Thigh
circumference, Satiety and Dietary Intake were analyzed using
Kruskal-Wallis for comparison among Dose Levels, Wilcoxon Mann
Whitney for comparison between Dose Levels. Comparisons within time
periods for each Dose Level were assessed using Wilcoxon Signed
Ranks Test or Sign Test.
Clinical Results
[0524] After completing the treatment of all subjects, there were
no significant changes from baseline until the end of treatment at
all three doses for all three compositions on all CBC,
comprehensive metabolic Penal (CMP), EKG, systolic and diastolic
blood pressure, body temperature, pulse rate, and respiratory rate
measurements. There were no reported serious adverse events (SAEs)
during the study.
[0525] Other clinical studies have shown that modest weight losses
are sufficient to produce clinically significant improvements in
cardiovascular risk factors in overweight and obese subjects. The
improvements in blood pressure, glycemic control, and lipids, with
the notable exception of LDL cholesterol, have been observed in
multiple weight loss studies. These same positive trends were
observed in the instant clinical pilot test of Compositions 1A, 2A
and 3 at all three dose levels, particularly in the improvement of
lipid profile with increased high density lipoprotein and reduced
triglycerides, LDL and cholesterol. The reduction of fasting
glucose and insulin level from the three different compositions
demonstrated improved glucose metabolism. The positive changes in
lipid and glucose metabolism observed with all three compositions
are consistent with the observations in the animal efficacy studies
described herein.
[0526] The compositions with the major changes in the evaluation
parameters were observed for Composition 2A--high dose (Table C),
Composition 1A--low dose (Table B), and Composition 3--high dose
(Table D).
[0527] After 4 weeks of treatment with Composition 1A (6 weeks
total including two week stabilization with placebo), subjects in
all three dose groups showed increased high density lipoprotein;
decreased triglycerides and glucose. Subjects treated with 1,500 mg
% day and 2,250 mg/day also experienced reduced low density
lipoprotein. Low dose group subjects had a tendency to present with
decreased body weight and reduced total cholesterol.
[0528] After 4 weeks of treatment with Composition 2A (6 weeks
total including two week stabilization with placebo), subjects
treated with 2,250 mg/day showed the most positive changes with
increased high density lipoprotein; and decreased total
cholesterol, low density lipoprotein, triglycerides, insulin; and a
tendency to present with decreased body weight. Subjects treated
with 1,500 mg/day of Composition 2A showed increased high density
lipoprotein; and decreased triglycerides, glucose; and tendency to
present with decreased body weight. Subjects treated with 750
mg/day of Composition 2A showed increased high density lipoprotein;
decreased glucose; and tendency to present with decreased body
weight.
[0529] After 4 weeks of oral treatment with Composition 3, subjects
treated with 2,250 mg/day showed increased high density
lipoprotein; and decreased low density lipoprotein, triglycerides,
glucose and insulin. Subjects treated with 1,500 mg/day of
Composition 3 showed increased high density lipoprotein; and
decreased total cholesterol, glucose; and tendency to present with
decreased body weight. Subjects treated with 750 mg/day of
Composition 3 showed increased high density lipoprotein; decreased
glucose and insulin; and tendency to present with decreased body
weight.
Example 82
Mutamba EtOH Extracts from Various Plant Parts and Various
Sources
[0530] Plant material from Mutamba (Guazuma ulmifolia) was ground
to a particle size of no larger than two millimeters (mm), and
transferred (150 g) to a one liter round bottom flask. An
approximate 5-fold volume of either 95% ethyl alcohol or 70% ethyl
alcohol in water (v/v) was added to the flask. The extraction was
carried out with reflux for one hour, filtered to remove biomass,
and then subjected to reflux two more times. The filtrates were
combined and concentrated with a rotary evaporator at 50.degree. C.
under vacuum to remove the ethanol, and then vacuum freeze-dried to
obtain the extract. Table 165 lists the extraction results of
Mutamba from various plant parts and various different
countries.
TABLE-US-00167 TABLE 165 Extraction Yield from Different Mutamba
Plant Parts and Source Extract % EtOH Plant Part Plant Origin Yield
82P1 95 Stem Bark Panama 16% 82P2 95 Bark Brazil 14% 82D1 70 Stem
Belize 9% 82D2 70 Bark Belize 17% 82D3 70 Stem Belize 7% 82D4 70
Bark Belize 12% 82D5 70 Bark Belize 17% 82D6 70 Stem Belize 7% 82D7
70 Bark Belize 15% 82D8 70 Stem Belize 7% 82D9 70 Bark Belize 12%
82D10 70 Stem Belize 5% 82D11 70 Stem Bark Peru Highland 10% 82D12
70 Stem Bark Peru Highland 10% 82D13 70 Stem Bark Peru Highland 15%
82D14 70 Bark Peru Highland 9% 82D15 70 Bark Peru Lowland 16% 82D16
70 Bark Peru Lowland 20% 82D17 70 Bark Peru Lowland 6% 82D18 70
Stem Peru Lowland 16% 82D19 70 Stem Peru Lowland 15%
[0531] Plants from Peru tended to provide the best yield of active
ingredients from Mutamba, regardless of which plant part was used
(e.g., the highest yield being 20% from bark from the Peru
lowland). For Mutamba from Belize, a greater yield was obtained
from bark as compared to stems. Use of 70% ethanol was as effective
as 95% ethanol to extract active ingredients from Mutamba.
Example 83
HPLC Analysis of Mutamba EtOH Extract Active Ingredients
[0532] High performance liquid chromatography coupled to a
photodiode-array (HPLC/PDA) was used with a Cl 8 reversed-phase
column (Agilent, USA, Eclipse 3.5 urn, 150 mm.times.4.6 mm) to
detect and quantify components of Mutamba EtOH extracts, such as
Procyanidin B2, Epicatechin, Procyanidin C1, and other minor
components (including tetramer Arecatannin A2). A binary gradient
of 0.05% trifluoroacetic acid in purified water (mobile phase A)
and acetonitrile (mobile phase B) was used to elute Mutamba extract
components as described in Table 166. The flow rate was set to 0.8
ml/min passing through the Eclipse C18 column at a column
temperature of 35.degree. C. The UV detector was set to read
absorbance at 275 nm.
TABLE-US-00168 TABLE 166 Mutamba HPLC Gradient Elution Scheme Time
(min) Mobile phase A % Mobile phase B % 0 3 97 3 3 97 8 9 91 25 16
84 35 60 40 45 100 0 50 100 0 52 3 97 60 97 3
[0533] The quantification standards included pure reference samples
epicatechin, procyanidin B2, and procyanidin C1 (Sigma-Aldrich Co.,
USA; Chendu Biopurify Phytochemicals, Ltd., China; and ChromaDex
Inc., USA, respectively). The highest concentration level of
epicatechin was 0.05 mg/ml and diluted to L5 from L1 (0.003 mg/ml)
using 50% methanol in water. The highest concentration ranges of
procyanidin B2 and procyanidin C1 were 0.05 mg/ml and diluted to L5
from L1 (0.003 mg/ml) using 50% methanol in water. The
concentration of Mutamba extract samples were adjusted to about 2
mg/ml in 50% methanol in water and sonicated until dissolved
(approximately 20 minutes), cooled to room temperature, mixed well,
and then filtered through a 0.45 um nylon syringe filter. A 10 dl
volume of each sample was examined by HPLC. HPLC quantification
results for procyanidin B2, epicatechin and procyanidin C1 content
for Mutamba extracts are provided in Tables 167 and 168.
TABLE-US-00169 TABLE 167 HPLC of Mutamba Extracts from Different
Plant Parts, Age and Gender Contents (%) Extract Plant Procyanidin
Procyanidin No. Part Gender (Age) B2 Epicatechin C1 Total 82D1 Stem
Female (1 yr) 0.7 0.5 0.26 1.46 82D2 Bark Female (1 yr) 1.24 0.69
0.66 2.59 82D2 Bark Female 4.67 2.47 1.80 8.94 82D2 Stem Female
1.07 0.63 0.42 2.12 82D2 Bark Male (mature) 4.12 2.15 1.56 7.83
82D2 Stem Male (mature) 0.33 0.13 0.11 0.57 82D2 Stem bark Female
(young) 0.61 0.18 0.34 1.13 82D2 Stem bark Female (mature) 1.73
0.72 0.98 3.43 82D2 Stem bark Male (young) 1.26 0.42 0.82 2.50
[0534] As shown in Table 167, a total of 9 samples were analyzed to
compare extract content from Mutamba plants at different ages,
gender, and plant part.
TABLE-US-00170 TABLE 168 HPLC of Mutamba Extracts from Different
Plant Parts and Countries Contents (%) Extract Plant Procyanidin
Procyanidin No. Part Origin B2 Epicatechin C1 Total 36 Bark Belize
1.12 0.89 0.49 2.50 82D20 Bark Peru 1.14 0.62 0.38 2.14 82D21 Bark
Mexico 0.98 0.36 0.20 1.54 82D22 Bark Belize 1.31 0.62 0.38 2.31
82P3 Bark Belize 0.71 0.43 0.19 1.33 82P3 Bark Belize 2.21 1.51
0.67 4.39 82P1 Stem Panama 1.31 0.86 0.34 2.51 bark 82D23 Bark
Belize 1.7 0.36 0.66 2.72 82D24 Bark England 0.31 0.05 0.27 0.63
82D25 Bark Peru 1.36 0.61 0.68 2.65
[0535] Further quantification analyses of Mutamba extracts
collected from plants originating in different countries/regions
and extracted from different plant parts are provided in Table 168
to identify the best raw material source.
Example 84
Preparation of Mutamba Stem Bark EtOH Extract and Extract
Fractions
[0536] Mutamba (Guazuma ulmifolia) EtOH extract 84 was produced as
follows: 97.4 kg of dried Mutamba stem bark was cut, crushed and
extracted with approximately 8-fold volume (800 L) of 70% ethyl
alcohol in water (v/v); the extraction was performed at 100.degree.
C. for 4 hours. The residue was filtered to obtain the extraction
solution. The above-described procedure was repeated 2 times. The
extraction solutions were combined and concentrated with a rotary
evaporator at 40.degree. C. under vacuum until the volume was 1/25
volume, and then the concentrated solution was dried by vacuum
freeze-drying process to obtain 70% EtOH extract powder from
Mutamba stem bark. A total of 13.1 kg of Mutamba EtOH extract 84
was obtained from 97.4 kg of raw material, and the extraction yield
was approximately 13.47% (w/w).
[0537] This crude Mutamba stem bark EtOH extract 84 (200 g) was
suspended in 0.6 liter of 20% aqueous ethyl alcohol, and then
loaded onto pre-conditioned HP-20 gel column (10.times.150 cm
column with 7 L of HP-20 resin, Diaion.TM., Mitsubishi Chemical,
Japan) with 20% aqueous ethyl alcohol solution. The column was
eluted with a stepwise gradient solvent mixture and a total of 20
fractions was collected as follows: 10 L of 20% EtOH elution to
collect 2 fractions (F1-2), 10 L of 40% EtOH elution to collect 5
fractions (F3-7), 10 L of 60% EtOH elution to collect 5 fractions
(F8-12). 10 L of 80% EtOH elution to collect 3 fractions (F13-15),
10 L of 100% EtOH elution to collect 2 fractions (F16-17), 10 L of
acetone-MeOH (1:1) washing to collect 3 fractions (F18-20).
TABLE-US-00171 TABLE 169 HP-20 Column fractions of Mutamba EtOH
Extract Fraction Solvent gradient Dry yield(g) Fraction ratio
Fractions 1-5 20%, 40% 89 g 47.0% Fraction 6 40% 47.6 g 25.1%
Fraction 7 40% 13.87 g 7.3% Fraction 8 60% 13.12 g 6.9% Fractions
9-12 60% 14.5 g 7.7% Fractions 13-17 80%, 100% 5.15 g 2.7%
Fractions 18-20 Acetone/MeOH(1:1) 6.24 g 3.3% Total yield 189.5 g
(~95%) 100%
[0538] As shown in Table 169, the fractions were combined based on
HPLC chemical profiling, and the most of weight is distributed at
early elution portion with an excellent recovery yield of about 95%
in the mass balance. Fractions 7, 8 and 9-12 were further combined
for in vivo assays. Three Mutamba stem bark EtOH extract 84
fractions of 84/F1-5, 84/F6 and 84/F7-12 were examined for in vivo
efficacy in a diet induced obesity (DIO) mice model.
Example 85
HPLC Analysis of Additional Mutamba EtOH Extract Fractions
[0539] Mutamba (Guazuma ulmifolia) 70% EtOH extracts (2 g) were
dissolved in 4 ml 20% EtOH/water, mixed with 13.5 g of HP-20 resin
(Sigma), and loaded onto the top space of a pre-packed SNAP HP-20
column (Biotage, 38.times.157 mm). Before sample loading, the HP-20
column was conditioned with 20% EtOH/water for 30 min at 20 ml/min
flow rate delivered by a Hitachi High Throughput Purification (HTP)
system. The columns were eluted with a linear gradient ranging from
20% EtOH/water to 100% EtOH in 50 min, and kept with 100% EtOH for
additional 16 minutes to wash all components off the column. A
total of 97 fractions were collected. Similar fractions in chemical
profile and color were combined to give equivalent pre-F6, F6 and
post-F6 best pools. The column recovery yields ranged from 88 to
100%.
TABLE-US-00172 TABLE 170 HPLC Analysis of Fraction 6 from Various
Mutamba EtOH Extracts Fraction Weight Distribution Content (%) in
F6 Extract Pre- Post- Procyanidin Procyanidin No. F6 F6 F6 B2
Epicatechin C1 82D1 46% 35% 18% 0.38 0.37 0.23 82D2 56% 35% 9% 1.53
0.93 1.07 82D3 51% 28% 21% 1.06 0.91 0.63 82D4 44% 42% 14% 1.96
1.25 1.00 82D5 42% 45% 12% 4.88 2.80 2.27 82D6 61% 27% 11% 1.71
1.16 0.94 82D26 47% 44% 9% 4.01 2.08 2.32 82D27 33% 50% 17% 1.49
0.96 0.87 82D7 40% 48% 12% 4.52 2.96 2.41 82D8 59% 26% 16% 0.63
0.35 0.33 82D9 44% 45% 11% 4.57 2.70 2.20 82D10 58% 28% 14% 0.43
0.44 N.D. 82D28 50% 40% 10% 2.74 0.63 1.83 82D29 47% 43% 11% 2.96
1.36 1.78 82D15 45% 45% 10% 2.26 0.81 1.26 82D14 37% 50% 13% 1.86
0.46 1.07 82D30 77% 16% 7% 0.97 0.49 0.78
Example 86
NMR Analysis of Mutamba EtOH Extract Fractions
[0540] NMR data were collected for HP-20 fractions F1-F20 on a
Varian VNMRS-500 MHz spectrometer to further understand what active
components are present in each fraction. The .sup.1H-NMR spectra
were acquired by VNMR-J 2.2 c with IH-1.9F/l 5N-31P 5 mm PFG AutoX
DB probe and processed by ACD/Labs 10.0 software. Based on NMR data
analysis, fractions 1-5 contained mainly oligosaccharides and
polysaccharides with huge overlapping peaks around 3-5 ppm region.
The proton NMR spectra of fractions 6, 7, and 8 showed very similar
patterns and these match with procyanidin-type compounds (e.g.,
condensed tannins). Proton data analysis of fractions 9, 10 and 11
indicated the presence of a mixture of condensed tannins and
hydrolyzable tannins with the increasing proton signals in the
3.5-4.5 ppm region and decreasing signals around 6.0 ppm. The
proton NMR spectra of fractions 12, 13, and 14 showed that the main
components of these fractions are the hydrolyzable tannins.
Fractions 17 and 18 contained mainly fat molecules.
[0541] Based on the NMR data, fractions 6, 7, and 8 are the three
main fractions that contain condensed tannins with a total ratio of
39.3%, wherein these components are the major active ingredients of
the Mutamba extracts.
Example 87
MALDI-TOF Mass Spectrometry Analysis of Mutamba Fraction 84/F6
[0542] The MALDI-TOF mass spectrometry (MS) spectra were recorded
on Bruker Autoflex II MALDI. The sample was dissolved in MeOH,
while CsCl was dissolved in (about 1.5 mg/ml.). Sample solution (20
.mu.L) was mixed with the CsCl solution (5 uL), and then a 0.5
.mu.l of solution mixture was spotted on the MALDI plate followed
by 2 .mu.L of a saturated solution of DHB in acetonitrile. After
drying, the plate was put into the MALDI. Acquisition was done in
linear positive mode. The MALDI-TOF mass spectra of Mutamba
fraction 84/F6 produced in Example 84, which was recorded as both
[M+Cs].sup.+ and [M+H].sup.+ in the positive mode, showed a series
of peaks grouped at intervals of 288 Da, corresponding to addition
of one catechin/epicatechin unit (FIG. 3).
TABLE-US-00173 TABLE 171 MALDI-TOF Data of Mutamba Extract Fraction
84/F6 Polymer [M + H].sup.+ [M + Cs].sup.+ Trimer 867 999 Tetramer
1155 1287 Pentamer 1465 1575 Hexamer 1753 1863 Heptamer 2041
2151
[0543] The results indicate that the active components in Mutamba
bark contains primarily procyanidins made up of dimers, trimers,
tetramers, pentamers, hexamers, heptamers, octamers, and nonamers
(up to decamers or even higher molecular weight polymers).
Example 88
Thiolysis and Chromatography of Mutamba Extract Active
Fractions
[0544] Thiolysis was carried out according to the methods of Torres
and Selga (Chromatographia 57:441, 2003) with modification.
Cysteamine hydrochloride was chosen as alternative thiol reagent
for the depolymerization reaction. Mutamba fraction 84/F6 produced
in Example 84 or Mutamba fraction 82D4/F6 produced in Example 85
were each individually dissolved in methanol to yield a 10 mg/mL
solution. An aliquot (50 .mu.L) was placed in a vial with 50 .mu.L
hydrochloride in methanol (3.3%, v/v), and then cysteamine
hydrochloride in methanol (50 mg/mL, 100 .mu.L) was added. The
mixture was heated at 65.degree. C. for 20 minutes, the reaction
was quenched with 0.1% (v/v) aqueous TFA 0.3 mL, and then cooled to
room temperature. After filtration through a 0.45 .mu.m membrane
filter, the solutions were analyzed by HPLC chromatography.
[0545] Analytical HPLC chromatography was performed on an LC-MS
(Hitachi M-8000) and PDA (Hitachi L-4500A) system with a ODS column
(Phenomex, Luna C18, 4.6.times.250 mm, 10 .mu.m) with mobile phase
A (0.1% FA in water) and B (acetonitrile). The gradient elution was
3% B for 5 minutes, 3 to 9% over 10 minutes, 9 to 16% over 30
minutes, 16-100% in 1 minute, and washed with 100% B for 7 minutes
at a flow rate of 1 mL/min with UV wavelength 280 nm. Catechin,
epicatechin, catechin-cysteamine and epicatechin-cysteamine
derivative peaks were identified. The mDP of the procyanidin
fractions was calculated based on the peak areas. The calculated
mDP (average degree of polymerization) value was 4.12 for Mutamba
fraction 84/F6 and 6.27 for Mutamba fraction 82D4/F6.
[0546] This result indicates that catechin and epicatechin served
as both terminal and extension units of the Mutamba procyanidins
and epicatechin was the major component in the polymeric
procyanidins from Mutamba bark. The active components in Mutamba
bark primarily include procyanidins with average number of four
epicatechin/catechin units, but also including dimers, trimers,
tetramers, pentamers, hexamers, heptamers, octamers, nonamers and
up to decamers, or even higher molecular weight polymers.
Example 89
Fractionation of Mutamba Extract Fraction 84/F6 and Thiolysis
[0547] Active fraction 6 obtained from Mutamba extract 84, referred
to as 84/F6, was further fractionated by size exclusion
chromatography. Fraction 84/F6 (100.5 mg) was loaded onto a LH-20
gel column with 5 g LH-20 Sephadex gel beads pre-conditioned with
water. The column was eluted with 125 mL water, 125 mL 20% MeOH in
water, 200 mL 50% MeOH, and finally washed off with 200 mL 70%
acetone in water. Five subfractions (84/F6-01-84/F6-05) were
collected. Each fraction was depolymerized with cysteamine-HCl as
described in Example 88 to determine the average degree of
polymerization.
TABLE-US-00174 TABLE 172 Subfractions of Mutamba Extract Fraction
84/F6 Subfraction Weight (mg) Ratio mDP 84/F6-01 6.2 6.17% 8.33
84/F6-02 12.1 12.04% 6.87 84/F6-03 16.7 16.62% 1.71 84/F6-04 64.8
64.48% 6.23 84/F6-05 5.0 4.98% 8.44
[0548] Subfraction 84/F6-01 (6.17%) and 84/F6-02 (12.04%) contained
polymeric procyanidins with an mDP value of 8.33 and 6.87,
respectively. Subfraction 84/F6-03 (16.6%) eluted with 50% MeOH and
contained mainly epicatechin, procyanidin B2, procyanidn C1, and a
small portion of oligomeric procyanidins with a mDP value at 1.71.
The major fraction 84/F6-06 (64.48%) was eluted by 70%
Me.sub.2CO--H.sub.2O and contained procyanidins with an mDP value
at 6.23.
Example 90
Fractionation of Mutamba Extract Fraction 82D4/F6 and Thiolysis
[0549] Active fraction 6 equivalent obtained from Mutamba extract
82D4 (see Example 82), referred to as 82D4/F6, was further
fractionated by size exclusion chromatography. Fraction 82D4/F6
(620.8 mg) was loaded onto an LH-20 gel column with 5 g LH-20
Sephadex gel beads pre-conditioned with water. The column was
eluted with 150 mL water, 250 mL 20% MeOH in water, 100 mL 40%
MeOH, and finally 300 mL 100% MeOH (recovery yield was 77.6%). Five
subfractions (82D4/F6-01-82D4/F6-05) were collected. About 22% of
substances from the fraction remained on the LH-20 column. Each
fraction was depolymerized with cysteamine-HCl as described in
Example 88 to determine the average degree of polymerization.
TABLE-US-00175 TABLE 173 Subtractions of Mutamba Extract Fraction
82D4/F6 Subfraction Weight (mg) Ratio mDP 82D4/F6-01 67.9 10.94%
13.43 82D4/F6-02 112.3 18.09% -- 82D4/F6-03 66.4 10.70% 5.87
82D4/F6-04 163.2 26.29% 4.24 82D4/F6-05 71.9 11.58% 7.38
[0550] Subfraction 82D4/F6-01 contained 10.9% polymeric
procyanidins with an mDP value at 13.43.
Example 91
Analysis of Condensed Tannin Content in Mutamba Extracts
[0551] The Butanol-HCl assay is one colorimetric method commonly
used to determine the amount of condensed tannins, particularly
procyanidins, in a sample. The method described by Porter et al.
(Phytochemistry 25:223, 1986) was followed as a standard method for
determination of the condensed tannin content in Mutamba extracts.
Butanol-HCl reagent was prepared by mixing 950 mL of n-Butanol with
50 mL concentrated HCl. Ferric reagent was prepared by dissolving
0.5 g FeNH.sub.4(SO.sub.4) in 25 mL of 2N HCl. In a tube and in
duplicate, 1 ml Mutamba extract at a 0.2 mg/mL concentration in 70%
acetone was added to 6 mL acid butanol reagent, then 0.2 mL iron
reagent was added and absorbance at 550 nm measured. The tubes were
capped, shaken, and then put in boiling water bath for 50 minutes.
After cooling the tubes, the absorbance at 550 nm was measured. The
absorbance of each sample before heating was subtracted from the
absorbance of the heated mixtures as a blank. Subfraction 84/F6-04
(tannin fraction) was used as the standard for quantification.
Condensed tannins (% in dry matter) as compared to the tannin
standard equivalent were calculated based on the absorbance at 550
nm.
TABLE-US-00176 TABLE 174 Condensed Tannin Content in Mutamba EtOH
Extracts Extract No. Extract Content (%)* Bark Content (%)* 36
48.68% 7.9% 84 38.09% 5.0% 82P2 68.93% 10.3% 82P1 72.77% 11.8% 82D5
66.90% 11.4% 82D27 38.90% 3.5% 82D7 60.45% 9.1% 82D9 57.95% 7.0%
82D11 22.46% 2.2% 82D31 48.67% 5.8% 82D32 55.58% 10.6% 82D33 62.98%
10.1% 82D34 33.92% 4.4% 82D35 71.62% 13.6% 82D36 38.21% 7.3% 82D37
57.08% 5.7% 82D38 50.08% 6.0% 82D38 50.58% 5.6% *Expressed as
tannin standard compound equivalents
[0552] The condensed tannin content of the Mutamba EtOH extracts
varied greatly, ranging from 20% up to as much as 73% (see Table
174), depending on the plant part, age of the plant, plant
collection location, and season. Mature bark and stem bark tend to
have the highest levels of condensed tannins. Based on the measured
condensed tannin content in Mutamba extracts and corrected for
extraction yield, the content in the raw plant material was
calculated to range from about 2% to about 14%.
[0553] Using this method on two batches of Composition 2
(Magnolia:Morus:Mutamba) produced according to the Example 42, the
condensed tannin content in each of the two batches was 35.26% and
31.63%, respectively.
Example 92
Analysis of Condensed Tannin Content in Mutamba Extract
Fractions
[0554] Mutamba fractions from Mutamba 70% EtOH extract 84 were
prepared at a concentration of 0.1 mg/mL in 70% acetone for use in
the butanol-HCl assay described in Example 91. The condensed tannin
content was quantified by the same method as described in Example
91, with data shown in the Table 175.
TABLE-US-00177 TABLE 175 Condensed Tannin Content in Mutamba EtOH
Extract Fractions Fraction No. Content* 84/F1-5 11.22% 84/F6 91.61%
84/F7 77.67% 84/F8 71.74% 84/F9 67.21% *Expressed as tannin
standard compound equivalents
[0555] The most active fraction, 84/F6, has a higher content of
condensed tannins as compared with other the other fractions.
Example 93
Short Term In Vivo Efficacy of Mutamba EtOH Extract Fraction
[0556] A 14-day study was used to evaluate the effect of Mutamba
EtOH extract fraction 84/F6 on body weight gain in high-fat-diet
(HFD) fed C57Bl/6J mice. Male C57BL/6J mice at the age of 6 weeks
were purchased from Charles River Laboratories (Wilmington, Mass.)
and acclimated for one week. On the day 0 (treatment start), body
weights were taken for all the mice and randomly assigned to a
group of four treatment groups as follows: (1) 8 mice/group
received positive control=Alli; (2) 15 mice/group received Mutamba
fraction 84/F6; (3) 8 mice/group received vehicle only (0.5%
carboxymethyl cellulose) given to HFD control; and (4) 8 mice/group
received vehicle only given to untreated normal diet control. Mice
received a daily oral dose of Alli at 30 mg/kg and Mutamba 84/F6 at
1 g/kg for 14 days. Once mice received their first respective dose,
they were provided ad libitum a 60% kcal high fat-diet, except for
the normal diet group. Body weights were taken daily for the
duration of study except on weekends. At the end of the study (on
the 15.sup.th day), mice were fasted for 5 hours and blood glucose,
triglyceride and total cholesterol levels were taken. Blood glucose
levels were measured, using the Contour blood glucose monitoring
kit (Bayer Health Care). Total triglyceride and cholesterol levels
were measured using the CardioChek Analyzer with PTS panel test
strips (Polymer Technology System, Inc, Indianapolis, Ind.).
TABLE-US-00178 TABLE 176 Effect of Mutamba EtOH Extract Fraction on
Body Weight Gain Body weight (g).dagger. Group Day 1 Day 2 Day 3
Day 4 Day 7 Day 8 Day 9 Day 10 Day 11 Day 14 HFD 21.1 .+-. 0.72
21.5 .+-. 1.0 21.3 .+-. 1.14 21.8 .+-. 0.88 22.9 .+-. 1.07 23.1
.+-. 1.06 23.1 .+-. 0.98 23.3 .+-. 1.06 23.6 .+-. 1.09 24.5 .+-.
1.06 84/F6 21.2 .+-. 0.74 21.3 .+-. 1.03 21.7 .+-. 0.94 21.7 .+-.
1.18 22.4 .+-. 1.26 22.1 .+-. 1.09 21.7 .+-. 1.07* 22.2 .+-. 1.25*
22.6 .+-. 1.08** 23.0 .+-. 1.62* 1 g/kg Alli 21.3 .+-. 0.72 21.5
.+-. 1.00 21.8 .+-. 1.14 22.2 .+-. 0.88 22.6 .+-. 1.07 22.7 .+-.
1.06 22.4 .+-. 0.98 22.3 .+-. 1.06 22.3 .+-. 1.09 22.8 .+-. 1.06*
30 mg/kg .dagger.Data are expressed as Mean .+-. SD. *P .ltoreq.
0.05; **P .ltoreq. 0.07
[0557] As seen in Table 176, mice receiving Mutamba 84/F6 showed
statistically significant less weight gain beginning day 9 after
treatment as compared to the vehicle treated HFD group. On the
other hand, the positive control. Alli, treated mice showed the
decrease in weight gain only on day 14 of treatment as compared to
the vehicle treated HFD group.
TABLE-US-00179 TABLE 177 Effect of Mutamba EtOH Extract Fraction on
Metabolism.dagger. Group Dose (mg/kg) Cholesterol (mg/dl)
Triglyceride (mg/dl) Glucose (mg/dl) HFD 0 117.3 .+-. 14.7 79.4
.+-. 9.5 212.3 .+-. 37.2 Alli 30 104.5 .+-. 10.2 (P = 0.06) 109.5
.+-. 18.2 (P = 0.001) 164.3 .+-. 46.8 (P = 0.04) Mutamba 84/F6 1000
102.4 .+-. 76.7 (P = 0.02) 81.3 .+-. 7.8 (P = 0.67) 156.9 .+-. 42.1
(P = 0.01) Normal diet 0 100.3 .+-. 0.7 (P = 0.01) 107.6 .+-. 21.6
(P = 0.001) 175.0 .+-. 25.1 (P = 0.03) .dagger.Data are expressed
as Mean .+-. SD. P-values for each group are indicated in
parenthesis
[0558] Similarly, after day 14 of oral treatments, a statistically
significant decrease in fasting blood total cholesterol and glucose
levels were observed for mice treated with Mutamba 84/F6 as
compared to the vehicle treated HFD group (Table 177). These low
levels of fasting blood glucose and total cholesterol were
comparable to the levels observed for mice fed a normal diet. No
difference in triglyceride levels were observed between Mutamba and
vehicle treated HFD groups. In contrast, the Alli and vehicle
treated regular diet fed mice showed statistically significant
higher levels of fasting triglyceride as compared to the HFD
group.
Example 94
In Vivo Efficacy of Mutamba Stem Bark EtOH Extract and
Fractions
[0559] Mutamba 70% EtOH extract 84 and three column fractions of
Mutamba 84/F1-5, 84/F6, and 84/F7-12 produced according to the
Example 84 were tested in the DIO mouse model as described in
Example 48. The Mutamba 70% EtOH extract treatment group (G1),
Mutamba fraction 84/F1-5 (G2), Mutamba fraction 84/F6 (G3) and
Mutamba fraction 84/F7-12 (04) were orally administered a dose of
1000 mg/kg by gavage two times per day.
TABLE-US-00180 TABLE 178 Effect of Mutamba EtOH Extract and
Fractions on Body Weight Animal Body Weight (gram) Group 0 1 2 3 4
5 6 7 ND Mean 28.99 28.56 28.48 28.35 28.51 29.10 29.42 29.66 SD
1.641 1.341 1.350 1.403 1.420 1.542 1.695 1.886 p value* 0.0000
0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 HFD Mean 41.47
40.78 40.46 41.51 42.48 44.00 45.91 47.39 SD 3.417 3.595 3.838
3.778 4.175 3.887 3.609 3.080 ORI Mean 41.44 40.17 36.20 34.80
36.09 37.06 38.91 40.22 SD 3.405 3.079 3.338 3.399 3.886 3.448
4.111 4.372 G1 Mean 41.41 41.51 41.39 42.05 43.61 45.30 46.92 47.81
SD 3.308 3.313 3.802 4.001 4.219 4.637 4.552 4.131 p value 0.9708
0.6602 0.5070 0.7730 0.5744 0.5301 0.6075 0.8070 G2 Mean 41.63
40.90 40.92 41.71 43.00 44.64 45.90 46.59 SD 3.361 3.339 3.523
3.581 3.829 3.523 3.493 2.917 p value 0.9215 0.9435 0.6707 0.9115
0.7843 0.7196 0.9958 0.5794 G3 Mean 41.61 40.72 38.72 39.09 39.46
39.41 39.31 39.72 SD 3.339 3.946 3.668 3.739 4.301 4.550 4.731
4.485 p value 0.9283 0.9725 0.4277 0.2051 0.1630 0.0402 0.0053
0.0008 G4 Mean 41.73 40.96 40.54 41.38 42.81 44.14 45.18 45.46 SD
3.318 3.556 3.954 4.278 4.739 4.878 4.837 5.108 p value 0.8709
0.9178 0.8424 0.9438 0.8762 0.9497 0.7218 0.3472 *p value: compare
to HFD by t-test
[0560] Table 178 shows that the 1000 mg/kg treatment group G3
showed significantly decreased body weight after weeks 5, 6 and 7
of treatment.
TABLE-US-00181 TABLE 179 Effect of Mutamba EtOH Extract and
Fractions on Weight Gain Weeks Group 1 2 3 4 5 6 7 NC Mean -0.08
-0.21 -0.06 0.53 0.86 1.10 1.51 SD 0.640 0.812 1.002 0.864 1.235
1.208 1.308 p value 0.843 0.0126 0.0113 0.0002 0.0000 0.0000 0.0000
HFD Mean -0.61 0.73 1.70 3.22 5.13 6.61 7.25 SD 0.631 0.645 1.646
1.516 1.453 1.310 1.651 ORI Mean -3.97 -5.37 -4.08 -3.11 -1.26 0.05
0.72 SD 0.538 1.150 1.333 1.519 1.544 1.798 1.954 p value 0.0000
0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 G1 Mean -0.12 0.54 2.10
3.79 5.41 6.31 6.85 SD 0.827 0.944 1.099 1.343 1.342 1.280 1.443 p
value 0.1748 0.6216 0.5485 0.4154 0.6720 0.6260 0.5949 G2 Mean 0.02
0.81 2.10 3.75 5.00 5.69 6.76 SD 0.815 0.828 1.160 1.070 1.242
1.539 2.189 p value 0.0835 0.8259 0.5520 0.4118 0.8449 0.1919
0.6017 G3 Mean -1.99 -1.46 -1.09 -1.14 -1.24 -0.83 -0.21 SD 2.773
1.634 1.220 1.253 2.048 1.967 1.903 p value 0.1807 0.0063 0.0014
0.000 0.000 0.000 0.000 G4 Mean -0.42 0.42 1.85 3.18 4.22 4.51 5.30
SD 0.777 1.315 1.753 1.986 2.110 2.298 2.381 p value 0.5681 0.5307
0.8475 0.9591 0.3047 0.0298 0.0603 * p value: compare to HFD by
t-test
[0561] The body weight gains from 1000 mg/kg Mutamba fraction 84/F6
group (G3) was significantly decreased after the second week of
treatment and such effect lasted until the end of the treatment
period.
TABLE-US-00182 TABLE 180 Effect of Mutamba EtOH Extract and
Fractions on Food Intake and FER Weight Gain Food intake FER* Group
(g/day) (g/day) (Food efficiency ratio) NC Mean 0.031 3.309 0.009
SD 0.027 0.450 0.008 p value.sup..dagger. 0.0000 0.0000 0.0000 HFD
Mean 0.151 2.548 0.059 SD 0.034 0.442 0.014 ORI Mean 0.015 2.675
0.006 SD 0.041 0.690 0.015 p value 0.0000 0.0737 0.0000 G1 Mean
0.143 2.532 0.056 SD 0.030 0.546 0.012 p value 0.5949 0.7917 0.6363
G2 Mean 0.141 2.365 0.060 SD 0.046 0.489 0.019 p value 0.6017
0.0014 0.9684 G3 Mean -0.004 1.994 -0.002 SD 0.040 0.692 0.020 p
value 0.0000 0.0000 0.0000 G4 Mean 0.110 2.349 0.047 SD 0.050 0.560
0.021 p value 0.0603 0.0013 0.1611 *FER (Feed efficacy ratio) =
Weight Gain (g/day)/Food intake (g/day) .sup..dagger.p value as
compared to HFD by Student's t-test
[0562] Food intake and Food Efficiency Ratio (FER) were
significantly reduced by oral treatment of DIO mice with Mutamba
fraction 84/F6 (G3) at 1000 mg/kg when compared to the high fat
diet group treated with vehicle only.
TABLE-US-00183 TABLE 181 Effect of Mutamba EtOH Extract and
Fractions on Absolute Organ Weight. Retroperitoneal Group Liver
Epididymal Fat Fat PeriRenal Fat Total Fat* NC Mean 1.091 0.740
0.216 0.113 1.069 SD 0.078 0.202 0.072 0.034 0.275 p
value.sup..dagger. 0.0038 0.0000 0.0000 0.0000 0.0000 HFD Mean
1.785 2.335 0.505 0.634 3.474 SD 0.519 0.510 0.025 0.145 0.507 ORI
Mean 1.184 1.998 0.567 0.388 2.952 SD 0.215 0.330 0.086 0.091 0.405
p value 0.0086 0.1182 0.0571 0.0008 0.0288 G1 Mean 1.973 2.259
0.542 0.616 3.417 SD 0.462 0.455 0.082 0.137 0.428 p value 0.4292
0.7418 0.2140 0.7891 0.7989 G2 Mean 1.696 2.230 0.512 0.582 3.323
SD 0.472 0.674 0.108 0.098 0.710 p value 0.7080 0.7134 0.8611
0.3869 0.6113 G3 Mean 1.196 1.949 0.495 0.396 2.841 SD 0.228 0.346
0.058 0.193 0.306 p value 0.0101 0.0919 0.6424 0.0111 0.0073 G4
Mean 1.850 1.938 0.484 0.544 2.966 SD 0.688 0.375 0.084 0.150 0.333
p value 0.8254 0.0779 0.4886 0.2153 0.0251 *Total fat is the sum of
the three fat pads (epididymal, retroperitoneal, perirenal)
.sup..dagger.p value as compared to HFD by Students' t-test
[0563] In Mutamba fraction 84/F6 treated group (G3), absolute
weights of liver, peri-renal fat and total fat pads were
significantly decreased when compared with the high fat diet
control group.
TABLE-US-00184 TABLE 182 Effect of Mutamba EtOH Extract and
Fractions on Biochemistry Parameters ALT AST ALP Glu T-chol TG TP
LDL-C HDL-C Group U/L U/L U/L mg/dL mg/dL mg/dL g/dL mg/dL mg/dL NC
Mean 17.45 35.74 214.53 226.00 125.60 17.50 5.04 4.61 64.91 SD 2.37
7.29 20.26 35.79 9.67 5.17 0.26 0.81 5.01 p value* 0.0001 0.0008
0.7258 0.1744 0.0004 0.0091 0.2168 0.0003 0.3780 HFD Mean 96.63
112.57 224.07 253.67 210.67 27.00 4.91 12.52 68.11 SD 31.09 44.95
76.68 48.92 45.05 8.66 0.16 4.08 9.88 ORI Mean 35.12 62.03 186.51
267.22 175.44 46.78 4.89 7.56 74.22 SD 15.54 16.20 24.08 38.57
17.85 18.56 0.25 2.52 5.31 p value 0.0001 0.0099 0.1926 0.5232
0.0530 0.0312 0.8243 0.0068 0.1217 G3 Mean 62.15 74.50 149.83
206.50 168.13 30.13 4.79 6.64 71.26 SD 40.16 27.86 18.12 40.01
17.96 17.20 0.24 1.21 3.33 p value 0.0259 0.0303 0.0094 0.0190
0.0211 0.4542 0.2175 0.0012 0.3565 G4 Mean 145.60 125.01 208.07
255.00 200.67 25.11 5.11 9.92 67.76 SD 96.63 63.40 45.39 25.92
32.52 8.15 0.33 3.11 3.97 p value 0.2222 0.7534 0.4193 0.7637
0.5493 1.0000 0.1344 0.0977 0.9704 *p value as compared to HFD by
Students' t-test
[0564] Mutamba fraction 84/F6 treatment (G3) reduced total
cholesterol and LDL-cholesterol significantly when compared with
the high fat diet group.
[0565] In summary, the 1000 mg/kg Mutamba fraction 84/F6 treatment
group (G3) showed significantly decreased body weight, body weight
gain, food efficiency ratio (FER), total cholesterol,
LDL-cholesterol and absolute organ weight of peri-renal fat and
total fat pads. These data, taken together, indicate that Mutamba
fraction 84/F6 contains the major active anti-obesity components
from. Mutamba stem bark, which can be used as is or standardized in
a Mutamba extract for managing body weight, blood cholesterol, body
fat, or any combination thereof,
[0566] The various embodiments described above can be combined to
provide further embodiments. All of the U.S. patents. U.S. patent
application publications, U.S. patent applications, foreign
patents, foreign patent applications and non-patent publications
referred to in this specification or listed in the Application Data
Sheet are incorporated herein by reference, in their entirety.
Aspects of the embodiments can be modified, if necessary to employ
concepts of the various patents, applications and publications to
provide yet further embodiments. These and other changes can be
made to the embodiments in light of the above-detailed
description.
[0567] In general, in the following claims, the terms used should
not be construed to limit the claims to the specific embodiments
disclosed in the specification and the claims, but should be
construed to include all possible embodiments along with the full
scope of equivalents to which such claims are entitled.
Accordingly, the claims are not limited by the disclosure.
* * * * *
References