U.S. patent application number 09/919966 was filed with the patent office on 2002-05-16 for compositions containing hypoglycemically active stilbenoids.
Invention is credited to Hopp, David C., Inman, Wayne D..
Application Number | 20020058707 09/919966 |
Document ID | / |
Family ID | 26919916 |
Filed Date | 2002-05-16 |
United States Patent
Application |
20020058707 |
Kind Code |
A1 |
Hopp, David C. ; et
al. |
May 16, 2002 |
COMPOSITIONS CONTAINING HYPOGLYCEMICALLY ACTIVE STILBENOIDS
Abstract
The use of isolated or purified stilbenoid compounds including
longistyline A-2-carboxylic acid as hypoglycemic agents or to lower
serum glucose levels is described. The invention also relates to
the use of such stibenoid compounds in combination with other
hypoglycemic agents.
Inventors: |
Hopp, David C.; (Mill Creek,
WA) ; Inman, Wayne D.; (Belmont, CA) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX PLLC
1100 NEW YORK AVENUE, N.W., SUITE 600
WASHINGTON
DC
20005-3934
US
|
Family ID: |
26919916 |
Appl. No.: |
09/919966 |
Filed: |
August 2, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60225800 |
Aug 16, 2000 |
|
|
|
Current U.S.
Class: |
514/568 ;
514/532; 514/533; 514/733 |
Current CPC
Class: |
A23L 33/105 20160801;
A61P 3/10 20180101; A61P 3/08 20180101 |
Class at
Publication: |
514/568 ;
514/733; 514/532; 514/533 |
International
Class: |
A61K 031/235; A61K
031/192; A61K 031/05 |
Claims
What is claimed is:
1. A dietary supplement useful for treating hyperglycemia or
reducing blood glucose levels comprising a carrier and a
hypoglycemically effective amount of an isolated compound having
the formula (I): 30wherein, A is selected from the group consisting
of a single bond and a double bond in trans conformation; R.sub.1
is selected from the group consisting of H, OH, C.sub.1-6alkoxy,
COOH, and COOC.sub.1-6alkyl; R.sub.2 is selected from the group
consisting of H, OH, and C.sub.1-10alkoxy; R.sub.3 is selected from
the group consisting of H, C.sub.1-10alkyl, C.sub.2-10alkenyl,
C.sub.2-10alkynyl, and C.sub.1-8cycloalkyl; R.sub.4 is selected
from the group consisting of H, OH, and C.sub.1-10 alkoxy; R.sub.5
are selected from the group consisting of H, C.sub.1-10alkyl,
C.sub.2-10alkenyl, C.sub.2-10alkynyl, and C.sub.1-8cycloalkyl;
R.sub.6 is selected from the group consisting of H, OH,
C.sub.1-6alkoxy, COOH, and COOC.sub.1-6alkyl; R.sub.7 is selected
from the group consisting of H, OH, C.sub.1-6alkoxy, COOH, and
COOC.sub.1-6alkyl; and wherein at least one of R.sub.3 and R.sub.5
is selected from the group consisting of C.sub.1-10alkyl,
C.sub.2-10alkenyl, C.sub.2-10alkynyl, and C.sub.1-8cycloalkyl; or a
pharmaceutically acceptable salt thereof.
2. The dietary supplement of claim 1, wherein said compound has the
formula (II): 31wherein, A is selected from the group consisting of
a single bond and a double bond in trans conformation; R.sub.1 is
selected from the group consisting of H and --COOH; R.sub.2 is
selected from the group consisting of H, OH and C.sub.1-2alkoxy;
R.sub.3 is selected from the group consisting of H,
3-methyl-2-butenyl, 3-methylbutyl, 3,7-dimethyl-2,6-octadienyl, and
3,7-dimethyloctadyl, R.sub.4 is selected from the group consisting
of H, OH and C.sub.1-2alkoxy; R.sub.5 are selected from the group
consisting of H, 3-methyl-2-butenyl, 3-methylbutyl,
3,7-dimethyl-2,6-octadienyl, and 3,7-dimethyloctadyl, R.sub.6 is
selected from the group consisting of H, OH, and C.sub.1-2alkoxy;
and R.sub.7 is selected from the group consisting of H, OH and
C.sub.1-2alkoxy; wherein, at least one of R.sub.3 and R.sub.5 is
selected from the group consisting of 3-methyl-2-butenyl,
3-methylbutyl, 3,7-dimethyl-2,6-octadienyl, and
3,7-dimethyloctadyl; or a pharmaceutically acceptable salt
thereof.
3. The dietary supplement of claim 1, wherein said compound has the
formula (III): 32wherein, A is selected from the group consisting
of a single bond and a double bond in trans conformation; R.sub.1
is selected from the group consisting of H and COOH; R.sub.2 is
selected from the group consisting of H, OH, and C.sub.1-2alkoxy;
R.sub.3 is selected from the group consisting of
--CH.sub.2CH.dbd.C(CH.sub.3).sub.2 and
--CH.sub.2CH.sub.2CH(CH.sub.3).sub.2; and R4 is C.sub.1-2alkoxy; or
a pharmaceutically acceptable salt thereof.
4. The dietary supplement of claim 1, wherein said compound has the
formula (IV): 33wherein, A is selected from the group consisting of
a single bond and a double bond in trans conformation; R.sub.1 is
selected from the group consisting of H and COOH; R.sub.2 is
selected from the group consisting of H, OH, and C.sub.1-2alkoxy;
R.sub.4 is C.sub.1-2alkoxy; and R.sub.5 is selected from the group
consisting of --CH.sub.2CH.dbd.C(CH.sub.3).sub.2 and
--CH.sub.2CH.sub.2CH(CH.sub.3).sub- .2; or a pharmaceutically
acceptable salt thereof.
5. The dietary supplement of claim 1, wherein said compound is
selected from the group consisting of: (1) Longistyline C(Cmd.A);
(2) Longistyline A(Cmd.C); (3) longistyline A-6-carboxylic acid
(Cmd.D); (4) 7,8-dihydrolongistyline C (Cmd.E); (5)
7,8,2",3"-tetrahydrolongistyline C (Cmd.F); (6)
7,8,2",3"-tetrahydrolongistyline A-6-carboxylic acid (Cmd.H); (7)
3-hydroxy-4isoprenyl-5-methoxystilbene-2-carboxylic acid; (8)
3-hydroxy-4-(3-methylbutyl)-5-methoxy-7,8-dihydrostilbene-2-carboxyli-
c acid; (9) 4-isopentenylresveratrol
(3,4',5-trihydroxy-4-(3-methyl-2-bute- nyl)stilbene; (Cmd.AA); (10)
3,5-dimethoxy-4-(3-methyl-2-butenyl)stilbene (Cmd.AB); (11)
3,4',5-trimethoxy-4-(3-methyl-2-butenyl)stilbene 27 (Cmd.AC); (12)
chlorophorin (Cmd.AD); (13) 3,5-dimethoxy-4-(3-methyl-2-bu-
tenyl)(Cmd.AE); (14)
3,5-dihydroxy-4-(3-methyl-2-butenyl)bibenzene(Cmd.AF)- ; (15)
3,5-dihydroxy-4-(3-methyl-2-butenyl)bibenzyl-2-carboxylic acid
(Cmd.AG); (16) 3-hydroxy-5-methoxy-4-(3-methyl-2-butenyl)bibenzene
(Cmd.AH); (17) 5-hydroxy-3-methoxy-4-(3-methyl-2-butenyl)bibenzene
(Cmd.AI); (18) 3,5-dihydroxy-2-(3-methyl-2-butenyl)bibenzene
(Cmd.AJ); (19) 3-hydroxy-5-methoxy-2-(3-methyl-2-butenyl)bibenzene
(Cmd.AK); (20) 5-hydroxy-3-methoxy-2-(3-methyl-2-butenyl)bibenzene
(Cmd.AL); (21)
3,5-dihydroxy-4-(3,7-dimethyl-2,6-octadienyl)-bibenzene (Cmd.AM);
(22) 3,5-dimethoxy-4-(3,7-dimethyl-2,6-octadienyl)-bibenzene
(Cmd.AN); (23)
3,5-diacetyl-4-(3,7-dimethyl-2,6-octadienyl)-bibenzene (Cmd.AO);
(24) 3,4',5-trihydroxy4-(3 ,7-dimethyl-2,6-octadienyl)-bibenzene
(Cmd.AP); (25) 3,5-dihydroxy-4-(3,7-dimethyloctyl)bibenzene
(Cmd.AQ); (26) 3,5-dimethoxy-4-(3,7-dimethyloctyl)bibenzene
(Cmd.AR); (27) 2-geranyl-3,5-dihydroxybibenzene (Cmd.AS); (28)
2-geranyl-3,5-dimethoxybi- benzene (Cmd.AT); (29)
2-geranyl-3-hydroxy-5-methoxybibenzene (Cmd.AU); and (30)
3-methoxy-4'-hydroxy-4-(3-methyl-2-butenyl)bibenzene (Cmd.AV).
6. A method of supplementing the diet a mammal suffering from
elevated blood glucose levels, comprising administering to said
mammal a dietary supplement comprising a carrier and a
hypoglycemically effective amount of an isolated compound having
the formula (I): 34wherein, A is selected from the group consisting
of a single bond and a double bond in trans conformation; R.sub.1
is selected from the group consisting of H, OH, C.sub.1-6alkoxy,
COOH, and COOC.sub.1-6alkyl; R.sub.2 is selected from the group
consisting of H, OH, and C.sub.1-10alkoxy; R.sub.3 is selected from
the group consisting of H, C.sub.1-10alkyl, C.sub.2-10alkenyl,
C.sub.2-10alkynyl, and C.sub.1-8cycloalkyl; R.sub.4 is selected
from the group consisting of H, OH, and C.sub.1-10alkoxy; R.sub.5
are selected from the group consisting of H, C.sub.1-10alkyl,
C.sub.2-10alkenyl, C.sub.2-10alkynyl, and C.sub.1-8cycloalkyl;
R.sub.6 is selected from the group consisting of H, OH,
C.sub.1-6alkoxy, COOH, and COOC.sub.1-6alkyl; R.sub.7 is selected
from the group consisting of H, OH, C.sub.1-6alkoxy, COOH, and
COOC.sub.1-6alkyl; and wherein, at least one of R.sub.3 and R.sub.5
is selected from the group consisting of C.sub.1-10alkyl,
C.sub.2-10alkenyl, C.sub.2-10alkynyl, and C.sub.1-8cycloalkyl; or a
pharmaceutically acceptable salt thereof.
7. The method of claim 6, wherein said hypoglycemically effective
amount of said compound is about 1 to about 1000 mg/kg/day.
8. The method of claim 7, wherein said hypoglycemically effective
amount of the compound is between about 50 to about 350
mg/kg/day.
9. The method of claim 6, wherein said dietary supplement is
administered orally.
10. The method of claim 6, wherein said compound is isolated from
Cajanus cajan.
11. The method of claim 6, wherein said dietary supplement is in
the form of a nutrition bar, a cereal, an energy bar, a soup, or a
pet food.
12. The method of claim 6, wherein said carrier is selected from
the group consisting of a dried bakery product, wheat flour, rice
flour, oat flour, corn flour, soy flour, wheat bran, rice bran, oat
bran, corn bran, wheat middlings, whole ground wheat, corn gluten
meal, whole ground corn, soybean meal, barley, sorghum, meat and
bone meals, poultry meal, fish meal and dry pet food.
13. The method of claim 6, wherein said mammal is suffering from
diabetes.
14. The method of claim 6, wherein said mammal is suffering from
hyperglycemia.
15. A method of supplementing the diet a mammal suffering from
elevated blood glucose levels, comprising administering to said
mammal a dietary supplement comprising a carrier and a
hypoglycemically effective amount of an isolated compound selected
from the group consisting of: (A) Longistyline C; (B) Longistyline
A; (C) longistyline A-6-carboxylic acid; (D)
7,8-dihydrolongistyline C; (E) 7,8,2",3"-tetrahydrolongistyline C;
(F) 7,8,2",3"-tetrahydrolongistyline A-6-carboxylic acid; (G)
3-hydroxy-4isoprenyl-5-methoxystilbene-2-carboxylic acid; (H)
3-hydroxy-4-(3-methylbutyl)-5-methoxy-7,8-dihydrostilbene-2-carboxylic
acid; (I) 4-isopentenylresveratrol
(3,4',5-trihydroxy-4-(3-methyl-2-buten- yl)stilbene; (J)
3,5-dimethoxy-4-(3-methyl-2-butenyl)stilbene; (K)
3,4',5-trimethoxy-4-(3-methyl-2-butenyl)stilbene; (L) chlorophorin;
(M) 3,5-dimethoxy-4-(3-methyl-2-butenyl)bibenzene; (N)
3,5-dihydroxy-4-(3-methyl-2-butenyl)bibenzene; (O)
3,5-dihydroxy-4-(3-methyl-2-butenyl)bibenzyl-2-carboxylic acid; (P)
3-hydroxy-5-methoxy-4-(3-methyl-2-butenyl)bibenzene; (Q)
5-hydroxy-3-methoxy-4-(3-methyl-2-butenyl)bibenzene; (R)
3,5-dihydroxy-2-(3-methyl-2-butenyl)bibenzene; (S)
3-hydroxy-5-methoxy-2-(3-methyl-2-butenyl)bibenzene; (T)
5-hydroxy-3-methoxy-2-(3-methyl-2-butenyl)bibenzene; (U)
3,5-dihydroxy-4-(3,7-dimethyl-2,6-octadienyl)-bibenzene; (V)
3,5-dimethoxy-4-(3,7-dimethyl-2,6-octadienyl)-bibenzene; (W)
3,5-diacetyl-4-(3,7-dimethyl-2,6-octadienyl)-bibenzene; (X)
3,4',5-trihydroxy4-(3,7-dimethyl-2,6-octadienyl)-bibenzene; (Y)
3,5-dihydroxy-4-(3,7-dimethyloctyl)bibenzene; (Z)
3,5-dimethoxy-4-(3,7-di- methyloctyl)bibenzene; (AA)
2-geranyl-3,5-dihydroxybibenzene; (AB)
2-geranyl-3,5-dimethoxybibenzene; (AC)
2-geranyl-3-hydroxy-5-methoxybiben- zene; and (AD)
3-methoxy-4'-hydroxy-4-(3-methyl-2-butenyl)bibenzene.
16. The method of claim 15, wherein said compound is selected from
the group consisting of: (1) Longistyline C (Cmd.A); (2)
Longistyline A (Cmd.C); and (3) longistyline A-6-carboxylic acid
(Cmd.D).
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 60/225,800, filed Aug. 16, 2000.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to stilbenoids that exhibit
hypoglycemic and/or antidiabetic activity in mammals. Provided
herein are processes for obtaining such stilbenoids, particularly
from Cajanus cajun; compositions comprising the stilbenoids and
methods for their use in treating diabetes mellitus and lowering
blood glucose.
[0004] 2. Related Art
[0005] Uses of Cajanus SPP
[0006] Plants of Cajanus spp. (Leguminoseae), particularly C.
cajun, also known as pigon pea or redgram, are herbaceous members
of the family Leguminoseae that grows widely throughout Africa,
Asia and South and Central America. Cajanus spp. have been used in
traditional medicine to treat stomach aches for women suspected of
being pregnant, wounds and scalds, toothache, gonorrhoea, bad
vision and heart diseases (Hedberg,H, et al., J Ethnopharmacol, 9
(2/3), 237-260 (1983).
[0007] In addition to its use by traditional healers, these plants
may also be included in the normal diet as a food plant. Canjanus
spp, for example, are consumed by people in India. To that end,
studies have reported redgram and blackgram consumption's effect on
blood glucose levels and glucose tolerance. (Srinivasan, M., Lancet
1957, 317 (1957)).
[0008] Extracts of Cajanus cajan have been reported to show
hyypoglycemic activity. Dhar reported a 50% ethanolic extract of
Cajanus cajun as exhibiting hypoglycemic activity. Dhar, M. L., et
al., Indian J. Exp. Biol., 6, 232 (1968).
[0009] While extracts of the genus Cajanus have been used
medicinally, such use is not without potential drawbacks. First, in
addition to containing one or more compounds having a "desired"
biological activity, plant materials often contain a myriad of
naturally-occurring organic compounds among which one or more can
elicit a physiological or pharmacological response that
contraindicate use for the desired activity. Secondly, when
administered in the form of a plant extract, the actual dosage of
the unknown active compound(s) is impossible to regulate, which can
result in an ineffective amount, i.e, too low a concentration, or a
toxic amount, too high a concentration, of active compound
administered.
[0010] Thus, there remains a need for an isolated or a purified,
hypoglycemically active compound, compositions comprising
therapeutically effective amounts of such a compound and methods
for their use.
[0011] Compounds Isolated From Cajanus SPP Stilbenoids
[0012] The term stilbenoid refers to stilbenes, bibenzyls and
phenyldihydroisocoumarins together with a number of nitrogen free
phenathrenols, which are thought to be products of the same
metabolic pathway that which leads to stilbenes. See generally
Gorham, J., Progress in Phytochemistry, Vol. 6, Reinhold, et al.,
eds, Pergamon press, New York, 1980, pp 203-252. Stilbenes
(dihydrostilbenes) generally have the basic two stereoisomeric
forms, a trans- and a cis-skeleton: 1
[0013] Generally, naturally occurring stilbenes and bibenzyls are
hydroxy and/or methoxy substituted at the 3,3',4,4',5, and 5'
positions. Some naturally occuring stilbenes and bibenzyls include
pinosylvin (3,5-dihydroxy stilbene), piceatannol
(3,3',4,5'-tetrahydroxlystilbene), piceid
(3,4',5-trihydroxystilbene-3-O-.beta.-D-glucopyranoside) and
resveratrol (3,4',5-trihydroxystilbene). Mono-
(3-hydroxy-5-methoxystilbe- ne) and di-methyl
(3,5-dimethoxystilbene) ethers of trans-pinosylvin and their
respective dihydroderivatives have been reported isolated from the
heartwood of Pinus armandi, P. morrisonicola, and P. parviflorai.
Fang, J-M, et al Phytochemistry 27(5): 1395-1397 (1988).
[0014] Stilbenoids may also be prenylated or homogeranylated at the
ortho (C-2 or C-6) or para (C-4) positions. Longistylines A
(3-hydroxy-5-methoxy-4-(3-methyl-2-butenyl)stilbene), B
(3,5-dihydroxy-2,4-di(3-methyl-2-butenyl)stilbene), C
(3-hydroxy-5-methoxy-2-(3-methyl-2-butenyl)stilbene, and D
(3,5-dihydroxy-2,6-di(3-methyl-2-butenyl)stilbene) were isolated
from the bark and root of Lonchocarpus longistylus. Monache, F. D.,
et al. (Lloydia 40(2), 201-208 (1977)). 4-isopentenylresveratrol
(3,4',5-trihydroxy-4-(3-methyl-2-butenyl)stilbene) was isolated
from Arachis hypogea (Keen, N. T., et al., Phytochemistry 15, 1794
(1976)). A prenylated pinosylvin dimethyl ether
(3,5-dimethoxy-4-(3-methyl-2-butenyl- )stilbene) was isolated from
Derris rariflora (Braz Filho, R., et al., Phytochemistry 14, 261
(1975a)) and D. floribunda (Braz Filho, R., et al., Phytochemistry
14, 1454 (1975b)). A prenylated resveratrol trimethyl ether
(3,4',5-trimethoxy-4-(3-methyl-2-butenyl)stilbene) was also
reported isolated from D. floribunda (Braz Filho, R., et al.,
1975b). Chlorophorin (4-homogeranyl-2,3',4,5'-tetrahydoxystilbene)
was isolated from Chlorophora excelsa (Grundon, M. F., et al.,
Nature (Lond.) 163, 154 (1949)). The occurrence in plants of
isoprenice chains substituted stilbenes has also been reported by
King and Grundo (J. Chem. Soc. 1950, 3547 (1950)); and Cooksey
(Cooksey, C. J., et al., Phytochemistry 21(12), 2935 (1982)).
[0015] Prenylated bibenzyls have been isolated from Radula spp.
Asakawa, Y, et al., reported the isolation of
3,5-dihydroxy-4-(3,7-dimethyl-2,6-oc- tadienyl)-bibenzyl from R.
variabilis (Phytochemistry 17, 2005 (1978a)) as well as the
synthesis of mono and dimethyl ethers and corresponding
tetrahydroderi-vatives. Asakawa reported the isolation from Radula
complanata of bibenzyls prenylated or geranylated at 4 position.
Asakawa, Y. et al., Phytochemistry 17, 2115 (1978b). Asakawa also
reported the isolation of prenyl bibenzyls from Radula kojana.
Asakawa, Y., et al., Phytochemistry 30(1), 219 (1991).
[0016] Four isoprenylated stilbene 2-carboxylic acid phytoalexins
(3-hydroxy-5-metloxy-6-(3-methyl-2-butenyl) stilbene-2-carboxylic
acid,
3-hydroxy-5-methoxy-4-(3-methyl-2-butenyl)stilbene-2-carboxylic
acid, 3,5-dimethoxy-6-(3-methyl-2-butenyl)stilbene-2-carboxylic
acid, and 3,5-dimethoxy-4-(3-methyl-2-butenyl)stilbene-2-carboxylic
acid) were reported isolated from the leaves of Canjanus cajan
challenged with Botrytis cinerea (Cooksey, C J, et al.,
Phytochemistry 21(12):2935-2938 (1982).
[0017] Various biological activities have been reported of the
stilbenoids. For example, stilbenoids have been reported to show
antioxidant activities [resveratrol] (A. Fauconneau, B., et al.,
Life Sci. 61(21): 2103 (1997)); antifungal activity
[3,3',4,5'-tetrahydroxysti- lbene](Inamori, Y., et al., Chem. Phar.
Bull. 33(7):2904-09 (1985)); antiplatelet aggregation activity
[resveratrol] (Chung, M-I, et al., Planta Med. 1992 58:274-275; and
Kimura, Y., et al., Biochim.Biophys. Acta 1995 175, 275-278);
coronary vasodilator activity (Inamori, Y., et al., Chem. Pharm.
Bull. 35, 887-89 (1987); anti-leukemia activity (Mannila, E.,
Phytochemistry, 1003, 33, 813-816) and protein-tyrosine kinase
inhibitory activity (Orsini, F., et al., J. Nat. Prods. 60,
1082-1087 (1997)).
[0018] Pterostilbene (3,4',5-trimethoxystilbene) isolated from
Pterocarpus marsupium reportedly significantly decreased the plasma
glucose level and body weights of of STZ-induced diabetic rats.
Manickam, M., et al., J. Nat. Prods. 60, 609-610 (1996). Both the
aqueous and alcoholic extracts of the heartwood of Pterocarpus
marsupium were reported to produce a reduction in blood sugar
level. Shah, D. S., Ind. J. Med. Res. 55(2), 166-168 (1967).
[0019] International patent application WO00/69430 by Nag et al.
and published on Nov. 23, 2000 describes diphenylethylene compounds
having the formula: 2
[0020] wherein,
[0021] R is hydrogen or --CO.sub.2Z, Z is hydrogen or a cation;
[0022] R.sub.1, R.sub.2, and R.sub.3 are each independently H,
--OH, or OR.sub.4, wherein R.sub.4 is linear or branched alkyl of
1-12 carbon atoms; with the proviso that when R is hydrogen and
R.sub.2.dbd.R.sub.3.dbd.0Me, then R.sub.2 is not OH.
[0023] These compounds are allegedly useful for the treatment of
diabetes.
[0024] Citation or identification of any reference in the
Background of this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
SUMMARY OF THE INVENTION
[0025] The present invention provides pharmaceutical compositions
useful for treating hyperglycemia or reducing blood glucose levels
comprising a pharmaceutically acceptable carrier and an effective
amount of an isolated compound having the formula (I): 3
[0026] wherein,
[0027] A is selected from the group consisting of a single bond and
a double bond in trans conformation, as noted by ____;
[0028] R.sub.1 is selected from the group consisting of H, OH,
C.sub.1-6alkoxy, COOH, and COOC-.sub.1-6alkyl;
[0029] R.sub.2 is selected from the group consisting of H, OH, and
C.sub.1-10alkoxy;
[0030] R.sub.3 is selected from the group consisting of H,
C.sub.1-10alkyl, C.sub.2-10alkenyl, C.sub.2-10alkynyl, and
C.sub.1-8cycloalkyl;
[0031] R.sub.4 is selected from the group consisting of H, OH, and
C.sub.1-10alkoxy;
[0032] R.sub.5 are selected from the group consisting of H,
C.sub.1-10alkyl, C.sub.2-10alkenyl, C.sub.2-10alkynyl, and
C.sub.1-8cycloalkyl;
[0033] R.sub.6 is selected from the group consisting of H, OH,
C.sub.1-6alkoxy, COOH, and COOC.sub.1-6alkyl;
[0034] R.sub.7 is selected from the group consisting of H, OH,
C.sub.1-6alkoxy, COOH, and COOC.sub.1-6alkyl; and
[0035] wherein at least one of R.sub.3 and R.sub.5 is selected from
the group consisting of C.sub.1-10alkyl, C.sub.2-10alkenyl,
C.sub.2-10alkynyl; C.sub.1-8cycloalkyl;
[0036] or a pharmaceutically acceptable salt thereof.
[0037] The present invention also provides methods for treating
hyperglycemia or reducing blood glucose levels to a mammal
comprising administering to said mammal a hypoglycemically
effective amount of an isolated or pure compound of formula (I). In
particular embodiments, the invention provides methods of treatment
for type I diabetes, type II diabetes, hyperthermia, trauma,
sepsis, burns, severe head injury, cerebral-thrombosis,
encephalitis, heat stroke, congenital metabolic glycogen storage
diseases, and hyperglycemia that occurs as a adverse advent of
anesthesia.
BRIEF DESCRIPTION OF THE FIGURES
[0038] FIG. 1 is a bar graph showing the plasma glucose levels
(mg/dL) of db/db mice treated with vehicle only, metformin, or 1000
mg/kg b.i.d. of enriched extracts of Cajanus cajun (EE1, EE2, EE3,
or EE4) as described herein. The relevant extract or compound was
administered to the animals at 0, 8, 24, 32, 48, 56, 72, 80, and 96
hours. Plasma glucose levels were measured at 0 (pre), 3 (Day 1),
51 (Day 3), and 99 (Day 5) hours post initial dosing. All data
points n=8 *P<0.05; **P<0.01; ***P<0.001 (analysis of
variance, one factor).
[0039] FIG. 2 is a bar graph showing the plasma glucose levels
(mg/dL) of db/db mice treated with vehicle only or 250 mg/kg q.d.
of Compound A, Compound B, or Compound C. The relevant compound was
administered to the animals at 0, 8 and 24 h, and plasma glucose
levels were measured at 0, 3, 6, 8, 24, and 30 hours. All data
points N=8. *P<0.05; **P<0.01; ***P<0.0001 (analysis of
variance (ANOVA); one factor).
[0040] FIG. 3 is a bar graph showing the plasma glucose levels
(mg/dL) of db/db mice treated with vehicle only; 250 mg/kg q.d. of
metformin; and 62.5, 125 and 250 mg/kg q.d. of longistyline C
(Compound A). The relevant compound was administered to the animals
at 0, 24 and 48 h, and plasma glucose levels were measured at 0, 6,
30 and 54 h. All data points N=8. *P<0.05; **P<0.01;
***P<0.0001 (analysis of variance (ANOVA); one factor).
[0041] FIG. 4 is a bar graph showing the plasma glucose levels
(mg/dL) of db/db mice treated with vehicle only; 250 mg/kg q.d. of
metformin; and 62.5, 125 and 250 mg/kg q.d. of longistyline
A-2-carboxylic acid (Compound C). The relevant compound was
administered to the animals at 0, 24 and 48 h, and plasma glucose
levels were measured at 0, 6, 30 and 54 h. All data points N=8.
*P<0.05; **P<0.01; ***P<0.0001 (analysis of variance
(ANOVA); one factor).
[0042] FIG. 5 is a bar graph showing the plasma glucose levels
(mg/dL) of db/db mice treated with vehicle only; 250 mg/kg q.d. of
metformin; and 125 mg/kg q.d. of 7,8-dihydrolongistyline C
(Compound E), 125 mg/kg q.d. of 7,8-dihydropinosylvin monomethyl
ether (Compound G), and 125 mg/kg q.d. of 7,8-dihydrolongistyline
A-2-carboxylic acid (Compound H). The relevant compound was
administered to the animals at 0, 24 and 48 h, and plasma glucose
levels were measured at 0, 6, 30 and 54 h. All data points N=8. *P
<0.05; **P<0.01; ***P<0.0001 (analysis of variance
(ANOVA); one factor).
[0043] FIG. 6 is a bar graph showing the plasma glucose levels
(mg/dL) of db/db mice treated with vehicle only; and 125 mg/kg q.d.
of longistyline C (Compound A), 125 mg/kg q.d. of longistyline A
(Compound D), 125 mg/kg q.d. of 7,8-dihydrolongistyline C (Compound
E), and 125 mg/kg q.d. of 2",3",7,8-tetrahydrolongistyline C
(Compound F). The relevant compound was administered to the animals
at 0, 24 and 48 h, and plasma glucose levels were measured at 0, 6,
30 and 54 h. All data points N=8. *P<0.05; **P<0.01;
***P<0.0001 (analysis of variance (ANOVA); one factor).
DETAILED DESCRIPTION OF THE INVENTION
[0044] Definitions
[0045] As used herein, the term "independently" or the equivalents
thereof is employed to described an instance were two or more
groups may be the same or different from each other and the
occurrence of one group does not impact or influence the occurrence
of the other group.
[0046] The term "alkyl" refers to a monovalent alkane (hydrocarbon)
derived radical containing from 1 to 10 carbon atoms unless
otherwise defined. It may be straight or branched. Preferred
straight or branched alkyl groups include methyl, ethyl, propyl,
isopropyl, butyl, 3-butyl, and t-butyl. Alkyl also includes a
straight or branched alkyl group that contains or is interrupted by
a cycloalkylene portion.
[0047] The term "cycloalkyl" refers to cyclic monovalent alkanes.
Preferred cycloalkyl groups include cyclopentyl and cyclohexyl.
[0048] The term "alkenyl" refers to a hydrocarbon radical straight
or branched containing from 2 to 10 carbon atoms and at least one
carbon to carbon double bond. Preferably one carbon to carbon
double bond is present, and up to four non-aromatic
(non-resonating) carbon-carbon double bonds may be present.
Preferred alkenyl groups include ethenyl, propenyl, butenyl and
geranyl.
[0049] The term "alkynyl" refers to a hydrocarbon radical straight
or branched containing from 2 to 10 carbon atoms and at least one
carbon to carbon triple bond. Up to three carbon-carbon triple
bonds may be present. Preferred alkynyl groups include ethynyl,
propynyl and butynyl.
[0050] The term "alkoxy" represents an alkyl group of indicated
carbon atoms attached through an oxygen linkage.
[0051] The present invention provides pharmaceutical compositions
useful for treating hyperglycemia or reducing blood glucose levels
comprising a pharmaceutically acceptable carrier and an effective
amount of an isolated compound having the formula (I): 4
[0052] wherein,
[0053] A is selected from the group consisting of a single bond and
a double bond in trans conformation;
[0054] R.sub.1 is selected from the group consisting of H, OH,
C.sub.1-6alkoxy, COOH, and COOC.sub.1-6alkyl;
[0055] R.sub.2 is selected from the group consisting of H, OH, and
C.sub.1-10alkoxy;
[0056] R.sub.3 is selected from the group consisting of H,
C.sub.1-10alkyl, C.sub.2-10alkenyl, C.sub.2-10alkynyl, and
C.sub.1-8cycloalkyl;
[0057] R.sub.4 is selected from the group consisting of H, OH, and
C.sub.1-10alkoxy;
[0058] R.sub.5 are selected from the group consisting of H,
C.sub.1-10alkyl, C.sub.2-10alkenyl, C.sub.2-10alkynyl, and
C.sub.1-8cycloalkyl;
[0059] R.sub.6 is selected from the group consisting of H, OH,
C.sub.1-6alkoxy, COOH, and COOC.sub.1-6alkyl;
[0060] R.sub.7 is selected from the group consisting of H, OH,
C.sub.1-6alkoxy, COOH, and COOC.sub.1-6alkyl; and
[0061] wherein at least one of R.sub.3 and R.sub.5 is selected from
the group consisting of C.sub.1-10alkyl, C.sub.2-10alkenyl,
C.sub.2-10alkynyl; C.sub.1-8cycloalkyl;
[0062] or a pharmaceutically acceptable salt thereof.
[0063] In one embodiment of the present invention are
pharmaceutical compositions and methods of treatment comprising a
hypoglycemically effective amount of a compound having the formula
(II): 5
[0064] wherein,
[0065] A is selected from the group consisting of a single bond and
a double bond in trans conformation;
[0066] R.sub.1 is selected from the group consisting of H and
--COOH;
[0067] R.sub.2 is selected from the group consisting of H, OH and
C.sub.1-2alkoxy;
[0068] R.sub.3 is selected from the group consisting of H,
3-methyl-2-butenyl, 3-methylbutyl, 3,7-dimethyl-2,6-octadienyl, and
3,7-dimethyloctadyl,
[0069] R.sub.4 is selected from the group consisting of H, OH and
C.sub.1-2alkoxy;
[0070] R.sub.5 are selected from the group consisting of H,
3-methyl-2-butenyl, 3-methylbutyl, 3,7-dimethyl-2,6-octadienyl, and
3,7-dimethyloctadyl,
[0071] R.sub.6 is selected from the group consisting of H, OH, and
C.sub.1-2alkoxy; and
[0072] R.sub.7 is selected from the group consisting of H, OH and
C.sub.1-2alkoxy; wherein at least one of R.sub.3 and R.sub.5 is
selected from the group consisting of 3-methyl-2-butenyl,
3-methylbutyl, 3,7-dimethyl-2,6-octadienyl, and
3,7-dimethyloctadyl;
[0073] or a pharmaceutically acceptable salt thereof.
[0074] In a second embodiment of the present invention are
pharmaceutical compositions and methods of treatment comprising a
hypoglycemically effective amount of a compound having the formula
(III): 6
[0075] wherein:
[0076] A is selected from the group consisting of a single bond and
a double bond in trans conformation;
[0077] R.sub.1 is selected from the group consisting of H and
COOH;
[0078] R.sub.2 is selected from the group consisting of H, OH, and
C.sub.1-2alkoxy;
[0079] R.sub.3 is selected from the group consisting of
--CH.sub.2CH.dbd.C(CH.sub.3).sub.2 and
--CH.sub.2CH.sub.2CH(CH.sub.3).sub- .2; and
[0080] R.sub.4 is C.sub.1-2alkoxy; or a pharmaceutically acceptable
salt thereof.
[0081] In a third embodiment of the present invention are
pharmaceutical compositions and methods of treatment comprising a
hypoglycemically effective amount of a compound having the formula
(IV): 7
[0082] A is selected from the group consisting of a single bond and
a double bond in trans conformation;
[0083] R.sub.1 is selected from the group consisting of H and
COOH;
[0084] R.sub.2 is selected from the group consisting of H, OH, and
C.sub.1-2alkoxy;
[0085] R.sub.4 is C.sub.1-2alkoxy; and
[0086] R.sub.5 is selected from the group consisting of
--CH.sub.2CH.dbd.C(CH.sub.3).sub.2 and
--CH.sub.2CH.sub.2CH(CH.sub.3).sub- .2; or a pharmaceutically
acceptable salt thereof.
[0087] Preferred stilbenoid compounds of formula (I) are selected
from the group consisting of:
[0088] Longistyline C (Cmd.A);
[0089] Longistyline A (Cmd.C);
[0090] longistyline A-6-carboxylic acid (Cmd.D);
[0091] 7,8-dihydrolongistyline C (Cmd.E);
[0092] 7,8, 2",3"-tetrahydrolongistyline C (Cmd.F);
[0093] 7,8,2",3"-tetrahydrolongistyline A-6-carboxylic acid
(Cmd.H);
[0094] 3-hydroxy-4isoprenyl-5-methoxystilbene-2-carboxylic
acid;
[0095]
3-hydroxy-4-(3-methylbutyl)-5-methoxy-7,8-dihydrostilbene-2-carboxy-
lic acid;
[0096] 4-isopentenylresveratrol
(3,4',5-trihydroxy-4-(3-methyl-2-butenyl)s- tilbene; (Cmd.AA);
[0097] 3,5-dimethoxy-4-(3-methyl-2-butenyl)stilbene (Cmd.AB);
[0098] 3,4',5-trimethoxy-4-(3-methyl-2-butenyl)stilbene 27
(Cmd.AC);
[0099] chlorophorin (Cmd.AD);
[0100] 3,5-dimethoxy-4-(3-methyl-2-butenyl)bibenzene (Cmd.AE);
[0101] 3,5-dihydroxy-4-(3-methyl-2-butenyl)bibenzene (Cmd.AF);
[0102] 3,5-dihydroxy-4-(3-methyl-2-butenyl)bibenzyl-2-carboxylic
acid (Cmd.AG);
[0103] 3-hydroxy-5-methoxy-4-(3-methyl-2-butenyl)bibenzene
(Cmd.AH);
[0104] 5-hydroxy-3-methoxy-4-(3-methyl-2-butenyl)bibenzene
(Cmd.AI);
[0105] 3,5-dihydroxy-2-(3-methyl-2-butenyl)bibenzene (Cmd.AJ);
[0106] 3-hydroxy-5-methoxy-2-(3-methyl-2-butenyl)bibenzene
(Cmd.AK);
[0107] 5-hydroxy-3-methoxy-2-(3-methyl-2-butenyl)bibenzene
(Cmd.AL);
[0108] 3,5-dihydroxy-4-(3,7-dimethyl-2,6-octadienyl)-bibenzene
(Cmd.AM);
[0109]
3,5-dimethoxy-4-(3,7-dimethyl-2,6-octadienyl)-bibenzene(Cmd.AN);
[0110] 3,5-diacetyl-4-(3,7-dimethyl-2,6-octadienyl)-bibenzene
(Cmd.AO);
[0111] 3,4',5-trihydroxy4-(3,7-dimethyl-2,6-octadienyl)-bibenzene
Cmd.AP);
[0112] 3,5-dihydroxy-4-(3,7-dimethyloctyl)bibenzene (Cmd.AQ);
[0113] 3,5-dimethoxy-4-(3,7-dimethyloctyl)bibenzene (Cmd.AR);
[0114] 2-geranyl-3,5-dihydroxybibenzene (Cmd.AS);
[0115] 2-geranyl-3,5-dimethoxybibenzene (Cmd.AT);
[0116] 2-geranyl-3-hydroxy-5-methoxybibenzene (Cmd.AU); and
[0117] 3-methoxy-4'-hydroxy-4-(3-methyl-2-butenyl)bibenzene
(Cmd.AV).
[0118] Pharmaceutical compositions contain a pharmaceutically
acceptable carrier or vehicle, a hypoglycemically effective amount
of a compound of formula (I) and optionally another hypoglycemic,
anti-diabetic, or anti-lipidemic agent useful for lowering blood
glucose or lowering fatty acids.
[0119] Specific Embodiments of the Present Invention
[0120] The following compounds illustrate the structure and
nomenclature of the compounds of Formula (I) and other compounds
described herein. 8
[0121] Compound A may also be known as Longistyline C;
3-hydroxy-5-methoxy-6-isoprenylstilbene;
5-hydroxy-3-methoxy-2-isoprenyls- tilbene;
5-hydroxy-3-methoxy-2-(3-methyl-2-butenyl)stilbene and
3-methoxy-4-(3-methyl-2-butenyl)-5-(trans-styryl)phenol. 9
[0122] Compound B may also be known as pinosylvin monomethyl ether;
3-hydroxy-5-methoxystilbene; and 3-methoxy-5-(trans-styryl)phenol.
10
[0123] Compound C may also be known as Longistyline A-6-carboxylic
acid; Longistyline A-2-carboxylic acid;
3-hydroxy-4-isoprenyl-5-methoxystilbene- -2-carboxylic acid;
3-hydroxy-4-(3-methyl-2-butenyl)-5-methoxystilbene-2-c- arboxylic
acid; 2-hydroxy-4-methoxy-3-(3-methyl-2-butenyl)-6-(trans-styryl-
)benzoic acid. 11
[0124] Compound D (Compound 3) may also be known as Longistyline A;
3-hydroxy-4-isoprenyl-5-methoxystilbene;
3-hydroxy-5-methoxy-4-(3-methyl-- 2-butenyl)stilbene;
3-methoxy-4-(3-methyl-2-butenyl)-5-(trans-styryl)pheno- l. 12
[0125] Compound E (compound 7, A) may also be known as
7,8-dihydrolongistyline C;
5-hydroxy-2-(3-methyl-2-butenyl)-3-methoxybibe- nzene;
3-hydroxy-6-isoprenyl-5-methoxybibenzene;
5-hydroxy-2-isoprenyl-3-m- ethoxybibenzene;
1-(3-methoxy-6-(3-methyl-2-butenyl))benzyl-4-benzylethane- . 13
[0126] Compound F (Compound 6, IA) may also be known as
7,8,2",3"-tetrahydrolongistyline C;
5-hydroxy-3-methoxy-2-(3-methylbutyl)- bibenzene;
5-hydroxy-3-methoxy-2-isopentenyl-bibenzene;
3-hydroxy-6-isopentenyl-5-methoxybibenzene;
3-hydroxy-5-methoxy-2-(3-meth- ylbutyl) bibenzene;
1-(5-hydroxy-3-methoxy-2-(3-methylbutyl)benzyl)-4-benz- ylethane.
14
[0127] Compound G (Compound 5, IA) may also be known as
7,8-dihydropinosylvin monomethyl ether;
5-hydroxy-3-methoxybibenzene; 3-hydroxy-5-methoxybibenzene; and
3-methoxy-5-(trans-styryl)phenol;
1-(3-methoxy-5-methoxybenzyl)-4-benzylethane. 15
[0128] Compound H (Compound 8) may also be known as
7,8,2",3"-tetrahydrolongistyline A-2-carboxylic acid;
3-hydroxy-4-isopentenyl-5-methoxybibenzyl-2-carboxylic acid;
3-hydroxy-5-methoxy-4-(3-methylbutyl)bibenzyl-2-carboxylic acid;
1-(3-hydroxy-5-methoxy-4-[3-methyl-2-butenyl])phenol-4-phenolethane.
[0129] The hypoglycemically active stilbenoids of formulae
described herein can be isolated directly from Cajanus spp.,
preferably from C. cajun, or chemically synthesized and isolated
from a reaction mixture. The hypoglycemically active stilbenoids of
formulae described herein, e.g., Compounds A, B, C, E, and H can
also be isolated directly from other species, e.g., Lonchocarpus
longistylus (Compound A); Pinus spp (Compound B); Lonchocarpus
violaceus (Jack) DC (Lonchocarpus longistylus Pittier) (Compound
C); Radula kojana (Compound E); and Pinus strobus var. chiapensis
(Compound G). The isolated hypoglycemically active stilbenoids of
formulae described herein can be obtained in purified form,
preferably in substantially purified form, via column
chromatography, recrystallization or other means known to those
skilled in the art. In an additional embodiment, the
hypoglycemically active stilbenoids of formulae described herein
can be obtained in a manner which results in an enriched extract or
isolate having the desired stilbenoid level in a convenient dosage
form. The hypoglycemically active stilbenoids of formulae described
herein, e.g., Compounds AA-AV, can also be isolated directly from
other plants, including, for example, Lonchocarpus violaceus
(Monache 1977); Arachis hypogea (Ingham, J. L., Phytochemistry 15,
1791); Derris rariflora (Braz Filho, R., et al., Phytochemistry 14,
261 (1975a)); D. floribunda (Braz Filho, R., et al., Phytochemistry
14, 1454 (1975b; Chlorophora excelsa (Grundon, M. F., Nature
(lond.) 163, 564 (1949)); and Radula complanata and R. kojana
(Asakawa, Y., et al., 1978(a), 1978(b), and (1991)) Keen, N. T., et
al., Phytochemistry 15, 1794 (1976) as described more fully in the
following paragraphs.
[0130] Processes for Isolating Hypoglycemically Active
Stilbenoids
[0131] Compound A-H can be isolated from Cajanus spp., preferably
C. cajun using the illustrative methods described below or other
standard extraction and purification techniques known to those of
ordinary skill in the art
[0132] Compounds A-H can be isolated from other sources by
techniques known to those of ordinary skill in the art. Compound A
has previously been reported isolated from Lonchocarpus violaceus
(Monache, et al., 1977) and from Cajanus cajun (Chung Ts'ao Yao,
1985, 18, 2). Compound B has been reported isolated from Alnus
sieboldiana (Betulaceae) (Asakawa, Y., et al., Bull.Chem.Soc.Jpn
44, 2671 (1971) (1991)). Compound C was reported isolated from
Cajanus cajun (Cooksey, C. J., et al., 1982). Compound D
(longistyline A) has been reported isolated from Lonchocarpus
violaceus (Monache, et al. (1977). Compound E (dihydrolongistyline
C) was reported isolated from Radula kojana ( Asakawa, et al,
Phytochemistry 30(1), 219-234 (1991); and Compound G has been
previously reported isolated from Pinus strobus var. chiapensis (J.
Braz. Chem. Soc. 1996, 7, 1996). Compounds F and H were prepared by
hydrogenation of Compounds A and C respectively. Compounds AA-AP
have been reported isolated by Asakawa 1978a, 1978b, 1991; Keen, N.
T., et al., 1976; Braz Filho, R., et al., 1975a; and 1975b;
Grundon, et al. 1949; and Monache, 1977. In addition, the Compounds
A-H and AA-AV so obtained can be purified by chromatography,
recrystallization or other purification methods known to those
skilled in the art.
[0133] Isolation and Purification of Hypoglycemically Active
Stilbenoids
[0134] Plant material from the natural source, for example Cajanus
spp., preferably C. cajun (Leguminoseae), is initially extracted
with a solvent to provide a crude extract containing the identified
stilbenoids. By "plant material" is meant any part of the plant,
such as bark, leaves, flowers, roots and stems. The plant material
may optionally be shredded, ground, macerated, or otherwise treated
prior to extraction. Alternatively, the plant material may already
be in a powdered, shredded, ground, macerated, or comminuted state
when used herein. Suitable extraction solvents include polar
solvents, non-polar solvents, or mixtures thereof. Useful polar
solvents include, but are not limited to, acetonitrile, methanol,
ethanol, isopropanol, acetone, butanol, ethyl acetate, water and
mixtures thereof. Useful non-polar solvents include, pentane,
hexane, heptane, higher alkane and other hydrocarbon solvents, such
as petroleum ether.
[0135] Preferably, the plant material is extracted with a polar
solvent, so as to maximize the amount of stilbenoids that can be
extracted from the plant material. More preferably, the plant
material is washed with a mixture of polar solvent and water,
wherein the ratio of water to polar solvent ranges from 1:99 to
99:1 volume/volume (v/v). Most preferably, the polar solvent is an
organic alcohol, such as methanol, ethanol, isopropanol, butanol
and the like. When the organic alcohol is ethanol, the ratio of
water to organic alcohol is preferably about 5:95 to about 95:5
(v/v), more preferably from about 10:90 to about 30:70 (v/v) and
most preferably about 20:80 (v/v).
[0136] Washing the plant material with solvent can be performed at
a temperature of about room temperature to about the reflux
temperature of the chosen solvent or solvent system, preferably at
room temperature, for between about 2 hours and 72 hours,
preferably for between about 24 hours, in order to maximize the
amount of stilbenoids that can be isolated from the plant
material.
[0137] The plant material may also be agitated, soaked-or otherwise
exposed to the solvent to facilitate the extraction process. For
example, the plant material can be mechanically mixed, sonicated,
or otherwise agitated in the solvent by methods known by those
skilled in the art.
[0138] The resulting crude extract can then filtered to remove
undesired solid or waste plant materials, e.g., spent plant
residue/materials therefrom and to afford a crude filtrate
containing the stilbenoids. Suitable filtering methods include
passing the crude extract through diatomaceous earth, e.g.,
diatomaceous earth sold under the trademark "CELITE.TM." by Fisher
Scientific Inc. (Los Angeles, Calif.); or a fritted funnel.
Centrifugation of solutions or diluted solutions of the crude
extract can also be employed to remove undesired solid
therefrom.
[0139] The crude filtrate is concentrated, preferably in vacuo, and
the resulting residue further purified by being partitioned between
two partitioning solvents, so as to enhance the yield and overall
purity of the isolated stilbenoids. It is important that the
partitioning solvents are immiscible in each other. Preferably, one
of the partitioning solvents is a non-aqueous solvent such as
toluene, diethyl ether, ethyl methyl acetate, chloroform, carbon
tetrachloride, ethyl acetate, pentane, hexane, heptane, higher
alkane (C<7) solvents, dichloromethane and other hydrocarbon
solvents, such as petroleum ether, known by those skilled in the
art to be immiscible in water or capable of dissolvating
stilbenoids. The aqueous solvent should preferably be capable of
dissolving impurities found in the plant material.
[0140] The organic phase, containing the stilbenoids, is separated,
optionally combined, and then concentrated to dryness to afford a
crude concentrate, which is enriched in stilbenoids. The previously
described extraction and filtering steps can be repeated to
increase the yield and overall purity of the isolated stilbenoids.
The crude concentrate can be further purified by standard
techniques known to those skilled in the art to ultimately afford
isolated stilbenoids. Exemplary purification techniques include
recrystallization and chromatography. Preferably, the crude
concentrate is purified using liquid chromatography, for example
high performance liquid chromatography, vacuum flash chromatography
and adsorption chromatography.
[0141] Various resin types can be utilized to achieve the desired
chromatographic effect. For example, in order to remove polar
impurities therefrom, the crude concentrate can be passed through
an adsorption resin (HP-20 grade resin sold by Mitsubishi-Kasei,
located in White Plains, N.Y.; C-18 (octadecyl) resin sold by J. T.
Baker of Phillipsburg, N.J., or Supelco of Bellefonte, Pa.; or
other silica gel) to selectively retain or pass the stilbenoids
according to polarity. Size, molecular weight, or cellulosic
characteristics of the desired resin material may be used to
separate the stilbenoids by selective use of molecular exclusion or
cellulose based resins.
[0142] An appropriate gradient solution is used to wash and
separate the stilbenoids from the crude concentrate on the column
filled with the desired resin. A suitable gradient may include an
initial solvent wash followed by an elution solvent. Suitable
elution solvents contain a high percentage of acetonitrile (ACN),
methanol, acetone, dichloromethane, ether/hexane or any other
organic solvent or mixtures thereof that can release stilbenoids
from the resin material, and into an enriched fraction. The
enriched fraction will be stilbenoids, or mixtures thereof. The
elution solvent can contain up to 50% water, so as to adjust or
optimize the polarity thereof. The type of elution solvent can
depend upon the type of resin used. For example, for HP-20 resin
equilibrated in methanol, the elution solvent can be
dichloromethane; for C-18 resin equilibrated in 70% ACN/30% water
(v/v), a gradient of increasing acetonitrile concentration is
appropriate; or for silica gel resin equilibrated in hexane, the
elution solvent can be a gradient of increasing ether in a
ether/hexane concentration solution.
[0143] High performance liquid chromatography (HPLC), thin layer
chromatography (TLC) and nuclear magnetic resonance (NMR) analysis
can be used to determine which of the eluting: fractions is an
enriched fraction, and which enriched fractions contain the desired
stilbenoids. Optionally, different eluting fractions can be
combined and subjected to the TLC and NMR analyses described above.
The enriched fractions can optionally be repurified using either
the same or a different eluent system.
[0144] The resulting fractions containing the stilbenoids are
concentrated, optionally in vacuo. The fractions containing the
stilbenoids from the chromatography methods described above can be
combined and further purified by successive iterations of the
above, or by recrystallization or other types of chromatography.
Optionally, successive recrystallization or chromatography
purifications may be performed to obtain purified stilbenoids.
[0145] Using the above purification techniques, the isolated
stilbenoids can be purified or substantially purified. By
"substantially purified" is meant that the stilbenoids of formulae
described herein have a degree of purity of at least about 95%. By
"purified" is meant that the stilbenoids of formulae described
herein have a degree of purity of at least about 97%.
[0146] Organic Synthesis of Hypoglycemically Active Stilbenoids
[0147] There are two main methods for synthesizing stilbenoids, the
earliest being variations on the Perkin condensation of a
phenylacetic acid with a benzaldehyde to form a
stilbene-.alpha.-carboxylic acid, followed by decarboxylation.
Funk, C, et al., Chem. Ber., 38, 939 (1905); Buckles, R. E., et
al., J. Am. Chem. Soc. 73, 4972 (1951); and Letcher, R. M.,
Phytochemistry 12, 2789 (1973). Moreover, the Wittig reaction
between a benzyltriphenylphos-phonium chloride or a
diethylbenzylphosphonate and a benzaldehyde has been used to give a
higher yield of a predominantly trans-stilbene. Gorham, J.,
Phytochemistry, 16, 249 (1977); Wheeler, O. H., et al., J. Org.
Chem. 30, 1473 (1965). Bibenzyls are also readily prepared from
stilbenes by catalytic hydrogenation with hydrogen in the presence
of palladium on carbon.
[0148] The compounds of formulae described herein may also be
semi-synthesized from other isolated stilbenoids.
3-hydroxy-5-methoxy-4-i- soprenylstilbene-2-carboxyolic acid
(Compound C) and
3-hydroxy-5-methoxy-6-isoprenylstilbene-2-carboxylic acid were
reported isolated from Cajanus cajun (Cooksey, C. J., et al.,
(1982)). Methylation of these compounds with diazomethane gave
3,5-dimethoxy-6-isoprenylstilbe- ne-2-carboxylic acid and
3,5-dimethoxy-4-isoprenylstilbene-2-carboxylic acid, respectively.
Cooksey, C J, et al. (1982).
[0149] Prenylated bibenzyls have been semi-synthesized from
pinosylvin and pinosylvin mono-methyl ether by hydrogenation,
treatment with sodium methoxide in methanol and then
2,2-dimethylallylbromide to give
3-hydroxy-5-methoxy-2-(3-methyl-2-butenyl)bibenzene,
3-hydroxy-5-methoxy-4-(3-methyl-2-butenyl)bibenzene, and
3-hydroxy-2,4-di(3-methyl-2-butenyl)bibenzene. Asakawa also
described synthesizing 2-geranyl-3-hydroxy-5-methoxybibenzene,
4-geranyl-3-hydroxy-5-methoxybibenzene,
2,4-digeranyl-3-hydroxy-5-methoxy- bibenzene,
2-geranyl-3,5-dihydroxybibenzene; and 2-geranyl-3,5-dimethoxybi-
benzene. Asakawa (1991).
[0150] Hypoglycemically active stilbenoids may also be synthesized
in vivo by a phenylpropanoid-polymalonate pathway. Pryce, R. J.,
Phytochemistry, 10, 2679 (1971).
[0151] Once the hypoglycemically active stilbenoids of formulae
described herein have been synthesized, they can be purified or
substantially purified, using conventional chromatography,
recrystallization or other purification techniques known to those
skilled in the art.
[0152] Derivatives of Hypoglycemically Active Stilbenoids
[0153] Also included within the scope of the present invention are
ether and acetate derivatives of stilbenoids that are useful for
lowering blood glucose. For example, the carboxylic acid groups of
the stilbenoid-2-carboxylic acids can be methylated with
CH.sub.2N.sub.2 and ether to produce the methyl ether thereof. The
hydroxy groups of the stilbenoids can also be methylated by adding
MeI in the presence of K.sub.2CO.sub.3 to a compound in Me.sub.2CO
to give mono- and di-methyl ethers. (Asakawa, 1991).
[0154] In addition, the hydroxyl groups of these stilbenoids can be
acetylated by methods well known to those skilled in the art, for
example, using acetyl chloride (Greene, Protective Groups in
Organic Synthesis 101, (1981)).
[0155] It is to be pointed out that any hydroxyl groups not so
methylated or acetylated can participate in the formation of those
pharmaceutically acceptable salts of stilbenoids described
above.
[0156] Pharmaceutical Compositions of Hypoglycemically Active
Stilbenoids
[0157] The stilbenoids of formulae described herein may be
compounded, for example with a pharmaceutically acceptable carrier
for solid compositions such as tablets, pellets or capsules;
capsules containing liquids; suppositories; solutions; emulsions;
suspensions or any other form suitable for use. Suitable carriers
include, for example, sterile water, sterile physiological saline,
gum acacia, gelatin, starch paste, talc, keratin, colloidal silica,
urea and the like. In addition, auxiliary, stabilizing, thickening,
lubricating and coloring agents may be used. The stilbenoids of
formulae described herein are present in the compositions in an
amount sufficient to produce a desired effect upon diabetes, blood
glucose levels; or hyperglycemia.
[0158] Compositions for oral administration may be in the form of
tablets, troches, lozenges, aqueous or oily suspensions, granules
or powders, emulsions, capsules, syrups or elixirs. Orally
administered compositions may contain one or more agents, such, as
sweetening agents such as fructose, aspartame or saccharin;
flavoring agents such as peppermint, oil of wintergreen, or cherry,
coloring agents and preserving agents to provide a pharmaceutically
palatable preparation. Moreover, compositions in tablet form may be
coated to delay disintegration and absorption in the
gastrointestinal tract thereby providing a sustained action over an
extended period of time. Selectively permeable membranes
surrounding an osmotically active driving compound are also
suitable orally administered compositions. In these later
platforms, fluid from the environment surrounding the capsule is
imbibed by the driving compound, which swells to displace the agent
or agent composition through an aperture. These delivery platforms
can provide an essentially zero order delivery profile as opposed
to the spiked profiles of immediate release formulations. A time
delay material such as glycerol monosterate or glycerol stearate
may also be used.
[0159] Aqueous suspensions containing the stilbenoids of formulae
described herein may also contain one or more preservatives, such
as, for example, ethyl or n-propyl-p-hydroxy-benzoate, one or more
coloring agents, flavoring agents or sweetening agents.
[0160] Dietary Supplements Containing Hypoglycemically Active
Stilbenoids
[0161] The stilbenoids of formulae described herein can be used in
the form of a food additive, food supplement, dietary supplement
for example, in solid, semisolid or liquid form, which contains at
least one of the stilbenoids of formulae described herein,
preferably longistyline C (Compound A); longistyline A-6-carboxylic
acid (Compound D); longistyline A (Compound C);
7,8-dihydrolongistyline C (Compound E);
7,8,2",3"-tetrahydro-longistyline C (Compound F); and
7,8,2",3"-tetrahydrolongistyline A-6-carboxylic acid (Compound H),
including their therapeutically active salts, as a bioactive
component. When incorporated into foodstuffs, the hypoglycemically
active stilbenoid may be used as an isolated compound or may be
contained in an enriched fraction of plant extract.
[0162] The compounds of the present invention may be incorporated
into foodstuffs alone or in combination with another antidiabetic,
antihyperglycemic (blood glucose lowering), or anti-lipidemic
compound, in admixture with a carrier or an excipient suitable for
oral administration.
[0163] Compositions for oral administration may be in the form of
foodstuffs comprising the compositions of this invention.
[0164] Any conventional food processing technique may be used to
achieve a product comprising the effective amount of the stilbenoid
compound of formula (I). There is much information on the art and
technology of the various conventional food processing techniques
and their practices in both the pet food and food industries, and
it is accordingly assumed that the general principals of these
techniques are understood by the person skilled in the art.
[0165] With the addition of a stilbenoid to a carrier material, the
method of assimilating the stilbenoid compound is not limited to
simple baking or dehydration, but may also include such techniques
as extrusion processing, coextruding, and canning. Additionally,
the process by which granola bars and food bars are prepared may be
used to prepare the present foodstuffs. Thus, various types of food
products may be produced in the practice of the invention in
addition to powder ingredients for finished foods. For example,
foodstuffs produced in the practice of the invention include dry
pet foods that serve as a complete nutritional diet for pets, as
well as biscuits and treats for pets. Additionally, the present
foodstuffs may be formed into cereals, snacks, and nutrition bars
for humans. Regardless of the method by which the present
foodstuffs are prepared or the components therein, it is preferred
that the resulting foodstuffs will provide a stilbenoid
concentration of at least about 0.1 gram per dietary unit.
[0166] In the practice of the invention, the carrier material is
contemplated to be a dry material of proteinaceous or farinaceous
character. Nonexclusive examples of suitable carrier materials
include: dried bakery product, the flours of wheat, rice, oat,
corn, and soy; the brans of wheat, rice, oat, and corn; wheat
middlings; whole ground wheat, corn gluten meal, whole ground corn,
soybean meal, barley, sorghum, meat and bone meals, poultry meal,
fish meal, dry dog food, and the like of the various materials that
typify conventional commercial and premium pet food products.
[0167] The foodstuff produced in the practice of the invention may
take any form that is edible by humans or pets, including a
complete and balanced pet food; a dry or semi-dry product that is
an additive for pet food or human food; or granola-type bars,
nutrition bars or other snacks for humans. Specifically, if the
foodstuff comprises a stilbenoid of formula (I), it is contemplated
that it will be employed as an ingredient to be incorporated into
another foodstuff. Toward that end, the stilbenoid can be in the
form of a powder or fine meal that may then serve as an ingredient
to other foods. Additional examples of foodstuffs contemplated for
human consumption that may include, as an ingredient, stilbenoids
processed in accordance with the invention include fillings or
puddings (similar to gelatins and JELLO products), as well as
performance foods in liquid gel form and canned soups.
[0168] Methods for Use of the Hypoglycemically Active
Stilbenoids
[0169] Due to the activity of the stilbenoids of the present
invention, the stilbenoids of formulae described herein, including
longistyline C (Compound A); longistyline A-6-carboxylic acid
(Compound D); longistyline A (Compound C);7,8-dihydrolongistyline C
(Compound E); 7,8, 2",3"-tetrahydrolongistyline C (Compound F); and
7,8,2",3"-tetrahydrolong- istyline A-6-carboxylic acid (Compound
H)or pharmaceutically acceptable salts thereof are advantageously
useful pharmaceutical compositions and dietary supplements. Such
compositions and dietary supplements may be used to treat mammals
suffering from hyperglycemia or diabetes or in veterinary and human
medicine for therapeutic treatment of complications that result in
hyperglycemia. In one embodiment of the present invention the
pharmaceutical compositions or dietary supplements are used to
lower blood glucose in mammals with type I or type II diabetes.
Additionally, the stilbenoids of formulae described herein can be
advantageously used as hypoglycemic agents to reduce blood glucose
in situations of acute stress such as experienced by animals or
patients with hyperthermia, trauma, sepsis, burns or those
undergoing general anesthesia. Hyperglycemia sometimes associated
with severe head injury, cerebral-thrombosis, encephalitis and heat
stroke can also be therapeutically treated with the stilbenoids of
formulae described herein. Additionally, the stilbenoids of
formulae described herein are useful as hypoglycemic agents for
rare congenital metabolic glycogen storage disease associated with
hyperglycemia. The stilbenoids of formulae described herein used in
the methods described herein are particularly suited to control
hyperglycemia in patients whose blood glucose cannot be controlled
by diet alone. Furthermore, the stilbenoids of formulae described
herein are capable of lowering blood glucose levels without an
accompanying increase in urine glucose levels.
[0170] When administered to a mammal for veterinary use or to a
human for clinical use, the stilbenoids of formulae described
herein are administered in isolated form. By "isolated" is meant
that the stilbenoids of formulae described herein are separated
from other components of either (a) a natural source such as a
plant or cell culture, or (b) a synthetic organic chemical reaction
mixture. Preferably, via conventional techniques, the stilbenoids
of formulae described herein are substantially purified, preferably
purified.
[0171] When administered to a mammal for veterinary use or to a
human for clinical use, the stilbenoids of formulae described
herein can be used alone or in combination with any physiologically
acceptable carrier or excipient suitable for enteral or parenteral
administration. Where used for parenteral administration, the
physiologically acceptable carrier must be sterile and suitable for
in vivo use in a human, or for use in a veterinary clinical
situation.
[0172] The compositions of this invention may be administered by a
variety of methods including orally, intramuscularly,
intravenously, subcutaneously, transdermally, rectally or by
inhalation. While the preferred mode of administration is through
the oral mode, the precise mode of administration is left to the
discretion of the practitioner. They are advantageously effective
when administered orally.
[0173] This invention comprises the use of a stilbenoid, preferably
in isolated or purified form administered at a dose of about 1 to
about 1,000 mg per kg of body weight per day, preferably from about
2 to about 500 mg per kg of body weight per day, more preferably
about 5 to about 350 mg per kg of body weight per day, still more
preferably about 50 to about 350 mg per kg of body weight per day.
In still a further embodiment, the invention comprises the use of a
stilbenoid of formulae described herein at a dose of about 5 to
about 350 mg/kg body weight/day of compound to be utilized in an
amount which results in the compositions exhibiting a
therapeutically effective hypoglycemic, antihyperglycemic or
antidiabetic activity. The dosage of the present compositions for
treatment or prevention of hyperglycemia or diabetes or for
reducing blood glucose levels, depends on the route and frequency
of administration was well as the age, weight and physical
condition of the patient. Generally the daily dosage is in the
range of about 1 to about 1,000 mg per kg of body weight per day,
preferably from about 2 to about 500 mg per kg of body weight per
day, more preferably about 5 to about 350 mg per kg of body weight
per day, still more preferably about 50 to about 350 mg per kg of
body weight per day. Treatment can be repeated as needed, depending
upon the dosage and need, for example, a dosage of about 62.5, 125
or 250 mg/kg body weight/day of patient animal can be administered
in dividing doses to prevent or treat diabetes or hyperglycemia or
to lower blood glucose. Treatment can be continued, for example,
reduced to the desired until the blood glucose level is level or to
be maintained at a desired level. The appropriate dosage of the
compositions can be readily determined by the skilled medical
practitioner.
[0174] For the treatment of diabetes, hyperglycemia effecting a
lowering of blood glucose, a composition of present invention may
be administered which contains a stilbenoid of formulae described
herein or a pharmaceutically or the acceptable salt thereof as
described above, together with another antidiabetic,
antihyperglycemic or blood glucose lowering agent including, but
not limited to insulin; a biguanide such as metformin or buformin;
a sulfonylurea such as acetohexamide, chlorpropamide, tolazamide,
tolbutamide, glyburide, glypizide or glyclazide; a PPAR.gamma.
agonist including but not limited to the thiazolidinediones (such
as troglitazone); an .alpha.-glucosidase inhibitor such as acarbose
or miglitol; a Pradrenoceptor agonist such as PL-316, 243, etc.,
cholestyramine, clofibrate, colestipol, fluvastatin, gemfibrozil,
lovastatin, niacin, pravastatin, probucol, psyllium hydrophilic
muccilloid, simvastatin, and sodium dichloroacetate. Alternatively,
the compositions comprising a hypoglycemically active stilbenoid or
a pharmaceutically acceptable salt thereof can be administered in
combination with, prior to, concurrent with or subsequent to the
administration of another antidiabetic, antihyperglycemic, or
anti-lipidemic agent as described supra.
[0175] Although the present inventors do not wish to be limited to
any particular mechanism of action to explain the hypoglycemic
activity of the stilbenoids of formulae described herein, it is
envisaged that they may advantageously be useful for treatment of
both insulin-dependent or type I diabetes (formerly termed
juvenile-onset or ketosis prone diabetes) and non-insulin-dependent
or type II diabetes (formerly termed adult-onset, maturity onset or
nonketotic diabetes). The stilbenoids of formulae described herein
can optionally be administered in an effective amount as
pharmaceutically acceptable carboxylate or phenolate salts using
counter ions such as sodium, potassium, lithium, calcium,
magnesium, zinc and iron.
[0176] The following examples are set forth to assist in
understanding the invention and should not, of course, be construed
as specifically limiting the invention described and claimed
herein. Such variations of the inventions which would be within the
purview of those in the art, including the substitution of all
equivalents now known or later developed, including changes in
formulation or minor changes in experimental design, are to be
considered to fall within the scope of the invention incorporated
herein.
EXAMPLES
Example Isolation and Characterization of Stilbenoid Compounds
[0177] Materials and Methods
[0178] Analytical high performance liquid chromatography (HPLC) was
performed on a Hitachi Model D-6500 Chromatography Data Station
equipped with a D-6000 interface, L-6200A pump, AS-2000
autosampler, L4500A diode array detector and a Sedex 55 light
scattering detector connected in series, and a Primesphere C18 HC,
4.times.50 mm (5 .mu.m) HPLC column. All chromatographic runs were
performed at ambient temperature. HPLC grade solvents were used
without further purification.
[0179] Nuclear magnetic resonance (NMR) spectra were recorded on a
Varian Unity Plus 400 or a Varian Unity 400 spectrometer. NMR
spectra of compounds were recorded in deuterated chloroform. One
and two-dimensional NMR experiments, including .sup.1H NMR,
.sup.13C NMR, Heteronuclear Multiple Quantum Correlation (HMQC),
and Heteronuclear Multiple Bond Correlation (HMBC) provided
molecular structure information. Mass spectra were recorded on a
Kratos MS-50 in high resolution power electron impact scanning
mode, 70 eV. Resolution was set to 2000 with a scanning rate of 10
sec/decay. Samples were run on a temperature gradient from 500 to
300.degree. C., increased at a rate of 50.degree./min. Infrared
spectra were recorded on a Perkin-Elmer 1600 Series FTIR.
Ultraviolet spectra were taken directly from the Hitachi
diode-array UV detector on the HPLC system.
Isolation of Stilbene Compounds Using Solvent Extraction
[0180] Small Scale Extraction and Isolation
[0181] Ground Leaf material of Cajanus cajun (4.2 kg) was extracted
with 50 L of 80% ethanol (EtOH) (80% ethanol:20% water v/v) by
stirring mechanically at room temperature for 24 hours. The
solution was then filtered through CELITE.TM. and the filtrate was
evaporated to dryness under vacuum. The residue was subsequently
dried in a vacuum oven overnight to give 521 g. This was
partitioned between DCM (10 L) and H.sub.2O (10 L). The DCM layer
was passed through CELITE.TM. and evaporated to dryness under
vacuum to give 225 g of material. From this, 30.0 g was adsorbed
onto 37 g Si gel and added to an open column containing 180 g Si
gel. The column was eluted with hexane and increasing percentages
of ethyl acetate (EtOAc). A total of seven fractions were
collected. Compounds B (0.59 g), Compound A (1.05 g) and Compound C
(1.21 g) from fraction four were obtained by preparative HPLC
separation (MeCN/H.sub.2O gradient, 60 mL/min). Preparative HPLC
(MeCN/H.sub.2O gradient, 60 mL/min.) of fraction two yielded
Compound D (0.17).
[0182] Preparation of Enriched Stilbenoid Extracts of Cajanus
spp
[0183] Ground Leaf material of Cajanus Cajun (1245 kg) was stirred
in 10 L of ethanol/water (80:20 v:v) for 24 hours at room
temperature with constant mixing. The ethanol solution was filtered
through a fritted funnel ("coarse" pore size) under vacuum,
evaporated in vacuo to dryness and left in a vacuum oven to dry
overnight to provide 200 g of a dark brown material (Enriched
Extract 1 (EE1)).
[0184] 185 g of EE1 was sonicated and stirred into 3 L of
dichloromethane (DCM) and 3 L water to dissolve. This mixture was
transferred to a separatory funnel and left overnight at room
temperature. Any clear lower layer was removed and retained for
latter pooling. In the presence of an emulsion within the DCM
fraction or at the DCM/water interface, the cloudy emulsion portion
of the lower layer (DCM) was removed. The cloudy lower layer
portion was removed, centrifuged, transferred back to the
separatory funnel and allowed to separate again. This process was
repeated to complete the separation between the water and DCM
layers.
[0185] The lower layer (DCM) was evaporated to dryness under vacuum
to yield pre-Enriched Extract 2 (pre-EE2). After concentration, the
water layer was freeze dried to yield Enriched Extract 3 (EE3).
[0186] The resulting pre-EE2 was dissolved in 2 L of 90%
ethanol/water and added to 1.5 L petroleum ether (pet ether). This
mixture was transferred to a separatory funnel and allowed to
separate into an upper layer (Pet ether) and lower layer (90%
ethanol). The lower layer was repeatedly subjected to biphasic
separation until the pet ether layer was light in color. In one
instance, the lower layer was extracted in 800 ml pet ether and
then again in 500 ml pet ether. All of the pet ether fractions were
combined and evaporated under vacuum to yield an enriched extract
compound. The remaining ethanol fraction was evaporated under
vacuum to yield and the final Enriched Extract 2 (EE2).
[0187] A water-soluble decoction, Enriched Extract 4 (EE4) was
prepared as follows:
[0188] 300 g of unground leaves in a water permeable bag (tea bag)
was immersed in boiling water allowed to boil for 15 minutes. At
the end of 15 minutes, the heater was turned off and the mixture
was allowed to stand overnight at room temperature. The supernatant
was passed through cheese cloth and then filter paper. The bag was
squeezed to remove residual fluid, which was centrifuged and
filtered. The filtrate was concentrated to about 375 ml. The
resulting filtrate was precipitated by adding 1.6 L isopropanol to
the filtrate and incubating the mixture in a refrigerator (4 C.)
overnight. The mixture was then filtered through a flitted funnel
("coarse") under vacuum. The supernatant was evaporated to dryness,
redissolved in water and freeze-dried to yield 24.16 g of Enriched
Extract 4 (EE4). 16
[0189] Ground leaf material of Cajanus cajun (11 kg) was stirred in
110 L of methanol (MeOH). The MeOH solution was filtered through 1
kg of CELITE.TM., and evaporated in vacuo to give 1.39 kg of a
green oily material. This material was triturated with 20 L of
acetone for four hours. The acetone mixture was vacuum filtered
through 1 kg of CELITE.TM. and the filtrate evaporated to dryness
giving 721 g of solids. The solids (538 g) were extracted with 4 L
MeOH to which 1 L of H.sub.2O was slowly added with mixing. The
resulting milky suspension was pumped onto an 18.times.92 cm column
containing HP 20 (Mitsubishi) sorbent. The column was eluted with
95:5 MeOH/H.sub.2O). Fourteen 10 L fractions were collected.
Fractions 7-10 were pooled and evaporated in vacuo giving 64 g of
an oily solid. This material was dissolved in 6.5 L of MeOH to
which the 3.5 L of H.sub.2O was added along with 10 mL of HOAc.
After 30 minutes, the mixture was filtered through Whatman #2
filter paper. Both the MeOH precipitation solids and MeOH
precipitation filtrate were kept for further processing. The MeOH
precipitation solids were dried in a vacuum oven overnight at
40.degree. C. This gave 10 g of a white amorphous powder, which was
placed into an Erlenmeyer flask along with 500 mL on n-hexane. With
good mixing, acetone was added until the solution became clear. One
gram of decolorizing charcoal was added to the stirred solution,
which was then vacuum filtered through a bed (2 g) of CELITE.TM..
The filtrate was allowed to stand open in a hood overnight
resulting in the precipitation of solids. The supernatent was
decanted and 40 mL of hexane and 5 mL of acetone were added back to
the solids, which formed two layers upon standing at -10.degree. C.
This gave 10 g of a white amorphous powder, which was placed into
an Erlenmeyer flask along with 500 mL of n-hexane. With good
mixing, acetone was added until the solution became clear. One gram
of decolorizing charcoal was added to the stirred solution, which
was then vacuum filtered through a bed (2 g) of CELITE.TM.. The
filtrate was allowed to stand open in a hood overnight resulting in
the precipitation of solids. The supernatant was decanted and 40 mL
of hexane and 5 mL of acetone were added back to the solids, which
formed two layers upon standing at -10.degree. C. for two hours.
The layers were separated and the top layer was allowed to stand
uncovered overnight, yielding colorless crystalline solids. The
crystalline material was triturated with a minimal amount of 10:1
hexane/acetone and allowed to stand in a closed container at room
temperature overnight. The supernatant was decanted and the
crystals dried in a vacuum oven giving 6 g of material identified
as Compound C from NMR and HPLC diode array data. Yield from plant
was 0.07%. 17
[0190] The MeOH precipitation filtrate (10 L) was passed through a
small amount of C18 (Bakerbond, 40 mm) pumped onto a 100.times.5 cm
C18 chromatography column which had been washed with MeOH and
equilibrated with 65:35:0.1 MeOH/water/HOAc. An additional 500 mL
of 65:35:0.1 MeOH/water/HOAc was pumped through the column in order
to complete the loading. The column was eluted with 80:20:0.1
MeOH/water/HOAc. Thirty-two fractions, each containing 1 L, were
collected. Fractions 12-18 were pooled and evaporated in vacuo to
give 18 g of solids. The solids (11.1 g) were dispersed in 700 mL
of hexane with stirring. To the dispersion was added enough DCM
(200 mL) to produce a clear solution. To this solution was added 1
g of decolorizing charcoal. The suspension was stirred for 1 hour
then filtered through a bed of CELITE.TM. (39 g). The CELITE.TM.
bed was washed with an additional 100 mL of 7:2 hexane/CH2Cl2. The
combined solutions (1000 mL) were slowly (3 Hours) evaporated using
a stream of nitrogen to a volume of 300 mL. The solids were
filtered, dried in a vacuum oven and recrystallized as follows. The
dried material (8 g) was dissolved into 10 mL of DCM to which was
added 70 mL of hexane with stirring. The solution was allowed to
stand uncovered until solids formed. The container was then covered
and allowed to stand overnight at -10.degree. C. The resulting
first crop of crystals was filtered and set aside. The supernatant
was concentrated using a stream of nitrogen and a second crop of
crystals was collected. The first and second crops were combined
and dried in a vacuum oven overnight to give 5.4 g of Compound A as
an off-white solid. Identification was made from NMR and HPLC
diode-array data. Yield from plant materials was 0.1%. 18
Semi-Synthetic Preparation of Stilbenoid Compounds
[0191] All agents were used as received. All moisture sensitive
reactions were done under a nitrogen atmosphere, using dry
solvents; air sensitive reactions were done under a nitrogen
atmosphere. Evaporation of solvents were done at room temperature
unless otherwise noted. Column chromatography was performed on C-18
preparative HPLC (Bakerbond). .sup.1H NMR and .sup.13C NMR were
obtained at 400 MHz and 100 Mhz, respectively. Elemental analyses
were performed by the Analytical Services Department at the
University of California, Berkeley. Melting points are uncorrected.
19
[0192] A solution of Compound A (394 mg) in MeOH (10 mL) was
hydrogenated over 10% Pd charcoal (41 mg) at room temperature for 3
hours under 50 psi H.sub.2. The catalyst was then removed by
filtration. The product was purified by C-18 preparative HPLC
(Bakerbond), using a gradient of acetonitrile and water as the
solvent system, to yield 289 mg of Compound E as an off-white
solid. UV (MeCN/H2O, 8:2) .lambda..sub.max 284 nm; IR (thin film)
.nu. 3354, 2929, 1604, 1456, 1304, 1189, 1140, 1086 cm.sup.-1;
.sup.1H NMR and .sup.13C NMR; EIMS m/z 296, 239, 191, 163, 137;
HREIMS m/z 296.1761 (M+, .DELTA.5.2 ppm from calcd) and 26 mg of
Compound F; UV (MeCN/H2O, 8:2) .lambda..sub.max 282 nm; IR (thin
film) .nu.3354, 2951, 1604, 1467, 1304, 1195, 1146 cm.sup.-1;
.sup.1H NMR and .sup.13C NMR; EIMS m/z 298, 241, 193, 151, 137;
HREIMS m/z 298.1927 (M+, .DELTA.2.0 ppm from calcd). 20
[0193] A solution of Compound B (500 mg) in MeOH (15 mL) was
hydrogenated over 20% Pd charcoal (99 mg) at room temperature for 3
hours under 50 psi H.sub.2. The catalyst was then removed by
filtration. The product was purified by C-18 preparative HPLC,
using a gradient of acetonitrile and water as the solvent system,
to yield 237 mg of Compound G as a brown oil. UV (MeCN/H2O, 8:2)
.lambda..sub.max 278 nm; IR (thin film) .nu.3365, 2929, 1598, 1456,
1195, 1146, 1059 cm.sup.-1; .sup.1H NMR and .sup.13C NMR; EIMS m/z
228, 137, 91; HREIMS m/z 228.1167 (M+, .DELTA.7.4 ppm from calcd)
21
[0194] A solution of Compound C (212 mg) in MeOH (10 mL) was
hydrogenated over 20% Pd charcoal (43 mg) at room temperature for 3
hours under 50 psi H.sub.2. The catalyst was then removed by
filtration. The product was purified by C-18 preparative HPLC,
using a gradient of acetonitrile and water as the solvent system,
to yield 175 mg of Compound H as a white solid. UV (MeCN/H2O, 8:2)
.lambda..sub.max 269 nm; IR (thin film) .nu.3398, 2951, 1609, 1456,
1271, 1140 cm.sup.-1; .sup.1H NMR and .sup.13C NMR; EIMS m/z 298,
268, 241, 207, 151, 137, 91; HREIMS m/z 342.1834 (M+, .DELTA.0.9
ppm from calcd.).
[0195] Structure Elucidation of the Stilbene Compounds 22
[0196] Compound A (Longistyline C) was isolated previously from
Lonchocarpus violaceus (Lloydia, 1977, 40, 201) and Cajanus cajun
(Chung Ts'ao Yao, 1985, 18, 2). The molecular formula of Compound A
was established as C.sub.20H.sub.22O.sub.2 based on a peak in the
HREIMS at m/z 294.1634 (M.sup.+, .DELTA.4.8 ppm from calcd.). An IR
spectrum of Compound A revealed absorbances at .nu. (cm.sup.-1):
3337, 2926, 1594, 1455, 1430, 1355, and 1316. The structure of
Compound A was elucidated by careful interpretation of spectral
data. Assignments were based on one and two-dimensional NMR
experiments known to those skilled in the art of structure
elucidation and included .sup.1H NMR, .sup.13C NMR, Heteronuclear
Multiple Quantum Correlation (HMQC), and Heteronuclear Multiple
Bond Correlation (HMBC). Values for the .sup.1H NMR and .sup.13C
NMR chemical shifts of Compound A are given in the table below.
Long range proton-carbon correlations observed in the HMBC spectrum
are also listed.
1 NMR Data for Compound A Spectra obtained in CDCl.sub.3 .sup.13C
NMR @ 100 MHz: .delta.; .sup.1H NMR @ 400 MHz: .delta., integral,
multiplicity, J Position .sup.13C NMR .sup.1H NMR HMBC Correlations
1 137.9 -- 2 121.0 -- 3 158.5 -- 4 98.5 6.38 1H, d 2.8 Hz C-2, C-3,
C-5, C-6 5 154.3 -- 6 104.1 6.70 1H, d 2.8 Hz C-2, C-4, C-5, C-7 7
126.4 7.33 1H, d 16 Hz C-1', C-2, C-6 8 130.5 6.94 1H, d 16 Hz C-1,
C-2', (C-6') 1' 137.5 -- 2'/6' 126.5 7.49 2H, m C-8, C-4' 3'/5'
128.6 7.37 2H, m C-1' 4' 127.6 7.27 1H, m C-2' (C-6') 3-OCH.sub.3
55.7 3.81 3H, s C-3 1" 24.4 3.43 2H, d 7 Hz C-1, C-2, C-3, C-2",
C-3" 2" 123.4 5.12 1H, t 7 Hz C-4", C-5" 3" 130.9 -- 4" 17.9 1.81
3H, s C-2", C-3", C-5" 5" 25.7 1.69 3H, s C-2", C-3", C-4"
[0197] 23
[0198] Compound B (Pinosylvin monomethyl ether) was isolated
originally in 1939 from the heartwood of Pine (Liebigs Ann. Chem.,
1939, 539, 116). Subsequently, it has been found in over 60
different Pinus species (Prog. in Phytochemistry, 1980, 6, 203) and
bovine urine (J. Nat. Prod., 1983, 46, 852). However, this compound
has not been reported previously from Cajanus cajan. The molecular
formula for Compound B was determined to be C.sub.15H.sub.14O.sub.2
by the appearance of a peak in the HRFABMS at m/z 294.1633
(M.sup.+, .DELTA.4.5 ppm from calcd.). The IR spectrum of Compound
B revealed absorbances at .nu. (cm.sup.-1): 3435, 1609, 1451, 1422,
and 1086. The structure of Compound B was elucidated by careful
interpretation of the spectral data. Values for the .sup.1H NMR and
.sup.13C NMR chemical shifts of Compound B are given in the table
below. Long range proton-carbon correlations observed in the HMBC
spectrum are also listed.
2 NMR Data for Compound B Spectra obtained in CDCl.sub.3 .sup.13C
NMR @ 100 MHz: .delta.; .sup.1H NMR @ 400 MHz: .delta., integral,
multiplicity, J Position .sup.13C NMR .sup.1H NMR HMBC Correlations
1 139.7 -- 2 105.0 6.68 1H, dd 2.0 Hz C-3, C-4, C-6, C-7 3 161.1 --
4 101.0 6.40 1H, dd 2.0 Hz C-2, C-3, C-5, C-6 5 156.8 -- 6 106.0
6.65 1H, dd 2.0 Hz C-2, C-4, C-5, C-7 7 128.3 7.00 1H, d 16 Hz C-2,
C-6, C-8, C-1' 8 129.4 7.06 1H, d 16 Hz C-1, C-7, C-2' (C-6') 1'
137.0 -- 2'/6' 126.6 7.50 2H, m C-8, C-4' 3'/5' 128.7 7.37 2H, m
C-1' 4' 127.8 7.29 1H, m C-2' (C-6') 3-OCH.sub.3 55.4 3.82 3H, s
C-3
[0199] 24
[0200] Compound C was reported previously from Cajanus cajan
(Phytochemistry, 1982, 21, 2935). Compound C was determined to have
a molecular formula of C.sub.21H.sub.22O.sub.4 based on the
presence of a peak at m/z 337.1473 (.DELTA.9.9 ppm from calcd.) in
the HRFABMS corresponding to [M-H].sup.-. The IR spectrum of
Compound C showed absorbances at .nu. (cm.sup.-1): 3422, 2968,
1702, 1629, 1451, 1277, 1170, and 1117. The structure of Compound C
was elucidated through careful examination of the spectral data.
Values for the .sup.1H NMR and .sup.13C NMR chemical shifts of
Compound C are given in the table below. Long range proton-carbon
correlations observed in the HMBC spectrum are also listed.
3 NMR Data for Compound C Spectra obtained in CDCl.sub.3 .sup.13C
NMR @ 100 MHz: .delta.; .sup.1H NMR @ 400 MHz: .delta., integral,
multiplicity, J Position .sup.13C NMR .sup.1H NMR HMBC Correlations
1 141.9 -- 2 103.3 6.67 1H, s C-1", C-1, C-3, C-4, C-6, C-7, 6-COOH
3 162.4 -- 4 116.7 -- 5 162.2 -- 6 103.1 -- 7 130.4 7.87 1H, d 16
Hz C-1', C-1, C-2, C-6, C-8 8 130.7 6.84 1H, d 16 Hz C-1, C-7,
C-1', C-2' (C-6') 6-COOH 175.7 4.70 1H, br s 3-OCH.sub.3 55.7 3.96
3H, s C-3 5-OH -- 11.58 1H, s C-4, C-5, C-6, 1' 137.3 -- 2'/6'
126.8 7.55 2H, m C-8, C-4' 3'/5' 128.7 7.40 2H, m C-1' 4' 127.8
7.30 1H, m C-2' (C-6') 1" 22.1 3.39 2H, d 7 Hz C-3, C-4, C-2", C-3"
2" 121.9 5.23 1H, t 7 Hz C-1", C-4", C-5" 3" 132.0 -- 4" 17.8 1.81
3H, s C-2", C-3", C-5" 5" 25.8 1.70 3H, s C-2", C-3", C-4"
[0201] 25
[0202] Compound D (Longistyline A) was isolated previously from
Lonchocarpus violaceus (Lloydia, 1977, 40, 201) and Cajanus cajun
(Chung Ts'ao Yao, 1985, 18, 2). The molecular formula of Compound D
was established as C.sub.20H.sub.22O.sub.2 on the basis of a peak
in the HREIMS at m/z 226.0981 (M.sup.+, .DELTA.5.6 ppm from
calcd.). The IR spectrum showed absorbances at .nu. (cm.sup.-1):
3328, 3027, 2940, 2839, 1592, 1497, 1454, and 1347. The structure
of Compound D was elucidated through comparison with literature and
careful examination of the spectral data including .sup.1H NMR,
.sup.13C NMR, Heteronuclear Multiple Quantum Correlation (HMQC),
and Heteronuclear Multiple Bond Correlation (HMBC). Values for the
.sup.1H NMR and .sup.13C NMR chemical shifts of Compound D are
given in the table below. Long range proton-carbon correlations
observed in the HMBC spectrum are also listed.
4 NMR Data for Compound D Spectra obtained in CDCl.sub.3 .sup.13C
NMR @ 100 MHz: .delta., .sup.1H NMR @ 400 MHz: .delta., integral,
multiplicity, J Position .sup.13C NMR .sup.1H NMR HMBC Correlations
1 136.6 -- 2 101.6 6.65 1H, d 1.6 Hz C-3, C-4, C-6, C-7 3 158.1 --
4 115.1 -- 5 155.5 -- 6 107.2 6.68 1H, d 1.2 Hz C-2, C-4, C-5, C-7
7 128.7 7.03 1H, d 16 Hz C-1', C-8 8 128.4 7.06 1H, d 16 Hz C-1,
C-2' (C-6') 1' 137.3 -- 2'/6' 126.5 7.52 2H, m C-8, C-4' 3'/5'
128.6 7.37 2H, m C-1' 4' 127.5 7.27 1H, m C-2' (C-6') 5-OH -- 5.38
1H, br s 3-OCH.sub.3 55.8 3.88 3H, s C-3 1" 22.4 3.43 2H, d 7 Hz
C-3, C-4, C-5, C-2", C-3" 2" 121.9 5.26 1H, t 7 Hz C-4", C-5" 3"
134.4 -- 4" 17.8 1.83 3H, s C-2", C-3", C-5" 5" 25.8 1.76 3H, s
C-2", C-3", C-4"
[0203] 26
[0204] Compound E (Dihydrolongistyline C) was prepared by
hydrogenation of Compound A. Compound E was reported previously as
a natural product isolated from Radula kojana (Phytochemistry,
1991, 30, 219). The molecular formula, C.sub.20H.sub.24O.sub.2, was
confirmed by a peak at m/z 296.1761 (M.sup.+, .DELTA.5.25 ppm from
calcd.) in the HREIMS of SP-36306. The IR spectrum of Compound E
showed absorbances at .nu. (cm.sup.-1): 3354, 2929, 1604, 1456,
1034, 1190, 1140, and 1086. The structure of Compound E was
elucidated through comparison with literature and careful
interpretation of the spectral data. Values for the .sup.1H NMR and
.sup.13C NMR chemical shifts of Compound E are given in the table
below.
5 NMR Data for Compound E Spectra obtained in CDCl.sub.3 .sup.13C
NMR @ 100 MHz: .delta., .sup.1H NMR @ 400 MHz: .delta., integral,
multiplicity, J Position .sup.13C NMR .sup.1H NMR 1 .sup.a141.97 --
2 120.6 -- 3 158.6 -- 4 97.0 6.32 1H, d 2.4 Hz 5 154.2 -- 6 107.9
6.29 1H, d 2.4 Hz 7 35.2 2.86 4H, s 8 37.5 2.86 4H, s 1'
.sup.a142.02 -- 2'/6' 128.3 7.20-7.35 5H, m 3'/5' 128.3 7.20-7.35
5H, m 4' 125.9 7.20-7.35 5H, m 3-OCH.sub.3 55.6 3.80 3H, s 1" 24.4
3.32 2H, d 6.4 Hz 2" 123.8 5.08 1H, t 7 Hz 3" 130.7 -- 4" 17.9 1.76
3H, d 0.8 Hz 5" 25.7 1.69 3H, d 1.2 Hz .sup.a: values
interchangeable
[0205] 27
[0206] Compound F (Tetrahydrolongistyline C) was prepared by
hydrogenation of Compound A and possesses a molecular formula of
C.sub.20H.sub.26O.sub.2 as evidenced by a peak in the HREIMS of
Compound F at m/z 298.1927 (M.sup.+, .DELTA.2.05 ppm from calcd.).
The IR spectrum of Compound F showed absorbances at .nu.
(cm.sup.-1): 3354, 2951, 1604, 1467, 1304, 1195, and 1146. The
structure of Compound F was elucidated through careful
interpretation of the spectral data, including .sup.1H NMR and
.sup.13C NMR. Values for the .sup.1H NMR and .sup.13C NMR chemical
shifts of Compound F are given in the table below.
6 NMR Data for Compound F Spectra obtained in CDCl.sub.3 .sup.13C
NMR @ 100 MHz: .delta., .sup.1H NMR @ 400 MHz: .delta., integral,
multiplicity, J Position .sup.13C NMR .sup.1H NMR 1 .sup.a141.5 --
2 122.1 -- 3 158.7 -- 4 96.9 6.28 1H, d 2.4 Hz 5 154.0 -- 6 107.8
6.31 1H, d 2.4 Hz 7 35.2 2.85 4H, s 8 37.9 2.85 4H, s 1'
.sup.a142.0 -- 2'/6' .sup.b128.3 7.20-7.35 5H, m 3'/5' .sup.b128.4
7.20-7.35 5H, m 4' 126.0 7.20-7.35 5H, m 3-OCH.sub.3 55.5 3.79 3H,
s 1" 28.6 2.57 2H 2" 39.6 1.33 2H 3" 23.4 1.64 1H 4" 22.6 0.98 3H
5" 22.6 0.96 3H .sup.a-b: values interchangeable
[0207] 28
[0208] Compound G (Dihydropinosylvin monomethyl ether) has been
reported previously as a natural product isolated from Pinus
strobus var. chiapensis (J. Braz. Chem. Soc. 1996, 7, 187).
Compound G was prepared by hydrogenation of Compound B. The
molecular formula for Compound G, C.sub.15H.sub.16O.sub.2, was
established based on a peak at m/z 228.1167 (M.sup.+, .DELTA.7.36
ppm from calcd.) in the HREIMS of SP-36308. The IR spectrum of
Compound G showed absorbances at .nu. (cm.sup.-1): 3365, 2929,
1598, 1456, 1195, 1146, and 1059. The structure of Compound G was
elucidated through comparison with the literature and careful
interpretation of the spectral data, including .sup.1H NMR and
.sup.13C NMR. Values for the .sup.1H NMR and .sup.13C NMR chemical
shifts of SP-36308 are given in the table below.
7 NMR Data for Compound G Spectra obtained in CDCl.sub.3 .sup.13C
NMR @ 100 MHz: .delta., .sup.1H NMR @ 400 MHz: .delta., integral,
multiplicity, J Position .sup.13C NMR .sup.1H NMR 1 144.5 -- 2
106.8 6.28 2H, m 3 160.8 -- 4 99.1 6.34 1H, m 5 156.5 -- 6 107.9
6.28 2H, m 7 37.5 2.87 4H, m 8 37.9 2.87 4H, m 1' 141.6 -- 2'/6'
.sup.a128.3 7.19-7.32 5H, m 3'/5' .sup.a128.4 7.19-7.32 5H, m 4'
125.9 7.19-7.32 5H, m 3-OCH.sub.3 55.2 3.76 3H, s .sup.a: values
interchangeable
[0209] 29
[0210] Compound H was prepared by hydrogenation of Compound C. The
molecular formula of Compound H was determined to be
C.sub.21H.sub.26O.sub.4 based on a peak in the HREIMS at m/z
342.1834 (M.sup.+, .DELTA.0.89 ppm from calcd.). The IR spectrum of
Compound H showed absorbances at .nu. (cm.sup.-1): 3398, 2951,
1609, 1457, 1271, and 1140. The structure of Compound H was
elucidated through careful examination of the spectral data. Values
for the .sup.1H NMR and .sup.13C NMR chemical shifts of Compound H,
along with the long range proton-carbon correlations, are given in
the table below.
8 NMR Data for Compound H Spectra obtained in CDCl.sub.3 .sup.13C
NMR @ 100 MHz: .delta., .sup.1H NMR @ 400 MHz: .delta., integral,
multiplicity, J Position .sup.13C NMR .sup.1H NMR HMBC Correlations
1 145.3 -- 2 103.5 6.21 1H, s C-3, C-4, C-6, C-7, 6-COOH 3 163.0 --
4 116.8 -- 5 162.3 -- 6 106.1 -- 7 39.2 3.27 2H, m C-1', C-1, C-2,
C-6, C-8 8 38.2 2.94 2H, m C-1, C-7, C-1', C-2' (C-6') 6-COOH 175.4
-- 3-OCH.sub.3 55.5 3.79 3H, s C-3 5-OH -- 11.62 1H, s C-4, C-5,
C-6, 1' 141.9 -- 2'/6' 128.5 7.21 2H, m C-4', C-8 3'/5' 128.3 7.31
2H, m C-1', C-2' (C-6') 4' 125.9 7.22 1H, m 1" 20.7 2.57 2H, m C-3,
C-4, C-5, C-2", C-3" 2" 37.9 1.38 2H, m C-4, C-1", C-3", C-4", C-5"
3" 28.3 1.60 1H, m C-1", C-4", C-5" 4" 22.6 0.97 3H, s C-2", C-3",
C-5" 5" 22.6 0.95 3H, s C-2", C-3", C-4"
[0211] Hypoglycemic Activity of the Stilbenoids
[0212] The following examples illustrate the effectiveness of the
stilbenoids of formulae described herein in reducing plasma glucose
levels in C57BL/ks diabetic (db/db) mice, i.e., an art-recognized
model of non-insulin dependent diabetes mellitus (NIDDM).
[0213] A representative sampling of stilbene analogues were tested
in the in vivo mouse model described below.
[0214] In vivo Experiments; General Protocols
[0215] The following experiments are set forth to assist in
understanding the invention and should not, of course, be construed
as specifically limiting the invention described and claimed
herein. Such variations of the inventions which would be within the
purview of those in the art, including the substitution of all
equivalents now known or later developed, including changes in
formulation or minor changes in experimental design, are to be
considered to fall within the scope of the invention as hereinafter
claimed.
[0216] This example illustrates the effectiveness of the
stilbenoids of formulae described herein, e.g. longistyline A
(Compound A); pinosylvin monomethyl ether (Compound B),
longistyline A-2-carboxylic acid (Compound C); longistyline
A(Compound D); 7,8-dihydrolongistyline C(Compound E);
7,8,2",3"-tetrahydrolongistyline C(Compound F);
7,8-dihydropinosylvin monomethyl ether (Compound G); and
7,8,2",3"-tetrahydrolongistyline A-2-carboxylic acid (Compound H),
in reducing plasma glucose levels in C57BL/ks diabetic (db/db)
mice, i.e., an art recognized model of non-insulin dependent
diabetes mellitus (NIDDM).
[0217] Materials and Methods
[0218] Genetically altered obese diabetic mice (designated C57BL/ks
diabetic or db/db) were purchased from the Jackson Laboratory (Bar
Harbor, Me., USA), and served as experimental animals. Male animals
between the ages of 8-9 weeks were employed in the studies
described here. Animals were housed (4 mice/cage) under standard
laboratory conditions at 22.degree. C. and 50% relative humidity,
and were maintained on a diet of Purina rodent chow and water ad
libitum. Prior to treatment, blood was collected from the tail vein
of each animal. Mice that had plasma glucose levels between 350 and
600 mg/dL were used. Each treatment group consisted of eight mice
that were distributed so that the mean glucose levels were
equivalent in each group at the start of the study. Db/db mice
received, orally by gavage: the experimental compound administered
at 62.5, 125, or 250 mg/kg/day (unless otherwise noted), or
metformin administered at 250 mg mmol/kg for 1-3 days. Test
compounds were delivered in a liquid vehicle containing 0.25% (w/v)
carboxymethyl-cellulose, 1% (v/v) Tween 60.RTM. (polyoxyethylene
sorbitan monosterate), and up to 10% (v/v) dimethyl sulfoxide
(DMSO) in a volume of 10 ml/kg. Blood was sampled from the tail
vein at three hours, six, eight, twenty-four, and thirty hours
post-administration (the first administration) of the particular
compound in non-fasted conditions. Experiments 3-5 also sampled
blood at fifty-four hours post-administration (the first
administration) of the particular compound in non-fasted
conditions. Blood samples were analyzed for plasma glucose levels.
Individual body weights and mean food consumption (each cage) were
also measured daily.
[0219] The isolated test substances were prepared as described
previously (vide supra). Metformin (1,1-dimethylbiguanide),
carboxymethyl cellulose and Tween 60 were purchased from Sigma
Chemical Co. (St. Louis, Mo., USA; catalog #'s D-5035, C-4888, and
p-1629, respectively). Plasma glucose levels were determined
colorimetrically using glucose oxidase (Sigma Chemical Co.; Sigma
catalog #315). Significant differences between groups (comparing
compound-treated to vehicle-treated) were evaluated using analysis
of variance and Fisher's post-hoc test.
[0220] Results
[0221] Enriched Extracts
[0222] As shown in FIG. 1 and in Table 1a, oral administration of
Enriched Extract 2 at a dose of 1000 mg/kg produced statistically
significant reductions in plasma glucose levels in db/db mice.
Interestingly, EE1 (an ethanolic extract) produced hypoglycemic
effects that were significant after 5 days. This extract was most
similar that those described in the art but significantly less
efficacious than the solvent extracted EE2 (Dhar, M. L., et al.,
Indian J. Exp. Biol., 6, 232 (1968)).
[0223] Vehicle, metformin (250 mg/kg) or enriched extract (1000
mg/kg) were given to db/db mice at 0(initial), 8, 24, 32, 48, 56,
72, 80, and 96 hours post initial dose.
[0224] Plasma glucose levels were measured at baseline, 3 (day 1),
51 hours (Day 3), and 99 hours (Day 5).
9 TABLE 1a Day 1 glucose Day 3 glucose Day 5 glucose Treatment
mg/dL P value mg/dL P value mg/dL P value Vehicle -80.9 -77.1
+102.1 Metformin -180.5 0.0022 -207.5 0.0018 -86.4 0.0002 EE1 -97.2
Ns -97.4 Ns -40.7 0.0020 EE2 -82.8 Ns -274.2 <0.0001 -144.3
<0.0001 EE3 -55.6 Ns -104.1 Ns +134.2 NS EE4 -31.2 Ns -54.0 Ns
+230.9 Ns Ns = not significant
[0225] Compounds A, B and C
[0226] As shown in FIG. 2 and in Tables 1 and 2, below, oral
administration of Compounds A and C at a dose level of 250 mg/kg to
db/db mice produced statistically significant reduction in plasma
glucose, relative to vehicle (control). The following test
substances were evaluated in db/db mice for the ability to lower
blood glucose: Longistyline C
(5-hydroxy-3-methoxystilbene-2-carboxylic acid) (Compound A);
pinosylvin monomethyl ether (5-hydroxy-3-methoxystilbene) (Compound
B); and Longistyline A-2-carboxylic acid
(3-hydroxy-5-methoxy-4-(3-methyl- -2-butenyl)stilbene-2-carboxylic
acid) (Compound C). The test substances were evaluated in a series
of experiments, which are summarized in Tables 1 and 2 below.
[0227] A single initial dose of Compound A and additional
subsequent doses of Compound C (250 mg/kg) given to db/db mice at
eight and twenty-four hours after the initial oral administration
resulted in statistically significant reductions in plasma glucose
relative to vehicle controls at either six, eight, twenty-four or
thirty hour timepoints after initial oral administration. Six hours
after the initial dosing, mean glucose levels for the active
experimental Compound A and C declined 130.6 mg/dL (p<0.0001)
and 8.4 mg/dL (p=0.0295), respectively, from the baseline value.
Eight hours after the initial dosing, mean glucose levels for the
active experimental Compounds A [02] and C [04] declined 129.1
mg/dL (p<0.0001) and rose 4.5 mg/dL (p=0.0466), respectively,
from the baseline values. Twenty-four hours after the initial
dosing, just after the third dosing, mean glucose levels for the
active experimental Compound C declined 122.8 mg/dL (p=0.0049) from
the baseline values.
[0228] Single doses of Compound A (250 mg/kg) in vehicle also
showed a trend in reducing plasma glucose relative to vehicle
controls at 3 hours after initial oral administration. Three hours
after dosing, mean glucose levels for the active experimental
Compound C suspended in vehicle declined 80.3 mg/dL (p=0.0797) from
the baseline value.
[0229] Doses of Compound B (250 mg/kg) in Formulation IV also
showed a trend in reducing plasma glucose relative to vehicle
controls at thirty hours after initial oral administration. Thirty
hours after dosing, mean glucose levels for the active experimental
Compound C suspended in Formula (IV) rose only 36.5 mg/dL
(p=0.1088) from the baseline value.
[0230] As shown in Table 3, below, the antihyperglycemic effect of
Compounds A and C at dosage regimes of 250 mg/kg occurred in the
absence of any significantly adverse effect on food intake or body
weight. Body weights were not affected in animals treated during
the test period (Table 2). It was noted that the food intake of
those animals administered compounds A (250 mg/kg) and C (250
mg/kg) was less than that of the normal food intake range (5-6
g/day/mouse).
[0231] The data in Tables 1, 2 and 3 indicate that the
aforementioned stilbenoids are effective hypoglycemic agents in a
rodent model of insulin resistance, obesity, and NIDDM.
10TABLE 1 Effects of test substances on glucose-lowering in
diabetic db/db mice. Change in Change Glucose (mg/dL) in Glucose
(mg/dL) Treatment 3 h P Value* 6 h P Value* Vehicle -8.0 64.5
Compound A -80.3 0.0797 -130.6 <0.0001 250 mg/kg Compound B
-35.3 NS +29.3 NS Compound C -10.4 NS -8.4 0.0295 *Statistical
significance evaluated using unpaired t-test and Fisher's post-hoc
test. NS-not significant at p = 0.05 level
[0232]
11TABLE 2 Effects of test substances on glucose-lowering in
diabetic db/db mice. Change in Change in Change in Glucose (mg/dL)
Glucose (mg/dL) Glucose (mg/dL) Treatment 8 h P Value* 24 h P
Value* 30 h P Value* Vehicle 79.5 18.6 Compound A -129.1 <0.0001
-61.9 NS Compound B +52.2 NS -50.6 NS +36.5 0.1008 Compound C +4.5
0.0466 -122.8 0.0049 *Statistical significance evaluated using
unpaired t-test and Fisher's post-hoc test. NS - not significant at
p = 0.05 level
[0233]
12TABLE 3 Effects of test substances on body weights and food
consumption in diabetic db/db mice. Body weight Body weight
(g/mouse) (g/mouse) Food Intake (mean) (mean) (g/mouse) Dosage
TREATMENT 0 h 24 hr 0-24 h VEHICLE 38.0 .+-. 0.8 37.8 .+-. 0.8 6.8
250 mg/kg Compound A 38.8 .+-. 0.7 38.3 .+-. 0.7 4.2 250 mg/kg
Compound B 37.8 .+-. 0.4 37.8 .+-. 0.4 5.9 250 mg/kg Compound C
38.7 .+-. 0.8 38.0 .+-. 0.7 3.3
[0234] Compound A
[0235] As shown in FIG. 3 and in Table 4, below, oral
administration of Compound A at dose levels of 62.5, 125, and 250
mg/kg to db/db mice produced statistically significant reduction in
plasma glucose, relative to vehicle (control).
[0236] Longistyline C (Compound A) was evaluated in db/db mice for
the ability to lower blood glucose. The test substance was
evaluated in a series of experiments, which are summarized in Table
4 below.
[0237] Single doses of Compound A (62.5, 125 and 250 mg/kg) were
given to db/db mice at twenty-four and forty-eight hours after the
initial oral administration resulted in statistically significant
reductions in plasma glucose relative to vehicle controls at six,
thirty or fifty-four h or at all timepoints after oral
administration. Six hours after the initial dosing, mean glucose
levels of the animals dosed with 125 mg/kg and 250 mg/kg of the
active experimental Compound A declined 89.9 mg/dL (p=0.0233) and
79.3 mg/dL (p=0.0367), respectively, from the baseline value.
Thirty hours after the initial dosing, six hours after the second
dosing, mean glucose levels of animals dosed with 62.5, 125 and 250
mg/kg of the active experimental Compound A declined 62.8 mg/dL
(p=0.0153), 44.9 mg/dL (p=0.0460) and 158.5 mg/dL p=0.0001),
respectively, from the baseline values. Fifty-four hours after the
initial dosing, six hours after the third dosing, mean glucose
levels of animals dosed with 250 mg/kg of the active experimental
Compound A declined 144.5 mg/dL (p=0.0001) from the baseline
values.
[0238] 62.5 and 125 mg/kg doses of Compound A suspended in vehicle
also showed a trend in reducing plasma glucose relative to vehicle
controls at fifty-four hours after initial oral administration.
Fifty-four hours after initial dosing, mean glucose levels of the
animals dosed with 62.5 and 125 mg/kg doses of the active
experimental Compound A suspended in vehicle declined 31.0 mg/dL
(p=0.1234) and 34.3 mg/dL (p=0.1106), respectively, from the
baseline value.
[0239] By comparison, the known hypoglycemic agent metformin, given
at 250 mg/kg, lowered plasma glucose levels by approximately 180.4
mg/dL (p=0.0003), six hours after the initial dose; and 112.4 mg/dL
(p=0.0016), thirty hours after the initial dose, six hours after
the second dose. Metformin suspended in vehicle also showed a trend
in reducing plasma glucose relative to vehicle controls at
fifty-four hours after initial oral administration. Fifty-four
hours after initial dosing, six hours after the dosing at
forty-eight hours, mean glucose levels of the animals dosed with
the metformin suspended in vehicle declined 41.3 mg/dL p=0.0882)
from the baseline value.
[0240] As shown in Table 5, below, the antihyperglycemic effect of
Compound A at dosage regimes of 62.5 mg/kg, 125 mg/kg, and 250
mg/kg occurred in the absence of any significantly adverse effect
on food intake or body weight. Body weights were not affected in
animals treated during the test period (Table 6). It was noted that
the food intake of those animals administered 250 mg/kg Compound A
was less than that of the normal food intake range (5-6
g/day/mouse).
[0241] These data indicate that the aforementioned stilbenoids are
effective hypoglycemic agents in a rodent model of insulin
resistance, obesity, and NIDDM.
13TABLE 4 Effects of test substances on glucose-lowering in
diabetic db/db mice. Change in Change in Change in Glucose (mg/dL)
Glucose (mg/dL) Glucose (mg/dL) 6 H P VALUE 30 H P VALUE 54 H P
VALUE Vehicle 35.9 79.6 60.3 Metformin -180.4 0.0003 -112.4 0.0016
-41.3 0.0882 Compound A -8.0 NS -62.8 0.0153 -31.0 0.1234 Compound
A -89.9 0.0233 -44.9 0.0460 -34.3 0.1106 Compound A -79.3 0.0367
-158.5 0.0001 -144.5 0.0012 *Statistical significance evaluated
using unpaired t-test and Fisher's post-hoc test. NS - not
significant at p = 0.05 level
[0242]
14TABLE 5 Effects of test substances on body weights and food
consumption in diabetic db/db mice. Body weight Body weight Food
(g/mouse) (g/mouse) Intake (mean) (mean) (g/mouse) Dosage TREATMENT
0 h 48 hr 0-48 h Vehicle 39.2 .+-. 0.5 39.5 .+-. 0.5 6.3 250 mg/kg
Metformin 37.3 .+-. 0.7 37.2 .+-. 0.6 5.1 62.5 mg/kg Compound A
38.6 .+-. 0.6 39.1 .+-. 0.5 6.1 125 mg/kg Compound A 39.0 .+-. 0.8
38.8 .+-. 1.0 5.1 250 mg/kg Compound A 38.2 .+-. 0.7 38.1 .+-. 0.7
4.1
[0243] Compound C
[0244] As shown in FIG. 4 and in Table 6, below, oral
administration of Compound C at dose levels of 62.5, 125, and 250
mg/kg to db/db mice produced statistically significant reduction in
plasma glucose, relative to vehicle (control).
[0245] Longistyline A-2-carboxylic acid(Compound C) was evaluated
in db/db mice for the ability to lower blood glucose. The test
substance was evaluated in a series of experiments, which are
summarized in Table 6 below.
[0246] Single doses of Compound C (62.5, 125 and 250 mg/kg) were
given to db/db mice at twenty-four and forty-eight hours after the
initial oral administration resulted in statistically significant
reductions in plasma glucose relative to vehicle controls at six,
thirty or fifty-four hours or at all timepoints after oral
administration. Six hours after the initial dosing, mean glucose
levels of the animals dosed with 250 mg/kg of the active
experimental Compound A declined 120.8 mg/dL (p=0.0019) from the
baseline value. Thirty hours after the initial dosing, six hours
after the second dosing, mean glucose levels of animals dosed with
62.5, 125 and 250 mg/kg of the active experimental Compounds 02
declined 40.7 mg/dL (p=0.0322), 69.7 mg/dL (p=0.0131) and 221.4
mg/dL (p<0.0001), respectively, from the baseline values.
Fifty-four hours after the initial dosing, six hours after the
third dosing, mean glucose levels of animals dosed with 125 mg/kg
and 250 mg/kg of the active experimental Compound 04 declined 60.4
mg/dL p=0.0409) and 122.0 mg/dL p=0.0086) from the baseline
values.
[0247] 25 mg/kg doses of Compound C suspended in vehicle also
showed a trend in reducing plasma glucose relative to vehicle
controls at six hours after initial oral administration. Six hours
after initial dosing, mean glucose levels of the animals dosed with
the active experimental Compound C suspended in vehicle declined
34.3 mg/dL (p=0.1287) from the baseline value.
[0248] By comparison, the known hypoglycemic agent metformin, given
at 250 mg/kg, lowered plasma glucose levels by approximately 180.4
mg/dL (p<0.0001), six hours after the initial dose; and 112.4
mg/dL (p=0.0009), thirty hours after the initial dose, six hours
after the second dose.
[0249] Metformin suspended in vehicle also showed a trend in
reducing plasma glucose relative to vehicle controls at fifty-four
hours after initial oral administration. Fifty-four hours after
initial dosing, mean glucose levels of the animals dosed with
metformin suspended in vehicle declined 41.3 mg/dL (p=0.0696) from
the baseline value.
[0250] As shown in Table 7, below, the antihyperglycemic effect of
Compound C at dosage regimes of 62.5 mg/kg, 125 mg/kg, and 250
mg/kg occurred in the absence of any significantly adverse effect
on food intake or body weight. Body weights were not affected in
animals treated during the test period (Table 7). It was noted that
the food intake of those animals administered 250 mg/kg Compound C
was less than that of the normal food intake range (5-6
g/day/mouse).
[0251] The data in Tables 6 and 7 indicate that the aforementioned
stilbenoids are effective hypoglycemic agents in a rodent model of
insulin resistance, obesity, and NIDDM.
15TABLE 6 Effects of test substances on glucose-lowering in
diabetic db/db mice. Change in Change in Change in Glucose (mg/dL)
Glucose (mg/dL) Glucose (mg/dL) 6 H P VALUE 30 H P VALUE 54 H P
VALUE Vehicle 35.9 79.6 60.3 Metformin -180.4 <0.0001 -112.4
0.0009 -41.3 0.0696 Compound C -18.6 NS -40.7 0.0322 65.4 NS
Compound C -34.3 0.1287 -69.7 0.0131 -60.4 0.0409 Compound C -120.8
0.0019 -221.4 <0.0001 -122.0 0.0086 *Statistical significance
evaluated using unpaired t-test and Fisher's post-hoc test. NS -
not significant at p = 0.05 level
[0252]
16TABLE 7 Effects of test substances on body weights and food
consumption in diabetic db/db mice. Body weight Body weight Food
(g/mouse) (g/mouse) Intake (mean) (mean) (g/mouse) Dosage TREATMENT
0 h 48 hr 0-48 h Vehicle 39.2 .+-. 0.5 39.5 .+-. 0.5 6.3 250 mg/kg
Metformin 37.3 .+-. 0.7 37.2 .+-. 0.6 5.1 62.5 mg/kg Compound C
37.4 .+-. 0.4 38.2 .+-. 0.5 5.9 125 mg/kg Compound C 39.4 .+-. 0.4
39.7 .+-. 0.3 5.6 250 mg/kg Compound C 39.1 .+-. 0.8 38.4 .+-. 0.1
4.3
[0253] Compounds E, G and H
[0254] As shown in FIG. 5 and in Table 8, below, oral
administration of Compounds E (7,8-dihydrolongistyline C), G
(7,8-dihydropinosylvin monomethyl ether), and 11
(7,8-dihydrolongistyline A-2-carboxylic acid), at dose levels of
125 mg/kg to db/db mice produced statistically significant
reduction in plasma glucose, relative to vehicle (control).
[0255] Longistyline C (Compound A) was evaluated in db/db mice for
the ability to lower blood glucose. The test substance was
evaluated in a series of experiments, which are summarized in Table
8 below.
[0256] Single doses of Compounds E (7,8-dihydrolongistyline C), G
(7,8-dihydropinosylvin monomethyl ether), and H
(7,8-dihydrolongistyline A-2-carboxylic acid) were given to db/db
mice at twenty-four and forty-eight hours after the initial oral
administration resulted in statistically significant reductions in
plasma glucose relative to vehicle controls at thirty or fifty-four
hours or at both timepoints after oral administration. Thirty hours
after the initial dosing, mean glucose levels of the animals dosed
with 250 mg/kg of the active experimental Compounds E and G
declined 47.0 mg/dL (p=0.0136) and 74.6 mg/dL (p=0.0023) from the
baseline value. Fifty-four hours after the initial dosing, six
hours after the third dosing, mean glucose levels of animals dosed
with 125 mg/kg of the active experimental Compounds E and H
declined 54.7 mg/dL (p<0.0001) and 33.3 mg/dL (p<0.0001),
respectively, from baseline values. Fifty-four hours after the
initial dosing, six hours after the third dosing, mean glucose
levels of animals dosed with 125 mg/kg of the active experimental
Compound G rose only 12.0 mg/dL (p=0.0071) from the baseline
values.
[0257] 125 mg/kg doses of Compound G suspended in vehicle also
showed a trend in reducing plasma glucose relative to vehicle
controls at thirty hours after initial oral administration. Thirty
hours after initial dosing, mean glucose levels of the animals
dosed with the active experimental Compound C suspended in vehicle
declined 12.2 mg/dL (p=0.0926) from the baseline value.
[0258] By comparison, the known hypoglycemic agent metformin, given
at 250 mg/kg, lowered plasma glucose levels by approximately 113.6
mg/dL (p<0.0001), six hours after the initial dose, and 25.1
mg/dL (p=0.0471), thirty hours after the initial dose, six hours
after the second dose. At fifty-four hours after the initial dose,
six hours after the third dose, glucose levels of animals given
metformin at 250 mg/kg rose only 6.8 mg/dL (p=0.0033).
[0259] As shown in Table 9, below, the antihyperglycemic effect of
Compounds E, G and H at dosage regimes of 125 mg/kg occurred in the
absence of any significantly adverse effect on food intake or body
weight. Body weights were not affected in animals treated during
the test period (Table 9).The data in Tables 8 and 9 indicate that
the aforementioned stilbenoids are effective hypoglycemic agents in
a rodent model of insulin resistance, obesity, and NIDDM.
17TABLE 8 Effects of test substances on glucose-lowering in
diabetic db/db mice. Change in Change in Change in Glucose (mg/dL)
Glucose (mg/dL) Glucose (mg/dL) 6 H P VALUE 30 H P VALUE 54 H P
VALUE Vehicle 15.5 56.6 88.6 Metformin -113.6 <0.0001 -25.1
0.0471 6.8 0.0033 Compound E -2.1 NS -47.0 0.0136 -54.7 <0.0001
Compound G 35.0 NS -12.2 0.0926 12.0 0.0071 Compound H 31.4 NS
-74.6 0.0023 -33.3 <0.0001 *Statistical significance evaluated
using unpaired t-test and Fisher's post-hoc test. NS - not
significant at p = 0.05 level
[0260]
18TABLE 9 Effects of test substances on body weights and food
consumption in diabetic db/db mice. Body weight Body weight Food
(g/mouse) (g/mouse) Intake (mean) (mean) (g/mouse) Dosage TREATMENT
0 h 48 hr 0-48 h Vehicle 39.8 .+-. 0.7 39.7 .+-. 0.6 6.0 250 mg/kg
Metformin 40.6 .+-. 0.4 40.5 .+-. 0.4 5.4 125 mg/kg Compound E 39.5
.+-. 0.5 39.7 .+-. 0.6 5.7 125 mg/kg Compound G 39.0 .+-. 1.1 39.0
.+-. 1.1 5.9 125 mg/kg Compound H 38.7 .+-. 0.5 38.9 .+-. 0.5
5.6
[0261] Compounds A, D, E and F
[0262] As shown in FIG. 6 and in Table 10, below, oral
administration of Compounds A (Longstyline C), D (Longstyline A), E
(7,8-dihydrolongistylin- e C) and F
(7,8,2",3"-tetrahydrolongistyline C) at a dose level of 125 mg/kg
to db/db mice produced statistically significant reduction in
plasma glucose, relative to vehicle (control).
[0263] The following test substances were evaluated in db/db mice
for the ability to lower blood glucose: Compound A (Longstyline C),
Compound D (Longstyline A), Compound E (7,8-dihydrolongistyline C)
and Compound F (7,8,2",3"-tetrahydrolongistyline C). The test
substances were evaluated in a series of experiments, which are
summarized in Table 10 below.
[0264] Single doses of Compounds A (Longstyline C), D (Longstyline
A), E (7,8-dihydrolongistyline C) and F
(7,8,2",3"-tetrahydrolongistyline C) were given to db/db mice at
twenty-four and forty-eight hours after the initial oral
administration resulted in statistically significant reductions in
plasma glucose relative to vehicle controls at six, thirty or
fifty-four hours or at all timepoints after oral administration.
Six hours after the initial dosing, mean glucose levels of the
animals dosed with 125 mg/kg of the active experimental Compounds
A, D, E and F declined 78.5 mg/dL (p=0.0070), 76.4 mg/dL
(p=0.0083), 53.8 mg/dL (p=0.0409) and 80.3 mg/dL (p=0.0062) from
the baseline value. Thirty hours after the initial dosing, mean
glucose levels of the animals dosed with 125 mg/kg of the active
experimental Compounds A, D, and E declined 116.0 mg/dL (p=0.0008),
130.6 mg/dL (p=0.0002) and 109.9 mg/dL (p=0.0020) from the baseline
value. Fifty-four hours after the initial dosing, six hours after
the third dosing, mean glucose levels of animals dosed with 125
mg/kg of the active experimental Compounds A, D and E declined 99.2
mg/dL (p=0.0331) and 99.5 mg/dL (p=0.0277), respectively, from
baseline values.
[0265] 125 mg/kg doses of Compound E suspended in vehicle also
showed a trend in reducing plasma glucose relative to vehicle
controls at fifty-four hours after initial oral administration.
Fifty-four hours after initial dosing, mean glucose levels of the
animals dosed with the active experimental Compound E suspended in
vehicle declined 86.6 mg/dL (p=0.0730) from the baseline value.
[0266] As shown in Table 11, below, the antihyperglycemic effect of
Compounds A, E, G and H at dosage regimes of 125 mg/kg occurred in
the absence of any significantly adverse effect on food intake or
body weight. Body weights were not affected in animals treated
during the test period (Table 11).
[0267] The data in Tables 11 indicate that the aforementioned
stilbenoids are effective hypoglycemic agents in a rodent model of
insulin resistance, obesity, and NIDDM.
19TABLE 10 Effects of test substances on glucose-lowering in
diabetic db/db mice. Change in Change in Change in Glucose (mg/dL)
Glucose (mg/dL) Glucose (mg/dL) 6 H P VALUE 30 H P VALUE 54 H P
VALUE Vehicle 20.0 0.1 -20.2 Compound A -78.5 0.0070 -116.0 0.0008
-99.2 0.0331 Compound D -76.4 0.0083 -130.6 0.0002 -99.5 0.0277
Compound E -53.8 0.0409 -109.9 0.0020 -86.6 0.0730 Compound F -80.3
0.0062 -37.5 NS -10.2 NS *Statistical significance evaluated using
unpaired t-test and Fisher's post-hoc test. NS - not significant at
p = 0.05 level
[0268]
20TABLE 11 Effects of test substances on body weights and food
consumption in diabetic db/db mice. Body weight Body weight Food
(g/mouse) (g/mouse) Intake (mean) (mean) (g/mouse) Dosage TREATMENT
0 h 48 hr 0-48 h Vehicle 39.4 .+-. 0.4 40.1 .+-. 0.4 6.1 125 mg/kg
Compound A 40.3 .+-. 0.6 41.2 .+-. 0.6 6.0 125 mg/kg Compound D
39.3 .+-. 0.6 39.7 .+-. 0.7 5.7 125 mg/kg Compound E 39.6 .+-. 0.3
40.2 .+-. 0.4 5.7 125 mg/kg Compound F 39.3 .+-. 0.8 39.8 .+-. 0.8
5.8
[0269] The present invention is not to be limited in scope by the
specific embodiments disclosed in the examples, which are intended
as illustrations of a few aspects of the invention and any
embodiments which are functionally equivalent are within the scope
of this invention. Indeed, various modifications of the invention
in addition to those shown and described herein will become
apparent to those skilled in the art and are intended to fall
within the appended claims.
[0270] All publications, patents and patent applications mentioned
in this specification are indicative of the level of skill of those
skilled in the art to which this invention pertains, and are herein
incorporated by reference to the same extent as if each individual
publication, patent or patent application was specifically and
individually indicated to be incorporated by reference.
* * * * *