U.S. patent application number 12/155743 was filed with the patent office on 2009-12-10 for extract of polygonum multiflorum thunb. ex murray var. hypoleucum and compositions for improving metabolic syndrome.
Invention is credited to Yuarn-Yee Chang, Chi-Hua Chen, Shiow-Wen Chen, Wen-Kia Liu.
Application Number | 20090304822 12/155743 |
Document ID | / |
Family ID | 41400537 |
Filed Date | 2009-12-10 |
United States Patent
Application |
20090304822 |
Kind Code |
A1 |
Chen; Chi-Hua ; et
al. |
December 10, 2009 |
Extract of Polygonum multiflorum Thunb. ex Murray var. hypoleucum
and compositions for improving metabolic syndrome
Abstract
An extract of Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi) or of Polygonum hypoleucum (Ohwi) for improving
metabolic syndrome is prepared by the following method: (a)
providing fresh or dry Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi) or Polygonum hypoleucum (Ohwi) in partial or
whole plant ; (b) extracting the partial or whole plant by a
solvent to obtain a crude extract; (c) condensing and drying the
crude extract to obtain an extract product; and (d) collecting the
extract product. The present invention also relates to a
pharmaceutical and a food composition having the above extract for
improving the metabolic syndromes. The extract of the present
invention can be easily prepared and is demonstrated to modulate
the levels of both blood glucose and blood lipid, to inhibit the
activities of glycolytic enzymes and acetyl-CoA carboxylase, and to
stimulate acyl-CoA oxidase. The extract of the present invention is
capable of improving metabolic syndromes.
Inventors: |
Chen; Chi-Hua; (Hsinchu,
TW) ; Chen; Shiow-Wen; (Hsinchu, TW) ; Chang;
Yuarn-Yee; (Hsinchu, TW) ; Liu; Wen-Kia;
(Hsinchu, TW) |
Correspondence
Address: |
Andrews & Kurth, L.L.P.
Suite 1100, 1350 I Street N.W.
Washington
DC
20005
US
|
Family ID: |
41400537 |
Appl. No.: |
12/155743 |
Filed: |
June 9, 2008 |
Current U.S.
Class: |
424/725 |
Current CPC
Class: |
A61K 36/704 20130101;
A61P 3/00 20180101 |
Class at
Publication: |
424/725 |
International
Class: |
A61K 36/00 20060101
A61K036/00; A61P 3/10 20060101 A61P003/10 |
Claims
1. An extract of Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi) or of Polygonum hypoleucum (Ohwi) for improving
metabolic syndrome being prepared by the following method,
comprising the steps of: (a) providing fresh or dry Polygonum
multiflorum Thunb. ex Murray var. hypoleucum (Ohwi) or Polygonum
hypoleucum (Ohwi) in partial or whole plant; (b) extracting
Polygonum multiflorum Thunb. ex Murray var. hypoleucum (Ohwi) or
Polygonum hypoleucum (Ohwi) by a solvent to obtain a crude extract;
(c) condensing and drying the crude extract to obtain a Polygonum
multiflorum Thunb. ex Murray var. hypoleucum (Ohwi) or Polygonum
hypoleucum (Ohwi) extract product; and (d) collecting the extracts
product of Polygonum multiflorum Thunb. ex Murray var. hypoleucum
(Ohwi) or of Polygonum hypoleucum (Ohwi).
2. The extract of Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi) or of Polygonum hypoleucum (Ohwi) as claimed in
claim 1, wherein the fresh or dry Polygonum multiflorum Thunb. ex
Murray var. hypoleucum (Ohwi) or Polygonum hypoleucum (Ohwi) in
partial is stem or root of Polygonum multiflorum Thunb. ex Murray
var. hypoleucum (Ohwi) or of Polygonum hypoleucum (Ohwi).
3. The extract of Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi) or of Polygonum hypoleucum (Ohwi) as claimed in
claim 2, wherein the solvent is water, alcohol, aqueous alcohol
solution or ethyl acetate.
4. The extract of Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi) or of Polygonum hypoleucum (Ohwi) as claimed in
claim 3, wherein the solvent comprises 25-100 wt % of ethanol and
75-0 wt % of water.
5. The extract of Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi) or of Polygonum hypoleucum (Ohwi) as claimed in
claim 4, wherein the solvent comprises 70-100 wt % ethanol and 30-0
wt % of water.
6. The extract of Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi) or of Polygonum hypoleucum (Ohwi) as claimed in
claim 5, wherein the solvent is an acidic alcohol.
7. The extract of Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi) or of Polygonum hypoleucum (Ohwi) as claimed in
claim 6, wherein the acidic alcohol comprises 0.1% of hydrochloric
acid.
8. The extract of Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi) or of Polygonum hypoleucum (Ohwi) as claimed in
claim 7, wherein Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi) or Polygonum hypoleucum (Ohwi) and the solvent
have a weight to volume ratio between 1:5 to 1:20.
9. The extract of Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi) or of Polygonum hypoleucum (Ohwi) as claimed in
claim 8, wherein the step (b) extracting Polygonum multiflorum
Thunb. ex Murray var. hypoleucum (Ohwi) or Polygonum hypoleucum
(Ohwi) by a solvent to obtain a crude extract is proceeded at room
temperature.
10. A method for improving metabolic syndrome by administering a
therapeutically effective amount of extract of Polygonum
multiflorum Thunb. ex Murray var. hypoleucum (Ohwi) or of Polygonum
hypoleucum (Ohwi) to a subject in needed.
11. The method as claimed in claim 10, wherein the extract of
Polygonum multiflorum Thunb. ex Murray var. hypoleucum (Ohwi) or of
Polygonum hypoleucum (Ohwi) for improving metabolic syndrome is by
inhibiting glycolytic enzymes or acetyl-CoA carboxylase,
stimulating acyl-CoA oxidase, and modulating the levels of blood
glucose and blood lipid.
12. The method as claimed in claim 11, wherein the metabolic
syndrome is insulin resistance syndrome.
13. A pharmaceutical composition, comprising the extract of
Polygonum multiflorum Thunb. ex Murray var. hypoleucum (Ohwi) or of
Polygonum hypoleucum (Ohwi) as claimed in claim 1 and at least one
pharmaceutically acceptable carrier or excipient.
14. The pharmaceutical composition as claimed in claim 13, wherein
the pharmaceutically composition is formed as tablet, powder,
suspension, chyle fluid, capsule, particle, pill, fluid, alcohol
solution or medical syrup.
15. A food composition comprising the extract of Polygonum
multiflorum Thunb. ex Murray var. hypoleucum (Ohwi) or of Polygonum
hypoleucum (Ohwi) as claimed in claim 1.
Description
BACKGROLND OF THE MWNTIQN
[0001] 1. Field of the Invention
[0002] The present invention relates to an extract of Polygonum
multiflorum Thunb. ex Murray var. hypoleucum (Ohwi), especially to
an extract of Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi) for improving metabolic syndrome.
[0003] 2. Description of the Related Art
[0004] Metabolic syndrome is a combination of medical disorders
that increase a person's risk of developing cardiovascular diseases
and diabetes. Metabolic syndrome affects a large number of people
in a clustered fashion. Recently, with changing dietary habits and
lifestyle, more and more people are being diagnosed as having
metabolic syndrome that have symptoms including high blood glucose,
high blood pressure or high blood lipid level.
[0005] Metabolic syndrome may specifically refer to insulin
resistance syndrome.
[0006] Polygonum multiflorum Thunb. ex Murray var. hypoleucum
(Ohwi), or Polygonum hypoleucum (Ohwi), is a special, endemic
variety found in Taiwan. Unlike the typical variety of Polygonum
multiflorum , Polygonum multiflorum Thunb. ex Murray var hypoleucum
(Ohwi) lacks the characteristic enlarged root part and is commonly
used in folk medicine for treating cold, coughing or arthritis.
[0007] U.S. Pat. No. 5,531,991 teaches a composition and method for
treating hyperglycemia, which utilizes an extract of the Chinese
herb Polygonum multiflorum. The herb is extracted with 0.1N
NH.sub.4OH and centrifuged. A supernatant is applied to a Sephadex
G-25 column. Three fractions are collected. One fraction exhibits a
high insulin-potentiating activity in fat cell assays and is shown
to lower blood glucose levels.
[0008] U.S. Pat. No. 6,200,569 teaches a composition and method for
increasing insulin activity, which provides a composition
comprising an insulin potentiating agent; such composition
comprises one or more substances derived from a water extract of
Polygonum multiflorum, Agaricaceae or Cinnamomum mairei. The
invention also provides a method for treating hyperglycemia in a
patient as an insulin-potentiating agent. The method can be used to
decrease blood glucose, glycosylated hemoglobin or glucose
level.
[0009] The foregoing prior arts relate exclusively to use of
Polygonum multiflorum extract to control glucose level by
stimulating the activity of insulin. However, the foregoing prior
arts are not capable of use for modulating the blood lipid, nor
improving metabolic syndrome.
[0010] Moreover, EP 1 829 519 A1 teaches a pharmaceutical
composition comprising Polygonum multiflorum Thunb, which comprises
Polygonum multiflorum Thunb, Fructus Crataegi, Salvia miltiorrhiza
Bunge and Radix Notog inseng. The composition has an effect of
lowering blood lipid and therefore can be used in preparation of
drugs for treatment and prevention of hyperlipidemia. The invention
also teaches a method to treat and prevent hyperlipidemia. However,
the composition must be used as a whole. In addition, the foregoing
patent did not disclose how to modulate blood glucose level nor to
improve metabolic syndrome.
[0011] As disclosed herein, pharmaceutical compositions and food
additives merely provide a single effect on decreasing either blood
glucose or blood lipid; yet no related prior art has been taught
for using Polygonum multiflorum Thunb. ex Murray var. hypoleucum
(Ohwi), or Polygonum hypoleucum (Ohwi) for treating metabolic
syndrome. Furthermore, the foregoing prior arts use the typical
variety of Polygonum multiflorum.
[0012] The population of patients with metabolic syndrome has been
increasing. To discover a compound or composition, especially a
natural plant composition having medical effects in preventing
insulin resistance and further to improve metabolic syndrome is
important in the field.
SUMMARY OF THE INVENTION
[0013] The main objective of the present invention is to prepare an
extract of Polygonum multiflorum Thunb. ex Murray var. hypoleucum
(Ohwi), or of Polygonum hypoleucum (Ohwi) for improving metabolic
syndrome.
[0014] To achieve the foregoing objective, an extract of Polygonum
multiflorum Thunb. ex Murray var. hypoleucum (Ohwi), or Polygonum
hypoleucum (Ohwi) for improving metabolic syndrome in accordance
with the present invention being prepared by the following method,
comprising:
[0015] (a) providing fresh or dry Polygonum multiflorum Thunb. ex
Murray var. hypoleucum (Ohwi), or Polygonum hypoleucum (Ohwi) in
partial or whole plant;
[0016] (b) extracting Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi), or Polygonum hypoleucum (Ohwi) by a solvent to
obtain a crude extract;
[0017] (c) condensing and drying the crude extract to obtain a
Polygonum multiflorum Thunb. ex Murray var. hypoleucum (Ohwi), or
Polygonum hypoleucum (Ohwi) extract product; and
[0018] (d) collecting the Polygonum multiflorum Thunb. ex Murray
var. hypoleucum (Ohwi), or Polygonum hypoleucum (Ohwi) extract
product.
[0019] One preferred embodiment, the fresh or dry Polygonum
multiflorum Thunb. ex Murray var. hypoleucum (Ohwi), or Polygonum
hypoleucum (Ohwi) in partial is stem or root of Polygonum
multiflorum Thunb. ex Murray var. hypoleucum (Ohwi), or of
Polygonum hypoleucum (Ohwi).
[0020] Another preferred embodiment, the solvent is water, alcohol,
aqueous alcohol solution or ethyl acetate.
[0021] Preferably, the solvent comprises 25-100 wt % of ethanol and
75-0 wt % of water; more preferably, the solvent comprises 70-100
wt % ethanol and 30-0 wt % of water.
[0022] One preferred embodiment, the solvent is an acidic alcohol;
preferably, an acidic alcohol containing 0.1% of hydrochloric
acid.
[0023] Preferably, Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi), or Polygonum hypoleucum (Ohwi) and the solvent
have a weight to volume ratio between 1:5 to 1:20.
[0024] One preferred embodiment, the step (b) is proceeded at room
temperature.
[0025] The present invention also relates to a method for improving
metabolic syndrome by administering to a therapeutically effective
amount of extract of Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi), or of Polygonum hypoleucum (Ohwi) to a subject
in needed.
[0026] One preferred embodiment, the extract of Polygonum
multiflorum Thunb. ex Murray var. hypoleucum (Ohwi), or of
Polygonum hypoleucum (Ohwi) for improving metabolic syndrome is by
inhibiting glucosidic enzymes or acetyl-CoA carboxylase (ACC),
stimulating acyl-CoA oxidase (ACO), and modulating the levels of
both blood glucose and blood lipid.
[0027] Preferably, the metabolic syndrome is insulin resistance
syndrome; more preferably, the insulin resistance syndrome is high
blood glucose or blood lipid The present invention also relates to
an extract of Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi), or of Polygonum hypoleucum (Ohwi) for improving
metabolic syndrome prepared by the following method,
comprising:
[0028] (a) providing fresh or dry stem and root of Polygonum
multiflorum Thunb. ex Murray var. hypoleucum (Ohwi), or of
Polygonum hypoleucum (Ohwi);
[0029] (b) extracting Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi), or Polygonum hypoleucum (Ohwi) as a crude
extract using an alcohol solvent;
[0030] (c) condensing and drying the crude extract as a Polygonum
multiflorum Thunb. ex Murray var. hypoleucum (Ohwi), Polygonum
hypoleucum (Ohwi) extract product; and
[0031] (d) collecting the extract product of Polygonum multiflorum
Thunb. ex Murray var. hypoleucum (Ohwi), or of Polygonum hypoleucum
(Ohwi).
[0032] One preferred embodiment, the solvent comprises 25-100 wt %
of ethanol and 75-0 wt % of water; preferably, the solvent
comprises 70-100 wt % ethanol and 30-0 wt % of water.
[0033] Another preferred embodiment, the solvent is an acidic
alcohol.
[0034] Preferably, the solvent contains 0.1 % of hydrochloric
acid.
[0035] One preferred embodiment, the Polygonum multiflorum Thunb.
ex Murray var. hypoleucum (Ohwi), or Polygonum hypoleucum (Ohwi)
and the solvent have a weight to volume ratio between 1:5 to
1:20.
[0036] Another preferred embodiment, the step (b) is proceeded at
room temperature.
[0037] The present invention also relates to a pharmaceutical
composition comprising the extract of Polygonum multiflorum Thunb.
ex Murray var. hypoleucum (Ohwi), or of Polygonum hypoleucum (Ohwi)
obtained from the present invention and at least one
pharmaceutically acceptable carrier or excipient.
[0038] One preferred embodiment, the pharmaceutically composition
is formed as tablet, powder, suspension, chyle fluid, capsule,
particle, pill, fluid, alcohol solution or medical syrup.
[0039] The present invention also relates to a food composition
comprising the extract of Polygonum multiflorum Thunb. ex Murray
var. hypoleucum (Ohwi), or of Polygonum hypoleucum (Ohwi) obtained
from the present invention.
[0040] The extract of Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi), or of Polygonum hypoleucum (Ohwi) of the present
invention is prepared easily and can modulate blood glucose and
blood lipid levels by inhibiting the activities of glucosidic
enzymes , or acetyl-CoA carboxylase, stimulating acyl-CoA oxidase
activities. Other objectives, advantages and novel features of the
invention will become more apparent from the following detailed
description when taken in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 is a flow chart describing a method for preparing the
extract of Polygonum multiflorum Thunb. ex Murray var. hypoleucum
(Ohwi), or of Polygonum hypoleucum (Ohwi) in accordance with the
present invention;
[0042] FIG. 2 is a plot showing the inhibitory effects of the
extract of Polygonum multiflorum Thunb. ex Murray var. hypoleucum
(Ohwi), or of Polygonum hypoleucum (Ohwi) on acetyl-CoA carboxylase
(ACC) activity;
[0043] FIG. 3 shows the stimulatory effects of (A) clofibrate and
(B) 100 .mu.g/ml of the extract of Polygonum multiflorum Thunb. ex
Murray var. hypoleucum (Ohwi), or of Polygonum hypoleucum (Ohwi) on
acyl-CoA oxidase (ACO) activity.
DETAILED DESCRIPTION OF THF PREFERRED EMBODIMENTS
[0044] Polygonum multiflorum Thunb. ex Murray var. hypoleucum
(Ohwi) Liu, Yng & Lai, common name Taiwan tuber fleeceflower,
is also known as Polygonum hypoleucum (Ohwi), or Pleuropterus
hypoleucus Nakai). The enlarged root tuber of Polygonum multiflorum
is one of the most famous tonic medicine in China and Japan. As an
endemic variety of Polygonum multiflorum, Polygonum multiflorum
Thunb. ex Murray var. hypoleucum (Ohwi) lacks the characteristic
feature of enlarged root tuber, is often found from low to medium
altitudes of Taiwan. The whole plant are used in folk medicine for
treating cold, coughing and arthritis, the leaves for topical
application of treating cuts, too.
[0045] The fresh or dry Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi) in partial or whole plant as defined herein is
the leaves, stems, or roots of Polygonum multiflorum Thunb. ex
Murray var. hypoleucum (Ohwi).
[0046] The solvent as defined herein is a polar solvent and may be
water, alcohol, aqueous alcohol solution or ethyl acetate.
[0047] Metabolic syndrome as defined herein may specifically refer
to insulin resistance syndrome, which may have symptoms including
high blood glucose, dyslipidemia, and high blood pressure.
[0048] The pharmaceutically acceptable carrier or pharmaceutical
carrier as defined herein refers to materials suitable for
pharmaceutical administration. They are inert agents or vehicles
for delivering one or more active agents to a subject.
[0049] The excipient is substantially non-toxic and allows a
medical composition to remain stable and bio-available.
[0050] The extract ofPolygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi), or of Polygonum hypoleucum (Ohwi), as defined
herein are those active ingredients of the Polygonum multiflorum
Thunb. ex Murray var. hypoleucum (Ohwi), or of Polygonum hypoleucum
(Ohwi), which can be used to modulate the concentrations of blood
glucose and blood lipid by interacting with enzymes including
intestinal glucosidic and hepatic enzymes. More specifically,
Polygonum multiflorum Thunb. ex Murray var. hypoleucum (Ohwi), or
Polygonum hypoleucum (Ohwi) exhibits bio-chemical efficacies with
regards to inhibiting both alpha-glucosidase and ACC and
stimulating ACO. More specifically, the ectract of Polygonum
multiflorum Thunb. ex Murray var. hypoleucum (Ohwi), or of
Polygonum hypoleucum (Ohwi) can be implemented in a pharmaceutical
composition or food composition for improving metabolic
syndrome.
[0051] The term "a subject in needed" as used herein is any patient
suffering from metabolic syndrome. The term "subject" is known in
the art, and, as used herein, refers to a warm-blooded animal, more
preferably a mammal, including, e.g., non-human animals such as
rats, mice, cats, dogs, sheep, horses, cattle, in addition to
humans. In a preferred embodiment, the subject is a human.
[0052] With reference to FIG. 1, the extract of Polygonum
multiflorum Thunb. ex Murray var. hypoleucum (Ohwi), or of
Polygonum hypoleucum (Ohwi) for improving metabolic syndrome in
accordance with the present invention is prepared by a method
comprising steps of: (a) providing fresh or dry Polygonum
multiflorum Thunb. ex Murray var. hypoleucum (Ohwi) or Polygonum
hypoleucum (Ohwi) in partial or whole plant; (b) extracting
Polygonum multiflorum Thunb. ex Murray var. hypoleucum (Ohwi) or
Polygonum hypoleucum (Ohwi) by a solvent to obtain a crude extract;
(c) condensing and drying the crude extract to obtain a Polygonum
multiflorum Thunb. ex Murray var. hypoleucum (Ohwi) or Polygonum
hypoleucum (Ohwi) extract product; (d) collecting the extract
product of Polygonum multiflorum Thunb. ex Murray var. hypoleucum
(Ohwi), or of Polygonum hypoleucum (Ohwi). The extract product of
Polygonum multiflorum Thunb. ex Murray var. hypoleucum (Ohwi) or of
Polygonum hypoleucum (Ohwi) may further be added into a
pharmaceutically acceptable carrier or excipient for forming a
pharmaceutical composition for improving metabolic syndrome. The
extract product of Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi) or of Polygonum hypoleucum (Ohwi) may also be
added to form a food composition for improving metabolic
syndrome.
[0053] The condensing and drying methods as defined in the present
invention may comprise freezing and drying, depressing and
condensing or other condensing and drying methods known in the
art.
[0054] The pharmaceutical composition according to the present
invention may be dosed in oral administration, and the form of
dosage can be a pill, tablet, powder, suspension, chyle fluid,
capsule, particle, fluid, a solution including alcohol and aqueous,
or medical syrup.
EXAMPLE 1
Preparation of Extracts of Polygonum multiflorum Thunb. ex Murray
var. hypoleucum (Ohwi) or of Polygonum hypoleucum (Ohwi)
[0055] i) Acidic Alcohol Extract
[0056] Polygonum multiflorum Thunb. ex Murray var. hypoleucum
(Ohwi) or Polygonum hypoleucum (Ohwi) was machine-grounded, then
extracted with an acidic alcohol containing 0.1% HC1. The weight to
volume ratio of grounded Polygonum multiflorum Thunb. ex Murray
var. hypoleucum (Ohwi) or Polygonum hypoleucum (Ohwi)and acidic
alcohol was about 1:8. The mix was left at room temperature with or
without shaking for 72 hours, filtered through 2 layers of
cheesecloth and centrifuged at 6,000 rpm for 15 min. The solution
was collected and concentrated. The extraction rate is about
6.9%.
[0057] A 50 mg/ml stock solution was prepared with water.
[0058] ii) Hot Water Extract:
[0059] Polygonum multiflorum Thunb. ex Murray var. hypoleucum
(Ohwi) or Polygonum hypoleucum (Ohwi) was heated in water for 2
hours. The weight to volume ratio of Polygonum multiflorum Thunb.
ex Murray var. hypoleucum (Ohwi) or Polygonum hypoleucum (Ohwi) and
water was about 1:20. The extract was filtered through 2 layers of
cheesecloth and freeze-dried. The extraction rate is about 9.2%. A
50 mg/ml stock solution was prepared with water.
EXAMPLE 2
The Inhibitory Effects of Extracts of Polygonum multiflorum Thunb.
ex Murray var. hypoleucum (Ohwi) or of Polygonum hypoleucum
(Ohwi)on .alpha.-glucosidase
[0060] Extract #1 and #2 are different batches of acidic alcohol
extracts, and extract #3 is hot water extract, prepared as
described in example 1.
[0061] The inhibitory effects of the 3 extracts on
.alpha.-glucosidase was assayed as following:
[0062] 1. Both enzyme .alpha.-glucosidase (EC 3.2.1.20, G5003,
Sigma-Aldrich Co., USA) and substrate p-nitrophenyl
.alpha.-D-glucopyranoside (pNP-G) (Sigma-Aldrich Co., USA)
solutions were prepared with 100 mM, pH 7.0 phosphoric acid buffer
solution to reached a concentration of 1 U/mland 5.times.10.sup.-3
M, respectively.
[0063] 2. 40 .mu.l of 0.4 M, pH 7.0 phosphoric acid buffer, 10
.mu.l of .alpha.-glucosidase, 50 .mu.l of serial dilutions of the
extracts of Polygonum multiflorum Thunb. ex Murray var. hypoleucum
(Ohwi) or of Polygonum hypoleucum (Ohwi)and 170 .mu.l of deionized
water were added orderly into each well of a 96-well plate
[0064] 3. An aliquot of 30 .mu.l preheated substrate solution was
added into each well, mixed thoroughly and incubated at 37.degree.
C. for 10 minutes.
[0065] 4. Absorbance at 405 nm wavelength (Power Wave XS, Bio Tek
Co., USA) was recorded before and after incubation. IC.sub.50
represents the concentration needed to inhibit 50% of the activity
of .alpha.-glucosidase. Table 1 shows the results of IC.sub.50 for
extracts #1-3.
TABLE-US-00001 TABLE 1 IC.sub.50 for extracts #1-3 extract #1
extract #2 extract #3 IC.sub.50(.mu.g/ml) 5.5 .+-. 0.5 6.5 .+-. 0.7
17.1 .+-. 0.4
[0066] Both acidic alcohol extracts and hot water extracts
demonstrated inhibitory effects on the activity of
.alpha.-glucosidase. One preferred embodiment of the present
invention is acidic alcohol extract.
EXAMPLE 3
The Inhibitory Effects of Extracts of Polygonum multiflorum Thunb.
ex Murray var. hypoleucum (Ohwi) or of Polygonum hypoleucum (Ohwi)
on Different Glycosidic Enzymes
[0067] Rat intestinal acetone powder (Sigma Chemical Co., St.
Louis, Mo.) was provided as a source of glycosidic enzyme to test
the inhibitory effects of the acidic alcohol extracts with various
glycosidic substrates (maltose, starch and sucrose)(Table 2).
TABLE-US-00002 TABLE 2 Values of IC.sub.50 of the acidic alcohol
extract of Polygonum multiflorum Thunb. ex Murray var. hypoleucum
(Ohwi) for various glycosidic enzymes IC.sub.50(mg/ml) Maltase
.alpha.-amylase Sucrase Acidic alcohol extract 0.6 1.9 3.1
[0068] Table 2. Values of IC.sub.50 of the acidic alcohol extract
of Polygonum multifloum Thunb. ex Murray var. hypoleucum (Ohwi) for
various glycosidic enzymes
[0069] As shown in Table 2, the acidic alcohol extract was capable
of inhibiting the activities of maltase, .alpha.-amylase and
sucrase, with the strongest effects on maltase.
EXAMLE 4
Preparation of Lipid-Soluble Extracts of Polygonum multiflorum
Thunb. ex Murray var. hypoleucum (Ohwi) or of Polygonum hypoleucum
(Ohwi)
[0070] Polygonum multiflorum Thunb. ex Murray var. hypoleucum
(Ohwi) or Polygonum hypoleucum (Ohwi) was machine-grounded, 50wt %
of methanol was added at a weight to volume ratio about 1:10. The
mix was kept at room temperature with shaking for 24 hours, and
filtered. An equal volume of water: ethyl acetate (1:1, vol:vol)
was added to the filtrate for liquid-liquid extraction. The upper
layer was obtained. An equal volume of 5 wt % of sodium bicarbonate
(NaHCO.sub.3) was added to remove free fatty acids. The ethyl
acetate layer was collected, condensed and dried. An extraction
rate was about 0.91%.
[0071] The obtained lipid-soluble extract was dissolved in dimethyl
sulfoxide (DMSO) at a concentration of 10 mg/ml.
EXAMPLE 5
Inhibitory Effects of Extracts of Polygonum multiflorum Thunb. ex
Murray var. hypoleucum (Ohwi) or of Polygonum hypoleucum (Ohwi) on
acetyl-CoA carboxylase (ACC)
[0072] Acetyl-CoA carboxykase (ACC) is one of the key enzymes
involved in fatty acid synthesis. The product of ACC reaction,
malonyl-CoA, is both a substrate of fatty acid synthase (FAS) and
an inhibitor of camitin palmitoyl CoA transferase (CPT). Therefore,
inhibition of ACC not only reduce the synthesis of endogenous
lipid, but also increase the oxidation of mitochondrial fatty
acid.
[0073] ACC was isolated and purified from rat liver, and assayed as
previously described (Tanabe T., Nakanishi, S., Hashimoto T.,
Ogiwara, H., Nikawa J., Numa S. 1981. Acetyl-CoA carboxylase from
rat liver, Meth. Enzymol. 71 (pt C): 5-16).
TABLE-US-00003 TABLE 3 Inhibitory effects of extracts of Polygonum
multiflorum Thunb. ex Murray var. hypoleucum (Ohwi) or of Polygonum
hypoleucum (Ohwi) on ACC ACC inhibition (%) Acidic alcohol extracts
(0.05 mg/ml) 61.27 .+-. 5.4 Lipid-soluble extracts (0.05 mg/ml)
45.99 .+-. 11.9
[0074] As demonstrated in Table 3, both acidic alcohol extract and
lipid-soluble extract of Polygonum multiflorum Thunb. ex Murray
var. hypoleucum (Ohwi) or of Polygonum hypoleucum (Ohwi)exhibited
inhibitory effects on ACC.
[0075] FIG. 2 shows the inhibitory effects of different
concentrations of acidic alcohol extracts on ACC. The IC.sub.50 was
0.04 mg/ml.
EXAMPLE 6
The Stimulatory Effects of the Acidic Alcohol Extract of Polygonum
multiflorum Thunb. ex Murray var. hypoleucum (Ohwi) or of Polygonum
hypoleucum (Ohwi) on Acyl-CoA oxidase (ACO)
[0076] ACO is one of the key enzymes associated with .beta.
oxidation of fatty acid. It is also a downstream gene of
PPAR.alpha., and can be used as a marker of fatty acid
oxidation.
[0077] Suitable number of hepatic cells H4IIEC3 were seeded in a
12-well plate. Once the cells reached confluence, the media were
replaced with either clofibrate or 100 .mu.l/ml of the acidic
alcohol extract of Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi) or of Polygonum hypoleucum (Ohwi). The foregoing
mixture solution was incubated for a proper period of time. Then
the culture solution was removed, and the cells were collected for
ACO analysis.
[0078] The method of analyzing ACO is described by Small et al.
(Small, G. M., Burdett, K., and ConNock, M. J. 1985. A sensitive
spectrophotometric assay for peroxisomal acyl-CoA oxidase.
Biochemical Journal. 227: 205-210). FIG. 3a shows the
concentration-dependent stimulatory effects of clofibrate on ACO
activity for different time periods (48 and 72 hours). The folds of
increases of ACO activities by the acidic alcohol extract of
Polygonum multiflorum Thunb. ex Murray var. hypoleucum (Ohwi) or of
Polygonum hypoleucum (Ohwi) is shown in FIG. 3b.
EXAMPLE 7
The Effects of the Acidic Alcohol extract of Polygonum multiflorum
Thunb. ex Murray var. hypoleucum (Ohwi) or of Polygonum hypoleucum
(Ohwi) on C57BL/6J Mice Fed on High Fat Diet
[0079] C57BL/6J mice were fed with a high fat diet comprising 30%
(g/g) butter for 8 weeks to induce obese, hypertriglyceridemic and
hyperinsulinemic symptoms. The mice with the aforementioned
symptoms were divided into 2 groups. One group of the mice was
given 1500 mg/kg body weight of the acidic alcohol extract of
Polygonum multiflorum Thunb. ex Murray var. hypoleucum (Ohwi) or of
Polygonum hypoleucum (Ohwi) (HF+No. 11). The other group, the
control group (HF), was given the same volume of water. Both groups
were maintained on the same high fat diet. Another group of mice
fed on normal chow (LF) was also examined. Oral glucose tolerance
test (OGTT) was carried out after 5-week feeding (Table 4). The
mice were then sacrificed, and blood and tissues collected for
analyses (Table 5).
TABLE-US-00004 TABLE 4 Results of oral glucose tolerance test
(OGTT) after five-week feeding Time LF HF HF + No.11 (min) (n = 14)
(n = 6-7) (n = 6-7) Concen- 0 6.1 .+-. 1.32.sup.a 8.76 .+-.
2.78.sup.b 7.6 .+-. 1.27.sup.a tration of 30 10.37 .+-. 1.45.sup.a
12.27 .+-. 2.65.sup.b 10.99 .+-. 1.45.sup.b blood 60 9.97 .+-.
1.25.sup.a 12.16 .+-. 2.16.sup.c 10.63 .+-. 1.42.sup.b glucose 90
8.75 .+-. 1.52.sup.a 12.25 .+-. 3.34.sup.b 10.47 .+-. 1.65.sup.b
(mmol/L) 120 8.56 .+-. 1.55.sup.a 13.74 .+-. 3.01.sup.c 10.01 .+-.
1.42.sup.b AUC.sub.120 min 656.3 .+-. 93.3.sup.a 961.2 .+-.
76.3.sup.c 831.1 .+-. 76.3.sup.b (mmol*min/L)
Data are expressed as average .+-. standard deviation. Values not
sharing the same superscript letters in the same row are
significantly different (P<0.05).
[0080] Five-week supplementing with the acidic alcohol extract of
Polygonum multiflorum Thunb. ex Murray var. hypoleucum (Ohwi) or of
Polygonum hypoleucum (Ohwi) significantly decreased the fasting
blood glucose level (0 min) of C57BL/6J mice fed on high fat diet
Both the blood glucose levels (at 90 and 120 min) and the area
under curve of 120 min of (HF+No. 11) group were significantly
lower than those of HF group. These results indicate that the
acidic alcohol extract of Polygonum multiflorum Thunb. ex Murray
var. hypoleucum (Ohwi) or of Polygonum hypoleucum (Ohwi) not only
decreases fasting blood glucose but also improve glucose
intolerance.
[0081] Table 5 shows that the acidic alcohol extract of Polygonum
multiflorum Thunb. ex Murray var. hypoleucum (Ohwi) or of Polygonum
hypoleucum (Ohwi) decreases body weight, serum levels of total
cholesterol (TC), triglyceride (TG), insulin and leptin of C57BL/6J
mice fed on high fat diets.
TABLE-US-00005 LF HF HF + No.11 (n = 14) (n = 6-7) (n = 6-7) Final
26.2 .+-. 1.2.sup.c 37.0 .+-. 1.8.sup.a 35.1 .+-. 3.2.sup.b body
weight (g) Serum 0.99 .+-. 0.23.sup.b 1.38 .+-. 0.18.sup.a 1.14
.+-. 0.32.sup.b TG (mol/- ml) Serum 2.62 .+-. 0.43.sup.c 4.68 .+-.
0.21.sup.a 3.69 .+-. 1.08.sup.b TG (m- mol/l) Serum 0.39 .+-.
0.11.sup.b 0.599 .+-. 0.2.sup.a 0.462 .+-. 0.2.sup.b insulin
(ng/ml) NEFA 1.27 .+-. 0.16.sup.a 1.63 .+-. 0.45.sup.a 1.28 .+-.
0.25.sup.a (mmol/- l) Leptin 467 .+-. 1557.sup.c 5911 .+-.
1623.sup.a 4204 .+-. 2209.sup.b (ng/ml)
Data are expressed as average .+-. standard deviation. Values not
sharing the same superscript letters in the same row are
significantly different (P<0.05).
[0082] The extract of Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi) or of Polygonum hypoleucum (Ohwi) of the present
invention comprises a simple preparing method without special
solvents nor complicated procedures. Moreover, examples above show
that the extract of Polygonum multiflorum Thunb. ex Murray var.
hypoleucum (Ohwi) or of Polygonum hypoleucum (Ohwi) is able to
modulate the concentrations of blood glucose and lipid; inhibit the
activities of both glucosidic enzymes and ACC, and increase ACO
activities. Therefore, the extract of Polygonum multiflorum Thunb.
ex Murray var. hypoleucum (Ohwi) or of Polygonum hypoleucum (Ohwi)
is able to improve metabolic syndromes as described.
[0083] Even though numerous characteristics and advantages of the
present invention have been set forth in the foregoing description,
together with details of the structure and features of the
invention, the disclosure is illustrative only. Changes may be made
in the details, especially in matters of shape, size, and
arrangement of parts within the principles of the invention to the
full extent indicated by the broad general meaning of the terms in
which the appended claims are expressed.
* * * * *