U.S. patent application number 13/191619 was filed with the patent office on 2011-11-17 for composition comprising bamboo extract and the compounds isolated therefrom showing activity for the treatment and prevention of inflammatory and blood circulation disease.
This patent application is currently assigned to UNIGEN, INC.. Invention is credited to Ji Nyeo Cho, Seon-Gil Do, Young Moon Heo, Tae Hyung Jo, Dong Seon Kim, Mi Ran Kim, Tae Woo Kim, Ju Yeon Lee, Kang Woo Lee, Seoung Ho Lee, Young Chul Lee, Jeong Bum Nam, Mi-Sun Oh, Jong Ha Ryu, Ji Sook Song, Hee Sun Sung, Sookyoung Sung, Sun-Young Sung, Sung-Sick Woo.
Application Number | 20110280975 13/191619 |
Document ID | / |
Family ID | 36118391 |
Filed Date | 2011-11-17 |
United States Patent
Application |
20110280975 |
Kind Code |
A1 |
Woo; Sung-Sick ; et
al. |
November 17, 2011 |
COMPOSITION COMPRISING BAMBOO EXTRACT AND THE COMPOUNDS ISOLATED
THEREFROM SHOWING ACTIVITY FOR THE TREATMENT AND PREVENTION OF
INFLAMMATORY AND BLOOD CIRCULATION DISEASE
Abstract
The present invention relates to a composition comprising a
bamboo plant extract for the prevention and treatment of
inflammatory or blood circulation disease. The extracts from bamboo
plant have potent anti-inflammatory activity by inhibiting NO
production and circulation-improving activity by inhibiting
elastase activity and, healing the wound of vascular endothelial
cell, activating u-PA expression and inhibiting PAI-1 expression,
lowering cholesterol deposit and inhibiting neointima formation,
therefore it can be used as the therapeutics or health care food
for treating and preventing inflammatory or blood circulation
diseases.
Inventors: |
Woo; Sung-Sick; (Seoul,
KR) ; Jo; Tae Hyung; (Gyeonggi-do, KR) ; Kim;
Dong Seon; (Daejeon, KR) ; Sung; Sun-Young;
(Gyeonggi-do, KR) ; Do; Seon-Gil;
(Chungcheongbuk-do, KR) ; Lee; Young Chul;
(Daejeon, KR) ; Nam; Jeong Bum; (Daejeon, KR)
; Ryu; Jong Ha; (Daegu, KR) ; Lee; Kang Woo;
(Chungcheongnam-do, KR) ; Sung; Hee Sun;
(Gyeonggi-do, KR) ; Heo; Young Moon;
(Jeollabuk-do, KR) ; Oh; Mi-Sun;
(Chungcheongnam-do, KR) ; Cho; Ji Nyeo;
(Gyeonggi-do, KR) ; Sung; Sookyoung;
(Chungcheongnam-do, KR) ; Lee; Ju Yeon; (Daegu,
KR) ; Kim; Tae Woo; (Ulsan, KR) ; Song; Ji
Sook; (Gyeonggi-do, KR) ; Lee; Seoung Ho;
(Chungcheongbuk-do, KR) ; Kim; Mi Ran; (Seoul,
KR) |
Assignee: |
UNIGEN, INC.
Chungnam
KR
|
Family ID: |
36118391 |
Appl. No.: |
13/191619 |
Filed: |
July 27, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13013880 |
Jan 26, 2011 |
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13191619 |
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11872845 |
Oct 16, 2007 |
7897182 |
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13013880 |
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10522832 |
Feb 2, 2005 |
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PCT/KR2004/000708 |
Mar 27, 2004 |
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11872845 |
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Current U.S.
Class: |
424/750 |
Current CPC
Class: |
A61P 7/00 20180101; A61P
9/12 20180101; A61P 35/00 20180101; A61P 9/10 20180101; A61P 9/00
20180101; A61P 31/12 20180101; A61K 31/192 20130101; A23L 33/105
20160801; A61K 36/899 20130101; A61P 1/16 20180101; A61P 3/06
20180101; A23V 2002/00 20130101; A61P 29/00 20180101; A61P 13/12
20180101; A61P 25/28 20180101; A61P 1/04 20180101; A61P 19/02
20180101; A23V 2002/00 20130101; A23V 2250/21 20130101; A23V
2200/324 20130101; A23V 2250/70 20130101; A23V 2250/156
20130101 |
Class at
Publication: |
424/750 |
International
Class: |
A61K 36/899 20060101
A61K036/899; A61P 31/12 20060101 A61P031/12; A61P 1/04 20060101
A61P001/04; A61P 25/28 20060101 A61P025/28; A61P 19/02 20060101
A61P019/02; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 27, 2003 |
KR |
10-2003-0019229 |
Mar 27, 2003 |
KR |
10-2003-0019230 |
Claims
1. A method for the prevention and treatment of inflammatory
disease comprising administering to a subject in need thereof an
effective amount of a composition comprising the crude extract or
non-polar solvent soluble extract of Bamboo plant.
2. The method according to claim 1 wherein said crude extract is
extracted with the solvent selected from the group consisting of
water, lower alcohol and the mixture thereof.
3. The method according to claim 1 wherein said non-polar solvent
soluble extract is extracted with non-polar solvent selected from
the group consisting of hexane, ethyl acetate, chloroform and
dichloromethane.
4. The method according to claim 1 wherein said bamboo is selected
from the Sasa genus or the Phyllostachys genus.
5. The method according to claim 4 wherein said bamboo selected
from the group consisting of Sasa borealis Makino, Sasa coreana
Nakai, Sasa japonica Makino, Sasa borealis var. gracilis and Sasa
palmata Nakai.
6. The method according to claim 4 wherein said bamboo plant
selected from the group consisting of Phyllostachys bambusoides
SIEB. Et Zucc, Phyllostachys nigra MUNRO, Phyllostachys nigra MUNRO
var. henonis STAPF and Phyllostachys pubescens MAZEL ex H. de
LEH.
7. The method according to claim 1 wherein said inflammatory
disease is selected from the group consisting of arthritis,
atheriosclerosis, gastritis, colitis, nephritis, hepatitis, cancer
or degenerative disease.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/013,880, filed Jan. 26, 2011, which is a continuation of
U.S. application Ser. No. 11/872,845, filed Oct. 16, 2007, which is
a continuation of U.S. application Ser. No. 10/522,832, filed Feb.
2, 2005, which is a 35 U.S.C. .sctn.371 national phase application
of PCT/KR2004/000708 (WO 2004/098624), filed on Mar. 27, 2004, each
of which is entitled "Composition Comprising Bamboo Extract and the
Compounds Isolated Therefrom Showing Treating and Preventing
Activity for Inflammatory and Blood Circulation Disease."
PCT/KR2004/000708 (WO 2004/098624), filed on Mar. 27, 2004, claims
priority to Korean Application Serial Nos. KR 10-2003-0019229 and
KR 10-2003-0019230, filed Mar. 27, 2003. Each of these references
is incorporated herein by reference in its entirety.
DESCRIPTION
[0002] 1. Field of the Invention
[0003] The present invention relates to bamboo extract and the
compound isolated therefrom showing treating and preventing
activity for inflammatory and blood circulation disease.
[0004] 2. Background of the Invention
[0005] Blood circulation disorder occurred by the blockage of blood
flow caused by deposited cholesterol and increased thrombus on
blood vessel, and lessened elastic force of blood vessel. The
representative symptoms of blood circulation disorder are benumbed
feet or hand, crick of neck and shoulders, loss of memory,
lethargy, loss of concentration, vertigo and chronic fatigue etc.,
which often give difficulty in normal living life. Hyperlipemia, as
one example of blood circulation disorder, is a condition where the
blood lipid parameters are elevated in the blood. This condition
manifests an abnormally high concentration of fats in the blood.
The lipid component in the circulating blood is total cholesterol,
low density lipoproteins, very low density lipoproteins or
triglycerides.
[0006] Inflammation occurred by the invasion of outer contaminant,
for example, bacteria, fungus, virus, various allergens inducing
inflammatory response and a biophysical process against them. The
characteristic syndrome of inflammatory response is serial and
complex physiological response such as the enhancement of enzyme
activity, the release of inflammatory mediators, fluidal
infiltration, cell movement, tissue disruption together with
external syndromes such as erythema, edema, pyrexia, ache and so
on.
[0007] NO (nitric oxide), a factor of those inflammatory responses
is formed by NOS (nitric oxide synthase), which acts on L-arginine
to form final products, i.e., NO and citrulline through an
intermediate (hydroxyarginine). The substance has small molecular
weight and it has been found that it acts on blood vessel system to
induce vasodilation, platelet agglutination and adhesion, neuronal
transmission, gastrointestinal movement and plays an important
roles in controlling metabolic pathway and physiological reaction
such as neuronal transmission, blood coagulation, blood pressure
regulation and the immunity against cancer cell etc. It is highly
toxic because of its free radical structure and is prone to be
changed to stabilized final product i.e., NO.sub.3 and NO.sub.2 in
the air (Snyder S. H., et al, Scientific American, May pp 28-35,
1992).
[0008] NOS can be classified with cNOS (constitutive NOS) and iNOS
(inducible NOS) according to the dependence to calcium ion or
calmodulin; wherein cNOS dependent to calcium ion or calmodulin is
present mainly in brain, epithelial cell, neutrophil, stomach
mucous cell and; wherein iNOS independent to calcium ion or
calmodulin is present mainly in macrophage, hepatic cell, cancer
cell etc and is induced by several factors, for example, cytokines
such as IL-1.beta., IFN-.gamma., TNF-.alpha., or an endotoxin such
as bacterial LPS (Dinerman, J. L., et al, Circ. Res., 73, pp
217-222, 1993). iNOS expression is closely correlated with COX-2
expression, therefore, formed NO may affect on COX-2 expression
(Robert C., et al., J. Immunol., 165, pp 1582-1587, 2000; Daniela
S., et al., Proc. Nat'l. Acad. Sci. USA, 90, pp 7240-7244,
1993).
[0009] Additionally, there have been many reports on the
correlation between NO production caused by iNOS and various
inflammatory diseases such as atheriosclerosis, arthritis,
gastritis, colitis, nephritis, hepatitis, cancer or various
degenerative diseases (Gobert A. P. et al., J. Immunol. 168(12), pp
6002-6006, 2002; Dijkstra G. et al., Scand. J. Gastroenterol.,
37(5) pp 546-554, 2002; Sakac V. and Sakac M. Med. Pregl., 53, pp
463-474, 2000; Albrecht E. W. et al., Am. J. Transplant, 29(5), pp
448-453, 2002; Ramachandran A. et al., Free Radical Biol. Med.,
33(11), pp 1465-1474, 2002; Sartor L. et al., Biochemical
Pharmacol., 64, pp 229-237, 2002; Sadowska Krowicka H. et al.,
Proc. Soc. Exp. Biol. Med., 217(3), pp 351-357 1998; Lo A. H. et
al., Carcinogenesis, 23(6) pp 983-991, 2002)
[0010] Accordingly, there have been investigated to develop a
medicine, health care food or food addatives to treat and prevent
above described various inflammatory diseases by finding potent
inhibitors for the production of NO caused by iNOS.
[0011] Bamboo belonged to Bambusaceae or Poaceae is distributed in
Asian countries including Korea and Japan. There are about 1259
species of bamboo all over the world. Among them, the
representative ones belonged to Bambusaceae are Phyllostachys
bambusoides SIEB. Et Zucc, Phyllostachys nigra MUNRO, Phyllostachys
nigra MUNRO var. henonis STAPF and Phyllostachys pubescens MAZEL ex
H. de LEH, and the representative ones belonged to another Poaceae
are Sasa borealis Makino, Sasa coreana Nakai, Sasa japonica Makino,
Sasa borealis var. gracilis, Sasa palmata Nakai, Setaria viridis
BEAUV and Oryza sativa L.
[0012] There have been several reports on the use of bamboo
extract, for example, Korean Patent Application No. 10-2001-69130
discloses on the process for preparing the leave extract from Sasa
japonica Makino and the use of the same as a food preservative
using its antimicrobial activity; U.S. Pat. No. 3,418,311 discloses
the polysaccharide isolated from bamboo having anticancer
activity.
[0013] However, there has been not reported or disclosed about
therapeutic effect for inflammatory or blood circulation disease of
bamboo extract and the compound isolated therefrom in any of above
cited literatures, the disclosures of which are incorporated herein
by reference.
[0014] To investigate and confirm the treating or preventing effect
on inflammatory or blood circulation disease of bamboo extract and
the compound isolated therefrom through several biochemical
experiments, the inventors of the present invention have
intensively carried out several biological experiments i.e., in
vitro inhibition test on NO or PLA.sub.2 production induced by LPS
activated macrophage and an effects on the expression of several
gene such as u-PA, eNOS and VEGF known to play an important role in
thrombolytic activity, the control of blood flow and the cell
growth in blood vessel together with cytotoxicity test, as well as
animal model test using LDL receptor defected mouse and normal
mouse and finally completed present invention by confirming that
the extract and the compound isolated therefrom have the treating
and preventing activity on inflammatory or blood circulation
diseases.
[0015] These and other objects of the present invention will become
apparent from the detailed disclosure of the present invention
provided hereinafter.
SUMMARY
[0016] The present invention provides a pharmaceutical composition
comprising bamboo extract or the compound isolated therefrom as an
active ingredient in an effective amount to treat and prevent
inflammatory disease caused by the over-production of NO.
[0017] The present invention provides a pharmaceutical composition
comprising bamboo extract or the compound isolated therefrom as an
active ingredient in an effective amount to treat and prevent blood
circulation disease.
[0018] The present invention also provides a use of above extract
or compound for the preparation of pharmaceutical composition to
treat and prevent inflammatory disease and blood circulation
disease.
[0019] The present invention also provides a health care food
comprising above extract or compound for the prevention or
alleviation of inflammatory disease by inhibiting NO production and
blood circulation disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The above and other objects, features and other advantages
of the present invention will more clearly understood from the
following detailed description taken in conjunction with the
accompanying drawings, in which;
[0021] FIGS. 1 and 2 represent the HPLC analysis data of tricin and
p-coumaric acid, FIG. 1a is for standard group, FIG. 1b is for
inventive bamboo extract.
[0022] FIGS. 3 and 4 show the effects of various concentrations of
bamboo crude extract and fractions isolated therefrom on NO
inhibition, FIG. 3 is for 50 .mu.g/ml treated group, FIG. 4 is for
100 .mu.g/ml treated group, wherein
TABLE-US-00001 A: crude extract, B: n-hexane soluble extract, C:
dichloromethane soluble extract, D: n-butanol soluble extract, E:
ethyl-acetate soluble extract, F: water-soluble extract;
[0023] FIG. 5 shows the cellular toxicity of bamboo extract, tricin
and p-coumaric acid in the HUVEC, wherein the numbers described
above the graph bars denote the concentration of treated sample
(.mu.g/ml)
[0024] FIG. 6 shows the cell proliferation effect of bamboo extract
in the HUVEC; FIGS. 7 to 9 show the effect of bamboo extract,
tricin and p-coumaric acid in the HUVEC with complete media on mRNA
expression, FIG. 7 is for VEGF expression, FIG. 8 is for u-PA
expression and FIG. 9 is for eNOS expression, wherein the numbers
described above the graph bars denote the concentration of treated
sample (.mu.g/ml);
[0025] FIGS. 10 to 12 show the wound healing effect of the
inventive bamboo extract through in vitro wound healing assay using
HUVEC, FIG. 10 is for control group, FIG. 11 is for 10 .mu.g/ml
bamboo extract-treated group and FIG. 12 is for 50 .mu.g/ml of
bamboo extract-treated group;
[0026] FIGS. 13 to 15 show the blood vessel formation of the
inventive bamboo extract through in vitro tube formation assay
using HUVEC, FIG. 13 is for control group, FIG. 14 is for 10
.mu.g/ml of bamboo extract-treated group and FIG. 15 is for 100
.mu.g/ml of bamboo extract-treated group;
[0027] FIG. 16 shows the change of body weights in the high
cholesterol diet induced atherosclerosis mice with or without the
treatment of bamboo extract for 16-weeks (50 and 100 mg/kg) and
20-weeks (500 mg/kg);
[0028] FIGS. 17 and 18 show the morphometry of Oil red 0 stained
aortic valve lesion areas by computer-associated mage analysis in
the high cholesterol diet induced atherosclerosis mice with or
without the treatment of bamboo extract for 16-weeks (50 and 100
mg/kg) and 20-weeks (500 mg/kg);
[0029] FIG. 17 represents Oil red 0 stained photographs of frozen
sections of aortic valve lesion, wherein the left panel is for
control group, the middle panel is for positive control group
treated with Lovastatin and right panel is for bamboo
extract-treated group, and FIG. 18 represents morphometric results
of aortic valve lesion areas by computer-associated image
analysis.
DETAILED DESCRIPTION OF THE INVENTION
[0030] Accordingly, it is an object of the present invention to
provide a pharmaceutical composition comprising the crude extract,
polar solvent soluble or non-polar solvent soluble extract of
bamboo plant as an active ingredient for the treatment and
prevention of cardiovascular disease.
[0031] It is an object of the present invention to provide a
pharmaceutical composition comprising the crude extract, polar
solvent soluble or non-polar solvent soluble extract of Bamboo
plant as an active ingredient for the treatment and prevention of
blood circulation disease by inhibiting elastase activity, healing
the wound of vascular endothelial cell, activating u-PA expression,
inhibiting PAI-1 expression, lowering cholesterol deposit and
inhibiting neointima formation.
[0032] It is an object of the present invention to provide a
pharmaceutical composition comprising the crude extract, polar
solvent soluble or non-polar solvent soluble extract of bamboo
plant as an active ingredient for the treatment and prevention of
inflammation by inhibiting NO production and phospholipase A.sub.2
expression.
[0033] The term "crude extract" disclosed herein comprises the
extract prepared by extracting plant material with water, lower
alcohols such as methanol, ethanol, preferably methanol and the
like, or the mixtures thereof.
[0034] The term "polar solvent soluble extract" disclosed herein
can be prepared by extracting above crude extract with polar
solvent, for example, water, lower alcohol such as methanol,
ethanol, preferably butanol and the like, or the mixtures
thereof.
[0035] The term "non-polar solvent soluble extract" disclosed
herein can be prepared by extracting above crude extract with
non-polar solvent, for example, hexane, ethyl acetate or
dichloromethane, preferably ethyl acetate.
[0036] Accordingly, it is another object of the present invention
to provide a pharmaceutical composition comprising tricin and
p-coumaric acid isolated from bamboo plant extract as an active
ingredient for the treatment and prevention of cardiovascular
disease.
[0037] It is an object of the present invention to provide a
pharmaceutical composition comprising tricin and p-coumaric acid
isolated from bamboo plant extract as an active ingredient for the
treatment and prevention of blood circulation disease by inhibiting
elastase activity, healing the wound of vascular endothelial cell
and increasing VEGF, u-PA and eNOS gene expression.
[0038] It is another object of the present invention to provide a
pharmaceutical composition comprising tricin and p-coumaric acid
isolated from bamboo plant extract as an active ingredient for the
treatment and prevention of inflammation by inhibiting NO
production.
[0039] The term "bamboo plant" disclosed herein comprises the stem
or leaves of bamboo plant belonged to Bambusaceae or Poraceae.
Preferable plants belonged to Bambusaceae are Phyllostachys
bambusoides SIEB. Et Zucc, Phyllostachys nigra MUNRO, Phyllostachys
nigra MUNRO var. henonis STAPF and Phyllostachys pubescens MAZEL ex
H. de LEH and more preferable one is Phyllostachys nigra MUNRO var.
henonis STAPF. Preferable plants belonged to Poaceae are Sasa
borealis Makino, Sasa coreana Nakai, Sasa japonica Makino, Sasa
borealis var. gracilis, Sasa palmata Nakai, Setaria viridis BEAUV
and Oryza sativa L and more preferable one is Sasa borealis
Makino.
[0040] The term "u-PA" disclosed herein is urokinase type
plasminogen activator gene (fibrinolytic factor), "PLA.sub.2" is
phospholipase A.sub.2 gene, "VEGF" is vascular endothelial growth
factor gene, "eNOS" is endothelial nitrous oxide synthase gene and
"PAI-1" is plasminogen activator inhibitor 1 gene.
[0041] It is an object of the present invention to provide a use of
a crude extract, polar solvent soluble or non-polar solvent soluble
extract of bamboo plant for the preparation of therapeutic agent
for the treatment and prevention of inflammatory disease by
inhibiting NO production and PLA.sub.2 expression in human or
mammal.
[0042] It is an object of the present invention to provide a use of
a crude extract, polar solvent soluble or non-polar solvent soluble
extract of bamboo plant for the preparation of therapeutic agent
for treatment and prevention of blood circulation disease by
inhibiting elastase activity, healing the wound of vascular
endothelial cell, activating u-PA expression and inhibiting PAI-1
expression, lowering cholesterol deposit and inhibiting neointima
formation in human or mammal.
[0043] It is an object of the present invention to provide a method
of treating and preventing inflammatory disease by inhibiting NO
production in a mammal comprising administering to said mammal an
effective amount of crude extract, polar solvent soluble or
non-polar solvent soluble extract of bamboo extract, together with
a pharmaceutically acceptable carrier thereof.
[0044] It is an object of the present invention to provide a method
of treating and preventing blood circulation disease by inhibiting
elastase activity and healing the wound of vascular endothelial
cell, lowering cholesterol deposit and inhibiting neointima
formation in a mammal comprising administering to said mammal an
effective amount of crude extract, polar solvent soluble or
non-polar solvent soluble extract of bamboo extract, together with
a pharmaceutically acceptable carrier thereof.
[0045] It is another object of the present invention to provide a
health care food comprising above described extract or compound,
together with a sitologically acceptable additive for the
prevention and alleviation of inflammatory disease by inhibiting NO
production and PLA expression and blood circulation disease.
[0046] The term "cardiovascular disease" disclosed herein comprises
various cardiovascular diseases such as hypertension, heart
disease, brain stroke, peripheral blood disease, hyperlipemia,
arteriosclerosis, stenosis, thrombosis or cardiac infarction
etc.
[0047] The term "inflammatory disease" disclosed herein comprises
various inflammatory diseases such as atheriosclerosis, arthritis,
gastritis, colitis, nephritis, hepatitis, cancer or various
degenerative diseases.
[0048] The pharmaceutical composition of the present invention can
contain about 0.1.about.70% by weight of the above extract or
compound based on the total weight of the composition.
[0049] The health care food of the present invention comprises the
above extract or compound as 0.01 to 80%, preferably 1 to 60% by
weight based on the total weight of the composition.
[0050] Above health care food can be contained in health care food,
health beverage etc, and may be used as powder, granule, tablet,
chewing tablet, capsule, beverage etc.
[0051] An inventive extract and compound isolated from bamboo plant
may be prepared in accordance with the following preferred
embodiment.
[0052] Hereinafter, the present invention is described in
detail.
[0053] An inventive extract of bamboo plant can be prepared in
detail by following procedures,
[0054] The inventive crude extract of Phyllostachys nigra MUNRO
var. henonis STAPF or Sasa borealis Makino can be prepared by
follows; Phyllostachys nigra MUNRO var. henonis STAPF or Sasa
borealis Makino is dried, cut, crushed and mixed with 5 to 25-fold,
preferably, approximately 10 fold volume of distilled water, lower
alcohols such as methanol, ethanol, butanol and the like, or the
mixtures thereof, preferably methanol; the solution is treated with
hot water at the temperature ranging from 20 to 100.degree. C.,
preferably from 60 to 100.degree. C., for the period ranging from 1
to 24 hours with extraction method by the extraction with hot
water, cold water, reflux extraction, or ultra-sonication
extraction with 1 to 5 times, preferably 2 to 3 times,
consecutively; the residue is filtered to obtain the supernatant to
be concentrated with rotary evaporator, at the temperature ranging
from 20 to 100.degree. C., preferably from 50 to 70.degree. C. and
then dried by vacuum freeze-drying, hot air-drying or spray drying
to obtain dried crude extract powder of Phyllostachys nigra MUNRO
var. henonis STAPF or Sasa borealis Makino which can be soluble in
water, lower alcohols, or the mixtures thereof.
[0055] Additionally, polar solvent soluble and non-polar solvent
soluble extract of present invention can be prepared by following
procedure; the crude extract prepared by above step, is suspended
in water, and then is mixed with 1 to 100-fold, preferably, 1 to
5-fold volume of non polar solvent such as ethyl acetate,
chloroform, hexane and the like; the non-polar solvent soluble
layer is collected to obtain non-polar solvent soluble extract of
the present invention and remaining polar solvent soluble layer is
collected to obtain polar solvent soluble extract of the present
invention which is soluble in water, lower alcohols, or the
mixtures thereof. Also, above described procedures may be modified
or subjected to further step to fractionate or isolate more potent
fractions or compounds by conventional procedure well-known in the
art, for example, the procedure disclosed in the literature
(Harborne J. B. Phytochemical methods: A guide to modern techniques
of plant analysis, 3.sup.rd Ed. pp 6-7, 1998).
[0056] To investigate the effect of bamboo plant extract on
inflammation and blood circulation through several biochemical
experiments and to confirm whether the crude extract and non-polar
solvent soluble extract play an important role in inhibiting NO
production, main cause of inflammation, and in improving blood
circulation or not, and then it is confirmed that the crude
extract, polar solvent soluble and non-polar solvent soluble
extract inhibit the NO production, iNOS gene expression, elastase
activity and PAI-1 gene expression, promotes the u-PA gene
expression and shows in vitro wound healing, in vitro tube
formation activity and inhibit cholesterol deposit and neointima
formation.
[0057] In accordance with another aspect of the present invention,
there is provided a pharmaceutical composition comprising the crude
extract, polar solvent soluble or non-polar solvent soluble extract
of Phyllostachys nigra MUNRO var. henonis STAPF or Sasa borealis
Makino prepared by above preparation method for the treatment and
prevention of inflammation by inhibiting NO production as active
ingredients.
[0058] It is another of the present invention to provide a treating
method and preventing method comprising administering a
pharmaceutical composition comprising said extract prepared by
above preparation method to said mammals including human for
treating inflammation or blood circulation disease.
[0059] The inventive composition for treating and preventing
inflammation by inhibiting NO production and for improving blood
circulation may comprises above extracts as 0.1.about.70% by weight
based on the total weight of the composition.
[0060] The inventive composition may additionally comprise
conventional carrier, adjuvants or diluents in accordance with a
using method well known in the art. It is preferable that said
carrier is used as appropriate substance according to the usage and
application method, but it is not limited. Appropriate diluents are
listed in the written text of Remington's Pharmaceutical Science
(Mack Publishing co, Easton Pa.).
[0061] Hereinafter, the following formulation methods and
excipients are merely exemplary and in no way limit the
invention.
[0062] The composition according to the present invention can be
provided as a pharmaceutical composition containing
pharmaceutically acceptable carriers, adjuvants or diluents, e.g.,
lactose, dextrose, sucrose, sorbitol, mannitol, xylitol,
erythritol, maltitol, starches, acacia rubber, alginate, gelatin,
calcium phosphate, calcium silicate, cellulose, methyl cellulose,
polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy
benzoate, talc, magnesium stearate or mineral oil. The formulations
may additionally include fillers, anti-agglutinating agents,
lubricating agents, wetting agents, flavoring agents, emulsifiers,
preservatives and the like. The compositions of the invention may
be formulated so as to provide quick, sustained or delayed release
of the active ingredient after their administration to a patient by
employing any of the procedures well known in the art.
[0063] For example, the compositions of the present invention can
be dissolved in oils, propylene glycol or other solvents that are
commonly used to produce an injection. Suitable examples of the
carriers include physiological saline, polyethylene glycol,
ethanol, vegetable oils, isopropyl myristate, etc., but are not
limited to them. For topical administration, the extract of the
present invention can be formulated in the form of ointments and
creams.
[0064] Pharmaceutical formulations containing present composition
may be prepared in any form, such as oral dosage form (powder,
tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs
pill, powder, sachet, granule), or topical preparation (cream,
ointment, lotion, gel, balm, patch, paste, spray solution, aerosol
and the like), or injectable preparation (solution, suspension,
emulsion).
[0065] The composition of the present invention in pharmaceutical
dosage forms may be used in the form of their pharmaceutically
acceptable salts, and also may be used alone or in appropriate
association, as well as in combination with other pharmaceutically
active compounds.
[0066] The desirable dose of the inventive extract or compound
varies depending on the condition and the weight of the subject,
severity, drug form, route and a period of administration, and may
be chosen by those skilled in the art. However, in order to obtain
desirable effects, it is generally recommended to administer at the
amount ranging 10 g/kg, preferably, 1 to 3 g/kg by weight/day of
the inventive extract or compounds of the present invention. The
dose may be administered in single or divided into several times
per day. In terms of composition, the amount of inventive extract
should be present between 0.01 to 70% by weight, preferably 0.5 to
50% by weight based on the total weight of the composition.
[0067] The pharmaceutical composition of present invention can be
administered to a subject animal such as mammals (rat, mouse,
domestic animals or human) via various routes. All modes of
administration are contemplated, for example, administration can be
made orally, rectally or by intravenous, intramuscular,
subcutaneous, intracutaneous, intrathecal, epidural or
intracerebroventricular injection.
[0068] The term "health care food" disclosed herein comprises
dietary supplement, nutraceuticals, food or food additives
[0069] Also, the present invention provide a composition of the
health care food beverage for the prevention or improvement of
inflammation or blood circulation adding above described extracts
0.01 to 80% by weight, amino acids 0.001 to 5% by weight, vitamins
0.001 to 2% by weight, sugars 0.001 to 20% by weight, organic acids
0.001 to 10% by weight, sweetener and flavors of proper amount.
[0070] Above described extract of bamboo plant can be added to food
and beverage for the prevention and improvement of inflammation or
blood circulation.
[0071] To develop for health care food, examples of addable food
comprising above extracts of the present invention are various
food, beverage, gum, vitamin complex, health improving food and the
like, and can be used as power, granule, tablet, chewing tablet,
capsule or beverage etc.
[0072] Also, the extract of the present invention will be able to
prevent and improve allergic disease and non-allergic inflammation
disease by adding to child and infant food, such as modified milk
powder, modified milk powder for a growth period, modified food for
a growth period.
[0073] Above described composition therein can be added to food,
additive or beverage, wherein the amount of above described extract
in food or beverage may generally range from about 0.1 to 80 w/w %,
preferably 1 to 50 w/w % of total weight of food for the health
care food composition and 1 to 30 g, preferably 3 to 10 g on the
ratio of 100 ml of the health beverage composition.
[0074] Providing that the health beverage composition of present
invention contains above described extract as an essential
component in the indicated ratio, there is no particular limitation
on the other liquid component, wherein the other component can be
various deodorant or natural carbohydrate etc such as conventional
beverage. Examples of aforementioned natural carbohydrate are
monosaccharide such as glucose, fructose etc; disaccharide such as
maltose, sucrose etc; conventional sugar such as dextrin,
cyclodextrin; and sugar alcohol such as xylitol, and erythritol
etc. As the other deodorant than aforementioned ones, natural
deodorant such as taumatin, stevia extract such as levaudioside A,
glycyrrhizin et al., and synthetic deodorant such as saccharin,
aspartam et al., may be useful favorably. The amount of above
described natural carbohydrate is generally ranges from about 1 to
20 g, preferably 5 to 12 g in the ratio of 100 ml of present
beverage composition.
[0075] The other components than aforementioned composition are
various nutrients, a vitamin, a mineral or an electrolyte,
synthetic flavoring agent, a coloring agent and improving agent in
case of cheese chocolate et al., pectic acid and the salt thereof,
alginic acid and the salt thereof, organic acid, protective
colloidal adhesive, pH controlling agent, stabilizer, a
preservative, glycerin, alcohol, carbonizing agent used in
carbonate beverage et al. The other component than aforementioned
ones may be fruit juice for preparing natural fruit juice, fruit
juice beverage and vegetable beverage, wherein the component can be
used independently or in combination. The ratio of the components
is not so important but is generally range from about 0 to 20 w/w %
per 100 w/w % present composition. Examples of addable food
comprising aforementioned extract therein are various food,
beverage, gum, vitamin complex, health improving food and the
like.
[0076] The inventive composition may additionally comprise one or
more than one of organic acid, such as citric acid, fumaric acid,
adipic acid, lactic acid, malic acid; phosphate, such as phosphate,
sodium phosphate, potassium phosphate, acid pyrophosphate,
polyphosphate; natural anti-oxidants, such as polyphenol, catechin,
.alpha.-tocopherol, rosemary extract, vitamin C, green tea extract,
licorice root extract, chitosan, tannic acid, phytic acid etc.
[0077] The above extract of bamboo plant may be 20 to 90% high
concentrated liquid, power, or granule type.
[0078] Similarly, the above extract of bamboo plant can comprise
additionally one or more than one of lactose, casein, dextrose,
glucose, sucrose and sorbitol.
[0079] Inventive extract of the present invention have no toxicity
and adverse effect therefore; they can be used with safe.
[0080] It will be apparent to those skilled in the art that various
modifications and variations can be made in the compositions, use
and preparations of the present invention without departing from
the spirit or scope of the invention.
BEST MODE FOR CARRYING OUT THE INVENTION
[0081] It will be apparent to those skilled in the art that various
modifications and variations can be made in the compositions, use
and preparations of the present invention without departing from
the spirit or scope of the invention.
[0082] The present invention is more specifically explained by the
following examples. However, it should be understood that the
present invention is not limited to these examples in any
manner.
EXAMPLES
[0083] The following Reference Example, Examples and Experimental
Examples are intended to further illustrate the present invention
without limiting its scope.
Example 1
Preparation of the Crude Extract of Bamboo Plant
[0084] Bamboo plants of Sasa borealis Makino, Sasa coreana Nakai,
Sasa japonica Makino, Sasa borealis var. gracilis, Sasa palmata
Nakai, Phyllostachys nigra MUNRO var. henonis STAPF, Phyllostachys
bambusoides SIEB. Et Zucc., Phyllostachys nigra MUNRO, and
Phyllostachys pubescens MAZEL ex H. de LEH were washed, dried for
10 days at room temperature.
[0085] 10 kg of dried leaves or stem of bamboo plant were cut into
small pieces, mixed with 100 L of 70% ethanol and the mixture was
heated 3 times at 80.degree. C. for 7 hours, repeatedly. And the
extract was filtered with filter paper (Whatman Co., U.S.A.). The
filtrates were pooled and concentrated by rotary evaporator
(N-1000, Eyela Co. Japan) at 55.about.65.degree. C. under reduced
pressure and dried with freezing dryer (Speed Spec 3000, Bio-Rad,
U.S.A.) to obtain dried crude extract of each bamboo plant (See
Table 1).
TABLE-US-00002 TABLE 1 Leaf Stem Sasa borealis Makino 880 g 880 g
Sasa coreana Nakai 850 g 640 g Sasa japonica Makino 750 g 550 g
Sasa borealis var. gracilis 810 g 760 g Sasa palmata Nakai 9070 g
790 g Phyllostachys nigra MUNRO var. henonis STAPF 810 g 740 g
Phyllostachys bambusoides SIEB. Et Zucc. 1030 g 870 g Phyllostachys
nigra MUNRO 840 g 870 g Phyllostachys pubescens MAZEL ex H. de LEH
1160 g 840 g
Example 2
Preparation of Polar Solvent and Non-Polar Solvent Soluble Extract
of Phyllostachys nigra
[0086] 2-1. Preparation of n-Hexane Soluble Extract
[0087] 50 g of crude extract of Phyllostachys nigra prepared in
Example 1 was suspended in 1 liter of distilled water and the
suspension was mixed with 1 liter of n-hexane vigorously to divide
into n-hexane soluble fraction and water-soluble fraction. n-hexane
soluble fraction was collected and the residual solution was
subjected to the n-hexane extraction again. Above-described
procedure was repeated 3 times.
[0088] n-hexane soluble fraction was evaporated in vacuo to give
9.1 g of n-hexane soluble extract of Phyllostachys nigra.
[0089] 2-2. Preparation of Dichloromethane Soluble Extract
[0090] Water-soluble fraction of Phyllostachys nigra prepared in
Example 2-1 was mixed with equivalent volume of dichloromethane
vigorously to divide into dichloromethane soluble fraction and
water-soluble fraction. Dichloromethane soluble fraction was
collected and the residual solution was subjected to the
dichloromethane extraction again. Above-described procedure was
repeated 3 times.
[0091] Dichloromethane soluble fraction was evaporated in vacuo to
give 4.6 g of dichloromethane soluble extract of Phyllostachys
nigra.
[0092] 2-3. Preparation of Ethylacetate Soluble Extract
[0093] Water-soluble fraction of Phyllostachys nigra in Example 2-2
was mixed with equivalent volume of ethylacetate vigorously to
divide into ethylacetate soluble fraction and water-soluble
fraction. Ethylacetate soluble fraction was collected and the
residual solution was subjected to the ethylacetate extraction
again. Above-described procedure was repeated 3 times.
[0094] Ethyl acetate soluble fraction was evaporated in vacuo to
give 4.3 g of ethylacetate soluble extract of Phyllostachys
nigra.
[0095] 2-4. Preparation of n-Butanol and Water-Soluble Extract
[0096] Water-soluble fraction of Phyllostachys nigra in Example 2-3
was mixed with equivalent volume of n-butanol vigorously to divide
into n-butanol soluble fraction and water-soluble fraction.
n-butanol soluble fraction was collected and the residual solution
was subjected to the n-butanol extraction again. Above-described
procedure was repeated 3 times.
[0097] n-butanol soluble fraction and water-soluble fraction were
respectively evaporated in vacuo to give 7.1 g of n-butanol soluble
extract and 25.1 g of water-soluble extract of Phyllostachys
nigra.
Example 3
Preparation of Polar Solvent and Non-Polar Solvent Soluble Extract
of Sasa borealis (1)
[0098] As shown in the Table 2, each polar solvent and non-polar
solvent soluble extract was prepared according to the identical
method disclosed in the above Example 2.
TABLE-US-00003 TABLE 2 Amount Crude extract 57 g n-hexane soluble
extract 9.5 g Dichloromethane soluble extract 4.1 g Ethyl acetate
soluble extract 4.8 g n-butanol soluble extract 27.9 g
Water-soluble extract 27.9 g
Example 4
Preparation of Polar Solvent and Non-Polar Solvent Soluble Extract
of Sasa borealis (2)
[0099] The dried extract from the stem of Sasa borealis Makino
prepared in Example 1 was subject to fractionation as follows.
[0100] 100 g of the crude extract obtained in Example 1 was
suspended in 1000 ml of distilled water. 1000 ml of chloroform was
added thereto in separatory funnel and the mixture was shaken
vigorously to divide into chloroform soluble layer and water
soluble layer. Chloroform soluble fraction was collected and the
residual solution was subjected to the chloroform extraction
again.
[0101] Above-described procedure was repeated 3 times to separate
the chloroform soluble component and chloroform soluble fraction
was collected and dried under reduced pressure to obtain 17.8 g of
chloroform soluble fraction.
[0102] Above water soluble fraction was mixed with equivalent
amount of ethyl acetate and then divided into ethyl acetate soluble
layer and water-soluble layer. The fractionation process was
repeated 3 times.
[0103] Above ethyl acetate soluble layer was concentrated by rotary
evaporator, dried under reduced pressure to obtain 15.4 g of ethyl
acetate soluble extract.
[0104] Finally, water-soluble layer was also obtained to use as a
sample in the following experiments.
Example 5
Isolation of Tricin and p-Coumaric Acid
[0105] 10 g of ethyl acetate soluble fraction prepared in Example 4
was subjected to Silica gel column chromatography to isolate tricin
and p-coumaric acid.
[0106] 10 g of ethyl acetate fraction was loaded onto the Silica
gel column and the column was eluted with a stepwise application of
solvent mixture containing linear gradient of chloroform:acetone
(100:1.fwdarw.1:1) to give 7 sub-fractions. Among 7 fractions, the
4.sup.th fraction was recrystallized using methanol and 26 mg of
yellow crystal was isolated therefrom.
[0107] Above prepared yellow crystal was subjected to thin layer
chromatography using TLC plate (Silica gel 60 F254 plate, layer
thickness 0.2 mm, 20.times.20, Merck Co, Germany) and
chloroform:methanol(20:1) mixture as a developing solvent. The TLC
result showed that the crystal was detected as a yellow spot in
anisaldehyde-H.sub.2SO.sub.4 treatment and as a dark brown spot in
365 nm UV light (Power wave-XS, Bio-Tek, USA) with 0.4 of R.sub.f
(solvent system: CHCl.sub.3:MeOH=20:1).
[0108] And the result of .sup.1H and .sup.13C-NMR data by NMR
spectroscopy (.sup.1H: 300 MHz, .sup.13C: 75 MHz, DRX 300, Bruker,
Germany) showed that the yellow crystal was identified as a tricin
and the spectral data were shown as below.
[0109] Tricin: C.sub.17H.sub.14O.sub.7
[0110] .sup.1H-NMR (300 MHz, d6-DMSO): .delta. 12.96 (5-OH, 1H, s),
7.33 (H-2',H-6', 2H, s), 6.97 (H-3, 1H, s), 6.56 (H-8, 1H, d, J=2.0
Hz), 6.21 (H-6, 1H, d, J=2.0 Hz), 3.89 (--OCH.sub.3, 6H, s).
[0111] .sup.13C-NMR (75 MHz, d6-DMSO): .delta. 182.66 (C-4), 164.96
(C-2), 164.53 (C-7), 162.26 (C-5), 158.21 (C-9), 149.07 (C-3',5'),
140.74 (C-4'), 121.30 (C-1'), 105.32 (C-3), 104.61 (C-2',6'),
104.46 (C-10), 99.69 (C-6), 95.04 (C-8), 57.26 (--OCH.sub.3)
[0112] Among 7 fractions, the 7.sup.th fraction was subjected to
preparation HPLC to obtain 4.2 mg of phenyl propanoid compound and
the isolated phenylpropanoid compound was identified as p-coumaric
acid derivatives by the result of .sup.1H-NMR spectrum by NMR
spectroscopy (.sup.1H: 300 MHz, DRX 300, Bruker, Germany) shown as
below and the retention time of HPLC analysis compared with the
data of p-coumaric acid standard purchased from Sigma Company (See
FIGS. 1 and 2).
[0113] p-Coumaric Acid: C.sub.9H.sub.8O.sub.3
[0114] .sup.1H-NMR (300 MHz, d6-DMSO): .delta. 7.5 (H-2, H-6, 2H,
d., J=8.4 Hz), 7.48 (H-.gamma., 1H, d., J=16.2 Hz, 6.79 (H-3, H-5,
2H, d., J=8.4 Hz), 6.285 (H-.beta., 1H, d., J=16.2 Hz).
Example 6
Content Analysis of Tricin and p-Comaric Acid
[0115] Each 10 g of stem extract and leaf extract of Sasa borealis
Makino, Sasa coreana Nakai, Sasa japonica Makino, Sasa borealis
var. gracilis, Sasa palmata Nakai, Phyllostachys nigra MUNRO var.
henonis STAPF, Phyllostachys bambusoides SIEB. Et Zucc.,
Phyllostachys nigra MUNRO or Phyllostachys pubescens MAZEL ex H. de
LEH was used to analyze the content of tricin and p-coumaric acid
in the different part of plant by HPLC (Hitachi Co. L-7000 model).
HPLC analysis was performed on condition shown in Table 3.
TABLE-US-00004 TABLE 3 Time (min) A* B** C*** 0 100 0 0 30 0 100 0
60 0 100 0 65 0 0 100 75 0 0 100 80 100 0 0 85 100 0 0 A* solution:
0.1% H.sub.3PO.sub.4 in ACN:H.sub.2O (1:9) B** solution: 0.1%
H.sub.3PO.sub.4 in ACN:H.sub.2O (25:75) C*** solution: 100% CAN
Condition: Stationary phase(phenomenex C18, 4.6 .times. 250 mm, 5
.mu.m) at 35.degree. C., the wavelength at the detectors (330 nm),
10 .mu.l of samples were injected by 50000 ppm.
[0116] The content of tricin and p-coumaric acid in various bamboo
leaf extract and bamboo stem extract was shown in Table 4 and Table
5 respectively.
TABLE-US-00005 TABLE 4 Leaf (70% ethanol extract) tricin p-coumaric
acid Sasa borealis Makino 13 mg 26 mg Sasa coreana Nakai 15 mg 34
mg Sasa japonica Makino 14 mg 5 mg Sasa borealis var. gracilis 5 mg
7 mg Sasa palmata Nakai 3 mg 1.3 mg Phyllostachys nigra MUNRO var.
18 mg 34 mg henonis STAPF Phyllostachys bambusoides SIEB. 5 mg 1.5
mg Et Zucc. Phyllostachys nigra MUNRO 5 mg 39 mg Phyllostachys
pubescens MAZEL 3 mg 2.1 mg ex H. de LEH
TABLE-US-00006 TABLE 5 Stem (70% ethanol extract) tricin p-coumaric
acid Sasa borealis Makino 18 mg 52 mg Sasa coreana Nakai 28 mg 83
mg Sasa japonica Makino 23 mg 16 mg Sasa borealis var. gracilis 7
mg 17 mg Sasa palmata Nakai 5 mg 11 mg Phyllostachys nigra MUNRO
var. 26 mg 33 mg henonis STAPF Phyllostachys bambusoides SIEB. Et
12 mg 32 mg Zucc. Phyllostachys nigra MUNRO 13 mg 58 mg
Phyllostachys pubescens MAZEL ex 23 mg 41 mg H. de LEH
Reference Example 1
Cell Culture and Reagent
[0117] 1-1 Cell Culture
[0118] Murine macrophage cell line RAW 264.7 cell (ATCC, Rockville,
Md., USA) were grown in DMEM (Gibco BRL Co., Ltd., USA),
supplemented with 2.0 mM L-arginine, 100 .mu.g/ml
penicillin-streptomycin and 10% fetal bovine serum at 37.degree. C.
in 5% CO2 and 95% air condition in humidified incubator.
[0119] Medium was changed with 10 ml fresh DMEM 4 times per week
and cells were passaged 2 times per week.
[0120] HUVEC (Human umbilical vein endothelial cell) was cultured
on 0.2% gelatin coating flask (MTT Co.) in the EGM-2 media
(Clonetics Co.) supplemented with 20% FBS, 100.times.antibiotics
and 200.times.ECGF and the cells passaged 3 to 5 times were used in
the following experiment.
[0121] 1-2 Reagent and Instrument
[0122] Centrifuge (Hanil Centrifuge Co. Ltd, Korea), NMR
Spectroscopy (1H; 300 MHz, 13C; 75 MHz, DRX 300, Bruker Germany)
and UV Spectroscopy (Power wave-XS model, Bio-Tek Co. Ltd, USA)
were used in Korea Basic Science Institute located in Korea, Silica
gel 60H(230-400 mesh, Merck, Germany) was used as a column
chromatographic absorbent and Silca gel 60 F254 Plate (layer
thickness 0.2 mm, 20.times.20 cm, Art. 5554, Merck, Germany) was
used as a TLC plate. Anisaldehyde-sulfuric acid reagent was used as
a developer and all the organic solvent were purchased from Duksan
Chemical. Co. Ltd. in Korea.
Experimental Example 1
Animal Model Test
[0123] 1-1. Experimental Animal
[0124] To evaluate the efficacy of bamboo extract on the protection
of blood vessel and the improvement of blood circulation,
arteriosclerosis model mice were used in the experiment.
[0125] Six-weeks old male LDL receptor defected mouse (B6.
12957-LdlrtmlHer) procured from Jackson Co. Ltd. (USA) had been
acclimated to the experimental environment from 1 week ago by
administrating the increasing ratio of fatty feed, i.e., ratio of
normal feed to high fat feed was gradually increased (7:3 at 2nd
day, 5:5 at 4th day, 3:7 at 6th day). During the experiment, the
environment of the cage was maintained to the temperature of
23.+-.2.degree. C. and the relative humidity of 55.+-.10.degree. C.
under the artificial lamp for 12 hours, and less than five mice
were bred in each mouse cage providing with free access to water
(disinfected distilled water) and normal fatty feed (Harlan 2018S,
Indianapolis USA). After 8 weeks, only high fat feed (Harlan
TD88051, Artherosclerotic diet, overall fat content is about 15.8%;
cholesterol level of 1.25%, and sodium cholate at 0.5%, about 4
kcal/g, and 35% of kcal from fat. About half the fat come from
added cocoa butter, and half from the chow) were provided.
[0126] 1-2. Grouping and Administration Period
[0127] Eight-weeks old male LDL receptor defected mouse accumulated
to high fat feed were divided into two dose administration groups
and one high dosing group of which group consist of six mice per
group, Injectable distilled water was used as a negative control
group, lovastain well known to be as an atheriosclerosis treating
agent was administrated in an amount of 4 mg/kg per body weight as
a positive control group, Bamboo was administrated to two treatment
group in an amount of 50 and 100 mg/kg per body weight
respectively, and one high dosing treatment group in an amount of
500 mg/kg per body weight for 20 weeks by way of compulsory oral
administration after weighing the body weight twice a day (See
Table 6).
TABLE-US-00007 TABLE 6 The experimental groups Groups Diets Animal
No. Control High fat diet 6 Positive control High fat diet plus
lovastatin 4 mg/kg 6 bamboo-50 High fat diet plus sample dose 50
mg/kg 6 bamboo-100 High fat diet plus sample dose 100 mg/kg 6
bamboo-500 High fat diet plus sample dose 500 mg/kg 6
[0128] 1-3. Experimental Animal
[0129] To evaluate the efficacy of bamboo extract on the protection
of blood vessel and the improvement of blood circulation,
atherosclerosis model mice were used in the experiment.
[0130] Six-weeks old male C57BL/J6 mouse procured from Jackson Co.
Ltd. (USA) had been acclimated to the experimental environment from
1 week ago by administrating the increasing ratio of fatty feed,
i.e., ratio of normal feed to high fat feed was gradually increased
(7:3 at 2nd day, 5:5 at 4th day, 3:7 at 6th day). During the
experiment, the environment of the cage was maintained to the
temperature of 23.+-.2.degree. C. and the relative humidity of
55.+-.10.degree. C. under the artificial lamp for 12 hours, and
less than five mice were bred in each mouse cage providing with
free access to water (disinfected distilled water) and normal fatty
feed (Harlan 2018S, Indianapolis USA). After 8 weeks, only high fat
feed (Harlan TD88051, Atherosclerotic diet, overall fat content is
about 15.8%; cholesterol level of 1.25%, and sodium cholate at
0.5%, about 4 kcal/g, and 35% of kcal from fat. About half the fat
come from added cocoa butter, and half from the chow) were
provided.
[0131] 1-4. Grouping and Administration Period
[0132] Eight-weeks old male C57BL/J6 mouse accumulated to high fat
feed were divided into two dose administration consist of six mice
per group, Injectable distilled water was used as a negative
control group, bamboo was administrated to two treatment group in
an amount of 50 and 100 mg/kg per body weight for 6 months by way
of compulsory oral administration after weighing the body weight
twice a day (See Table 7).
TABLE-US-00008 TABLE 7 The experimental groups Groups Diets Animal
No. Control High fat diet 6 bamboo-50 High fat diet plus sample
dose 50 mg/kg 6 bamboo-100 High fat diet plus sample dose 100 mg/kg
6
Experimental Example 2
Effect of Bamboo Extract and the Compound Isolated Therefrom on NO
Production
[0133] To test the inhibiting activity of bamboo extract and the
compound isolated therefrom on nitric oxide (NO), which is one of
inflammatory factors, the increase of NO was determined in the
cells treated with extract or compound of the present
invention.
[0134] 200 .mu.l of RAW 264.7 cells (1.times.10.sup.6 cells/ml)
were seeded onto each well of 96-well microtiter plate (Nunc,
Sweden) and incubated for 3 hours in DMEM media containing 10% FBS.
After changing media to fresh DMEM, the cells were treated with 1
.mu.g/ml of LPS and 50 .mu.g/ml or 100 .mu.g/ml of bamboo extract
or tricin prepared in above Example 1-5, and cultured at 37.degree.
C. for 20 hours in 5% CO.sub.2 incubator.
[0135] And then 100 .mu.A of cell supernatant of each well was
transferred to new 96 well plate and 50 .mu.A of Griess reagent
(0.1% N-(1-naphtyl)ethylenediamine 2HCl, 1% sulfanilamide in 5%
conc. H.sub.3PO.sub.4 in H.sub.2O) was added thereto followed by
incubating for 10 mins at R.T. The absorbance was measured at 540
or 550 nm using ELISA reader (Power wave-XS, Bio-Tek, USA).
[0136] As shown in Table 8 and FIGS. 3 and 4, it was confirmed that
sample treatment group with and 100 and 50 .mu.g/ml of bamboo
extract inhibited NO production at the rate of 90% and 50%
respectively, therefore, the bamboo extract treatment group
inhibited effectively in dose dependent manner compared with
control. Non-polar solvent soluble extract-treated group showed
higher NO inhibition rate than polar solvent soluble
extract-treated group.
TABLE-US-00009 TABLE 8 Sample NO inhibition rate (%) LPS Crude
n-hexane Dichloro- Ethyl n-butanol Water Conc. (.mu.g/ml) Con
treated ext. fr. methane fr. aceteate fr. fr. soluble fr. P. nigra
50 0.34 0.10 33.1 51.0 53.6 39.1 12.5 0 100 0.38 0.12 73.2 91.1
90.8 85.5 48.5 14.5 S. borealis 50 0.23 0.15 21.5 45.2 48.5 30.1
9.4 0 100 0.25 0.14 60.3 84.3 82.4 80 35.1 9.4
[0137] On searching active ingredient from non-polar organic
solvent, we have found that tricin prepared from Example 5 is
active compound and it showed strong NO inhibition rate at the
concentration of 25, 12.5 and 6.5 .mu.g/ml of which concentration
showed no toxicity (See Table 9)
TABLE-US-00010 TABLE 9 Conc. of tricin (.mu.g/ml) NO inhibition
rate (%) Cell viability (%) 50 49.7 .+-. 0.007 49 25 74.6 .+-.
0.009 80 12.5 71.2 .+-. 0.003 92 6.5 52.0 .+-. 0.015 96 3.25 38.1
.+-. 0.005 99 1.625 25.9 .+-. 0.006 126
Experimental Example 2
Effect of Bamboo Extract on Elastase Activity
[0138] In order to test the effect of bamboo extract on blood
vessel, the inventive bamboo extract was treated with elastase
enzyme, which degrades elastin protein in charge of maintaining
elasticity and strength of blood vessel.
[0139] Each bamboo extract or fraction prepared in Example 1-5 was
diluted to 20, 2 and 0.2 mg/ml and aliquoted by 500 ml into each 96
well plate. Commercial elastase (Molecula probe Co.) was added
thereto at the concentration of 0.15 U/ml and elastin protein was
also added at the concentration of 50 .mu.g/ml. For determining the
enzyme activity, absorbance was detected using ELISA reader.
[0140] In the result of Table 10, the inventive bamboo extract of
P. nigra and S. borealis inhibited the elastase activity at 2 mg/ml
and dichloromethane, ethyl acetate and n-butanol soluble fractions
of bamboo extract showed more potent elastase-inhibiting
activity.
TABLE-US-00011 TABLE 10 Inhibition of elastase activity (%)
Phyllostachys nigra Sasa borealis Conc. (mg/ml) 20 2 0.2 20 2 0.2
Control 0 0 0 0 0 0 Crude ext. 92.3 53 84.5 47.6 n-hexane 34.2 0
21.2 0 Dichloromethane 61.6 15.7 54.6 15.7 Ethyl acetate 60.8 33 50
28 n-butanol 54.6 7.9 42 6.4 Water soluble 12.1 1.5 7.5 0
Experimental Example 3
Effect of Bamboo Extract on Wound Healing of the Endothelial Wall
of the Blood Vessel
[0141] 3-1. In Vivo Wound Healing Assay
[0142] HUVEC was grown confluently on 0.2% gelatin-coated 12-well
plate and then scratched by cell scraper to make original wound
edge. The cells were treated with 10 .mu.g/ml or 50 .mu.g/ml of
bamboo extract and cultured in 5% CO.sub.2 incubator. The
translocation of the cell was observed by the pictures.
[0143] In the result of FIGS. 10 to 12, the translocation of HUVEC
treated with 10-50 .mu.g/ml of bamboo extract was apparently
increased compared to that of control group, which confirmed that
the bamboo extract showed the wound healing effect on the
endothelial cell of blood vessel.
[0144] 3-2. In Vitro Tube Formation Assay
[0145] 200 ul/well of matrigel diluted with media (1:2) was plated
on 24-well plate, incubated at 37.degree. C. for at least 30 min to
polymerize and 1000 cells/well HUVEC were seeded thereon.
[0146] The cells were treated with 10 .mu.g/ml or 50 .mu.g/ml of
bamboo extract and cultured in 5% CO.sub.2 incubator. The
morphological change of the HUVEC was observed under microscope at
regular interval and taken the picture.
[0147] As shown in FIGS. 13 to 15, it was observed that the tube
formation of HUVEC treated with 10-50 .mu.g/ml of bamboo extract
was apparently increased compared to that of control group, which
confirmed that bamboo extract has the potential improving blood
circulation.
Experimental Example 4
Effect of Bamboo Extract and Tricin Compound on Gene Expression
[0148] To investigate the inhibiting effect on gene expression, the
extract or compound of the present invention was treated to the
cell and the RNA extracted therefrom was used in the RT-PCR to
evaluate the quantitative gene expression.
[0149] 4-1. Effect of Bamboo Extract on iNOS Gene Expression
[0150] To observe the effect of Bamboo extract on iNOS gene
expression, 1.times.10.sup.6 cells of RAW 264.7 cells were treated
with LPS and various concentrations (0.032.about.65 .mu.g/ml) of
the inventive crude extract of Phyllostachys nigra or Sasa
borealis, or curcumin and incubated for 24 hours. And RNA was
extracted by conventional extraction method using Trizol reagent
(Gibco BRL) to use in the following reverse
transcription-polymerase chain reaction.
[0151] RT-PCR was performed according to the RT reaction
(25.degree. C. 10 min, 48.degree. C. 30 min, 95.degree. C. 5 min,
4.degree. C. 10 min; 1 cycle) and subsequent PCR (50.degree. C. 2
min, 95.degree. C. 10 min, 95.degree. C. 15 sec, 60.degree. C. 1
min, 40 cycles) method well known in the art.
[0152] As an internal control, 18S ribosomal RNA was used.
[0153] As shown in Table 11, it was known through real time-gene
expression analysis that the concentrations, at which iNOS gene
expression is inhibited by 90%, are 4 .mu.g/ml for curcumin, 62.5
.mu.g/ml for Phyllostachys nigra and 65 .mu.g/ml for Sasa
borealis.
TABLE-US-00012 TABLE 11 Inhibition of Gene expression (%) Conc.
(.mu.g/ml) LPS Curcumin P. nigra S. borealis 0.032 40% 0.16 40%
0.625 60% 40% 0.8 40% 1.25 45% 40% 4 100% 90% 6.25 80% 58% 12.5 90%
70% 60% 25 70% 75% 62.5 90% 85% 65 90%
[0154] Based on the above results of Table 11, IC.sub.50 of each
sample on iNOS gene expression was calculated and presented as
below Table 12.
TABLE-US-00013 TABLE 12 Comparative iNOS expression (% of control,
DMSO) Conc. (.mu.g/ml) IC.sub.50 Sample 0.032 0.16 0.625 0.8 1.25
2.5 4 6.25 12.5 20 25 62.5 125 250 625 1250 2500 (.mu.g/ml)
Curcumin 80.5 75.7 64.4 49.9 1.3 5.3 P. nigra 115.6 125.9 118.5 125
114.1 49.6 31.6 27.2 25.4 23.6 30.2 25.1 25 S. borealis 100 111 105
92 87 44 25 23.1 22 18 21 15 30
[0155] 4-2. Effect of Bamboo Extract on PLA.sub.2 Gene
Expression
[0156] To observe the effect of Bamboo extract on PLA.sub.2 enzyme,
inflammatory factor related to inflammatory response,
1.times.10.sup.6 cells of RAW 264.7 cells were treated with LPS and
various concentrations (0.032.about.65 .mu.g/ml) of the inventive
bamboo extract and incubated for 24 hours. And RNA was extracted by
conventional extraction method using Trizol reagent (Gibco BRL) to
use in the following reverse transcription-polymerase chain
reaction.
[0157] The OD260/OD280 value of extracted RNA determined by
Spectrophotometer was more than 1.7 and the purity of RNA was
confirmed by Denaturing agarose gel electrophoresis. RT-PCR was
performed according to the RT reaction (10 min at 25.degree. C., 30
min at 48.degree. C., 5 min at 95.degree. C., 10 min at 4.degree.
C.; 1 cycle) and subsequent PCR (2 min at 50.degree. C., 10 min at
95.degree. C., 15 sec at 95.degree. C., 1 min at 60.degree. C., 40
cycles) method well known in the art.
[0158] As an internal control, 18S ribosomal RNA was used.
[0159] As shown in Table 13, it is confirmed that bamboo extract
inhibits PLA.sub.2 gene expression in a dose dependent manner
compared with that of control group.
TABLE-US-00014 TABLE 13 mRNA Relative Conc. of bamboo extract
(.mu.g/ml) level Control Rate LPS 0.625 1.25 2.5 6.25 12.5 25
PLA.sub.2 1 X 1.8 1.8 1.8 1.8 1.5 1.2 0.9
[0160] 4-3. Effect of Bamboo Extract on u-PA, PAI-1 Gene
Expression
[0161] RNA extraction and RT-PCR were performed according to the
method above described in Experimental Example 4-1.
[0162] In the result of Table 14 and 15, it was confirmed that the
extracts of Phyllostachys nigra and Sasa borealis increased the
expression of u-PA (urokinase type plasminogen activator) gene
related to thrombolysis, while those reduced the expression of
PAI-1 gene inhibiting the activity of plasminogen activator.
TABLE-US-00015 TABLE 14 u-PA (fold) PAI-1 (fold) DMSO treated
control group 1 1 LPS 3.65 (induction) 4.3 (induction) P. nigra (10
.mu.g/ml) 11.58 (induction) 9.56 (inhibition)
TABLE-US-00016 TABLE 15 u-PA (fold) PAI-1 (fold) DMSO treated
control group 1 1 LPS 3.81 (induction) 4.7 (induction) S. borealis
(10 .mu.g/ml) 8.7 (induction) 7.5 (inhibition)
[0163] 4-4. Effect of Tricin on VEGF, u-PA and eNOS Gene
Expression
[0164] RNA was isolated from HUVEC cell by Rneasy mini kit (cat#
74103, Qiagen Co.) according to the manufacturer's instruction. And
RT-PCT reaction was performed by using quantitative PCR method (SDS
7700, Applied biosystems Co., U.S.A.).
[0165] 5 .mu.l of cDNA product obtained in the reverse
transcription (RT) was aliquoted into each well of 96-well plate
and then the mixture containing 5.6 mM MgCl.sub.2, 1.times.PCR
buffer, 2 mM dNTP, 0.05% gelatin, 1 .mu.M of a pair of each target
gene primer or 0.16 .mu.M of house keeping gene primer, 0.5 .mu.M
of target gene probe or 0.025 .mu.M of housekeeping gene probe,
1.25 U of Taq polymerase was added thereto for polymerase chain
reaction (PCR) (50.degree. C. 2 min, 95.degree. C. 10 min,
95.degree. C. 15 sec, 60.degree. C. 1 min, 40 cycles).
[0166] As the result, final cT value was read and calculated.
[0167] In the result of Table 16 and FIG. 7.about.9, tricin
compound treatment to HUVEC had increased the expressions of VEGF
(vascular endothelial growth factor), u-PA gene and the expression
of eNOS (endothelial nitrous oxide synthase), which affects the
vascular expansion in atrophy.
TABLE-US-00017 TABLE 16 mRNA expression (fold) 0.5 .mu.g/ml 1.0
.mu.g/ml 5.0 .mu.g/ml Gene Control Tricin Tricin Tricin u-PA 1
1.619 1.103 2.962 VEGF 1 2.046 1.469 1.545 eNOS 1 3.024 4.711
0.452
Experimental Example 5
Effect of Bamboo Extract on the Change of General Symptom and Body
Weight in LDL Defected Mouse
[0168] To investigate the effect on the change of general symptom
and body weight in LDL defected mouse, the change of general
symptom was observed everyday more than once a day during the
treatment period and the change of body weight was determined at
the time of grouping, the moment of the sample treatment and
dislocation of mice after the end of experiment. At the result, we
did not observed the dead mouse as well as particular clinical
syndrome such as change of appearance and abnormal behavior etc
(See FIG. 16). Moreover, there has been not observed in the change
of body weight during the experimental period and the mean body
weight of the mice was increased by about 2.00.+-.0.6 g for each
groups.
Experimental Example 6
Effect of Bamboo Extract on the Change of Blood Lipid in LDL
Defected Mouse
[0169] To investigate the effect of bamboo extract on the change of
blood lipid in LDL defected mouse, following method was
performed.
[0170] At the end of experiment, all the mice were anesthetized
with 0.12% of avertin and exsanguinations was performed from
infraorbitalis plexus venosus with heparin treated capillary. And
then blood plasma was isolated by centrifugation at the speed of
11,000 g for 10 minutes and left alone at -70.degree. C. before
use. The value of blood lipid was determined by three categories
i.e., TC (Total cholesterol), HDL-C (High-density lipoprotein
cholesterol) and TG (Triglycerides) at KRIBB in Korea.
[0171] As can be seen in Table 17, the result showed that sample
treatment group treated with 50, 100, and 500 .mu.g/ml of bamboo
extract decreased all the values, TC (Total cholesterol), HDL-C
(High-density lipoprotein cholesterol) and TG (Triglycerides)
compared with control group in a dose dependent manner.
TABLE-US-00018 TABLE 17 Group TG TC LDL-C HDL-C NC 285 .+-. 119.02
3207.5 .+-. 562.64 3125 .+-. 533.26 25 .+-. 10.00 Lovastatin 300.00
.+-. 14.14 3750.00 .+-. 42.43 3485.00 .+-. 304.06 25.00 .+-. 7.07
Bamboo 50 313.33 .+-. 130.51 3063.33 .+-. 166.23 2963.33 .+-.
189.30 40.00 .+-. 26.46 Bamboo 100 240.00 .+-. 36.06 3000.67 .+-.
219.62 2903.33 .+-. 205.02 23.33 .+-. 5.77 Bamboo 500 186.67 .+-.
32.15 2930.00 .+-. 278.39 2870.00 .+-. 278.39 23.33 .+-. 5.77
Experimental Example 7
Effect of Bamboo Extract on the Change of Blood Lipid in C57BL/6J
Mouse
[0172] To investigate the effect of bamboo extract on the change of
blood lipid in C57BL/6J mouse, following method was performed.
At the end of experiment, all the mice were anesthetized with 0.12%
of avertin and exsanguinations was performed from infraorbitalis
plexus venosus with heparin treated capillary. And then blood
plasma was isolated by centrifugation at the speed of 11,000 g for
10 minutes and left alone at -70.degree. C. before use. The value
of blood lipid was determined by three categories i.e., TC (Total
cholesterol), HDL-C (High-density lipoprotein cholesterol) and TG
(Triglycerides) at KRIBB in Korea.
[0173] As can be seen in Table 18, the result showed that sample
treatment group treated with 50, 100 .mu.g/ml of bamboo extract
decreased all the values, TC (Total cholesterol), HDL-C
(High-density lipoprotein cholesterol) and TG (Triglycerides)
compared with control group in a dose dependent manner.
TABLE-US-00019 TABLE 18 Total cholesterol Triglycerides HDL-C LDL-C
Control 366.20 .+-. 71.06 73.80 .+-. 19.52 56.00 .+-. 8.34 84.80
.+-. 20.04 Bamboo 282.60 .+-. 35.52 64.80 .+-. 15.55 47.40 .+-.
7.89 62.40 .+-. 8.62 50 Bamboo 285.00 .+-. 12.25 52.50 .+-. 9.26
54.25 .+-. 8.42 57.75 .+-. 2.36 100
Experimental Example 8
Inhibition Effect of Bamboo Extract on Arteriosclerosis in LDL
Defected Mouse
[0174] To investigate the inhibiting effect of bamboo extract on
the occurrence of arteriosclerosis and the progress of the lesion
in LDL defected mouse, following method was performed.
[0175] At the end of experiment, the exsanguinated heart was fixed
with 4% para-formaldehyde dissolved in 0.1M phosphate buffer (pH
7.4) and delivered removing remaining blood and fixing with 10%
neutral formalin. And then it is embedded with OCT compound, sliced
into 0.6 .mu.m of thickness, stained with oil red 0 and
count-stained with Harris hematoxylin to observe the lesion.
[0176] The calculation of lesion area was performed by staining the
lesion formed at the position between 3.sup.rd cervical blood and
aortic valve and photocopying and then the lesion area was
calculated by using computer--assisted morphometry (TDI microscope
Image Analyzer, USA) comparing with control group.
[0177] At the result, sample treatment group inhibit the formation
of arteriosclerosis by about 17% compared with control group while
lovastatin used as a positive control inhibit by about 47% and
prevent the formation of neointima (See FIGS. 17 and 18).
Experimental Example 9
Cell Toxicity Test and Cell Proliferation Assay
[0178] The cell toxicity of tricin compound of Example 5 was tested
using modified MTT method (J. Immunological Methods, 119, pp
203-210, 1989).
[0179] 200 .mu.l of HUVEC (2.times.10.sup.5 cells/ml) on flat
bottom 96-well microtiter plates (Nunc, Sweden) were treated with
tricin prepared in various concentrations and cultured at
37.degree. C. for 24 hours.
[0180] 50 .mu.l of MTT solution (1 mg/ml) was added to each well
and incubated at 37.degree. C. for 4 hours. And then supernatant
was removed.
[0181] To detect formazan crystal, 100 .mu.l of DMSO was also added
to each well and the colorigenic analysis was performed at 550 nm
using microplate reader (Power wave-XS, Bio-Tek, USA).
[0182] As the result, the inventive tricin compound showed the 51%
of strong cellular toxicity at 5 .mu.g/ml, however, bamboo extract
or p-coumaric acid was no cellular toxicity (See FIG. 5).
[0183] The cell proliferation assay of bamboo extract was performed
using Cell Proliferation ELISA BrdU colorimetric kit (Roche). HUVEC
were seed 5.times.10.sup.3 cells/well in 96 well plate. Triplicate
plate of cells were measured using ELISA Reader.
[0184] As the result, bamboo extract enhanced strong cellular
proliferation by dose dependent manner (See FIG. 6)
Experimental Example 10
Animal Toxicity Test
[0185] Methods (1)
[0186] The acute toxicity tests on ICR mice (mean body weight
25.+-.5 g) and Sprague-Dawley rats (235.+-.10 g, Jung-Ang Lab
Animal Inc.) were performed using the extract of the Example 1.
Four group consisting of 10 mice or rats was administrated orally
intraperitoneally with 250 mg/kg, 500 mg/kg, 1000 mg/kg and 5000
mg/kg of test sample or solvents (0.2 ml, i.p.) respectively and
observed for 2 weeks.
[0187] Methods (2)
[0188] The acute toxicity tests on ICR mice and Sprague-Dawley rats
were performed using the extract of the Example 1. Four group
consisting of 10 mice or rats was administrated intraperitoneally
with 25 mg/kg, 250 mg/kg, 500 mg/kg and 725 mg/kg of test sample or
solvents (0.2 ml, i.p.), respectively and observed for 24
hours.
[0189] Results
[0190] There were no treatment-related effects on mortality,
clinical signs, body weight changes and gross findings in any group
or either gender. These results suggested that the extract prepared
in the present invention were potent and safe.
[0191] Hereinafter, the formulating methods and kinds of excipients
will be described, but the present invention is not limited to
them. The representative preparation examples were described as
follows.
[0192] Preparation of Powder
TABLE-US-00020 Dried powder of Example 1 50 mg Lactose 100 mg Talc
10 mg
Powder preparation was prepared by mixing above components and
filling sealed package.
[0193] Preparation of Tablet
TABLE-US-00021 Dried powder of Example 1 50 mg Corn Starch 100 mg
Lactose 100 mg Magnesium Stearate 2 mg
[0194] Tablet preparation was prepared by mixing above components
and entabletting.
[0195] Preparation of Capsule
TABLE-US-00022 Dried powder of Example 1 50 mg Corn starch 100 mg
Lactose 100 mg Magnesium Stearate 2 mg
[0196] Tablet preparation was prepared by mixing above components
and filling gelatin capsule by conventional gelatin preparation
method.
[0197] Preparation of Injection
TABLE-US-00023 Dried powder of Example 1 50 mg Distilled water for
injection optimum amount PH controller optimum amount
[0198] Injection preparation was prepared by dissolving active
component, controlling pH to about 7.5 and then filling all the
components in 2 ml ample and sterilizing by conventional injection
preparation method.
[0199] Preparation of Liquidpowder
TABLE-US-00024 Dried powder of Example 1 0.1~80 g Sugar 5~10 g
Citric acid 0.05~0.3% Caramel 0.005~0.02% Vitamin C 0.1~1%
Distilled water 79~94% CO.sub.2 gas 0.5~0.82%
[0200] Liquid preparation was prepared by dissolving active
component, filling all the components and sterilizing by
conventional liquid preparation method.
[0201] Preparation of Healthcare Food
TABLE-US-00025 Extract of Example 1 1000 mg Vitamin mixture optimum
amount Vitamin A acetate 70 .mu.g Vitamin E 1.0 mg Vitamin B.sub.1
0.13 mg Vitamin B.sub.2 0.15 mg Vitamin B6 0.5 mg Vitamin B12 0.2
.mu.g Vitamin C 10 mg Biotin 10 .mu.g Amide nicotinic acid 1.7 mg
Folic acid 50 .mu.g Calcium pantothenic acid 0.5 mg Mineral mixture
optimum amount Ferrous sulfate 1.75 mg Zinc oxide 0.82 mg Magnesium
carbonate 25.3 mg Monopotassium phosphate 15 mg Dicalcium phosphate
55 mg Potassium citrate 90 mg Calcium carbonate 100 mg Magnesium
chloride 24.8 mg
[0202] The above-mentioned vitamin and mineral mixture may be
varied in many ways. Such variations are not to be regarded as a
departure from the spirit and scope of the present invention.
[0203] Preparation of Health Beverage
TABLE-US-00026 Extract of Example 1 1000 mg Citric acid 1000 mg
Oligosaccharide 100 g Apricot concentration 2 g Taurine 1 g
Distilled water 900 ml
[0204] Health beverage preparation was prepared by dissolving
active component, mixing, stirred at 85.degree. C. for 1 hour,
filtered and then filling all the components in 1000 ml ample and
sterilizing by conventional health beverage preparation method.
[0205] The invention being thus described, it will be obvious that
the same may be varied in many ways. Such variations are not to be
regarded as a departure from the spirit and scope of the present
invention, and all such modifications as would be obvious to one
skilled in the art are intended to be included within the scope of
the following claims.
INDUSTRIAL APPLICABILITY
[0206] As described in the present invention, the bamboo plant
extract and the tricin compound therefrom have potent
anti-inflammatory activity by inhibiting NO production and PLA
expression, blood circulation-improving activity by inhibiting
elastase activity and healing the wound of vascular endothelial
cell, activating u-PA expression and inhibiting PAl-1 expression,
lowering cholesterol deposit and inhibiting neointima formation,
therefore, it can be used as a therapeutic, health care food for
treating and preventing inflammatory or blood circulation
diseases.
* * * * *