U.S. patent application number 14/549184 was filed with the patent office on 2015-06-25 for process for preparation of a grifola frondosa polysaccharide f2 and its hypoglycemic activity.
This patent application is currently assigned to Guangdong Institute of Microbiology. The applicant listed for this patent is Guangdong Institute of Microbiology, Guangdong Yuewei Edible Fungi Technology Co., Ltd.. Invention is credited to Senzhu Li, Qingping Wu, Chun Xiao, Yizhen Xie, Xiaobing Yang.
Application Number | 20150175715 14/549184 |
Document ID | / |
Family ID | 50448765 |
Filed Date | 2015-06-25 |
United States Patent
Application |
20150175715 |
Kind Code |
A1 |
Wu; Qingping ; et
al. |
June 25, 2015 |
Process for Preparation of a Grifola frondosa Polysaccharide F2 and
Its Hypoglycemic Activity
Abstract
A Grifola frondosa polysaccharide F2 with hypoglycemic activity,
process for preparation and use thereof. The process for
preparation of Grifola frondosa polysaccharide F2 is as follows:
The fruit bodies of Grifola frondosa were homogenized to a fine
powder and extracted with hot water. The mixture was filtered and
precipitated with absolute ethanol. The precipitation was obtained.
The said precipitation was applied on DEAE Sepharose Fast
chromatographic column, equilibrated with Tris-HCl(10 mM, pH=8.0),
collecting the efficient eluting peak to obtain the fraction F1;
eluted with Tris-HCl(10 mM, pH=8.0) which contains 0.1M NaCl,
fraction F2 was obtained; then concentrated under reduced pressure,
dialyzed and lyophilized, Grifola frondosa polysaccharide F2 was
obtained. This isolates a new Grifola frondosa polysaccharide F2
with hypoglycemic activity from the fruit bodies of Grifola
frondosa. The Grifola frondosa polysaccharide F2 can be used in
manufacturing a drug for treating diabetes. The polysaccharide
makes a foundation for developing new anti-diabetes agents.
Inventors: |
Wu; Qingping; (Guangzhou,
CN) ; Xiao; Chun; (Guangzhou, CN) ; Xie;
Yizhen; (Guangzhou, CN) ; Yang; Xiaobing;
(Guangzhou, CN) ; Li; Senzhu; (Guangzhou,
CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Guangdong Institute of Microbiology
Guangdong Yuewei Edible Fungi Technology Co., Ltd. |
Guangzhou
Guangzhou |
|
CN
CN |
|
|
Assignee: |
Guangdong Institute of
Microbiology
Guangdong Yuewei Edible Fungi Technology Co., Ltd.
|
Family ID: |
50448765 |
Appl. No.: |
14/549184 |
Filed: |
November 20, 2014 |
Current U.S.
Class: |
536/123.1 |
Current CPC
Class: |
C08L 5/00 20130101; C08B
37/006 20130101; A61K 31/715 20130101; A61P 3/10 20180101; C08B
37/0003 20130101; A61K 36/07 20130101; A61K 2236/00 20130101 |
International
Class: |
C08B 37/00 20060101
C08B037/00; A61K 31/715 20060101 A61K031/715; A61K 36/07 20060101
A61K036/07 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 25, 2013 |
CN |
201310733480.8 |
Claims
1. A process for preparation of Grifola frondosa polysaccharide F2,
comprising: (a) the fruit bodies of Grifola frondosa are
homogenized to a fine powder and extracted with hot water; the
mixture was filtered and precipitated with absolute ethanol; the
precipitation was obtained. (b) the said precipitation is applied
on DEAE Sepharose Fast chromatographic column, equilibrated with
Tris-HCl(10 mM, pH=8.0), collecting the efficient eluting peak to
obtain the fraction F1; eluted with Tris-HCl(10 mM, pH=8.0) which
contains 0.1M NaCl, fraction F2 was obtained; then concentrated
under reduced pressure, dialyzed and lyophilized, Grifola frondosa
polysaccharide F2 is obtained.
2. A process for preparation of Grifola frondosa polysaccharide F2
according to claim 1, characterized in that the said precipitation
in the step (a) is dissolved into hot water, filtered by a 0.45
.mu.m millipore filter and dialyzed in dialysis bag of 3000 D for
24 h to yield crude polysaccharides; the concentration of crude
polysaccharide in the Grifola frondosa crude polysaccharides
solution was adjusted, and then be used as the sample of
chromatographic separation in the step (b).
3. Grifola frondosa polysaccharide F2 prepared by the process for
preparation of Grifola frondosa polysaccharide F2 as claimed in
claim 1.
4. Use of Grifola frondosa polysaccharide F2 as claimed in claim 3
for manufacturing a drug for the treatment of diabetes.
5. The use according to claim 4, wherein, the said diabetes is type
2 diabetes.
6. A drug for the treatment of diabetes, characterized in that the
said medicament comprises Grifola frondosa polysaccharide F2 as
claimed in claim 3 as active ingredients.
7. The drug according to claim 6, wherein the said diabetes is type
2 diabetes.
Description
RELATED APPLICATION
[0001] This application claims the benefit of Chinese Patent
Application No. 201310733480.8, filed on Dec. 25, 2013, the
specification of which is incorporated herein with this
reference.
TECHNICAL FIELD
[0002] This invention relates to a Grifola frondosa polysaccharide
F2 with hypoglycemic activity, preparation method and use thereof,
belonging to the field of bio-medicine.
BACKGROUND
[0003] Diabetes is a metabolic disorder syndrome characterized by
hyperglycemia. The number of diabetes patients around the world has
been increasing at a speed of 6 percent each year. Between 2010 and
2030, the number of adults with diabetes was expected to increase
by 69% in developing countries and by 20% in developed countries.
Diabetes includes type 1 and type 2 diabetes, with type 2 diabetes
accounting more than 90% of all cases of diabetes. There is
currently no known method to conquer diabetes, and the patients'
conditions are generally controlled and alleviated by orally
administered drugs for a long-term. But most of current drugs for
diabetes are chemical or biochemical drugs, which have much side
effects. Based on this critical situation, safer and more effective
treatment modalities for diabetes are therefore needed to address
the increase in prevalence of diabetes.
[0004] A polysaccharide with hypoglycemic activity was obtained
from fruit body of cultivated Grifola frondosa by Xun
Ma--.alpha.-glucan with a molecular weight of 400-450 KD. In the
process of isolating polysaccharides from fruit body of Grifola
frondosa, Kubo obtained a glycoprotein after adding one volume of
ethanol into the hot water extract to generate suspension and
centrifugation. The glycoprotein named X-fraction
(polysaccharide:protein=65:35), with a molecular weight of
5.times.105 D, showed a significant hypoglycemic activity.
Structure analysis indicated that it was a .beta.-1,6-glucan with
.alpha.-1,4-branch.
[0005] The modern pharmacology studies reveals that blood serum
glucose can be lowered through increasing insulin levels,
regulating the activity of some enzymes related to glycometabolism
and sequentially accelerating the glucose oxidation utilization,
and through improving the insulin resistance or inhibition of
glucose absorption.
[0006] The possible action mechanism of MT-.alpha.-glucan from the
fruit body of Grifola frondosa was increasing sensitivity of
insulin and improving insulin resistance of the surrounding tissue
through increasing the number of insulin receptor. Water soluble
extract FXM from Grifola frondosa potentially decreased blood serum
glucose by improvement the insulin resistance. Glycoprotein SX from
fruit body of Grifola frondosa can improve glucose tolerance and
increase the body's sensitivity to insulin.
[0007] It has been found that Grifola frondosa and its active
ingredients have obvious hypoglycemic activity, however, the
compositions, structures and mechanisms of most bioactive
hypoglycemic ingredients are not fully understood, due to the
complex chemical structures. That directly hinders the process of
developing new anti-diabetes agents.
CONTENTS OF THE INVENTION
[0008] The object of this invention is to provide a new Grifola
frondosa polysaccharide F2 with hypoglycemic activity and
preparation method thereof.
[0009] A process for preparation of the above-mentioned Grifola
frondosa polysaccharide F2 comprising:
(a) The fruit bodies of Grifola frondosa are homogenized to a fine
powder and extracted with hot water. The mixture was filtered and
precipitated with absolute ethanol, and the resulting precipitate
of is obtained; (b) The said precipitation is applied on DEAE
Sepharose Fast Flow chromatographic column, equilibrated with
Tris-HCl(10 mM, pH=8.0), collecting the efficient eluting peak to
obtain the fraction F1; eluted with Tris-HCl(10 mM, pH=8.0) which
contains 0.1M NaCl, fraction F2 is obtained; then concentrated
under reduced pressure, dialyzed and lyophilized, Grifola frondosa
polysaccharide F2 is obtained.
[0010] Preferably, the said precipitation in the step (a) is
dissolved into hot water, filtered by a 0.45 .mu.m millipore filter
and then dialyzed in dialysis bag of 3000 D for 24 h to yield crude
polysaccharides solutions. The concentration of crude
polysaccharide in the Grifola frondosa crude polysaccharides
solution is adjusted, and then be used as the sample of
chromatographic separation in the step (b).
[0011] The said polysaccharide F2 from Grifola frondosa has a
molecular weight of 4.52.times.10.sup.5 D. The contents of
polysaccharides and protein are respectively 95.6% and 3.6%. The
said polysaccharides is mainly consisted of glucose, mannose,
xylose, galactose, and arabinose; the amino acids mainly comprises:
proline(Pro), serine(Ser), asparitic acid(Asp), lysine(Lys),
alanine(Ala), glutamic acid(Glu), threonine(Thr), glycine(Gly),
arginine(Arg), leucine(Leu) and valine(Val). The said
polysaccharide F2 is a .beta.-heteropolysaccharides composed of
uronic acids. Based on the above-mentioned parameters, there is no
known analogous polysaccharide in the existing technology, that is
to say, Grifola frondosa polysaccharide F2 of this invention is a
new Grifola frondosa polysaccharide.
[0012] Pharmacology experiment in vivo showed that when diabetic
rats were continuously intragastrical administered Grifola frondosa
polysaccharide F2 for 7 days, FBG (fasting blood glucose) can be
decreased significantly. The mechanism of lower blood glucose
levels is mainly through improvement insulin resistance.
[0013] Accordingly, the second object of the invention is to
provide the use of Grifola frondosa polysaccharide F2 in
manufacturing a drug for the treatment of diabetes, especially for
type 2 diabetes.
[0014] Another object of the invention is to provide a drug for the
treatment of diabetes, characterized in that the drug comprises
Grifola frondosa polysaccharide F2 as active ingredients.
[0015] The said diabetes is type 2 diabetes.
[0016] This invention isolates a new Grifola frondosa
polysaccharide F2 with hypoglycemic activity from the fruit body of
Grifola frondosa. It can be used in manufacturing a drug for the
treatment of diabetes, especially for type 2 diabetes. The
invention makes a foundation for developing new anti-diabetes
agents in the future, and actively promotes the study on active
ingredients of natural medicine for treating diabetes.
DESCRIPTION OF THE FIGURES
[0017] FIG. 1 shows the elution curve of the precipitation on DEAE
Sepharose Fast Flow chromatography.
[0018] FIG. 2 shows the calibration curve of the standard
polysaccharides on Gel permeation chromatography.
[0019] FIG. 3 shows the HPLC chromatogram of Grifola frondosa
polysaccharide F2.
[0020] FIG. 4 shows the Infrared spectrum (IR) of Grifola frondosa
polysaccharide F2.
[0021] FIG. 5 shows the .sup.1H-NMR spectrum of Grifola frondosa
polysaccharide F2.
[0022] FIG. 6 shows the .sup.13C-NMR spectrum of Grifola frondosa
polysaccharide F2.
EXAMPLES
[0023] The examples below further illustrate the invention, rather
than limiting the scope thereof.
Example 1
[0024] I. Preparation of Grifola frondosa Polysaccharide F2
[0025] The fruit bodies of Grifola frondosa were homogenized to a
fine powder and extracted with hot water(5000 mL, 80) for 8 hours.
The mixture was filtered and the filtrate was concentrated to 1000
mL under reduced pressure, then precipitated with 4 volume of
absolute ethanol. The precipitation was obtained by centrifugation
at a speed of 5000 rpm. The precipitation was dissolved into hot
water, filtered by a 0.45 .mu.m millipore filter and dialyzed in
dialysis bag of 3000 D for 24 h to yield crude polysaccharides
solution. Finally the concentration of the solution was adjusted to
be 10 mg/mL.
[0026] The above-mentioned crude polysaccharides solution (100 mL)
was filtrated and applied on DEAE Sepharose Fast Flow
chromatographic column (4.5.times.30 cm), F1 was obtained by
collecting and concentrating the fraction equilibrated with 300 mL
Tris-HCl (10 mM, pH=8.0), F2 was obtained by collecting and
concentrating this fraction eluted with 300 mL Tris-HCl buffer (10
mM, pH=8.0) which contains 0.1M NaCl. The elution curve was shown
in FIG. 1.
[0027] II. Determination of Purity and Molecular Weight of Grifola
frondosa Polysaccharide F2
[0028] Grifola frondosa polysaccharide F2 (10 mg) was dissolved
into the ultrapure water (1 mL) and analyzed by HPLC.
[0029] Chromatographic conditions: TSK-GEL G3000SW.sub.XL column
(300 mm.times.718 mm); column temperature: 35.degree. C.; mobile
phase: 0.05M NaH.sub.2PO.sub.4--Na.sub.2HPO.sub.4 buffer (pH 6.7)
containing 0.05% NaN.sub.3; flow rate: 0.5 mL/min; differential
refractive index detector and constant temperature at 35.degree.
C.; injection volume: 20 .mu.L.
[0030] Establishment GPC calibration curve: 10 mg polysaccharides
with Mr of 738, 5800, 1.22.times.10.sup.4, 2.37.times.10.sup.4,
4.80.times.10.sup.4, 1.00.times.10.sup.5, 1.86.times.10.sup.5,
3.80.times.10.sup.5, 8.53.times.10.sup.5 D were used as standards,
dissolved into 0.05M NaH.sub.2PO.sub.4--Na.sub.2HPO.sub.4 buffer (1
mL, pH 6.7) containing 0.05% NaN.sub.3, filtered by a 0.45 .mu.m
millipore filter and analyzed by GPC. The retention times of the
standard polysaccharides with the known Mr were shown in table 1.
GPC calibration curve was established with elution volume of the
standard polysaccharides as the abscissa and the value of Mr as the
vertical coordinates, see FIG. 2.
[0031] Determination of purity and molecular weight of Grifola
frondosa polysaccharide F2: 10 mg Grifola frondosa polysaccharide
F2 was dissolved in 0.05M NaH.sub.2PO.sub.4--Na.sub.2HPO.sub.4
buffer (1 mL, pH 6.7) containing 0.05% NaN.sub.3, filtered by a
0.45 .mu.m millipore filter and analyzed by GPC. The average
molecular weight of Grifola frondosa polysaccharide F2 is
4.52.times.10.sup.5 D (as shown in FIG. 3), calculated
automatically by GPC chromatographic working station.
TABLE-US-00001 TABLE 1 The retention times of standard
polysacchrides Mr (D) Retention time (min) Elution volume (mL) 738
22.6 11.3480 5800 20.1 10.4296 1.22 .times. 10.sup.4 18.3 9.4976
2.37 .times. 10.sup.4 16.3 8.4592 4.80 .times. 10.sup.4 13.8 7.0456
1.00 .times. 10.sup.5 12.3 6.1120 1.86 .times. 10.sup.5 11.9 5.7824
3.80 .times. 10.sup.5 11.7 5.6615 8.53 .times. 10.sup.5 11.7
5.6416
[0032] III. Physicochemical Property of Grifola frondosa
Polysaccharide F2
[0033] 1. Determination of the Contents of Polysaccharides and
Protein
[0034] The content of polysaccharides of F2 is 95.6% determined by
phenol sulfuric acid method, and that of protein is 3.6% determined
by Bradford method.
[0035] 2. Amino Acid Composition Analysis
[0036] HPLC (HP1050, America), consisted of quaternary gradient
pump, automatic sampler, column oven and HP1046A fluorescence
detector; Full-automatic high Speed Freezing Centrifuge (2000 rpm,
GL20A, Japan).
[0037] Standard amino acid, derivatization reagent OPA, FMOC from
SIGMA firm; Na.sub.2HPO.sub.4 was of analytical grade; methyl
alcohol and acetonitrile were of HPLC grade.
[0038] Chromatographic conditions: Hypersil ODS column
(4.0.times.125 mm, particle size 5 .mu.m); mobile phase (A): 10
mmolL.sup.-1 pH 7.2 Na.sub.2HPO.sub.4 buffer (PB); mobile phase
(B): the mixture of PB, methyl alcohol and acetomitrile (volume
percents of them were respectively 50%, 35% and 15%); linear
gradient: volume fraction of mobile phase B rise to 40% from 0%
linearly in 0-10 min; volume flow rate: 1.0 mLmin.sup.-1; column
temperature: 40; determine wavelength: excitation wavelength 340 nm
and emission wavelength 450 nm.
[0039] Samples Treatments:
[0040] Standard solution: standard amino acids were dissolved into
0.1 molL.sup.-1 HCl solution, and diluted to standard solution, in
which the concentration of each amino acid was 250 nM.
[0041] Hydrolysis: putting Grifola frondosa polysaccharide F2 into
a hydrolysis tube, adding 6 M HCl (10.about.15mL) and two drops of
newly-distilled phenol. The hydrolysis tube was freezed for 3-5 min
in refrigerant, and then linked to the exhaust tube of vacuum pump.
Vacuum pumping to be closed to 0 Pa and filling with high-purity
nitrogen, the treatment was repeated three times and the hydrolysis
tube was sealed under nitrogen. The sealed hydrolysis tube was put
into a constant temperature drying oven (110.+-.1), after 23 hours,
taken out and cooled. Opening and washing the hydrolysis tube with
deionized water, all the hydrolysate was transferred into a 100 mL
volumetric flask and diluted with deionized water to volume. The
hydrolysate was properly diluted to be detected.
[0042] Contrasting to HPLC chromatogram of the standard amino
acids, it was inferred that the amino acids of Grifola frondosa
polysaccharide F2 mainly comprises: proline(Pro), serine(Ser),
asparitic acid(Asp), lysine(Lys), alanine(Ala), glutamic acid(Glu),
threonine(Thr), glycine(Gly), arginine(Arg), leucine(Leu) and
valine(Val). See table 2.
TABLE-US-00002 TABLE 2 Amino acid composition of Grifola frondosa
polysaccharide F2 Analysis items Results Unit Phenylalanine (Phe)
<0.10 g/100 g Alanine (Ala) 0.19 Methionine (Met) <0.10
Proline (Pro) 0.38 Glycine (Gly) 0.15 Glutamic acid (Glu) 0.19
Arginine (Arg) 0.11 Lysine (Lys) 0.19 Tyrosine (Tyr) 0.10 Leucine
(Leu) 0.10 Serine (Ser) 0.23 Threonine (Thr) 0.17 Aspartic acid
(Asp) 0.21 Isoleucine (Ile) 0.10 Histidine (His) <0.10 Valine
(Val) 0.10 Total 2.1
[0043] 3. Monosaccharide Composition of Grifola frondosa
polysaccharide F2 analysis by GC-MS
[0044] Hydrolysis: Grifola frondosa polysaccharide F2 was dissolved
in 2 M H.sub.2SO.sub.4, heated to reflux for 6 hours, cooled and
then neutralized with saturated Ba (OH).sub.2 to be neutral. The
mixed liquid was filtrated and the filtrate was collected.
[0045] Acetylation: the above-mentioned hydrolysate was evaporated
to dryness. The residue sample was converted to acetylated
derivatives with 70 mg hydroxylamine hydrochloride and 5 mL
pyridine for 1 hour at 90.degree. C. by water bath heating. After
slightly cooling, 5 mL acetic anhydride was then added with heating
at 90.degree. C. After 1 hour, 10 mL water was added to break the
anhydride and the acetylated products was extracted with
chloroform. The extract liquor was washed with water and then
subsequently dehydrated with anhydrous Na.sub.2SO.sub.4. The
supernate was concentrated to 1 mL under nitrogen and analyzed by
using GC-MS.
[0046] GC-MS operation conditions: SE230 elastic quartz capillary
column (15 m.times.012 mm.times.0133 Lm); the temperature program
was set to increase to 280 from 100 at a rate of 10/min, then
holding for 10 min at 280; carrier gas: Helium; column pressure: 70
kPa; split ratio:10:1; solvent delay:2 min; electron ionization
mode: EI; electron energy:70 eV; quadrupole rod temperature:150;
temperature of ion source:230; voltage of electron
multiplier:2300V; interface temperature of GC-MS:280; Mass scanned
range (m/z): 29.about.500.
[0047] The monosaccharide composition of Grifola frondosa
polysaccharide F2 was analyzed by GC-MS after hydrolysis and
acetylation, as control, the standard monosaccharides was
acetylated simultaneously. According to the total ions chromatogram
of standard monosaccharides, it can be inferred that Grifola
frondosa polysaccharide F2 is a heteropolysaccharide mainly
consisted of glucose, mannose, xylose, galactose, arabinose and
ribose. The relative content of the monosaccharides were shown in
table 3.
TABLE-US-00003 TABLE 3 Monosaccharide composition of Grifola
frondosa polysaccharide F2 Relative content by peak area Sample
Monosaccharide normalization Names Composition (%) Methods Grifola
Ribose 1.96 The samples were frondosa Arabinose 3.22 analyzed by
GC-MS polysaccharide Xylose 8.02 after hydrolysis and F2 Mannose
16.72 acetylation Glucose 63.74 Galactose 6.75
[0048] 4. Infrared Spectrum(IR) Analysis of Grifola frondosa
polysaccharide F2
[0049] 1 mg of Grifola frondosa polysaccharide F2 and 100 mg of
dried KBr were porphyrizied and tabletted, the infrared profile of
Grifola frondosa polysaccharide F2 was scaned in the range of 400
to 4000 cm.sup.-1. Table 4 was the analysis of absorption peaks in
the infrared spectrum (FIG. 4). According to the infrared spectrum
analysis, Grifola frondosa polysaccharide F2 is mainly consisted of
pyranoid rings linked by .beta.-glucosidic bonds.
TABLE-US-00004 TABLE 4 Analysis of absorption peaks in the infrared
spectrum of Grifola frondosa polysaccharide F2 Trans- Wave- mit-
Num- number tance ber (cm.sup.-1) (%) Analysis of the peaks 1
3272.3 15 hydroxy 2 2924.5 16 Stretching vibration of C--H 3 1670.5
18 Stretching vibration of C.dbd.O 4 1408 20 Stretching vibration
of COO.sup.- 5 1064 10 pyranoid rings linked by .beta.-glucosidic
bonds 6 885-900 .beta.-glucosidic bonds
[0050] 5. NMR Spectrum of Grifola frondosa Polysaccharide F2:
.sup.1H-NMR and .sup.13C-NMR
[0051] The .sup.1H-NMR spectr of Grifola frondosa polysaccharide F2
was shown in FIG. 5 and the .sup.13C-NMR spectrum in FIG. 6.
[0052] The signal at 4.395 ppm of .sup.1H-NMR indicated that
Grifola frondosa polysaccharide F2 is mainly consisted of pyranoid
rings, in accordance with the results from infrared spectrum
analysis.
[0053] In .sup.13C-NMR, the chemical shifts of anomeric carton are
generally in the range of 90-110 ppm, and the number of glycoside
residues depends on the number of signal peaks. So Grifola frondosa
polysaccharide F2 is consisted of five glycoside residues. The
results of GC-MS showed it contains six glycoside residues, wherein
the fraction of ribose is just 1.96%, thus, Grifola frondosa
polysaccharide F2 is mainly consisted of glucose, mannose, xylose,
galactose, and arabinose.
[0054] According to .sup.13C-NMR, the number of glycoside residues
and their relative contents can be determined by the number of
peaks in the resonance area of anomeric carton (90-110 ppm). In
general, the chemical shifts of anomeric carbons from
.alpha.-glucosides are in the range of 95-101 ppm, while that of
.beta.-glucosides in the range of 101-105 ppm. The chemical shifts
of Grifola frondosa polysaccharide F2 are respectively at 97.94
ppm, 101.30 ppm, 102.26 ppm, 102.92 ppm and 115.47 ppm, it is
consequently considered as a .beta.-heteropolysaccharide.
[0055] In addition, the characteristic signals of .sup.13C-NMR can
determine some glycoside residues and groups, for example, the
signals of carboxyls-C of uronic acids are in the range of 170-180
ppm, and the chemical shifts of Grifola frondosa polysaccharide F2
are respectively at 171.81 ppm, 173.32 ppm, 174.37 ppm, 177.68 ppm
and 177.53 ppm, as a consequence, Grifola frondosa polysaccharide
F2 contains uronic acids.
[0056] In conclusion, Grifola frondosa polysaccharide F2 of the
invention has a molecular weight of 4.52.times.10.sup.5 D. The
contents of polysaccharides and protein are respectively 95.6% and
3.6%. The said polysaccharides is mainly consisted of glucose,
mannose, xylose, galactose, and arabinose; the amino acids mainly
comprises: proline(Pro), serine(Ser), asparitic acid(Asp),
lysine(Lys), alanine(Ala), glutamic acid(Glu), threonine(Thr),
glycine(Gly), arginine(Arg), leucine(Leu) and valine(Val). The said
polysaccharide F2 is a .beta.-pyran heterocyclic polysaccharide
containing uronic acids. Based on the above-mentioned parameters,
there is no known analogous polysaccharide in the existing
technology. Therefore, Grifola frondosa polysaccharide F2 of this
invention is a new Grifola frondosa polysaccharide.
[0057] IV. Pharmacology Experiment
[0058] Hypoglycemic experiment of Grifola frondosa polysaccharide
F2 on type 2 diabeticrats:
(1) Induction of diabetes rats: SD rats at age of 6 weeks (weight:
140-160 g), male, were marked and kept in separate cages. They were
adapted for 7 days and then fasted overnight before an
intraperitoneal injection of freshly prepared STZ (Sigma, 35 mg/kg
body weight [BW], dissolved in citrate buffer, pH 4.5). Then, the
rats were fed a high-fat diet. fter 4 weeks, the rats were fasted
for 5 h and fasting serum glucose levels were determined. Rays with
fasting serum glucose levels>11.1 mM were considered to be
diabetic and were used in the study. (2) Assessment of hypoglycemic
activity: Except normal control (NC), the diabetic rats (DM) were
randomly divided into three groups: model control group (MC);
low-dose group of Grifola frondosa polysaccharide F2 (50 mg/kg BW,
Intragastric(ig)); high-dose group of Grifola frondosa
polysaccharide F2 (100 mg/kg BW, Intragastric(ig)). The rats of
low-dose and high-dose groups were administered different doses of
Grifola frondosa polysaccharide F2 by intragastric infusion,
meanwhile, rats of NC and MC were administered saline. The blood
glucose levels were measured after continuously giving drugs for 2
weeks. According to table 5, FBG were decreased significantly
(*P<0.05) afterdiabetic rats were continuously administered
Grifola frondosa polysaccharide F2 (100 mg/kg BW) for 2 weeks by
intragastric infusion. Thus, Grifola frondosa polysaccharide F2 of
the invention can be used to manufacture medicaments for the
treatment of diabetes, especially for type 2 diabetes.
TABLE-US-00005 TABLE 5 Effects of Grifola frondosa polysaccharide
F2 on FBG in type 2 diabetic rats(ig). Dose Fasting Serum Glucose
FSG(mmol/L) Groups (mg/kg/d) 0 w 1 w 2 w NC / 5.68 .+-. 0.39 5.78
.+-. 0.61 5.94 .+-. 0.42 MC / 20.65 .+-. 4.56 25.22 .+-. 3.94 24.77
.+-. 4.27 F2 50 21.41 .+-. 4.29 23.74 .+-. 2.67 23.43 .+-. 2.98 F2
100 21.41 .+-. 4.23 23.62 .+-. 2.97 21.03 .+-. 3.19* ps: compared
to MC: *P < 0.05, ** P < 0.01.
* * * * *