CN111892566A

CN111892566A - Ampelopsis grossedentata water extract, preparation method thereof and application of water extract as acetylcholinesterase inhibitor

Info

Publication number: CN111892566A
Application number: CN201910367888.5A
Authority: CN
Inventors: 唐静成; 吕昕航; 张欣逸; 李家奇; 邵建群; 杨一帆
Original assignee: Capital Medical University
Current assignee: Capital Medical University
Priority date: 2019-05-05
Filing date: 2019-05-05
Publication date: 2020-11-06

Abstract

The invention discloses a ampelopsis grossedentata water extract, a preparation method thereof and application of the ampelopsis grossedentata water extract as an acetylcholinesterase inhibitor. The invention pulverizes Ampelopsis grossedentata, screens, extracts with purified water at 80 ℃, stands, centrifuges, and freezes and dries the supernatant to obtain the water extract of Ampelopsis grossedentata. The HPLC-MS method is adopted to clarify the main chemical composition of the ampelopsis grossedentata water extract, and the HPLC-MS chemical fingerprint spectrum of the ampelopsis grossedentata water extract is obtained. Then, the water extract of Ampelopsis grossedentata and the main active substances are detected to inhibit the activity of acetylcholinesterase, and the results show that: the ampelopsis grossedentata aqueous extract has a good effect of inhibiting the activity of acetylcholinesterase, and dihydromyricetin has the best effect of inhibiting the activity of acetylcholinesterase, and the second is myricetin. The present invention provides a novel acetylcholinesterase inhibitor, and provides a new technical means for treating Alzheimer's disease.

Description

Ampelopsis grossedentata water extract, preparation method thereof and application of water extract as acetylcholinesterase inhibitor

Technical Field

The invention relates to a ampelopsis grossedentata water extract, a preparation method thereof and application thereof as an acetylcholinesterase inhibitor. The invention belongs to the technical field of medicines.

Background

Alzheimer's Disease (AD) is a degenerative brain Disease occurring in the elderly and pre-senile stages characterized by progressive neurodegeneration, and is a degenerative Disease of the central nervous system, which is clinically manifested by progressive cognitive decline accompanied by behavioral disorders and emotional disorders, and the like, and severe patients lose their ability to self-care.

The pathogenesis of AD has not been fully elucidated, and the cholinergic, oxidative stress and free radical damage theories are one of its important pathogenesis [ Terry AV Jr, Buccafusco JJ. the Choline organic principles of agents and Alzheimer's disease-related cognitive details: receptors and derivatives for novel drug delivery [ J ] Pharmacol Exp Ther,2003,306: 821-. AchE is an enzyme inhibitor target of AD, and can catalyze acetylcholine cleavage reaction, cause acetylcholine deletion, and directly cause nerve signal transmission failure, and cause Alzheimer's disease [ Leqian, Chusanxia, Sunwaran. Currently, clinical drug therapy for AD is primarily directed to increasing acetylcholine levels in patients by inhibiting AChE. Oxidative stress also plays an important role in the pathogenesis of AD [ Vina J, Lloret A, AlonsoD, et al molecular bases of the treatment of Alzheimer's disease with reactive species: prevention of oxidative stress [ J ]. Mol assays Med, 2004; 25(1-2):117-23.]. Excessive oxidative stress can lead to lipid peroxidation and/or protein nitration and nucleic acid destruction on the nerve cell membrane or organelle membrane, affecting synaptic ability of nerve cells and even leading to nerve cell apoptosis [ Jiang T, Sun Q, Chen S.oxidative stress: A major pathogenic and pore-biological therapeutic target of oxidative agents in Parkinson's disease and dAlzheimer's disease [ J ]. Prog Neurobiol, 2016; 147(1):1-19.]. Oxidative stress and apoptosis are common pathways for the pathogenesis of AD patients. The amyloid (A beta) is irreversibly deposited in brain tissues at first, oxygen free radicals are further activated, oxidative stress, inflammatory reaction and apoptosis are caused, and finally, neuron function loss, metabolic disturbance, and remarkable decline of learning, cognition and memory functions are caused [ Fanwei, Hu nationality, Liu Nengjie, Alzheimer disease and oxidative stress research progress [ J ]. China journal of Others, 2017,37(20):5205-5207 ]. Studies have shown that antioxidants inhibit amyloid aggregation [ Meske V, AlbertF, Ohm TG.coupling of a large target of rapamycin with phosphoside 3-kinase signaling pathway regulation proteins-phase 2A-and glycogenosylkinase-3-dependent phosphorylation of tau [ J ]. J Biol Chem, 2008; 283(1):100-9.].

Ampelopsis grossedentata (Ampelopsis grossedentata) is a Vitaceae Ampelopsis plant, commonly called Maoyan strawberry (MYM) and Ampelopsis grossedentata, and young stems and leaves of the Ampelopsis grossedentata are used as health-care tea and herbal medicines by the Zhuang nationality and the Yao nationality for hundreds of years. The plant is mainly distributed in areas in south of Yangtze river such as Fujian, Yunnan, Guangdong, Guangxi, Guizhou and Hunan, and is a plant with homology of medicine and food. Vine tea has been spread for a long time in these areas, has a wide public base, and people think that the vine tea has the efficacy of clearing away heat and toxic materials, and can treat sore throat, cold, toothache, hepatitis and the like when being drunk after being soaked in water. Previous studies indicate that vitis vinifera mainly contains flavonoid compounds, wherein Dihydromyricetin (DMY) accounts for about 86% of the total flavonoid compound content in the extract [ gaojiahua, rhoaojing, ningzhengxiang ] purification and crystal morphology research of plant flavone dihydromyricetin [ J ] research and development of natural products, 2006, 18 (1): 81-84.]. Ampelopsis grossedentata adopts an aqueous extraction process, dihydromyricetin is easy to separate out under the condition of cooling or concentrating an extracting solution [ Dingminlong, Penggu, Yangling, Wangxiang ] ampelopsis grossedentata antioxidant activity and main component correlation research [ J/OL ]. natural product research and development, 2019,04 (05): 1-12]. The research on various pharmacological actions of Ampelopsis grossedentata is more and more, and mainly comprises the actions of diminishing inflammation, relieving cough, inhibiting bacteria, resisting hypertension, reducing blood fat, protecting liver, resisting oxidation, regulating immunity, resisting tumor, relieving pain and reducing blood sugar.

The invention discloses a method for preventing and treating Alzheimer's disease by using Ampelopsis grossedentata, which comprises the steps of using an HPLC-MS method to clarify the main chemical composition of an aqueous extract of Ampelopsis grossedentata, obtaining an HPLC-MS chemical fingerprint of the aqueous extract of Ampelopsis grossedentata for the first time, and proving that the aqueous extract of Ampelopsis grossedentata and main active substances thereof have the effect of inhibiting the activity of acetylcholinesterase through experiments, thereby providing experimental basis and scientific basis for further development and utilization of the Ampelopsis grossedentata.

Disclosure of Invention

The invention aims to provide a ampelopsis grossedentata water extract, a preparation method thereof and a new application of the ampelopsis grossedentata water extract as an acetylcholinesterase inhibitor.

In order to achieve the purpose, the invention adopts the following technical means:

the invention is prepared by crushing Ampelopsis grossedentata, sieving, extracting the Ampelopsis grossedentata powder and purified water at 80 ℃, standing, centrifuging, and freeze-drying the supernatant to obtain the water extract of the Ampelopsis grossedentata. The HPLC-MS method is adopted to clarify the main chemical composition of the ampelopsis grossedentata water extract, and the HPLC-MS chemical fingerprint spectrum of the ampelopsis grossedentata water extract is obtained. Then, the water extract of Ampelopsis grossedentata and the inhibition of acetylcholinesterase activity of main active substances are detected by adopting an improved Ellman method, and the results show that: the ampelopsis grossedentata aqueous extract has a good effect of inhibiting the activity of acetylcholinesterase, and dihydromyricetin has the best effect of inhibiting the activity of acetylcholinesterase, and the second is myricetin.

On the basis of the research, the invention provides the application of the ampelopsis grossedentata water extract as an acetylcholinesterase inhibitor.

Wherein, preferably, the ampelopsis grossedentata water extract contains dihydromyricetin and/or myricetin.

Wherein, preferably, the ampelopsis grossedentata water extract contains 5 compounds, which are myricetin, dihydromyricetin, quercetin-3-O-alpha-L-rhamnoside, galloyl-beta-D-glucose and myricitrin respectively.

Wherein, preferably, the ampelopsis grossedentata water extract is prepared by the following method:

pulverizing Ampelopsis Grossdentata, sieving, extracting with purified water at 80 deg.C, standing, centrifuging, and freeze drying the supernatant to obtain water extract of Ampelopsis Grossdentata.

More preferably, the water extract of Ampelopsis grossedentata is prepared by the following method:

pulverizing Ampelopsis Grossdentata, sieving with 300 mesh sieve, extracting 1kg of Ampelopsis Grossdentata powder and purified water at 80 deg.C for 30min at a ratio of 1:10(W/V), standing, centrifuging, freezing the supernatant at-20 deg.C for 48 hr, and freeze-drying at-60 deg.C for 2 days to obtain water extract of Ampelopsis Grossdentata.

Currently, clinical drug therapy for AD is primarily directed to increasing acetylcholine levels in patients by inhibiting AChE. Experiments prove that the ampelopsis grossedentata aqueous extract has a good inhibition effect on the activity of acetylcholinesterase, so that the invention further provides the application of the ampelopsis grossedentata aqueous extract in preparing the medicine for treating the Alzheimer disease.

Compared with the prior art, the invention has the beneficial effects that:

the invention adopts an HPLC-MS method to clarify the main chemical composition of the ampelopsis grossedentata water extract, obtains the HPLC-MS chemical fingerprint of the ampelopsis grossedentata water extract for the first time, and proves that the ampelopsis grossedentata water extract and the main active substances thereof have the effect of inhibiting the activity of acetylcholinesterase through experiments.

Drawings

FIG. 1 is HPLC-MS fingerprint of Ampelopsis grossedentata aqueous extract (blue: ultraviolet 290nm detection, orange: ESI negative ion mode detection);

FIG. 2 shows the identification of galloyl- β -D-glucose cleavage;

FIG. 3 shows the identification of dihydromyricetin cleavage;

FIG. 4 shows the myricitrin cleavage assay;

FIG. 5 shows the identification of quercetin-3-O-alpha-L-rhamnoside cleavage;

FIG. 6 shows the myricetin cleavage assay;

FIG. 7 is a graph of inhibition of acetylcholinesterase activity by aqueous extracts of MYM as a function of concentration;

FIG. 8 is a graph showing the relationship between the inhibitory rate of myricitrin on acetylcholinesterase activity and the concentration;

FIG. 9 shows the relationship between the inhibitory rate of myricitrin and dihydromyricetin on acetylcholinesterase activity and the concentration.

Detailed Description

The invention will be further described with reference to specific embodiments and drawings, the advantages and features of which will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

The instruments and materials used in the embodiments of the present invention:

the instrument comprises the following steps: high performance liquid chromatography-mass spectrometry instrument (chromatograph Waters 2795-.

Materials: acetylcholinesterase (AchE, EC 3.1.1.7, Sigma), 5, 5' -dithiobis (2-nitrobenzoic acid) (dithiobisbutyrobenzoic acid, DTNB, Sigma), thiocholine iodide (acetoxythiocoline, ATCI, Sigma), Tacrine (Sigma), absolute ethanol (beijing chemical plant), sodium bicarbonate (beijing chemical plant), NaH₂PO₄·2H₂O (Beijing, modern Oriental Fine Chemicals Co., Ltd.), NaH₂PO₄·2H₂O (scientific and technological development Co., Ltd., Oriental China, Beijing), VC, DPPH, pure methanol, Ampelopsis grossedentata (commercially available from tea leaf market in Maliandao, Beijing).

Example 1 preparation of an aqueous extract of Ampelopsis grossedentata and HPLC-MS analysis

1. Preparation of Ampelopsis grossedentata aqueous extract

Pulverizing Ampelopsis Grossdentata, sieving with 300 mesh sieve, extracting 1kg of Ampelopsis Grossdentata powder and purified water at 80 deg.C for 30min at a ratio of 1:10(W/V), standing, centrifuging, freezing the supernatant at-20 deg.C for 48 hr, and freeze-drying at-60 deg.C for 2 days to obtain the final product, i.e. Ampelopsis Grossdentata water extract (MYM water extract).

The water extract of Ampelopsis grossedentata is light yellow, 220g in total, and the extraction rate is 22%.

2. HPLC-MS analysis of aqueous extract composition

2.1 sample preparation

The experiment adopts automatic sample injection, 0.0122mg of Ampelopsis grossedentata water extract is weighed, 1mL of purified water is dissolved to obtain the concentration of 0.0122mg/mL, the obtained solution is filtered through a 0.45 mu m filter membrane, the sample injection amount is 30 mu L, gradient elution is carried out, and the operation time is 320 min.

2.2 chromatographic conditions

A chromatographic column: agilent eclipse XDB-C18, 5 mu, 4.6 x 250mm

Mobile phase: a: water (0.1% formic acid), B: acetonitrile (0.1% formic acid)

Temperature: 30 deg.C

2.3 Mass Spectrometry conditions

ESI-Q-TOF-MS: an optimized small molecule analysis method is adopted, and the specific parameters are as follows:

ESI source parameters:

the mass spectrometer was calibrated with sodium formate before each analysis.

2.4 HPLC-MS analysis results of Ampelopsis grossedentata aqueous extract

FIG. 1 shows HPLC-MS fingerprint of Ampelopsis grossedentata aqueous extract.

5 compounds are separated from the ampelopsis grossedentata water extract, which are myricetin, dihydromyricetin, quercetin-3-O-alpha-L-rhamnoside, galloyl-beta-D-glucose and myricitrin respectively.

Peak 1 is galloyl-beta-D-glucose, peaks 2-7 are dihydromyricetin and isomers thereof, peaks 8-10 are myricitrin and isomers thereof, peak 11 is quercetin-3-O-alpha-L-rhamnoside, and peaks 12-13 are myricetin and isomers thereof.

The cracking identification modes of galloyl-beta-D-glucose, dihydromyricetin, myricitrin, quercetin-3-O-alpha-L-rhamnoside, and myricetin are shown in FIGS. 2-6.

The galloyl-beta-D-glucose cleavage identification mode comprises the following steps: m/z 331[ M-H]Fragments of-lose fragments of m/z 138 to fragments of m/z 193. M/z 331[ M-H]The fragment of (E) lost one molecule of sugar to give a fragment of m/z 169 and a fragment of m/z 169 lost one molecule of H₂O gave fragments of m/z 151. Fragments of m/z 169 lost one molecule of CO₂Fragments of m/z 125 were obtained.

The cracking identification mode of the dihydromyricetin is as follows: m/z 319[ M-H]Fragment of-loss of one molecule of H₂O to M/z301 [ M-H [)₂O]-fragments of (a). Fragments of m/z301 lost one molecule of CO₂Obtaining M/z 257[ M-H [)₂O-CO₂]Fragment of (E), loss of one molecule of H₂O to obtain M/z283[ M-H [)₂O-CO₂-H₂O]-fragments of (a). M/z 319[ M-H]Reverse RDA fragmentation of fragment-to yield fragment m/z153, fragment missing m/z 168 to yield fragment m/z 233. Fragment of m/z 233 lost one molecule of H₂O gave a fragment of m/z 215 and the fragment missing 52 gave a fragment of m/z 181. The m/z283 fragment loses the m/z 92 fragment to obtain the m/z 191 fragment. Fragments of m/z153 lose one molecule of CO to give fragments of m/z 125. M/z 319[ M-H]The fragment of-loses the fragment of m/z 126 to obtain the fragment of m/z 193. Fragment of m/z 193 lost one molecule of H₂O gave a fragment of m/z 175 and a fragment missing m/z 14 gave a fragment of m/z 179.

The myricitrin cracking identification mode comprises the following steps: m/z 463[ M-H]The fragment of-loses one molecule of sugar to give a fragment of m/z 316. The m/z 316 fragment was cleaved by reverse RDA to give m/z 169 fragment, and the m/z 169 fragment lost OH to give m/z 152 fragment. Fragments with m/z 152 lost H gave fragments with m/z 151 and fragments with m/z15 gave fragments with m/z 137. Fragments of m/z 151 lost fragments of m/z 26 to fragments of m/z 125. The m/z 316 fragment lost m/z 123 fragment to m/z 193 fragment, m/z29 fragment to m/z 287 fragment, and OH fragment to m/z299 fragment. The fragment of m/z299 lost one molecule of CO to give a fragment of m/z 271. Fragment of m/z 271 lost one molecule of CO₂The fragment with m/z 227 was obtained, and the fragment with m/z29 lost was obtained as a fragment with m/z 242. The fragment of m/z 287 loses one molecule of CO to yield a fragment of m/z 259. The m/z 193 fragment lost the m/z 14 fragment to the m/z 179 fragment. The fragment of m/z 179 loses the fragment of m/z 16 to the fragment of m/z 163.

The cracking identification mode of the quercetin-3-O-alpha-L-rhamnoside is as follows: m/z 447[ M-H ] -loss of a single sugar gives a fragment of M/z301, and M/z 447[ M-H ] -loss of M/z 130 gives a fragment of M/z 317. The fragment at m/z301 was subjected to reverse RDA fragmentation to give a fragment at m/z 151. Fragments of m/z 317 lost fragments of m/z 138 to fragments of m/z 179.

The myricetin cracking identification mode comprises the following steps: m/z 317[ M-H]CO loss of the fragments to M/z 289[ M-H-CO]A fragment of [ M-H-CO ]]The fragments of-are each deprived of one molecule of CO₂A molecule H₂Fragmentation of O, 110 to give M/z 245[ M-H-CO ]₂]Fragment of-217 [ M-H-CO-H ]₂O]Fragments of-and fragments of 179. [ M-H-CO ]₂]Fragment of-loss of one molecule of H₂O to M/z 227[ M-H-CO ]₂-H₂O]-fragments of (a). Fragment 179 lost fragment 42 and fragment 137. The m/z 317 fragment lost 125 fragment to 192 fragment. The m/z 317 fragment is cracked by reverse RDA to obtain m/z 169 fragment and m/z 109 fragment, then the 169 fragment is broken off OH to obtain m/z 152 fragment, and 152 fragment is lost H to obtain m/z 151 fragment. At the same time, the m/z 152 fragment lost 27 fragments to m/z 125 fragments, the 125 fragments lost H₂The fragmentation of O gave a fragmentation of m/z 107.

Example 2 inhibition of acetylcholinesterase Activity by an aqueous extract of Ampelopsis grossedentata

This embodiment employs a modified Ellman method. The Ellman method, which is an important method for detecting sulfydryl groups such as biological tissues and proteins, was created by Ellman in 1959 [ ELLMAN G D. tissue Sulfhydryl groups [ J ]. Archivesof biochemiry and Biophymics, 1959,82(1):70-77 ], and has been widely used and is representative of photometric analysis. The principle is that 5-5 ' -dithio-2-nitrobenzene reacts with sulfhydryl anion, and when the pH is 8, strong absorption is generated at 410nm, in the subsequent development, researchers use 5,5 ' -dithio-2-nitrobenzoic acid as Ellman reagent more, the principle of the method is the same as that of the method proposed by Ellman, 5 ' -dithio-2-nitrobenzoic acid (DTNB) reacts with free-SH to generate 5-sulfhydryl-2-nitrobenzoic acid (TNB), and the product has strong absorption at the wavelength of 410-420 nm.

The hydrolysis reaction process of acetylcholinesterase on iodothio-acetylcholine in the modified Ellman method is shown as the following formula:

the reaction procedure for detecting the hydrolysis reaction product is shown in the following formula [ Zhan, Shang Zhong Bo, Chang Zhi obviously, Qishichong, Lidell.thiol detection methods research progress [ J ]. proceedings of Henan university (Nature science edition), 2018,48(04): 430-:

1. inhibitory Activity of MYM aqueous extract on Acetylcholinesterase

Preparing a sample solution: weighing 20.00mg of dried MYM aqueous extract, dissolving in 2ml of distilled water to prepare 10mg/ml sample solution, filtering with 0.45 μm filter membrane, and diluting to 9.0, 8.0, 7.0, 6.0, 5.0, 4.0, 3.0, and 2.0mg/ml concentrations.

The modified Ellman method is adopted for inhibiting the activity of acetylcholinesterase, the positive control is Tacrine (3000 mu g/mL), and the reaction system is as follows: pH 8.0, 140. mu.l of Phosphate Buffered Saline (PBS), 20. mu.l of the sample solution, 15. mu.l of acetylcholinesterase (0.2U/ml), 96-well microplate were sequentially added thereto, the mixture was shaken and mixed, the mixture was incubated at 37 ℃ for 2min, 20. mu.l of iodothioacetylcholine (ATCI) (15mmol/L) was added thereto to start the reaction, and after incubation at 37 ℃ for 20min, 20. mu.l of DTNB (1mmol/L) was added thereto, and the absorbance was measured at 410nm using a microplate reader. Blank group replaced sample with PBS; the positive control group replaced the sample with Tacrine (3000. mu.g/mL); background controls replaced the enzyme solution with PBS. The inhibition of acetylcholinesterase activity by the sample was calculated using the formula:

inhibition rate 100% × [ (a blank-a positive) - (a sample-a background) ]/(a blank-a positive)

The inhibition result of the MYM aqueous extract on the acetylcholinesterase activity is shown in figure 7, the inhibition effect of the MYM aqueous extract on the acetylcholinesterase activity is measured by adopting a modified Ellman method, the result shows that the IC50 of the MYM aqueous extract is 0.376mg/ml, the inhibition on the acetylcholinesterase activity is in a concentration-dependent relation before the concentration is less than 0.558mg/ml, and the inhibition rate is kept relatively stable and is close to 100% after the concentration is more than 0.558 mg/ml. As MYM aqueous extract has a good inhibitory effect on acetylcholinesterase activity, the main active ingredients of the MYM aqueous extract are further studied.

2. Inhibitory Activity of the Standard on Acetylcholinesterase

Preparing a standard solution: weighing 1.30mg of dihydromyricetin, 1.30mg of myricitrin and 9.30mg of myricetin respectively, dissolving with 2ml of distilled water respectively, preparing into standard solutions of 0.650mg/ml, 0.650mg/ml and 4.65mg/ml, and diluting according to gradient to obtain series of concentrations. The experiments for inhibiting acetylcholinesterase activity were performed according to the methods described above.

The results of the inhibition of acetylcholinesterase activity by the standard are shown in FIGS. 8 and 9.

As shown in FIG. 8, it can be seen that the concentration of myricetin standard is less than 2.51 × 10²The inhibition of acetylcholinesterase activity at μ g/ml was concentration-dependent, and IC50 was determined to be 1.12X 10²Mu g/ml; at a concentration of greater than 2.51X 10²The inhibition remained relatively stable at μ g/ml and was close to 100%.

As shown in fig. 9, the inhibition effect of myricitrin and dihydromyricetin standard on acetylcholinesterase activity was determined by the modified Ellman method. Wherein the dihydromyricetin has concentration-dependent relationship on inhibition of acetylcholinesterase activity at concentration less than 36.4 μ g/ml, and at concentration greater than 2.51 × 10²The inhibition rate at μ g/ml remained relatively stable, approaching 100%.

The results of comparison between fig. 8 and fig. 9 show that the inhibitory effect of dihydromyricetin on acetylcholinesterase activity is the best, followed by myricetin, and the inhibitory rate of myricitrin on acetylcholinesterase activity cannot reach 100%.

Claims

1. The application of Ampelopsis grossedentata water extract as acetylcholinesterase inhibitor is provided.

2. The use of claim 1, wherein said aqueous extract of Ampelopsis grossedentata comprises dihydromyricetin and/or myricetin.

3. The use of claim 2, wherein said aqueous extract of Ampelopsis grossedentata comprises 5 compounds selected from the group consisting of myricetin, dihydromyricetin, quercetin-3-O- α -L-rhamnoside, galloyl- β -D-glucose, and myricitrin.

4. The use as claimed in claim 3, wherein said aqueous extract of Ampelopsis grossedentata is prepared by the following method:

5. The use of claim 4, wherein said aqueous extract of Ampelopsis grossedentata is prepared by the following method:

6. Use of water extract of Ampelopsis grossedentata in preparation of medicine for treating Alzheimer disease is provided.

7. The use of claim 6, wherein said aqueous extract of Ampelopsis grossedentata comprises dihydromyricetin and/or myricetin.

8. The use of claim 7, wherein said aqueous extract of Ampelopsis grossedentata comprises 5 compounds selected from the group consisting of myricetin, dihydromyricetin, quercetin-3-O- α -L-rhamnoside, galloyl- β -D-glucose, and myricitrin.

9. The use of claim 8, wherein said aqueous extract of Ampelopsis grossedentata is prepared by the following method:

10. The use of claim 9, wherein said aqueous extract of Ampelopsis grossedentata is prepared by the following method: