Active part of fructus aristolochiae, extraction method of active part and application of active part in prevention and treatment of alcoholic liver disease

Abstract

The invention belongs to the field of natural medicines, and particularly relates to a fructus aristolochiae active site, an extraction method thereof and application of the fructus aristolochiae active site in prevention and treatment of alcoholic liver diseases. The active part is obtained by the following extraction method: S1, extracting fructus aristolochiae powder with 50% ethanol, and concentrating an extracting solution under reduced pressure to obtain a 50% ethanol extract; s2, carrying out macroporous resin column chromatography on the 50% ethanol extract, and sequentially eluting with water and 30% ethanol; and S3, collecting 30% ethanol eluate, and concentrating to obtain the active site for preventing and treating the alcoholic liver disease. Experiments prove that the active part of fructus aristolochiae disclosed by the invention has a preliminary effect of preventing and treating the alcoholic liver disease, and has the potential of preparing the medicine for preventing and treating the alcoholic liver disease.

Description

Technical Field

[0001] The invention belongs to the field of natural medicines, and particularly relates to an active fraction of Cynanchum cyrtonema, an extraction method thereof, and application thereof in preventing and treating alcoholic liver disease. Background Art

[0002] Alcoholic liver disease (ALD) is a liver disease caused by long-term, excessive alcohol consumption. It typically begins with fatty liver disease and can progress to alcoholic hepatitis, liver fibrosis, and cirrhosis. Severe alcohol consumption can induce extensive hepatocellular necrosis and even lead to liver failure. In recent years, the incidence and severity of ALD have continued to rise, seriously endangering the health of patients and significantly increasing the global healthcare burden. Oxidative stress is one of the primary mechanisms of ALD, and substances with antioxidant properties have potential for the prevention and treatment of ALD.

[0003] Although research into therapeutic drugs for ALD has made some progress (for example, Patent Publication No. CN200810305398.4 discloses the use of a Herba Adenophorae extract in the preparation of a drug for treating ALD; Patent Publication No. CN201010106231.2 discloses a Herba Adenophorae extract for the prevention and / or treatment of liver diseases (including ALD)), no suitable drug for alcoholic liver disease has yet been developed. Therefore, the development of a therapeutic drug with effective efficacy for ALD remains a critical issue that urgently needs to be addressed.

[0004] Toona sinensis, also known as Toona sinensis, is a plant of the Meliaceae family. Toona sinensis The fruit of the A. Juss. Roem. is distributed in North, East, Central, South, and Southwest China, as well as Taiwan and Tibet. The fruit of the A. Juss. Roem. is warm in nature, bitter in taste, and non-toxic. It has the effects of clearing heat and detoxifying, promoting dampness and relieving jaundice. It is rich in polysaccharides and flavonoids, which have potential antioxidant properties.

[0005] Currently, there is no report on the use of Cynanchum cyrtonema, its extracts or its active fractions for preventing or treating liver diseases. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an active fraction of Cynanchum cyrtonema, an extraction method thereof and application thereof in preventing and treating alcoholic liver disease.

[0007] In order to solve the above problems, the technical solution adopted by the present invention is: Technical Topic 1 A method for extracting the active fraction of Cynanchum cyrtonema for preventing and treating alcoholic liver disease comprises the following steps: S1: Extract the powder of Cynanchum cyrtonema with 50% ethanol, and concentrate the extract to obtain a 50% ethanol extract; S2: The 50% ethanol extract was chromatographed on a macroporous resin column, eluted with water and then 30% ethanol; S3: Collect and concentrate the 30% ethanol eluate to obtain the active fraction for preventing and treating alcoholic liver disease.

[0008] As a further improvement of the present invention, the powder of the citronella sibiricum in S1 is obtained by crushing the citronella sibiricum and passing it through an 80-mesh sieve.

[0009] As a further improvement of the present invention, the extraction method in S1 is as follows: soaking the powder of Cynanchum cyrtonema and 50% ethanol in a mass ratio of 1:8-12 for 10-14 hours, heating and reflux for 25-35 minutes, filtering after cooling, adding the same amount of 50% ethanol as during the soaking to the filter residue, repeating the heating and reflux once, combining the filtrate to obtain an extract.

[0010] As a further improvement of the present invention, the extraction method in S1 is as follows: soaking the powder of Cynanchum cyrtonema and 50% ethanol in a mass ratio of 1:10 for 12 hours, heating and reflux for 30 minutes, filtering after cooling, adding the same amount of 50% ethanol as during the soaking to the filter residue, repeating the heating and reflux once, combining the filtrate to obtain an extract.

[0011] As a further improvement of the present invention, the concentration method in S1 is reduced pressure concentration.

[0012] As a further improvement of the present invention, the macroporous resin in S2 is D101 macroporous resin, and column chromatography is performed using a medium-pressure purification preparative chromatography system.

[0013] Technical Topic 2 An active fraction of Cynanchum cyrtonema extracted by the method described in technical subject one for preventing and treating alcoholic liver disease.

[0014] Technical Theme 3 A pharmaceutical composition comprising the active site described in Technical Subject 2, and optionally, one or more pharmaceutically acceptable carriers or excipients.

[0015] As used herein, "pharmaceutically acceptable carriers or excipients" include: diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, taste masking agents, coloring agents, anti-caking agents, humectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants and buffers. Those skilled in the art will understand that certain pharmaceutically acceptable excipients can be used in more than one function and in alternative functions, depending on how much of the excipient is present in the formulation and what other ingredients are present in the formulation. For example, when used for oral administration, oral preparations such as tablets, capsules, granules and pills can be prepared, containing fillers (e.g., sugar derivatives such as lactose, sucrose, glucose, mannitol and sorbitol; starch derivatives such as corn starch, potato starch, dextrin and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, carboxymethyl cellulose sodium; gum arabic; dextran; silicate derivatives such as magnesium aluminum metasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; sulfate derivatives such as calcium sulfate, etc.), binders (e.g., gelatin, polyvinyl pyrrolidone and polyethylene glycol), disintegrants (e.g., cellulose derivatives such as sodium carboxymethyl cellulose, polyvinyl pyrrolidone), lubricants (e.g., talc, calcium stearate, magnesium stearate, spermaceti, boric acid, sodium benzoate, leucine), stabilizers (methyl parahydroxybenzoate, propyl parahydroxybenzoate, etc.), flavoring agents (e.g., commonly used sweeteners, acidulants and spices, etc.). When used parenterally, the drug can be prepared as an injection, including sterile powder for injection and solvent for injection. The carrier or excipient used includes sterile water, Ringer's solution, and isotonic sodium chloride solution. Suitable additives such as antioxidants, buffers, and antibacterial agents may also be added depending on the properties of the drug. When used for rectal administration, the drug can be prepared as a suppository, etc. When used for pulmonary administration, the drug can be prepared as an inhaler or spray, etc. There are many resources available to those skilled in the art that describe pharmaceutically acceptable excipients and can be used to select suitable pharmaceutically acceptable excipients, such as books such as "Remington's Pharmaceutical Compendium," "Chinese Pharmaceutical Annals," and "Pharmaceutics."

[0016] Technical Theme 4 An application of the active site as described in technical theme 2 in the preparation of drugs for preventing and treating alcoholic liver disease.

[0017] Technical Topic 5 A use of the active site as described in technical theme 2 as the sole active ingredient in the preparation of a drug for preventing and treating alcoholic liver disease.

[0018] Technical Topic 6 An application of the active site as described in the second technical theme in the preparation of liver-protecting foods or health products.

[0019] The beneficial effects of adopting the above technical solution are: This application uses macroporous resin column chromatography technology to elute the 50% ethanol extract of Cynanchum wilfordii with water and 30% ethanol in sequence, and finally obtains an active fraction that has the effect of preventing and treating alcoholic liver disease. Experimental verification shows that the active fraction of Cynanchum wilfordii disclosed in this application has a preliminary effect of preventing and treating alcoholic liver disease, and has the potential to prepare drugs for preventing and treating alcoholic liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This figure shows the results of screening for active sites in the prevention and treatment of alcoholic liver disease using the 50% ethanol extract of Cynanchum cyrtonema prepared in Example 1 and its freeze-dried powders containing water, 30% ethanol, 70% ethanol, and anhydrous ethanol eluates; (* indicates P < 0.05 compared with the model group, ** indicates P < 0.01 compared with the model group, *** indicates P < 0.001 compared with the model group, and **** indicates P < 0.0001 compared with the model group); Figure 2 This is a graph showing the DPPH radical scavenging rate of the 50% ethanol extract of Cynanchum cyrtonema prepared in Example 1 and the freeze-dried powders of its eluates with water, 30% ethanol, 70% ethanol, and anhydrous ethanol; Figure 3 This is a graph showing the ABTS free radical scavenging rate of the 50% ethanol extract of Cynanchum cyrtonema prepared in Example 1 and the freeze-dried powders of its eluates in water, 30% ethanol, 70% ethanol, and anhydrous ethanol; Figure 4 is the ion chromatogram of the 30% ethanol solution of the lyophilized powder of the 30% ethanol eluate in Example 1 in positive ion mode, wherein B represents the corresponding intensity of the highest peak in the graph; Figure 5 This is the ion chromatogram in negative ion mode of a 30% ethanol solution of the lyophilized powder of the 30% ethanol eluate in Example 1, wherein B represents the corresponding intensity of the highest peak in the graph. DETAILED DESCRIPTION

[0021] In order to make the objectives, technical solutions and advantages of the present invention more clear, the invention is clearly and completely described below in conjunction with specific embodiments.

[0022] The angelica seeds used in this application were purchased from Hebei Renxin Pharmaceutical Co., Ltd. 96-well plates, PBS, and fetal bovine serum were purchased from Wuhan Sewell Biotechnology Co., Ltd.; Mouse hepatocytes NCTC1469 were purchased from Shanghai Biotechnology Co., Ltd. Anhydrous ethanol was purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.; DPPH was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; The total antioxidant capacity (T-AOC) colorimetric test kit (ABTS chemical method) was purchased from Wuhan Yilai Ruite Biotechnology Co., Ltd., with the catalog number: E-BC-K271-M; DMEM high glucose medium was purchased from Gibco, USA, batch number 6124636.

[0023] Example 1 S1: Crush the citronella seeds and pass through an 80-mesh sieve. Soak the powder in 50% ethanol for 12 hours at a mass ratio of 1:10. Heat extraction is performed using a condensation reflux apparatus. After the first drop of distillate drips from the nozzle of the spherical condenser, maintain constant temperature extraction for 30 minutes (approximately 80°C). Cool, filter, and pour off the extract. Repeat the extraction by adding the same amount of 50% ethanol as used during the soaking process. After cooling, filter, and combine the filtrates, then vacuum-evaporate to obtain a 50% ethanol extract of citronella seeds at a concentration of approximately 1 g / ml (crude drug amount).

[0024] S2: Using D101 macroporous resin in combination with a medium-pressure purification and preparative chromatography system, the 50% ethanol extract of Cynanchum cyrtonema prepared in S1 with a concentration of approximately 1 g / ml (crude drug amount) was loaded onto a pretreated macroporous resin column (column volume was 5 L, sample volume was 30 ml), and gradient elution was performed using water, 30% ethanol, 70% ethanol, and anhydrous ethanol as eluents in sequence (fractions were collected starting from the appearance of the chromatographic peak, and continued for 15 minutes after the peak completely disappeared, and then eluted with the next eluent). Four eluates were collected in the elution order.

[0025] S3: The four eluates were evaporated under reduced pressure, concentrated, and freeze-dried at -50°C overnight, in this application, for 12 hours, to obtain freeze-dried powders of water, 30% ethanol, 70% ethanol, and anhydrous ethanol eluates.

[0026] Take a portion of the 50% ethanol extract and evaporate under reduced pressure, concentrate, and freeze-dry at -50°C overnight, freeze-dry for 12 hours, to obtain 50% ethanol extract freeze-dried powder for use.

[0027] Example 2 The effects of the 50% ethanol extract of Fructus Caryophylli and the four eluates prepared in Example 1 on the viability of the alcoholic hepatocyte model were detected by CCK-8 method.

[0028] The 50% ethanol extract freeze-dried powder, water eluate freeze-dried powder, 30% ethanol eluate freeze-dried powder, 70% ethanol eluate freeze-dried powder solution or anhydrous ethanol eluate freeze-dried powder used in this example were prepared in Example 1.

[0029] NCTC1469 cells (normal mouse hepatocytes) in logarithmic growth phase were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were passaged when the cell density increased to a point where, as observed under a microscope, the cells occupied 80% to 90% of the available attachment area on the bottom of the culture vessel.

[0030] NCTC1469 cells were evenly seeded in 96-well plates and divided into the normal group (Control), model group (Model) and drug-treated group. The cell density per well was approximately 1×10 4 The cells were incubated in 100 μL of high-glucose DMEM medium containing 3% fetal bovine serum and 1% penicillin-streptomycin for 24 hours at 37°C in a 5% CO2 incubator. Then, 10 μL of the following solutions (10 μg / mL) were added to each well: lyophilized powder of the 50% ethanol extract, lyophilized powder of the water eluate, lyophilized powder of the 30% ethanol eluate, lyophilized powder of the 70% ethanol eluate, or lyophilized powder of the anhydrous ethanol eluate. The solvent for each of these solutions was high-glucose DMEM medium containing 1% penicillin-streptomycin. The model and treatment groups were each incubated in a 37°C, 5% CO2 incubator for 24 hours. The supernatant was discarded, and 100 μL of high-glucose DMEM medium containing 3% fetal bovine serum and 1% penicillin-streptomycin and 10% CCK-8 reagent was added to each well. The cells were incubated in the dark for 2 hours. The absorbance (OD value) was measured using a microplate reader at a wavelength of 450 nm, and the cell viability (%) was calculated according to the formula: cell viability (%) = (OD value of the experimental group - OD value of the blank group) / (OD value of the normal group - OD value of the blank group) × 100%.

[0031] The results are as follows Figure 1 As shown in the figure, the comparison of data shows that the 30% ethanol eluate group has the most obvious preventive and therapeutic effect on alcoholic liver disease.

[0032] Example 3 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) is a compound containing a stable nitrogen free radical. Its ethanol solution is purple with a strong absorbance peak at 517 nm. When an antioxidant donates electrons or hydrogen atoms, reducing it to its non-radical form (DPPH-H), the solution becomes lighter in color and its absorbance decreases. In this example, DPPH was used to test the antioxidant activity of the 50% ethanol extract prepared in Example 1, as well as the freeze-dried powders of the water, 30% ethanol, 70% ethanol, and anhydrous ethanol eluates.

[0033] 1) Preparation of DPPH solution and sample solution Prepare a 0.1 mM DPPH solution by dissolving 0.002 g DPPH in 50 mL ethanol and protect from light. Prepare sample solutions at concentrations of 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625, 0.003125, 0.001563, 0.000781, and 0.000391 mg / mL in 50% ethanol.

[0034] The samples are freeze-dried powder of 50% ethanol extract prepared in Example 1 and freeze-dried powder of water, 30% ethanol, 70% ethanol, and anhydrous ethanol eluates.

[0035] 2) Add sample Each group has 3 replicate wells. Pay attention to the light-proof operation. After the sample is added, keep it away from light at room temperature for 30 minutes and measure the absorbance. The amount added to each well and the distribution of the 96-well plate are as follows: Sample group: 100 μL sample solution + 100 μL DPPH alcohol solution (three replicate wells for each concentration); Blank group: 100 μL sample solution + 100 μL anhydrous ethanol (three replicate wells for each concentration); Control group: 100 μL of DPPH alcohol solution + 100 μL of 50% ethanol.

[0036] 3) Measure absorbance and calculate clearance rate Measure the absorbance at 517 nm, take the average value, calculate the DPPH clearance rate of each concentration, and use GraphPad to make a line graph based on the clearance rate to obtain the IC 50 ( Figure 2 Clearance rate = (1-(A sample-A black) / A control) × 100%.

[0037] According to the above experimental method, the DPPH radical scavenging rates of the four eluate freeze-dried powders and 50% ethanol extract freeze-dried powder were obtained, and their IC 50 The values are: 30% ethanol eluate (0.003733 mg / ml) < 50% ethanol extract (0.005969 mg / ml) < water eluate (0.006017 mg / ml) < 70% ethanol eluate (0.007831 mg / ml) < anhydrous ethanol eluate (0.03251 mg / ml). By comparison, the IC value of the free radical scavenging rate of the 30% ethanol eluate is 50 The lowest value indicates the strongest free radical scavenging effect.

[0038] Example 4 ABTS free radical scavenging rate In this example, the antioxidant activity of the 50% ethanol extract prepared in Example 1 and the lyophilized powders of the water, 30% ethanol, 70% ethanol, and anhydrous ethanol eluates was determined using a total antioxidant capacity (T-AOC) colorimetric test kit (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) chemical method).

[0039] The determination principle involves: ABTS will be oxidized into green ABTS·+ under the action of appropriate oxidants. The formation of ABTS·+ will be inhibited in the presence of antioxidants. By measuring the absorbance of ABTS·+ at 734nm, the total antioxidant capacity of the sample can be detected and calculated.

[0040] 1) Preparation of ABTS working solution Before testing, equilibrate the reagents to room temperature. Mix the ABTS solution and oxidant solution in the colorimetric test kit in a 1:1 volume ratio to prepare the ABTS working stock solution. Store it in the dark at room temperature for 12-16 hours before use. It will remain stable for 2 days. Before use, dilute the ABTS working stock solution with diluent (1× PBS) to ABTS working solution. Dilute to the blank well (10μL diluent + 200μL working solution). 734 (Absorbance at 734 nm compared to pure water) is 0.9-1.1.

[0041] 2) Preparation of sample solution The 50% ethanol extract freeze-dried powder prepared in Example 1 and the water, 30% ethanol, 70% ethanol, and anhydrous ethanol eluates freeze-dried powders were prepared in 1×PBS to prepare sample solutions of 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625, 0.003125, 0.001563, 0.000781, and 0.000391 mg / ml.

[0042] 3) Add sample and measure OD value Take 10 μL of sample solution of varying concentrations and add it to the corresponding wells of the microplate. Add 200 μL of ABTS working solution to each well. Incubate at room temperature for 2-6 minutes. Measure the OD value at 734 nm on a microplate reader. Use 200 μL of ABTS working solution + 10 μL of 1× PBS as the control.

[0043] 4) Result determination Each sample was tested in parallel three times, and the average value was taken. The ABTS scavenging rate was calculated according to the following formula, and the free radical scavenging rate-lyophilized powder concentration curve was drawn to obtain the IC 50 .

[0044] ABTS clearance rate (%) = [(Acontrol - Asample) / Acontrol] × 100%, where sample group refers to the sample group.

[0045] According to the above experimental method, the free radical scavenging rates of the four lyophilized powders of the eluate and the lyophilized powder of 50% alcohol extract were obtained, and their IC 50 The values are: 30% ethanol eluate (0.04689 mg / ml) < 50% ethanol extract (0.06930 mg / ml) < 70% ethanol eluate (0.07143 mg / ml) < water eluate (0.3140 mg / ml) < anhydrous ethanol eluate (0.6354 mg / ml). By comparison, it can be concluded that the IC value of the free radical scavenging rate of the 30% ethanol eluate is 50 The value was the lowest and the free radical scavenging effect was the strongest (n=3).

[0046] Example 5 The 30% ethanol solution of the lyophilized powder of the 30% ethanol eluate prepared in Example 1 was characterized by LC-MS.

[0047] 1. Chromatographic conditions are: Thermo Scientific U3000 fast liquid chromatography and reversed-phase chromatography columns were used for analysis.

[0048] Column: Waters HSS T3; Mobile phase: Positive ion mode: phase A (water containing 0.1% formic acid) and phase B (acetonitrile containing 0.1% formic acid); negative ion mode: phase A (water) and phase B (acetonitrile).

[0049] The elution gradient is shown in Table 1: The flow rate is 0.4 ml / min; The injection volume was 1 μL; The column temperature was 50°C.

[0050] The sample is a 30% ethanol solution of lyophilized powder of 30% ethanol eluate with a crude drug amount of 1 g / ml.

[0051] 2. Mass spectrometry conditions are: Mass spectrometry analysis was performed using a quadrupole-orbitrap mass spectrometer (Orbitrap Exploris480) equipped with a thermal electrospray ionization source.

[0052] (1) Ion source: Positive ion mode voltage (V): 3500; Negative ion mode voltage (V): 2500; Sheath gas flow (Arb): 50; Auxiliary gas flow (Arb): 10; Purge gas flow (Arb): 1; Ion transfer tube temperature (°C): 325; Atomizer temperature (℃): 350.

[0053] (2) Full Scan Mode: Orbital trap resolution: 60,000; Scan range (m / z): 67-1000; RF lens (%): 40; Normalized automatic gain control target value (%): 100; Maximum injection time (ms): 100; Intensity threshold: 5.0e3.

[0054] (3) Secondary mass spectrometry scanning (ddMS²): Isolation window (m / z): 1; Collision energy type: normalized; HCD collision energy (%): 20, 40, 60; Orbital trap resolution: 30,000; Maximum injection time (ms): 54; Number of micro scans: 1.

[0055] The results are as follows Figure 4 (positive ion mode ion chromatogram) and Figure 5 As shown in (ion chromatogram in negative ion mode), the results showed that the 30% ethanol eluate mainly contained flavonoids, phenols, and phenylpropanoid compounds such as quercetin and gallic acid.

[0056] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for extracting the active fraction of Cynanchum cyrtonema for preventing and treating alcoholic liver disease, characterized in that: It includes the following steps: S1: Extract the powder of Cynanchum cyrtonema with 50% ethanol, and concentrate the extract to obtain a 50% ethanol extract; S2: The 50% ethanol extract was chromatographed on a macroporous resin column, eluted with water and then 30% ethanol; S3: Collect and concentrate the 30% ethanol eluate to obtain the active fraction for preventing and treating alcoholic liver disease.

2. The extraction method according to claim 1, wherein The powder of the citronella sibiricum in S1 is obtained by crushing the citronella sibiricum and passing it through an 80-mesh sieve.

3. The extraction method according to claim 1, wherein The extraction method in S1 is as follows: soak the powder of Cynanchum cyrtonema and 50% ethanol at a mass ratio of 1:8-12 for 10-14 hours, heat under reflux for 25-35 minutes, filter after cooling, add the same amount of 50% ethanol as that used during the soaking to the filter residue, repeat the heating and reflux once, combine the filtrate, and obtain the extract.

4. The extraction method according to claim 1, wherein The extraction method in S1 is as follows: soak the powder of Cynanchum cyrtonema and 50% ethanol in a mass ratio of 1:10 for 12 hours, heat and reflux for 30 minutes, filter after cooling, add the same amount of 50% ethanol as that used for soaking to the filter residue, repeat heating and reflux once, combine the filtrate to obtain the extract.

5. The extraction method according to claim 1, wherein The macroporous resin in S2 is D101 macroporous resin, and column chromatography is performed using a medium-pressure purification preparative chromatography system.

6. An active fraction of Cynanchum cyrtonema extracted by the extraction method according to any one of claims 1 to 5 for preventing and treating alcoholic liver disease.

7. A pharmaceutical composition comprising the active moiety according to claim 6, and optionally, one or more pharmaceutically acceptable carriers or excipients.

8. Use of the active fraction according to claim 6 in the preparation of a drug for preventing and treating alcoholic liver disease.

9. Use of the active fraction according to claim 6 as the sole active ingredient in the preparation of a drug for preventing and treating alcoholic liver disease.

10. Use of the active fraction according to claim 6 in preparing liver-protecting food or health-care product.

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