Application of beer sterol in preparation of anti-hepatic fibrosis medicine

Abstract

The invention discloses an application of beer sterol in preparation of an anti-hepatic fibrosis drug. The method comprises the following steps: gradually separating a crude extract of an ethyl acetate part of a cordyceps sinensis sporocarp to obtain beer sterol; meanwhile, LX-2 cells are adopted to measure the in-vitro anti-hepatic fibrosis activity of the cordyceps sinensis sporocarp, it is proved that the cordyceps sinensis sporocarp can improve hepatic fibrosis, and a scientific basis is provided for reasonable development and utilization of medicinal resources of the cordyceps sinensis sporocarp.

Description

Technical Field

[0001] The invention belongs to the field of anti-liver fibrosis, and in particular relates to the application of beer sterol in anti-liver fibrosis drugs. Background Art

[0002] Cordyceps sinensis has many parasites and fungal spores attached to its surface. It is a dried complex of the fruiting body and larval corpse of the Cordyceps sinensis (BerK.) Sacc., a fungus of the genus Cordyceps of the family Ergotaceae, parasitizing the larvae of Lepidoptera bats and insects. It is composed of the insect body and the fungal fruiting body growing from the insect head. The insect body is like a silkworm, 3 to 5 cm long and 0.3 to 0.8 cm in diameter; the surface is dark yellow to yellow-brown, with 20 to 30 rings, and the rings near the head are thinner; the head is reddish brown; there are 8 pairs of legs, 4 pairs in the middle are more obvious; the texture is brittle, easy to break, the cross section is slightly flat, and the light yellow white. The fruiting body is slender and cylindrical, 4 to 7 cm long and about 0.3 cm in diameter; the surface is dark brown to brown, with fine longitudinal wrinkles, and the upper part is slightly swollen; the texture is flexible, and the cross section is off-white. The medicinal part is a complex of sclerotium and fruiting body, with a slightly fishy smell, sweet taste and warm nature, and the powder is brown. Cordyceps sinensis contains a variety of pharmacologically active substances, which have a wide range of pharmacological effects such as immunomodulation, anti-oxidation, anti-aging, anti-fatigue, anti-bacterial, anti-tumor, and liver and kidney protection.

[0003] Liver fibrosis is a pathophysiological process that refers to the abnormal proliferation of connective tissue in the liver caused by various pathogenic factors. Any liver damage will undergo a process of liver fibrosis during the process of liver repair and healing. If the damaging factors cannot be removed for a long time, the fibrosis process will continue for a long time and develop into cirrhosis. The pathological manifestations are the formation of fibrous tissue in the liver and the destruction of lobular structure. The clinical manifestations can be very mild, or there may be portal hypertension and liver decompensation.

[0004] Hepatic stellate cells (HSC) play a key role in the process of liver fibrosis. HSC activation and transformation into myofibroblast cells (MFC) and fibroblasts (Fibroblast) are the core links in the occurrence and development of fibrosis. Liver damage is the starting link in the development of liver fibrosis. Hepatocytes and other non-parenchymal liver cells paracrine release some cytokines to activate HSC. Activated HSC proliferate, synthesize extracellular matrix and express some cytokines (autocrine), jointly participating in the regulation process. Metalloproteinases related to matrix degradation and tissue inhibitors of metalloproteinases also change accordingly. Finally, excessive ECM deposition leads to fibrosis.

[0005] The beer sterol was disclosed as compound 5 in the literature Piccialli, V., & Sica, D. (1987). Four New Trihydroxylated Sterols from the Sponge Spongionella gracilis. Journal of Natural Products, 50(5), 915–920. Summary of the Invention

[0006] The object of the present invention is to provide the use of beer sterol in the preparation of anti - liver fibrosis drugs.

[0007] The present invention discovers that beer sterol has the effect of anti - liver fibrosis and has good application potential for improving liver fibrosis. Therefore, the present invention provides the use of beer sterol in the preparation of anti - liver fibrosis drugs;

[0008] The structural formula of the beer sterol is as follows:

[0009]

[0010] The anti - liver fibrosis drug is for treating liver system diseases, such as chronic hepatitis B. Long - term chronic hepatitis B may cause liver fibrosis and even liver cirrhosis.

[0011] The drug may further contain a pharmaceutically acceptable carrier.

[0012] The drug can be made into various dosage forms, such as powder, oral liquid or injection.

[0013] The second object of the present invention is to provide an anti - liver fibrosis drug containing the above - mentioned beer sterol as an active ingredient.

[0014] The present invention also provides a method for preparing beer sterol, which is prepared from the fruiting body of Cordyceps sinensis.

[0015] Preferably, the specific preparation method is as follows:

[0016] The fruiting bodies of Cordyceps sinensis were crushed and successively extracted with ethanol and ethyl acetate. The ethyl acetate extract was concentrated to obtain a crude extract. The crude extract was first dissolved in ethanol and then extracted with petroleum ether. The raffinate was further extracted with ethyl acetate. The ethyl acetate fraction was collected and separated by silica gel column chromatography using gradient elution with petroleum ether:ethyl acetate = 20:1, 10:1, 5:1, 2:1, 1:1 v / v; dichloromethane:methanol = 20:1, 10:1, 5:1, 2:1, 1:1 v / v. The fractions 2 - 6 eluted under the gradient of petroleum ether:ethyl acetate = 10:1 were collected and further separated by thin - layer chromatography using petroleum ether:ethyl acetate = 5:1 v / v as the developing agent to obtain the fraction with Rf = 0.5, which was cerevisterol.

[0017] In the present invention, cerevisterol was obtained by gradually separating the crude extract from the ethyl acetate fraction of the fruiting bodies of Cordyceps sinensis. Meanwhile, the in vitro anti - hepatic fibrosis activity was determined using LX - 2 cells, demonstrating that the fruiting bodies of Cordyceps sinensis can improve hepatic fibrosis and providing a scientific basis for the rational development and utilization of its medicinal resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the 1H - NMR spectrum of cerevisterol;

[0019] Figure 2 is the 13C - NMR spectrum of cerevisterol;

[0020] Figure 3 shows the effect of different concentrations of TGF - β1 on the proliferation of LX - 2 cells;

[0021] Figure 4 shows the morphological changes of LX - 2 cells after TGF - β1 intervention for 72 h. Note: The upper - left figure is normal cells at 10×, the upper - right figure is normal cells at 20×, the lower - left figure is cells after TGF - β1 intervention at 10×, and the lower - right figure is cells after TGF - β1 intervention at 20×;

[0022] Figure 5 shows the effect of different concentrations of cerevisterol on the proliferation of LX - 2 cells;

[0023] Figure 6 shows the effect of different concentrations of Compound Biejia Ruangan Tablets on the proliferation of LX - 2 cells;

[0024] Figure 7 shows the effect of cerevisterol on the proliferation of cells in the hepatic fibrosis model induced by TGF - β1. DETAILED DESCRIPTION OF THE INVENTION

[0025] The following examples are for further illustration of the present invention rather than limitations thereof.

[0026] Example 1:

[0027] I. Experimental methods and results:

[0028] (I) Compound Extraction and Isolation of Cordyceps sinensis Fruiting Bodies

[0029] 1. Source of materials: The Cordyceps sinensis fruiting bodies were provided by the Research Center for Resource Insects and Bioengineering, Institute of Zoology, Guangdong Provincial Hospital of Science.

[0030] 2. Extract the crude substance

[0031] Collect 700 g of Cordyceps sinensis fruiting bodies, cut the fruiting bodies into pieces with scissors, with a length of 1 - 3 mm. Place the cut fruiting bodies in a 5 L conical flask, pour in anhydrous ethanol and soak for 24 h. After extraction three times, remove the ethanol, and soak the remaining fruiting bodies with ethyl acetate for 24 h and extract by rotary evaporation to finally obtain the final crude substance.

[0032] 3. Separate the substances in the petroleum ether fraction, ethyl acetate fraction, and n-butanol fraction

[0033] a. Dissolve the sample: Dissolve the crude extract with 600 ml of 80% ethanol and add it to a separatory funnel.

[0034] b. Extract the petroleum ether fraction: Add 600 ml of petroleum ether (1∶1) to the separatory funnel, shake well, let it stand for layering. The petroleum ether is in the upper layer. Collect the petroleum ether fraction into a 250 ml rotary evaporation flask and repeat three times.

[0035] c. Extract the ethyl acetate fraction: Add the raffinate in b to the separatory funnel, then add 600 ml of ethyl acetate (1∶1 v / v), shake well. Since ethyl acetate and ethanol are miscible, an appropriate amount of water can be added for extraction. Let it stand for layering. The ethyl acetate is in the upper layer. Collect the ethyl acetate fraction into a 250 ml rotary evaporation flask and repeat three times.

[0036] d. Extract the n-butanol fraction: Add the raffinate in c to the separatory funnel, then add 600 ml of n-butanol (1∶1 v / v), shake well, let it stand for layering. The n-butanol is in the upper layer. Collect the n-butanol fraction into a 250 ml rotary evaporation flask and repeat three times.

[0037] e. Rotary evaporate the samples: Rotary evaporate the samples of each fraction with a rotary evaporator to obtain solid samples.

[0038] 4. Thin layer chromatography

[0039] The sample solution obtained by extraction is spotted at one end of a thin-layer plate with a capillary tube, placed in a closed tank, and an appropriate solvent is added as the mobile phase. Due to capillary action, the solvent is drawn up, moves along the plate, and drives each component in the sample forward. This process is called development. Since the properties of each component are different, the moving distances are different. After developing a certain distance, spots of separated components are obtained. Appropriate methods can be used to make each component show its position on the plate. For example, if the component itself has a color, it can be directly observed. Otherwise, methods such as spraying a color-developing reagent or observing fluorescence under an ultraviolet lamp can be used to determine it.

[0040] a. Spotting. Before spotting, first gently draw a horizontal line with a pencil 1 cm from the end of the chromatography plate, and then use a capillary tube to aspirate the sample solution and gently spot it on the horizontal line. If re-spotting is required, be sure to wait until the residual solvent from the previous spotting has evaporated before spotting again to avoid overly large spotting spots. Generally, the diameter of the spot is greater than 2 mm and should not exceed 5 mm. The bottom line is 1 - 2.5 cm from the baseline, the distance between spots is about 1 cm, and the distance between the sample spot and the glass edge is at least 1 cm.

[0041] b. Development. Place the spotted thin-layer plate in a developing tank containing the developing agent. Due to capillary action, the developing solvent slowly advances on the thin-layer plate. After advancing a certain distance, take out the thin-layer plate, and the sample components are separated from each other due to different moving speeds.

[0042] c. Detection of spots. After the developed thin-layer plate is dried, it is commonly irradiated with an ultraviolet lamp or developed with a color-developing agent to detect the spots. For colorless components, the vanillin sulfuric acid color-developing agent should be sprayed evenly and in an appropriate amount. After separation by development, the position of the compound on the thin-layer plate is represented by the retention factor (Rf value). The ratio of the distance from the center of the compound spot to the origin to the distance from the solvent front to the origin is the Rf value of the compound.

[0043] 5. Silica gel column chromatography

[0044] a. Weighing. For 200 - 300 mesh silica gel, weigh 30 - 70 times the sample loading amount.

[0045] b. Stir into a homogeneous slurry. Add petroleum ether with a volume one time that of the dry silica gel and stir well with a glass rod.

[0046] c. Column packing. Add about 1 / 3 volume of petroleum ether, install a reservoir ball, open the piston at the bottom of the column, and pour the homogeneous slurry into the reservoir ball at one time. As it settles, some silica gel will adhere to the reservoir ball, and use petroleum ether to wash it into the column.

[0047] d. Compaction. After settlement is completed, add more petroleum ether and pressurize with an air compressor until the flow rate is constant and the column is compacted. This can greatly improve the resolution and avoid cracking due to column bed shrinkage during column chromatography.

[0048] e. Sample mixing. Dissolve the sample in the ethyl acetate fraction with petroleum ether and stir well with a small amount of dry silica gel until there are no particles.

[0049] f. Sample loading. After sample loading, add some eluent, add a layer of silica gel as a buffer layer, and then stuff a ball of absorbent cotton close to the silica gel surface. Then a large amount of eluent can be added with confidence without damaging the silica gel surface.

[0050] g. Column chromatography and collection. In order to better elute the sample, gradient elution is used. Petroleum ether∶ethyl acetate = 20∶1, 10∶1, 5∶1, 2∶1, 1∶1 v / v; dichloromethane∶methanol = 20∶1, 10∶1, 5∶1, 2∶1, 1∶1 v / v; finally, elute completely with methanol. Collect one fraction every 10 ml during collection.

[0051] h. Combining. Collect fractions 2 - 6 under the gradient of petroleum ether:ethyl acetate = 10∶1.

[0052] g. Detection. Perform thin-layer chromatography on the fractions. The developing solvent is petroleum ether:ethyl acetate = 5:1 v / v. Use vanillin sulfuric acid for color development, irradiate with a portable ultraviolet analyzer, calculate the Rf value, and collect the fractions with Rf = 0.5.

[0053] h. Spectral submission. Concentrate the collected fractions to dryness to obtain Compound 6, and submit it for 1H NMR and 13C NMR detection.

[0054] 6. Structure identification of Compound 6:

[0055] Compound 6: Colorless needle-like crystals. 1 1H NMR(600MHz,Chloroform-d)δ:5.35(dt,J=4.9,2.2Hz,1H),5.29–5.12(m,2H),4.08(tt,J=10.7,4.7Hz,1H),3.62(s,1H),3.49(s,2H),2.18–2.10(m,1H),2.05(ddt,J=15.2,13.1,5.1Hz,2H),1.94(ddd,J=27.5,12.1,6.6Hz,2H),1.84(s,2H),1.82–1.67(m,2H),1.63–1.51(m,10H),1.46(dddd,J=23.5,21.0,10.7,5.3Hz,4H),1.39–1.19(m,4H),1.09(s,2H),1.03(d,J=6.6Hz,2H),0.92(d,J=6.8Hz,2H),0.83(dd,J=9.2,6.8Hz,4H),0.60(s,2H). 1313C NMR (600 MHz, CDCl3) δ: 144.01 (C-8), 135.36 (C-22), 132.17 (C-23), 117.52 (C-7), 76.75 (C-5), 73.66 (C-6), 67.72 (C-3), 55.97 (C-17), 54.74 (C-14), 43.75 (C-13), 43.46 (C-9), 42.80 (C-24), 40.39 (C-20), 39.45 (C-4), 39.20 (C-12), 37.13 (C-10), 33.06 (C-25), 32.95 (C-1), 30.84 (C-2), 27.89 (C-16), 22.87 (C-15), 22.03 (C-11), 21.10 (C-21), 19.93 (C-26), 19.63 (C-27), 18.83 (C-19), 17.58 (C-28), 12.32 (C-18), Figure 1 and Figure 2 ..

[0056] The above spectral data are basically consistent with the reported compound 9 in the literature GAO H, HONG K, ZHANG X, et al. New steryl esters of fatty acids from the mangrove fungus Aspergillus awamori [J]. Helvetica Chimica Acta, 2007, 90(6): 1165 - 1178. Therefore, it was identified as cerevisterol.

[0057] (2) Study on the anti - inflammatory activity of compounds in the fruiting body of Cordyceps sinensis

[0058] 1. Experimental materials: The experimental cell LX - 2 (CL - 0560) was provided by Wuhan Punosai Life Science Co., Ltd., batch number 240607032301.

[0059] 2. Cell culture

[0060] ① Cell resuscitation

[0061] a. Quickly take out the cryopreservation tube containing LX - 2 cells from the liquid nitrogen tank;

[0062] b. Quickly clamp the cryopreservation tube with hemostatic forceps and gently shake it in a 37 °C water bath to dissolve (about 1 min);

[0063] c. Wipe the cryopreservation tube with a 75% alcohol cotton ball at the workbench side, and then take it into the central operation area of the workbench;

[0064] d. Aspirate the liquid in the cryotube into a 15 mL centrifuge tube, then place it in a centrifuge for equilibration centrifugation at a speed of 1200 rpm for 3 min;

[0065] e. Aspirate and discard the supernatant, add 1 mL of complete DMEM culture medium, and pipette to form a cell suspension;

[0066] f. Transfer the cell suspension into a 25 mL sterile culture flask, supplement with complete DMEM culture medium to 4 mL, not exceeding 5 mL at most, and place it in an incubator for culture. The culture conditions are 95% air, 5% CO2, and a temperature of 37 °C.

[0067] ② Cell passage

[0068] a. Aspirate the original culture medium;

[0069] b. Add about 2 mL of PBS, gently shake the culture flask to rinse the cells, and aspirate and discard the PBS;

[0070] c. Add about 1 mL of 0.25% trypsin solution (containing EDTA), gently shake the culture flask to soak all the cells;

[0071] d. Place it in the incubator for digestion. When the cells in the middle of the cell mass are clearly rounded and there are gaps under the microscope, the digestion can be terminated. Do not tap the culture flask throughout the process;

[0072] e. Add 3 mL of serum-containing medium to terminate the digestion, pipette the cells to detach them from the wall, and pipette repeatedly in the liquid to make the cells into a suspension of single cells as much as possible;

[0073] f. Collect the cell suspension and centrifuge at 1200 rpm / min for 3 min. After centrifugation, aspirate and discard the supernatant;

[0074] g. Add fresh culture medium, pipette a few times to mix the cells evenly, inoculate them into a new culture flask at a ratio of 1:2 or 1:3, supplement the culture medium, and place it in an incubator at a temperature of 37 °C and 5% CO2 for culture.

[0075] ③ Cell cryopreservation

[0076] a. Prepare the cryopreservation solution. The ratio of the cryopreservation solution is: 55% basal medium + 40% FBS + 5% DMSO;

[0077] b. Take cells with good morphology and in the logarithmic growth phase, digest and centrifuge them (centrifuge at 1200 rpm for 3 min);

[0078] c. Discard the supernatant, add 1 mL of the prepared cryopreservation solution, and pipette to mix evenly;

[0079] d. Carefully open the cap of the cryotube with forceps, dispense 1 mL of the cell-containing cryopreservation solution into each tube, tighten the tube cap, and make good marks.

[0080] e. After the well - packed cryotubes are transferred into the programmable freezer box, directly place them at -80°C overnight.

[0081] f. If there is no programmable freezer box, cool down in the following order successively: room temperature → 4°C for 20 min → -20°C for 30 min → -80°C overnight. Note that during the transfer process, keep cold to avoid generating temperature differences or melting of the cryotubes.

[0082] g. Quickly transfer the cryotubes taken out from the -80°C refrigerator to liquid nitrogen for long - term storage.

[0083] 3. In vitro experiments

[0084] ① Preparation of complete medium

[0085] Add 56.2 mL of fetal bovine serum (FBS) and 5.62 mL of double - antibody (penicillin, streptomycin) to 500 mL of DMEM medium, gently shake and mix evenly to prepare the complete medium. Aliquot and place in the 4°C refrigerator for storage and standby.

[0086] ② Preparation of PBS buffer

[0087] Dissolve potassium dihydrogen phosphate (0.24 g), disodium hydrogen phosphate (1.44 g), potassium chloride (0.2 g), and sodium chloride (8 g) of the PBS standard in 1000 mL of double - distilled water, adjust the pH value to 7.35, aliquot with 4 250 - mL glass bottles, sterilize in an autoclave, and store at 4°C for standby.

[0088] ③ Establishing a model by TGF - β1 - induced LX - 2 cells

[0089] Seed LX - 2 cells at a cell concentration of 1×10 4 cells / mL in a 96 - well plate. After culturing for 4 h, the cells adhere to the wall. Discard the supernatant, wash with PBS, and re - add the complete medium containing TGF - β1 (5, 10, 20, 40 ng / mL) for culturing for 24, 48, 72, 96, 120, 144, 168 h. Use the CCK8 method to measure the absorbance, repeat 3 times, and take the average value to calculate the proliferation rate of LX - 2 cells at each concentration after TGF - β1 induction, and screen the optimal concentration of TGF - β1 required for modeling with a significantly increased proliferation rate.

[0090] Adjust the cell concentration to 1×10 5cells / mL were inoculated into culture flasks and divided into two groups: (1) normal control group; (2) model group (treated with 10 ng / mL TGF-β1). They were cultured in an incubator. After normal cells were resuscitated and passaged, cell attachment and growth were observed under a high-power inverted microscope. The cells were short fusiform, arranged relatively closely, of uniform size, with abundant cytoplasm, clear cell nuclei, and oval in shape. The time for establishing the in vitro model was determined by observing the morphological changes of the cells under the microscope every day. The time when LX-2 cells changed from small fusiform to spindle shape was the time for establishing the model.

[0091] ④ Screening of the induction concentration of beer sterol on LX-2 cells

[0092] LX-2 cells were inoculated into 96-well plates at a cell concentration of 1×10 5 cells / mL. After culturing for 4 h, the cells attached to the wall. The supernatant was discarded, and the cells were rinsed with PBS. Then, complete medium containing beer sterol (5, 10, 25, 50, 100 μM) was added and cultured for 24 h. The absorbance was measured by the CCK8 method, repeated 3 times, and the average value was taken to calculate the proliferation rate of LX-2 cells at each concentration after the influence of beer sterol, and the optimal concentration with the lowest influence on the cells was screened out.

[0093] ⑤ Screening of the induction concentration of Compound Biejiaruangan Tablets (positive group) on LX-2 cells

[0094] LX-2 cells were inoculated into 96-well plates at a cell concentration of 1×10 5 cells / mL. After culturing for 4 h, the cells attached to the wall. The supernatant was discarded, and the cells were rinsed with PBS. Then, complete medium containing the positive group (5, 10, 25, 75, 100 ng / mL) was added and cultured for 24 h. The absorbance was measured by the CCK8 method, repeated 3 times, and the average value was taken to calculate the proliferation rate of the cells after the influence of Compound Biejiaruangan Tablets, and the optimal concentration with the lowest proliferation rate compared with the control group was screened out.

[0095] ⑥ Effect of beer sterol on the proliferation of liver fibrosis model cells induced by TGF-β1

[0096] Cells were seeded at 2.5×10 4Cells were inoculated in 96-well plates at a concentration of cells / mL, with 200 μL of complete DMEM culture medium in each well. After 24 h, the culture medium was aspirated. The experiment was divided into a normal control group, a TGF-β1-induced model group (10 ng / mL), a beer sterol group (10 μM), and a positive group (75 ng / mL). The total volume in each well was 200 μL. After culturing in an incubator for 120 h, the culture medium of the model group was completely aspirated, and compounds at low, medium, and high concentrations (100, 200, 300 μg / mL) were added, followed by culturing for 48 h. The absorbance was measured using the CCK8 method. Inhibition rate (%) = [1 - (OD490 compound group - OD490 blank group)] / [OD490 model group - OD490 normal control group] × 100%.

[0097] 4. Data processing: The experimental data were processed using GraphPad Prism 8.0.2 263 software.

[0098] 5. Result analysis:

[0099] ① Screening of the optimal concentration and time of TGF-β1-induced LX-2 cells

[0100] As Figure 3 shown, compared with the control group, TGF-β1 at 5 ng / mL had no obvious effect on cell proliferation, while 10, 20, and 40 ng / mL all had obvious proliferation effects, and the proliferation rate was the highest at 10 ng / mL at 72 h. Therefore, 10 ng / mL TGF-β1 was finally selected to act on LX-2 cells.

[0101] As Figure 4 shown, after normal cells were resuscitated and passaged, they were observed under a high-power inverted microscope. The cells adhered to the wall and grew, arranged tightly, with uniform size, rich cytoplasm, clear cell nuclei, and were round. After 72 h of induction with 10 ng / mL TGF-β1, it was obvious that the cells gradually changed from the original round shape to a long spindle shape, as shown in the figure. Therefore, 72 h was determined as the optimal intervention time for constructing the LX-2 liver fibrosis cell model.

[0102] ② Inhibitory effect of beer sterol on the proliferation of LX-2 cells

[0103] As Figure 5 shown, compared with the control group, beer sterol had an inhibitory effect on cell growth at concentrations of 5, 25, and 50 μM. At 100 μM, the inhibitory effect was the most significant, while at 10 μM concentration, there was no significant effect on the cells. Therefore, the concentration of beer sterol at 10 μM was the optimal concentration for the cells.

[0104] ③ Inhibitory effect of Compound Biejia Ruangan Tablets (positive group) on the proliferation of LX-2 cells

[0105] As Figure 6As shown, compared with the control group, compound turtle shell and malan head soft liver tablets promoted cell growth at concentrations of 5, 10, and 25 ng / mL; at 100 ng / mL, compound turtle shell and malan head soft liver tablets inhibited cells; when the concentration of compound turtle shell and malan head soft liver tablets was 75 ng / mL, there was no significant effect on cells. Therefore, the optimal concentration of compound turtle shell and malan head soft liver tablets was 75 ng / mL.

[0106] ④ Effect of beer sterol on the proliferation of cells in a liver fibrosis model induced by TGF-β1

[0107] As Figure 7 shown, at 24 and 48 h, the cell growth rate of the positive group was higher than that of the other three groups, and there were no significant differences among the normal group, the model group, and the beer sterol group; at 72 h, there was no significant difference in the effects between the normal group and the beer sterol group and the positive group; at 96 h, there was no significant difference in the results between the beer sterol group and the positive group and the normal group. At 72 h and 96 h, there were significant differences between the normal group, the beer sterol group, the positive group and the model group. This indicates that beer sterol has a good anti-liver fibrosis effect.

Claims

1. Application of beer sterol in the preparation of anti-liver fibrosis drugs. The structural formula of the beer sterol is shown below:

2. The use according to claim 1, characterized in that: The anti-liver fibrosis drug is a drug for treating liver system diseases.

3. The use according to claim 1, characterized in that: The liver system disease is chronic hepatitis B, and chronic hepatitis B can cause liver fibrosis and cirrhosis.

4. The use according to claim 1, characterized in that: The drug may further contain a pharmaceutically acceptable carrier.

5. The use according to claim 1, characterized in that: The medicine is prepared into various dosage forms, such as powder, oral solution or injection.

6. An anti-liver fibrosis drug, characterized in that: Contains beer sterol as an active ingredient; The structural formula of the beer sterol is shown below:

7. A method for preparing beer sterol, characterized in that: It is prepared from the fruiting body of Cordyceps sinensis.

8. The method according to claim 7, characterized in that The specific preparation method is: The Cordyceps sinensis fruiting body was crushed and extracted with ethanol and ethyl acetate successively. The ethyl acetate extract was concentrated to obtain a crude extract. The crude extract was first dissolved with ethanol and then extracted with petroleum ether. The raffinate was extracted with ethyl acetate again. The ethyl acetate fraction was collected and subjected to silica gel column chromatography with gradient elution, petroleum ether: ethyl acetate = 20:1, 10:1, 5:1, 2:1, 1:1 v / v; Dichloromethane: methanol = 20: 1, 10: 1, 5: 1, 2: 1, 1: 1 v / v, the collected fractions 2-6 under the gradient of petroleum ether: ethyl acetate = 10: 1, and then subjected to thin layer chromatography, using petroleum ether: ethyl acetate = 5: 1 v / v as the developing solvent, to obtain the fraction with Rf = 0.5, which is beer sterol.

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