Application of two phenylethanoid glycoside compounds in ponsamie in anti-hepatitis B virus and liver protection

Abstract

The invention relates to the technical field of chemistry and medicine, in particular to application of two phenylethanoid glycoside compounds in Pensamie in anti-hepatitis B virus and liver protection, including application of forsythiaside F and forsythiaside B in preparation of anti-hepatitis B virus drugs and / or application of forsythiaside F and forsythiaside B in preparation of liver protection drugs. According to the invention, two compounds of forsythiaside F and forsythiaside B are obtained from 75% ethanol extract of separated and purified Tibesimie, the properties of the two compounds are researched, and the two compounds are found to be capable of reducing HBsAg and HBeAg and have a certain liver protection effect, so that data are provided for development and utilization of Tibesimie and research and development of anti-hepatitis B virus drugs, and the research and development of the Tibesimie and the research and development of the anti-hepatitis B virus drugs are facilitated. And a new way is provided for HBV prevention and treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of chemistry and medicine, and in particular to the application of two phenylethanol glycoside compounds in penthiabendazole in resisting hepatitis B virus and protecting the liver. Background Art

[0002] Hepatitis B replication requires reverse transcription catalyzed by polymerase protein. HBV polymerase is an enzymatically active protein that plays a key role in the viral replication process. It is crucial for the treatment of hepatitis B and is therefore an important target for the development of new hepatitis B drugs.

[0003] The envelope of hepatitis B virus (HBV) is composed of small (S-HBsAg), medium (M-HBsAg) and large (L-HBsAg) hepatitis B surface antigen (HBsAg), which surrounds the hepatitis B virus polymerase (Pol), nucleocapsid and core protein (HBc). The HBV genomic DNA contains a positive strand and a negative strand, and its genome has four open reading frames: C, X, P, and S. The three surface antigens come from S, the polymerase comes from P, and the E antigen (HBeAg) comes from the C open reading frame. During the immune tolerance period of HBV infection, the levels of HBsAg, HBeAg and HBV in the serum are elevated, and the levels of HBsAg, HBeAg and HBV decrease during the immune clearance period. The current treatment goal is to achieve functional cure, that is, HBsAg and DNA cannot be detected in the serum. Therefore, the quantitative hepatitis B surface antigen has been widely used as a marker for HBV infection, and HBsAg, HBeAg and HBV DNA are important indicators for drug screening.

[0004] Liver disease is a serious public problem worldwide, which can cause high morbidity and mortality. Studies have found that maintaining a low concentration of reactive oxygen in the body is the key to cell survival. When the human body is exposed to some chemicals and toxic substances, or in a diseased state, the body will produce excessive reactive oxygen through various pathways, which exceeds the body's cell antioxidant capacity, and the same is true for liver cells. In addition, excessive reactive oxygen can cause inflammation, and severe inflammation can cause cell damage or even death, which is one of the causes of liver damage and liver disease.

[0005] Natural products are characterized by high efficiency, low toxicity, and rich variety, and are currently a hot topic in the research of new anti-HBV drugs. In recent years, more and more domestic and foreign studies have found that natural products contain anti-HBV active ingredients. Pennellia ruellioides, also known as earring grass, sawtooth grass, menstrual regulation grass, duckbill, etc., is the whole herb of Lindernia ruellioides (Colsm.) Pennell, a plant of the Scrophulariaceae family. It is mainly produced in Guangxi, Taiwan, Hunan, Fujian and other regions, and is a commonly used folk medicine. It tastes sweet, light, and has a flat nature. It has the effects of detoxification, regulating qi, activating blood circulation, and reducing swelling. It is often used to treat snake and dog bites, mastitis, scrofula, traumatic injuries, irregular menstruation and other diseases. The research team found in the early stage that the substances extracted from Pennellia ruellioides may have anti-HBV or liver protection effects, but there are currently no relevant reports in this regard. Summary of the invention

[0006] The object of the present invention is to provide the use of two phenylethanoid glycosides in penthion in anti-hepatitis B virus and liver protection in response to the above-mentioned problems.

[0007] In order to achieve the above-mentioned purpose, the present invention adopts the following technical scheme: The present invention provides the use of two phenylethanol glycoside compounds in penjumia ulmoides in anti-hepatitis B virus and liver protection, wherein the two phenylethanol glycoside compounds are forsythiaside F and forsythiaside B, respectively, and the two phenylethanol glycoside compounds are obtained by separating and purifying the 75% ethanol extract of penjumia ulmoides. The specific extraction process can be referred to in the literature: Zhao Tongshiyao, Liang Chengqin, Liang Yancui, et al. Isolation and identification of chemical components of dryland grass. Chinese Medicinal Materials, 2022, 45(9): 20113-2117.

[0008] Furthermore, the present invention provides the use of the forsythiaside F and forsythiaside B in the preparation of anti-hepatitis B virus drugs.

[0009] Preferably, the anti-HBV drug includes a drug that inhibits hepatitis B surface antigen.

[0010] Preferably, the anti-HBV drug includes a drug that inhibits hepatitis B E antigen.

[0011] Furthermore, the present invention also provides the use of the forsythiaside F and forsythiaside B in the preparation of liver protection drugs.

[0012] Preferably, the liver protection drug includes a drug that improves the antioxidant capacity of cells.

[0013] In summary, due to the adoption of the above technical solution, the present invention has the following beneficial effects:

[0014] 1. The present invention uses enzyme-linked immunosorbent assay (ELISA) to detect the inhibitory effects of forsythiaside F and forsythiaside B on hepatitis B surface antigen and E antigen. It is found that both forsythiaside F and forsythiaside B have the effect of inhibiting hepatitis B surface antigen and E antigen. Among them, the IC 50 The IC values ​​of hepatitis B virus E antigen are 54.81mg / L and 128.10mg / L respectively. 50 They are 135.40 mg / L and 48.57 mg / L respectively.

[0015] 2. The present invention predicts the affinity and binding mode of two anti-HBV active compounds to the HBV polymerase protein through molecular docking. The results show that forsythiaside F and forsythiaside B interact with the amino acid residues of HBV polymerase through hydrogen bonds. The binding energy of forsythiaside F with HBV polymerase is -8.5kcal / mol, and the binding energy of forsythiaside B with HBV polymerase is -8.3kcal / mol, indicating that the ligand has a good affinity with the receptor, especially forsythiaside B interacts with 7 amino acid residues, and the effect is stable, indicating that the two compounds, forsythiaside F and forsythiaside B, can play an anti-HBV effect through interaction with HBV polymerase.

[0016] 3. The present invention uses the tetramethylthiazolyl blue method (MTT) to detect the effect of the compounds on the vitality of normal human liver cells (HL-7702). The experimental results show that when the concentration of the compounds forsythiaside F and forsythiaside B is 6.25 mg / L, the survival rate of HL-7702 cells is close to 100%, and when the concentration is 100 mg / L, the survival rate of HL-7702 cells is above 70%, indicating that forsythiaside F and forsythiaside B have less toxicity to normal human liver cells.

[0017] 4. The present invention uses normal human liver cells (HL-7702) as carriers, and studies the protective effects of two phenylethanol glycoside compounds on liver cells by constructing a hydrogen peroxide (H2O2) liver injury model. The results show that after hydrogen peroxide injury, the survival rate of HL-7702 cells in the model group was 50.72%, while after treatment with the Forsythiaside F drug group, the survival rate of HL-7702 cells increased to 63.08%; after treatment with the Forsythiaside B drug group, the survival rate of HL-7702 cells increased to 65.43%, indicating that Forsythiaside F and Forsythiaside B can improve the antioxidant capacity of cells and have a certain protective effect on liver cells in oxidative damage.

[0018] 5. The present invention obtains two compounds, forsythiaside F and forsythiaside B, from the 75% ethanol extract of separated and purified Penzimidium, and studies their properties. It is found that the two compounds can reduce HBsAg and HBeAg and have a certain liver protective effect, which provides information for the development and utilization of Penzimidium and the research and development of anti-hepatitis B virus drugs, and provides a new approach for the prevention and treatment of HBV. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a graph showing the inhibitory effects of two monomer compounds, forsythiaside F and forsythiaside B, in Penicillium truncatum on the growth of Hep G.2.15 cells, where (A) is forsythiaside F; (B) is forsythiaside B; 3TC is the positive control; compared with the negative group, *P<0.05, **P<0.0.

[0020] Figure 2 This is a graph showing the inhibitory effect of the compound forsythiaside F on HBsAg in the supernatant of Hep G2.2.15 cells, wherein 3TC is the positive control; compared with the negative group, *P<0.05, **P<0.01.

[0021] Figure 3 This is a graph showing the inhibitory effect of forsythiaside B on HBsAg in the supernatant of Hep G2.2.15 cells, wherein 3TC is the positive control; compared with the negative group, *P<0.05, **P<0.01.

[0022] Figure 4 This is a graph showing the inhibitory effect of the compound forsythiaside F on HBeAg in the supernatant of Hep G2.2.15 cells, wherein 3TC is the positive control; compared with the negative group, *P<0.05, **P<0.01.

[0023] Figure 5 This is a graph showing the inhibitory effect of forsythiaside B on HBeAg in the supernatant of Hep G2.2.15 cells, where 3TC is the positive control; compared with the negative group, *P<0.05, **P<0.01.

[0024] Figure 6 The molecular docking diagrams of the compounds forsythiaside F and forsythiaside B with hepatitis B virus polymerase, wherein in the 3D structure of the protein-ligand complex, the compounds are colored with green sticks, and the docked amino acid residues are colored with pink sticks. (A) is the docking diagram of forsythiaside F with hepatitis B virus polymerase; (B) is the docking diagram of forsythiaside B with hepatitis B virus polymerase.

[0025] Figure 7 This is a graph showing the inhibitory effects of two monomer compounds, forsythiaside F and forsythiaside B, in Pensambar formosana on the growth of HL-7702 cells. DETAILED DESCRIPTION

[0026] In order to express the present invention more clearly, the present invention is further described below through specific examples.

[0027] The two phenylethanol glycoside compounds of the present invention are forsythiaside F and forsythiaside B respectively, and the two phenylethanol glycoside compounds are obtained by separating and purifying the 75% ethanol extract of Penicilla ovata.

[0028] Furthermore, the forsythiaside F and forsythiaside B can be used to prepare anti-hepatitis B virus drugs; preferably, the anti-hepatitis B virus drugs include drugs that inhibit hepatitis B surface antigen and / or drugs that inhibit hepatitis B E antigen.

[0029] In addition, the forsythiaside F and forsythiaside B can also be used to prepare liver protection drugs; preferably, the liver protection drugs include drugs that improve the antioxidant capacity of cells.

[0030] In this embodiment, the above application is further described through a plurality of specific examples.

[0031] The materials used in the following specific embodiments are as follows:

[0032] 1. Cell lines

[0033] Hep G2.2.15 cell line is a human liver cancer cell stably transfected with HBV virus, which can secrete HBV antigen and complete HBV particles. It was donated by Su Heling, a teacher at Guilin Medical College. It is preserved and subcultured in our laboratory. HL-7702 cell line is a normal human liver cell purchased from the cell bank of the Chinese Academy of Sciences. It is preserved and subcultured in our laboratory.

[0034] 2. Experimental reagents and consumables

[0035] The main experimental reagents and consumables are shown in Table 1:

[0036] Table 1 Experimental reagent consumables and manufacturers

[0037]

[0038] 3. Experimental instruments

[0039] The main instruments are shown in Table 2:

[0040] Table 2.2Instruments and manufacturers

[0041]

[0042] Example 1: MTT assay to determine the toxicity of compounds to Hep G2.2.15 cells

[0043] In this example, the MTT method was used to detect the cell growth inhibitory effect of forsythiaside F and forsythiaside B on Hep G2.2.15 cells. The specific steps are as follows:

[0044] Hep G2.2.15 cells in the logarithmic growth phase with good vitality were used for seeding. According to the amount of 200 μL per well, 2.3×10 4 / mL Hep G2.2.15 cell suspension. Take out the 96-well plate, seed the plate with 7 concentrations of each drug and 3 replicates for each concentration, and set the wells without drug addition as negative wells and blank wells without cells. The outermost circle of the well plate is not seeded to avoid edge effects. After 24 hours, discard the supernatant in the well, and forsythiaside B, forsythiaside F and lamivudine are administered at 7 concentrations of 1.56mg / L, 3.13mg / L, 6.25mg / L, 12.5mg / L, 25mg / L, 50mg / L, and 100mg / L, respectively. Each time the drug is added, culture for 72 hours, and the drug is administered three times in succession, that is, 9 days. On the 9th day of drug action, the supernatant in the well is aspirated, and 20μL of the prepared 5mg / mL MTT solution and 180μL of culture medium are added to each well. Incubate in the dark for 4 hours, gently aspirate the supernatant, and do not aspirate to the bottom crystals. Then add 150 μL of DMSO to each well and shake evenly for 10 minutes. After all the crystals in the wells are completely dissolved, use an enzyme-labeled instrument to detect the absorbance of each well at 490 nm. Repeat the experiment three times. Record the results and use the formula to calculate the inhibition rate and TC of each drug concentration. 50 , and the inhibitory effects of each drug on Hep G2.2.15 cells were obtained.

[0045] Formula 1:

[0046] Test results such as Figure 1 As shown by Figure 1 It can be seen that the compounds forsythiaside F and forsythiaside B have less cytotoxicity to HepG2.2.15 cells. At a concentration of 100 mg / L on the ninth day, the survival rate was still above 77%. The half-inhibitory concentrations of the test substances on HepG2.2.15 cells are: Compound Forsythiaside F: TC 50 =1808.19 mg / L, compound forsythiaside B: TC 50 =1675.45mg / L.

[0047] Example 2: Cell supernatant treatment

[0048] The method of Example 1 was used to seed the plate. After 24 hours, the culture medium in the wells was discarded, and forsythiaside B, forsythiaside F and lamivudine were administered at 5 concentrations of 6.25 mg / L, 12.5 mg / L, 25 mg / L, 50 mg / L and 100 mg / L, respectively. The drug was administered once every 3 days, for a total of 3 times. The supernatant in the wells was collected on the 3rd, 6th and 9th days, respectively, and stored at -20°C for later use.

[0049] Example 3: ELISA method to determine the effects of forsythiaside F and forsythiaside B on the secretion of HBsAg and HBeAg in cell supernatants

[0050] 1. Detection of HBsAg in cell supernatant. The specific detection steps are as follows:

[0051] Before testing, first thaw the supernatant stored at -20°C in Example 2 at 4°C, and then equilibrate the supernatant sample and the hepatitis B surface antigen detection reagent at room temperature for 30 minutes. The specific process can be directly referred to the operation of this kit. Take 25× washing solution and dilute it 25 times according to the dosage. Prepare the washing solution with working concentration, mix well and set aside. Take out the microporous reaction plate, add 75μL of the sample to be tested and the positive and negative control to each well, and reserve 1 well as a blank control. Seal the reaction plate with sealing paper and incubate at 37°C for 1h. Take out the reaction plate, remove the sealing sheet, add 50μL of enzyme conjugate to each well, shake gently for 10s, and then continue to incubate at 37°C for 30min. Wash the plate with the prepared washing solution, discard the liquid in the well, fill each reaction well with washing solution and let it stand for a while, discard it, so that there is no residue in each well. Repeat this step 5 times, and finally pat dry on absorbent paper. Add 50 μL of colorimetric reagent A and colorimetric reagent B to each well, shake and mix, and then incubate at 37°C for 30 minutes. Add 50 μL of stop solution to each well, shake for a few seconds, and use an enzyme reader to detect the absorbance of each well at 450 nm. Finally, calculate the HBsAg and HBeAg inhibition rate and IC of each drug. 50 .

[0052] Formula 2:

[0053] Compounds forsythiaside F and forsythiaside B were used to act on Hep G2.2.15 cells. After the third, sixth, and ninth days, the cell supernatants of the drug-treated wells at 6.25 mg / L, 12.5 mg / L, 25 mg / L, 50 mg / L, and 100 mg / L were collected, respectively, and the expression level of HBsAg in the culture medium was detected. The detailed results are shown in Figure 2 and Figure 3 .

[0054] The results showed that both compounds have the effect of inhibiting HBsAg secretion, which is manifested as follows:

[0055] On the third day, the inhibition rates of various concentrations of forsythiaside F on HBsAg were 11.02%, 27.48%, 29.37%, 31.18%, and 34.11%, respectively; on the sixth day, the inhibition rates of various concentrations of forsythiaside F on HBsAg were 13.61%, 29.79%, 40.48%, 44.42%, and 50.63%, respectively; on the ninth day, the inhibition rates of various concentrations of forsythiaside F on HBsAg were 15.80%, 28.08%, 44.92%, 47.31%, and 56.74%, respectively. The IC value of HBsAg of forsythiaside F was calculated. 50 is 54.81 mg / L, so forsythiaside F TI HBsAg =TC 50 / IC 50 =32.99, TI>2, indicating that the drug Forsythiaside F has the advantages of high efficiency and low toxicity;

[0056] On the third day, the inhibition rates of various concentrations of forsythiaside B on HBsAg were 8.96%, 15.62%, 19.08%, 20.74%, and 28.68%, respectively; on the sixth day, the inhibition rates of various concentrations of forsythiaside B on HBsAg were 10.05%, 22.37%, 33.47%, 34.29%, and 37.79%, respectively; on the ninth day, the inhibition rates of various concentrations of forsythiaside B on HBsAg were 25.55%, 31.60%, 35.35%, 43.49%, and 46.96%, respectively. The IC value of HBsAg of forsythiaside B was calculated. 50 is 128.10 mg / L, so forsythiaside B TI HBsAg =TC 50 / IC 50 =13.08, TI>2, indicating that the drug Forsythiaside B has the advantages of high efficiency and low toxicity.

[0057] 2. Detection of HBeAg in cell supernatant

[0058] The specific detection steps are the same as those of "Detection of HBsAg in cell supernatant", except that the hepatitis B surface antigen detection reagent is changed to the hepatitis B e antigen detection reagent. The specific process can be directly referred to the kit operation of this reagent.

[0059] Compounds forsythiaside F and forsythiaside B were used to act on Hep G2.2.15 cells. After 3, 6, and 9 days, the supernatants of cells in the 6.25 mg / L, 12.5 mg / L, 25 mg / L, 50 mg / L, and 100 mg / L drug-treated wells were collected and the expression level of HBeAg in the culture medium was detected using an ELISA kit. The detailed results are shown in Figure 4 and Figure 5 .

[0060] The experimental results show that both compounds have the effect of inhibiting HBeAg secretion, which is specifically manifested as follows:

[0061] On the third day, the inhibition rates of various concentrations of forsythiaside F on HBeAg were 2.05%, 13.14%, 17.34%, 21.59%, and 36.43%, respectively; on the sixth day, the inhibition rates of various concentrations of forsythiaside F on HBeAg were 13.00%, 16.39%, 21.23%, 22.46%, and 41.50%, respectively; on the ninth day, the inhibition rates of various concentrations of forsythiaside F on HBeAg were 13.91%, 21.92%, 29.30%, 36.55%, and 44.87%, respectively. The IC value of forsythiaside F on HBeAg was calculated. 50 135.40mg / L, forsythiaside F TI HBeAg =TC 50 / IC 50 =13.35, TI>2, indicating that the drug Forsythiaside F has the advantages of high efficiency and low toxicity;

[0062] On the third day, the inhibition rates of various concentrations of forsythiaside B on HBeAg were 15.40%, 21.38%, 26.72%, 29.68%, and 32.96%, respectively; on the sixth day, the inhibition rates of various concentrations of forsythiaside B on HBeAg were 17.25%, 22.10%, 35.44%, 36.51%, and 39.42%, respectively; on the ninth day, the inhibition rates of various concentrations of forsythiaside B on HBeAg were 22.77%, 29.28%, 39.03%, 54.62%, and 58.47%, respectively. The IC value of forsythiaside B on HBeAg was calculated. 50 48.57mg / L, forsythiaside B TI HBeAg =TC 50 / IC 50 =34.50, TI>2, indicating that the drug Forsythiaside B has the advantages of high efficiency and low toxicity.

[0063] Through the above experiments, it can be seen that the compounds forsythiaside F and forsythiaside B have certain inhibitory effects on HBsAg and HBeAg, and the inhibition rate increases with the concentration, and the longer the time, the better the inhibition effect, indicating that there is a dose-effect and time-effect relationship between these drugs. Among them, the inhibition rate of HBsAg of forsythiaside F at 100 mg / L on the ninth day was 56.74%, and the inhibition rate of HBeAg was 44.87%; the inhibition rate of HBsAg of forsythiaside B at 100 mg / L on the ninth day was 46.96%, and the inhibition rate of HBeAg was 58.47%; Forsythiaside F and forsythiaside B are two caffeoylphenylethanol glycoside compounds with extremely similar structures. At 100 mg / L, the inhibition rate of these two compounds on HBV surface antigen and E antigen is about 40% to 60%, and in the toxicity experiment, the effect on growth is not obvious with the increase of dose, so these two compounds are worthy of further study.

[0064] In summary, in terms of inhibiting the secretion of hepatitis B surface antigen, the inhibitory effects of forsythiaside F and forsythiaside B are close to or slightly lower than the positive drug lamivudine; in terms of inhibiting the secretion of hepatitis B E antigen, the inhibitory effects of forsythiaside F and forsythiaside B are better than the positive drug lamivudine.

[0065] Example 4: Molecular docking analysis of compounds forsythiaside F and forsythiaside B with hepatitis B virus RNA polymerase

[0066] In order to further clarify the mechanism of action of active compounds and proteins at the molecular level, forsythiaside F and forsythiaside B were molecularly docked with hepatitis B virus polymerase, respectively. The specific operation was as follows: HBV RNA polymerase protein (PDB ID: 2hn7) was found in the PDB database and downloaded as a PDB file. The 3D structure of the active compound was drawn using Chem3D software. Active pockets were generated based on ligands and proteins, and smina was used to perform molecular docking of four active compounds and HBV RNA polymerase protein. Finally, the best conformation was selected and mapped using Pymol software. The results are shown in the figure. Figure 6 shown.

[0067] All four compounds can be docked in the substrate binding cavity of HBV polymerase, among which compound forsythiaside F binds to three amino acid residues (PRO-15, GLU-89, ARG-75) of HBV RNA polymerase through hydrogen bonds, and the best conformational binding energy is -8.5 kcal / mol. Compound forsythiaside B binds to seven amino acid residues (SER-88, GLY-91, ARG-82, GLU-89, LEU-78, ARG-75, GLU-19) of HBV polymerase through hydrogen bonds, and the best conformational binding energy is -8.3 kcal / mol.

[0068] Hepatitis B replication requires polymerase protein to catalyze reverse transcription. HBV polymerase is an enzyme active protein that plays a key role in the viral replication process. It is crucial for the treatment of hepatitis B and is therefore an important target for the development of new drugs for hepatitis B. In this example, molecular docking was used to predict the affinity and binding mode of two anti-hepatitis B active compounds to the hepatitis B polymerase protein, showing that forsythiaside F and forsythiaside B interact with the amino acid residues of HBV polymerase through hydrogen bonds, and the binding energies of forsythiaside F and forsythiaside B to hepatitis B virus polymerase are -8.5 and -8.3 kcal / mol, respectively, indicating that the ligand has a good affinity with the receptor, especially forsythiaside B interacts with 7 amino acid residues, and the action is stable, which shows that the two compounds separated and purified from pentaerythrocytes can play an anti-hepatitis B virus role by acting on HBV polymerase.

[0069] Example 5: MTT assay to determine the toxicity of the compound to normal hepatocytes HL-7702 cells

[0070] The compounds forsythiaside F and forsythiaside B were respectively applied to normal liver cells for 72 hours, and the cell survival rate after the action of each drug was detected. The specific steps are as follows:

[0071] Cells in logarithmic growth phase were collected and 3×10 4 The cells were seeded at a density of 100 μL / mL in a 96-well cell culture plate and cultured overnight. The drugs were added after the cells adhered to the wall. There were 3 replicates for each concentration, and a negative control group was set up. 200 μL of drug-containing culture medium was added to each well and placed in a CO2 incubator. MTT detection was performed after 72 hours. 20 μL of 5 mg / mL MTT was added to each well. After incubation for 4 hours, the supernatant of each well was discarded, 150 μL DMSO was added, and the plate was shaken for 10 minutes. After the crystals in the well plate were completely dissolved, the OD value was measured at 490 nm. The experiment was repeated 3 times, and the effect of the drug on the survival rate of the cells was used to reflect the toxic effect of the drug on normal liver cells. The survival rate calculation formula is as follows. The results are as follows Figure 7 shown.

[0072] Formula 3:

[0073] The experimental results showed that when the concentration of the compounds Forsythiaside F and Forsythiaside B was 6.25 mg / L, the cell survival rate was close to 100%, and at the concentration of 100 mg / L, the cell survival rate was above 70%, indicating that the compounds Forsythiaside F and Forsythiaside B had little effect on the growth of normal human liver cells.

[0074] Example 6: Protective effect of compounds on H2O2-induced liver cell damage

[0075] In order to study the protective effect of compounds forsythiaside F and forsythiaside B on liver cell damage, this example uses normal human liver cells (HL-7702) as carriers and constructs a reactive oxygen liver damage model through H2O2 damage. The details are as follows:

[0076] Cells were seeded in 96-well cell culture plates, and drug groups, model groups, and normal control groups were set up. The prepared drugs were added to the culture plates at 3 replicates per concentration. After 24 hours of drug action, H2O2 damage was performed. The drug group and model group were damaged by hydrogen peroxide at the optimal damage time and concentration screened. Finally, MTT was added for cell viability detection.

[0077] The results are shown in Table 3. The survival rate of the model group after hydrogen peroxide injury was 50.72%, while the survival rate of the 100 mg / L cells in the Forsythiaside F drug group increased to 63.08% after treatment; the survival rate of the 100 mg / L cells in the Forsythiaside B drug group increased to 65.43% after treatment. This indicates that the compounds Forsythiaside F and Forsythiaside B have a certain protective effect on liver cells in oxidative damage.

[0078] Table 3 Effects of forsythiaside F and forsythiaside B on the survival rate of HL-7702 cells damaged by H2O2 (n=3, )

[0079] Table 3Effects ofarenarioside and forsythoside B on the survival rate of HL-7702cells damaged by H2O2(n=3, )

[0081]

[0082]

[0083] Note: Compared with the negative control group, *P<0.05, **P<0.01

[0084] It should be noted that the above examples were statistically analyzed using SPSS 25.0 software, and the data were plotted using GraphPad Prism 9. *P<0.05 indicates that the result is statistically significant, **P<0.01 indicates that the result is statistically significant.

[0085] The above description is a detailed description of the preferred feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. Application of two phenylethanol glycoside compounds in penzimidazole in anti-hepatitis B virus and liver protection, characterized in that: The two phenylethanol glycoside compounds are forsythiaside F and forsythiaside B respectively, and the two phenylethanol glycoside compounds are obtained by separating and purifying the 75% ethanol extract of Penicilla ovata.

2. The use according to claim 1, characterized in that: The application of the forsythiaside F and forsythiaside B in the preparation of anti-hepatitis B virus drugs.

3. The use according to claim 2, characterized in that: The anti-HBV drugs include drugs that inhibit hepatitis B surface antigen.

4. The use according to claim 2, characterized in that: The anti-HBV drugs include drugs that inhibit hepatitis B E antigen.

5. The use according to claim 1, characterized in that: The application of the forsythiaside F and forsythiaside B in the preparation of liver protection drugs.

6. The use according to claim 1, characterized in that: The liver protection drugs include drugs that improve the antioxidant capacity of cells.

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