Application of flavonoids in morinda officinalis how in preparation of medicine or health care product for preventing and treating alcoholic liver injury

Abstract

The application belongs to the field of medicine and health care products, and particularly relates to application of flavonoid components in Abrus cantoniensis Hance in preparation of medicines or health care products for preventing and treating alcoholic liver disease, wherein the flavonoid components are flavonoid component enrichment parts, main flavonoid glycoside component compositions or monomer compounds. An in-vitro alcoholic liver injury model is simulated by stimulating AML12 cells with ethanol, and it is found that the flavonoid component enrichment parts, the main flavonoid glycoside component compositions and the monomer compounds in Abrus cantoniensis Hance can all significantly improve cell survival rate. The flavonoid components in Abrus cantoniensis Hance described in the application have outstanding curative effect and high safety, and are expected to develop into effective medicines for preventing and treating alcoholic liver injury.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and relates to application of flavonoids in Abrus cantoniensis Hance in preparation of medicines or health products for preventing and treating alcoholic liver injury. BACKGROUND

[0002] Excessive drinking is a global health care problem, which can cause the liver to suffer the most extensive tissue damage because the liver is the main site of ethanol metabolism. Alcoholic liver disease (ALD) is one of the most common liver diseases in the world, accounting for about 25% of liver cirrhosis deaths. ALD can cause extensive liver damage, from fatty degeneration, fatty hepatitis to fibrosis, cirrhosis and even hepatocellular carcinoma. However, there are few specific drugs for treating ALD, and glucocorticoids are usually used alone or in combination with pentoxifylline, but glucocorticoids have obvious side effects and are not suitable for long-term treatment. Metadoxine is usually used to treat acute alcoholism, but it cannot directly improve liver function. Natural antioxidant compounds, such as silymarin, can repair the liver by reducing lipid peroxidation, although they have low toxicity and side effects, but their low bioavailability and rapid metabolic rate make them quickly disappear from the body, so it is very urgent to find safe and effective drugs to prevent and treat alcoholic liver injury.

[0003] Abrus cantoniensis Hance is a dry whole plant of Abrus cantoniensis Hance in Leguminosae, which has the effects of removing dampness and reducing yellow, clearing heat and detoxifying, and soothing liver and relieving pain, and is often used to treat damp-heat jaundice, uncomfortable hypochondrium, stomachache, and mastitis. As a medicinal and edible traditional Chinese medicine, Abrus cantoniensis Hance is often used to cook soup for food therapy in spring and autumn. Abrus cantoniensis Hance mainly grows in Guangdong, Guangxi and other places. Modern research shows that Abrus cantoniensis Hance is rich in flavonoids, triterpenes, alkaloids, amides, polysaccharides, organic acids, anthraquinones, lignans, amino acids and other chemical components. The rich chemical components of Abrus cantoniensis Hance have a wide range of pharmacological effects. At present, Abrus cantoniensis Hance is clinically used in combination with other traditional Chinese medicines or compound prescriptions to treat hepatobiliary diseases. In recent years, researchers have gradually strengthened the research efforts on the extraction, separation, pharmacological research and new drug development of effective components of Abrus cantoniensis Hance, and many preparations mainly containing Abrus cantoniensis Hance have been marketed as medicines, such as compound Abrus cantoniensis Hance capsules, Abrus cantoniensis Hance hepatitis granules, liver friend capsules, liver delight capsules, liver soothing mixture, Abrus cantoniensis Hance pills, stone relieving tablets, and Abrus cantoniensis Hance tablets. The wide pharmacological effects of Abrus cantoniensis Hance, combined with its low cost and easy cultivation, make it have very high economic value and social benefits, and have great development potential and broad market prospects. Abrus cantoniensis Hance has diverse chemical components and extensive pharmacological research, but so far, no one has clearly proposed that flavonoids in Abrus cantoniensis Hance treat alcoholic liver disease as the pharmacodynamic material basis. SUMMARY

[0004] OBJECTIVE

[0005] The application provides an effect of flavonoid components in Merremia herb on resisting alcoholic liver injury.

[0006] Technical scheme

[0007] The application of the flavonoid components in Merremia herb in preparing medicines or health products for preventing and treating alcoholic liver injury is characterized in that the flavonoid components in Merremia herb are prepared by the following steps: whole plants of Merremia herb are dried, and then extracted by reflux extraction with 70% industrial ethanol; the extracted solution is filtered, and then concentrated under reduced pressure to obtain a crude extract; the crude extract is loaded according to a ratio of D101 macroporous resin filler weight: extract weight of 2:1, and then eluted with 10%, 30%, 40% and 70% ethanol in turn to obtain four parts Fr.A, Fr.B, Fr.C and Fr.D; the Fr.B part is further separated by an ODS column to obtain three separated sections Fr.B1, Fr.B2 and Fr.B3, wherein the Fr.B1 section is the flavonoid components in Merremia herb.

[0008] The application is characterized in that the flavonoid components in Merremia herb are three flavonoid glycoside monomer compounds 1-3 obtained by further separation of the Fr.B1 section.

[0009]

[0010] The application is characterized in that the flavonoid components in Merremia herb are a pharmaceutical composition formed by any one or several of the compounds 1-3 or pharmaceutically acceptable salts thereof and pharmaceutically acceptable excipients.

[0011] The application is characterized in that the pharmaceutical composition is in the form of oral administration or non-oral administration; the oral administration is in the form of tablets, capsules, powders or granules, and the non-oral administration is in the form of suppositories or injections.

[0012] The application provides a preparation method of flavonoid component enrichment parts, main flavonoid glycoside component compositions and monomer compounds in Merremia herb and the application of the monomer compounds in medicines or health products for preventing and treating alcoholic liver injury.

[0013] The application is characterized in that the pharmaceutical composition includes any one or several of the monomer compounds, solvates, salts of solvates or pharmaceutically acceptable salts thereof.

[0014] Further,

[0015] The preparation method of flavonoids in the Morinda officinalis in the application is as follows: the whole plant of Morinda officinalis is dried, and then extracted by reflux extraction with 70% industrial ethanol for three times, each time for 4 hours. The extract is filtered, and then concentrated under reduced pressure to obtain a crude extract. The crude extract is loaded according to the ratio of D101 macroporous resin filler weight: extract weight of 2:1, and then eluted with 10%, 30%, 40% and 70% ethanol concentration gradient in turn to obtain four parts of Fr.A, Fr.B, Fr.C and Fr.D, wherein the Fr.B part is the flavonoids-rich part. The Fr.B part is further separated by ODS column to obtain three separation sections of Fr.B1, Fr.B2 and Fr.B3, wherein the Fr.B1 section is the main flavonoid glycoside composition. The Silica-gel, Sephahex LH-20, ODS, MCI and Pre-HPLC chromatographic techniques are continuously used to separate the Fr.B1 section, and three flavonoid glycoside monomer compounds 1-3 are mainly obtained. The structural formulas of the compounds 1-3 are as follows:

[0016]

[0017] Beneficial effects

[0018] Although the prior art reports that Morinda officinalis has a protective effect on the liver, it does not clearly indicate which drug parts or compounds play a role. In the application, the ethanol-stimulated AML12 cell model is used to simulate the in-vitro alcoholic liver injury model, and the activity of flavonoids in Morinda officinalis is studied. It is found for the first time that the Fr.B2 section has the alcoholic liver injury protection activity, and three flavonoid glycoside monomer compounds 1-3 are the main active ingredients, wherein the compound 2 is the best, and the hepatoprotective effect of the above-mentioned compounds has not been reported in the literature. Specifically:

[0019] Firstly, the protective activity of Fr. A, Fr. B, Fr. C and Fr. D against alcoholic liver injury was investigated. It was found that Fr. B could significantly improve the survival rate of AML12 cells. Further investigation of Fr. B1, Fr. B2 and Fr. B3 showed that Fr. B1 could significantly improve the survival rate of AML12 cells. Further separation and purification of Fr. B1 yielded three flavonoid glycoside monomer compounds 1-3, which could significantly increase the survival rate of AML12 cells under ethanol stimulation. The above results showed that the flavonoid-rich fraction, the main flavonoid glycoside composition and monomer compounds 1-3 from M. kaempferi could improve the survival rate of AML12 cells under ethanol stimulation. Compound 2 was selected as a representative to further study its protective activity against alcoholic liver injury in vitro and in vivo. The results showed that compound 2 could dose-dependently improve the survival rate of AML12 cells under ethanol stimulation. By up-regulating the expression of alcohol dehydrogenase (Adh) and acetaldehyde dehydrogenase (Aldh) and down-regulating the expression of cytochrome P4502E1 (Cyp2e1), compound 2 could improve the ethanol-induced disorder of alcohol metabolism-related genes in AML12 cells. In addition, compound 2 could reduce the level of reactive oxygen species (ROS) in cells, improve the degree of oxidative stress in cells by increasing the levels of glutathione (GSH) and superoxide dismutase (SOD) and reducing the level of malondialdehyde (MDA). In the in vivo alcoholic liver injury model established by feeding Lieber-DeCarli alcohol diet to C57BL / 6J mice, compound 2 could reduce the liver index. The results of HE and oil red O staining of liver pathological sections showed that compound 2 could alleviate the pathological damage of mouse liver and reduce the accumulation of liver lipids. At the same time, compound 2 could reduce the levels of alanine transaminase (ALT), aspartate amino transferase (AST), triglyceride (TG), high density lipoprotein cholesterol (HDL) and low density lipoprotein cholesterol (LDL) in serum and reduce the content of TG in liver. In addition, consistent with the results of in vitro experiments, compound 2 could also improve the disorder of alcohol metabolism in mouse liver and reduce the level of oxidative stress in mouse liver.The above results show that flavonoids in M. alba as a drug or health care products for the prevention and treatment of alcoholic liver injury has good market prospects and clinical value. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 . Chicken grass extraction and separation flow chart.

[0021] Figure 2 . Different concentrations of ethanol stimulated AML12 cells, and the IC 50 value of ethanol on AML12 cells was detected.

[0022] Figure 3 . The flavonoid-rich fraction (Fr.B fraction) (A), the main flavonoid glycoside composition (Fr.B1 segment) (B) and the monomer compound (Compound 1-3) (C) in M. alba improved the survival rate of AML12 cells under ethanol stimulation.###p<0.001versus control group.**p<0.01,***p<0.001versus EtOH group.

[0023] Figure 4 . Compound 2 dose-dependently improved the survival rate of AML12 cells under ethanol stimulation.###p<0.001versus control group.*p<0.05,**p<0.01,***p<0.001versus EtOH group.

[0024] Figure 5 . Compound 2 improved the mRNA expression of alcohol metabolism-related genes Adh (A), Aldh (B) and Cyp2e1 (C) in AML12 cells. #p<0.05,##p<0.01versus control group.*p<0.05versus EtOH group.

[0025] Figure 6 . Compound 2 reduced alcohol-induced oxidative stress in AML12 cells. A is the flow cytometry result, B is the ROS quantification result in cells, C is the GSH content in cells, D is the SOD content in cells, and E is the MDA content in cells. #p<0.05,##p<0.01versus control group.*p<0.05versus EtOH group.

[0026] Figure 7Effect of Compound 2 on Lieber-DeCarli alcohol diet-induced liver index and liver morphology in mice. A is a schematic diagram of the construction of a mouse alcoholic liver injury model, B is the liver index (liver weight / body weight) result of mice, C is a representative mouse liver photo.

[0027] Figure 8 Effect of Compound 2 on Lieber-DeCarli alcohol diet-induced liver pathological staining results in mice. Figure 9 Effect of Compound 2 on Lieber-DeCarli alcohol diet-induced serum ALT (A), serum AST (B), serum TG (C), liver TG (D), serum LDL (E) and serum HDL (F) in mice. #p<0.05, ###p<0.001 versus pair-fed group. *p<0.05, **p<0.01, ***p<0.001 versus EtOH-fed group.

[0028] Figure 10 Effect of Compound 2 on Lieber-DeCarli alcohol diet-induced mRNA levels of alcohol metabolism-related genes Adh (A), Aldh (B) and Cyp2e1 (C) in the livers of mice. #p<0.05, ##p<0.01, ###p<0.001 versus pair-fed group. *p<0.05, **p<0.01, ***p<0.001 versus EtOH-fed group.

[0029] Figure 11 Effect of Compound 2 on Lieber-DeCarli alcohol diet-induced oxidative stress levels in the livers of mice. A is the GSH content in the livers of mice, B is the SOD content in the livers of mice, C is the MDA content in the livers of mice. #p<0.05, ##p<0.01 versus pair-fed group. *p<0.05, **p<0.01, ***p<0.001 versus EtOH-fed group. DETAILED DESCRIPTION

[0030] The present application is described in detail below with reference to Examples and drawings, but the content is an explanation of the present application rather than a limitation.

[0031] The experimental methods in the following examples that do not specify specific conditions are generally performed under conventional conditions, or under conditions recommended by the manufacturer.

[0032] Example 1: Extraction and preparation of flavonoid-enriched fraction from

[0033] Dry the whole grass of Merremia herb 20.0 kg, extract with 70% industrial ethanol by reflux for three times, 4h each time, filter the extract and concentrate under reduced pressure to obtain the crude extract 4.7 kg. Take one-third of the macroporous resin, load into the chromatographic column, add the extract, and adsorb statically for 12h, then open the valve and collect the effluent. Add the effluent into the chromatographic column again, and repeat the flow adsorption for 3 times. Add the remaining two-thirds of the macroporous resin (blank resin) to the column, and equilibrate with the first eluent (10% ethanol solution). Add the macroporous resin adsorbed with the sample above the blank resin, and elute with 10%, 30%, 40%, and 70% ethanol from low to high, respectively, to obtain four fractions Fr.A, Fr.B, Fr.C, and Fr.D (see Table 1). Figure 1 ), wherein the Fr.B fraction is the flavonoid-enriched fraction.

[0034] Example 2: Separation and purification of compounds 1-3

[0035] Further separate the Fr.B fraction by ODS column to obtain three separation sections Fr.B1, Fr.B2, and Fr.B3, wherein the Fr.B1 section is the main flavonoid glycoside composition. Separate the Fr.B1 section by Silica-gel, Sephahex LH-20, ODS, MCI, and Pre-HPLC chromatographic techniques to obtain compound 1 (900 mg), compound 2 (190 mg), and compound 3 (270 mg) Figure 1 ), and determine the structures of compounds 1-3 by H NMR and C NMR methods. 1 H NMR and 13 C NMR.

[0036] The structures of the above compounds are as follows:

[0037]

[0038] Compound 1: yellow powder, molecular formula C 26 H 28 O 14 .

[0039] 1 H NMR (600MHz, Methanol-d4) δ H7.98 (d, J = 7.8 Hz, 2H, H-2' / 6'), 6.93 (d, J = 7.9 Hz, 2H, H-3' / 5'), 6.63 (s, 1H, H-3), 5.03 (d, J = 9.4 Hz, 1H, H-1"), 4.87 (d, J = 8.6, 1H, H-1"), 4.09 (m, 1H, H-2"), 4.04 (m, 1H, H-5"), 4.02 (m, 1H, H-2"), 3.45 - 3.95 (m, 7H, H-4" / 5" / 3" / 4" / 5" / 6").

[0040] 13 C NMR (151 MHz, MeOD) δ C 184.28 (C-4), 166.72 (C-2), 162.98 (C-7), 162.85 (C-4'), 160.32 (C-5), 157.45 (C-9), 130.18 (C-2' / 6'), 123.42 (C-1'), 116.99 (C-3' / 5'), 108.35 (C-6), 105.75 (C-8), 105.50 (C-10), 103.77 (C-3), 82.98 (C-5"), 80.26 (C-3"), 76.54 (C-1"), 75.23 (C-2"), 75.06 (C-1"), 73.14 (C-2"), 72.38 (C-4"), 71.99 (C-3"), 71.23 (C-4"), 70.43 (C-5"), 63.07 (C-6").

[0041] Compound 2: yellow powder, molecular formula C 28 H 32 O 14 .

[0042] 1 H NMR (600 MHz, Methanol-d4) δ H8.29 (s, 1H, H-2), 7.93 (d, J = 9.1 Hz, 1H, H-5), 7.50 (d, J = 8.4 Hz, 2H, H-2' / 6'), 7.35 (d, J = 9.1 Hz, 1H, H-6), 7.00 (d, J = 8.6 Hz, 2H, H-3' / 5'), 5.55 (s, 1H, H-1'''), 5.26 (d, J = 7.7 Hz, 1H, H-1”), 4.14 (d, J = 9.7 Hz, 1H, H-4'''), 4.03 (s, 3H, 8-OCH3), 4.01 (d, J = 1.8 Hz, 1H, H-2'''), 3.91 (d, J = 12.2 Hz, 1H, H-4'''), 3.84 (s, 3H, 4'-OCH3), 3.79 (d, J = 7.9 Hz, 1H, H-2”), 3.74 - 3.65 (m, 2H, H-3” / 5”), 3.55 (s, 2H, H-5'''), 3.45 (t, J = 9.4 Hz, 1H, H-6”).

[0043] 13 C NMR (151 MHz, MeOD) δ C 178.03 (C-4), 161.24 (C-4'), 155.59 (C-7), 155.13 (C-2), 152.32 (C-9), 138.69 (C-8), 131.40 (C-2' / 6'), 125.70 (C-1'), 125.22 (C-3), 121.92 (C-5), 121.02 (C-10), 115.04 (C-6), 114.89 (C-3' / 5'), 110.70 (C-1'''), 100.32 (C-1”), 80.68 (C-3'''), 78.61 (C-5”), 78.45 (C-3”), 78.29 (C-2'''), 78.10 (C-2”), 75.37 (C-4'''), 71.24 (C-4”), 65.78 (C-5'''), 62.35 (C-8-OCH3), 62.28 (C-6”), 55.75 (C-4'-OCH3).

[0044] Compound 3: yellow powder, molecular formula C 23 H 24 O 10 .

[0045] 1 H NMR (500 MHz, Chloroform-d) δ H8.28 (s, 1H, H-2), 7.92 (d, J = 9.1 Hz, 1H, H-8), 7.49 (d, J = 8.9 Hz, 2H, H-2' / 6'), 7.39 (d, J = 9.1 Hz, 1H, H-7), 6.99 (d, J = 8.9 Hz, 2H, H-3' / 5'), 5.14 (d, J = 8.0 Hz, 1H, H-1'), 4.02 (s, 2H, H-11), 3.91 (d, J = 12.2 Hz, 1H, H-6”), 3.83 (s, 3H, 4'-OCH3), 3.72 (dd, J = 12.1, 5.5 Hz, 1H, H-6'), 3.58 (m, 1H, H-5”), 3.52 (m, 2H, H-2” / 3”), 3.44 (d, J = 9.1 Hz, 1H, H-4”).

[0046] 13 C NMR (126 MHz, CDC13) δ C 178.03 (C-4), 161.27 (C-4'), 155.85 (C-6), 155.17 (C-2), 152.28 (C-9), 138.97 (C-5), 131.40 (C-2' / 6'), 125.76 (C-1'), 125.24 (C-3), 121.98 (C-10), 121.25 (C-8), 115.70 (C-7), 114.90 (C-3' / 5'), 102.27 (C-1”), 78.44 (C-2”), 78.13 (C-3”), 74.92 (C-5”), 71.23 (C-4”), 62.45 (C-11), 62.36 (C-6”), 55.76 (C-4'-OCH3).

[0047] Example 3: Study on the protective activity of enriched flavonoid fraction in Morinda officinalis How against alcoholic liver injury

[0048] AML12 cells were stimulated with different concentrations of ethanol for 24 h to obtain IC50 of ethanol-stimulated cells 50 was 890 mM, and 800 mM was selected as the induction concentration Figure 2 ) in subsequent experiments.

[0049] CCK8 assay was used to detect the cell survival rate after adding 50 μg / ml Fr.A, Fr.B, Fr.C, and Fr.D fraction mixture into ethanol-treated AML12 cells for 24 h. The results showed that Fr.B fraction could significantly improve the survival rate of AML12 cells Figure 3 Fr.B fraction was analyzed, and it was found that the main component was flavonoids.

[0050] Example 4: Study on the protective activity of the main flavonoid glycoside composition in Merremia herb for alcoholic liver injury

[0051] Fr.B1, Fr.B2 and Fr.B3 were obtained by further separation of Fr.B. After 24 h, the cell survival rate was detected by CCK8 method. The results showed that Fr.B1 could significantly improve the survival rate of AML12 cells Figure 3 ), and 3 flavonoid glycoside monomer compounds 1-3 were obtained by further separation and purification of Fr.B1.

[0052] Example 5: Study on the protective activity of compounds 1-3 for alcoholic liver injury

[0053] The cytotoxicity of compounds 1-3 on AML12 cells was evaluated by CCK8 method, and the IC 50 values of compounds 1-3 were determined. The results showed that compounds 1-3 had no cytotoxicity (Table 1).

[0054] Table 1. IC values of compounds 1-3 in AML12 cells 50

[0055] Compound IC 50 (μM) 1 ＞100 2 ＞100 3 ＞100

[0056] AML12 cells were treated with 100 μM of compounds 1-3 combined with 800 mM of ethanol for 24 h, and the cell survival rate was detected by CCK8 method. The results showed that compounds 1-3 could significantly improve the survival rate of AML12 cells Figure 3 ).

[0057] According to the above results, compound 2 was selected for further study.

[0058] Example 6: Effect of compound 2 on the survival rate of ethanol-stimulated AML12 cells

[0059] AML12 cells were treated with 800 mM of ethanol for 24 h, and different concentrations of compound 2 were added for treatment. The cell survival rate was detected by CCK8 method. The results showed that compound 2 could dose-dependently improve the survival rate of AML12 cells, and the optimal administration concentration of compound 2 was determined to be 2.5, 5 and 10 μM Figure 4 ).

[0060] Example 7: Effect of compound 2 on the expression amount of alcohol metabolism-related genes in AML12 cells

[0061] ​The total RNA in the cells was extracted by using an RNA extraction kit, the RNA concentration was determined by NanoDrop, 1 μg of RNA was used to reverse transcribe into cDNA by using a kit, and the appropriate amount of cDNA was diluted 5 times with RNase Free ddH2O. The PCR reaction solution was prepared according to the instructions, and the reaction conditions were as follows:

[0062] Table 2. RT-qPCR reaction conditions

[0063] Step Temperature (°C) Time (s) Number of cycles Pre-denaturation 95 30 1 Denaturation 95 5 40 Annealing 60 10 40 Extension 72 15 40

[0064] The ethanol-induced AML12 cells were treated with positive drug silymarin (SM, 10 μM) and different concentrations of compound 2, and the expression levels of Adh, Aldh and Cyp2e1 genes in the cells were detected. The results showed that compound 2 can dose-dependently increase the expression levels of Adh and Aldh genes and reduce the expression level of Cyp2e1 gene, indicating that compound 2 can improve the alcohol metabolism disorder in ethanol-stimulated cells Figure 5 ).

[0065] The primer sequences used in the experiment are as follows:

[0066] Table 3. Primer sequences of related genes

[0067]

[0068] Example 8: Effect of compound 2 on the level of alcohol-induced oxidative stress in AML12 cells.

[0069] Flow cytometry was used to detect the ROS level in ethanol-induced AML12 cells, and the results showed that the ROS level in AML12 cells was significantly increased under ethanol induction, and the ROS level was significantly reduced after treatment with compound 2. The kit was used to detect the effect of compound 2 on the levels of SOD, GSH and MDA in AML12 cells, and the results showed that after treatment with compound 2, the activities of SOD and GSH in the cells were significantly increased, and the level of MDA was reduced. The above results show that compound 2 can alleviate the level of alcohol-induced intracellular oxidative stress Figure 6 ).

[0070] Example 9: Construction of a mouse alcoholic liver injury model induced by Lieber-DeCarli alcohol feed.

[0071] Male C57BL / 6J, 6-week-old (20g-22g) mice were randomly divided into 5 groups, 6 mice per group, namely control group, model group, drug administration group (positive drug group, low concentration group, medium concentration group, high concentration group). The control group of mice was initially fed with Lieber-DeCarli control feed for 2 days to adapt to liquid feed, and then the proportion of alcohol feed was increased every day to adapt to alcohol, except for the control group. After 7 days, the mice in the remaining groups were fed with Lieber-DeCarli alcohol feed containing 5% (v / v) ethanol every day for 10 days, while the control group of mice was fed with isocaloric control feed (same heat content). At the same time, the positive drug group was given SM 30 mg / kg, and the low, medium and high concentration groups were given compound 2 3, 10 and 30 mg / kg respectively, and the drug was administered by gavage every day. The drug was dissolved in normal saline. The body weight and food intake of each group of mice were recorded every day. On the 11th day, the ethanol-fed mice and the paired-fed mice were given a single dose of ethanol (large dose, 5 g / kg) or isocaloric maltodextrin by gavage at about 7 o'clock in the morning, and were euthanized 9 hours later Figure 7 A). The mouse liver and whole blood were collected for subsequent experiments.

[0072] Example 10: Effect of Compound 2 on Lieber-DeCarli alcohol feed-induced mouse liver index and liver morphology.

[0073] The results of mouse body weight and liver weight showed that the body weight of alcohol feed-fed mice gradually increased during the adaptation stage and then remained stable, while the body weight of control diet-fed mice continued to increase throughout the process. Compared with control feed-fed mice, the liver to body weight ratio of alcohol feed-fed mice was significantly increased, while the SM group and compound 2 administration group significantly reduced the liver to body weight ratio of mice. In addition, the results of liver morphology showed that the compound 2 administration group significantly improved the phenomenon of liver enlargement, whitening and loose texture of the model group mice Figure 7 ).

[0074] Example 11: Effect of Compound 2 on Lieber-DeCarli alcohol feed-induced mouse liver pathological staining results.

[0075] The paraformaldehyde-fixed liver tissue was sent to China Pharmaceutical University Pharmacology and PDX Efficacy Evaluation Platform for tissue wax block making and HE and oil red O staining. The digital pathology slide scanner (NanoZoomer S60) was used for scanning and NDP software was used for analysis. The results of HE staining showed that the compound 2 administration group could significantly improve the phenomenon of liver cell disorder, fat vacuoles and cell gap expansion in the model group, and restore the normal tissue structure of the liver. The results of oil red O staining showed that the compound 2 administration group could significantly improve the lipid accumulation in the liver Figure 8 ).

[0076] Example 12: Effect of compound 2 on serum ALT, AST, TG, HDL, LDL and liver TG of Lieber-DeCarli alcohol diet-induced mice.

[0077] The whole blood of mice was collected, and after standing at room temperature, the supernatant was collected after centrifugation at 4°C and 4000 r·min -1 After centrifugation for 10 min, the supernatant was collected as serum, and the contents of ALT, AST, TG, HDL and LDL in the serum of each group of mice and the TG content in the liver of mice were detected using the related kit. It was found that the compound 2 administration group could significantly reduce the serum ALT and AST levels of the model group mice, alleviate liver damage; significantly reduce the TG levels in serum and liver and the HDL and LDL levels in serum, and reduce the lipid deposition in liver Figure 9 ).

[0078] Example 13: Effect of compound 2 on alcohol metabolism-related genes in the liver of Lieber-DeCarli alcohol diet-induced mice.

[0079] The total RNA in the liver tissue of mice was extracted by using an RNA extraction kit, and after reverse transcription into cDNA, the RT-qPCR reaction was carried out, and the reaction conditions were consistent with Table 2. The experimental results showed that the compound 2 administration group could significantly increase the expression amounts of Adh and Aldh genes in the liver of mice, and reduce the expression amount of Cyp2e1 gene, indicating that the compound 2 could improve the alcohol metabolism level in the liver of alcohol diet-induced model mice Figure 10 ). The primer sequences used in the experiment were consistent with Table 3.

[0080] Example 14: Effect of compound 2 on the oxidative stress level in the liver of Lieber-DeCarli alcohol diet-induced mice.

[0081] An appropriate amount of mouse liver tissue was taken, ground, and the contents of GSH, SOD and MDA in the liver were detected using the corresponding kit. The experimental results showed that the compound 2 administration group could significantly increase the contents of GSH and SOD in the liver, and reduce the content of MDA, indicating that the compound 2 could significantly reduce the oxidative stress degree in the liver of model mice Figure 11 ).

Claims

1. A flavonoid component of Herba Ceratitise glutinosae for preventing or treating alcoholic liver damage, characterized in that: The flavonoid component is compound 2, and the structural formula of compound 2 is as follows:

2. The use of the flavonoids of Herba Ceratitise var. juncea according to claim 1 in the preparation of a drug for preventing and treating alcoholic liver damage.

3. The use according to claim 2, characterized in that The flavonoid component of the Herba Cercidiphyllum is prepared by the following steps: drying the whole Herba Cercidiphyllum with 70% industrial ethanol for reflux extraction, filtering the extract and concentrating under reduced pressure to obtain a crude extract; loading the sample at a ratio of 2:1 (weight ratio) of D101 macroporous resin filler to the extract weight, eluting with 10%, 30%, 40%, and 70% ethanol concentration gradients in sequence to obtain four fractions, Fr.A, Fr.B, Fr.C, and Fr.D, respectively; further separating the Fr.B fraction using an ODS column to obtain three separated segments, Fr.B1, Fr.B2, and Fr.B3, wherein the Fr.B1 segment contains the Herba Cercidiphyllum flavonoid component; and further separating the Fr.B1 segment to obtain three flavonoid glycoside monomer compounds 1-3:

4. The use according to claim 2 or 3, characterized in that The flavonoid components of the chordate thunbergii are added to pharmaceutically acceptable excipients to prepare a pharmaceutical composition.

5. The use according to claim 4, characterized in that The pharmaceutical composition is in an oral dosage form or a non-oral dosage form; the oral dosage form is a tablet, capsule, powder or granule, and the non-oral dosage form is a suppository or injection.

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