Application of erythrinine, soyasaponin Bb and composition of erythrinine and soyasaponin Bb in preparation of medicine for treating acute liver injury and hepatic fibrosis

Abstract

The invention discloses an application of an abrus herb component soybean saponin Bb (Soyasaponin Bb, C48H78O18), erythrinine (Hypaphorinine, C14H18N2O2) and a composition of the abrus herb component soybean saponin Bb and the Hypaphorinine in prevention and treatment of liver injury and liver fibrosis, and particularly discloses an application of the composition of the abrus herb component soybean saponin Bb, the Hypaphorinine and the composition of the abrus herb component soybean saponin Bb and the Hypaphorinine. Erythrinine and soyasaponin Bb are active components separated from traditional Chinese medicine abrus cantoniensis hance, have good effects of preventing and treating liver injury and hepatic fibrosis when being used independently or in combination, and have important significance in preventing and treating chronic diseases such as hepatic fibrosis when being prepared into health-care food or drugs.

Description

Technical Field

[0001] The present invention relates to the fields of medical technology and food, and in particular to application of soybean saponin Bb, erythrine and a composition thereof in the preparation of prevention, improvement and treatment of liver damage and liver fibrosis. Background Art

[0002] The liver is an important metabolic organ in humans and other animals, with functions such as detoxification, metabolism and immune defense. Long-term exposure of the liver to damaging environments, such as viral hepatitis infection, long-term alcohol consumption, exposure to chemical toxic substances, fatty hepatitis and cholestatic diseases, leads to apoptosis of hepatocytes, followed by activation of hepatic stellate cells (HSCs) and differentiation into fibroblasts. Fibroblasts are the main source of extracellular matrix components, and the extracellular matrix is ​​gradually deposited, leading to the formation of fibers. Liver fibrosis is a reversible wound healing response to acute or chronic cell damage. During acute injury, changes in liver structure are transient and reversible. In chronic injury, the liver parenchyma is gradually replaced by scar tissue, and persistent liver damage often leads to the slow development of cirrhosis in patients.

[0003] The occurrence of liver fibrosis is directly related to the activation and proliferation of hepatic stellate cells (HSC), and is closely related to the microenvironment formed by factors such as transforming growth factor-β1 (TGF-β1) and hypoxia inducible factor HIF-1α (hypoxia inducible factor-1). The treatment strategy for liver fibrosis is mainly to inhibit HSC activation and promote collagen degradation. However, existing drugs, including small molecule inhibitors of PDGF or VEGF, have unsatisfactory clinical effects on liver fibrosis and have safety issues.

[0004] Traditional Chinese medicine has a history of thousands of years in China and also plays an important role in the treatment of liver diseases. Traditional Chinese medicines with the effects of soothing liver and relieving depression, clearing liver and improving eyesight, calming liver and extinguishing wind are often used in the prevention and treatment of liver-related diseases, such as Artemisia capillaris, Lysimachia chinensis, Rhizoma Cibotii, Camellia sinensis, and Hedyotis diffusa. Studies have found that traditional Chinese medicine can improve liver microcirculation, scavenge oxygen free radicals, resist lipid peroxidation, promote bilirubin metabolism, promote liver glycogen and protein synthesis, and increase the content of liver microsomal cytochrome. The effective ingredients of traditional Chinese medicine for liver damage and liver fibrosis generally include flavonoids, saponins, and alkaloids. The mechanisms of anti-liver damage and liver fibrosis include inhibiting liver inflammation, resisting lipid peroxidation damage, inhibiting the activation and proliferation of hepatic stellate cells (HSCs), regulating the synthesis and secretion of profibrotic factors, and regulating the synthesis and degradation of extracellular matrix (ECM).

[0005] As a traditional Chinese medicine for both medicinal and edible purposes, the whole plant of Glechoma longituba is used as medicine. It has the effects of removing dampness and relieving jaundice, clearing away heat and detoxifying, soothing the liver and relieving pain. It is used to treat damp-heat jaundice, discomfort in the ribs, stomach distension and pain, mastitis and other diseases. It is used to treat liver cirrhosis ascites and acute and chronic hepatitis with definite efficacy. It has been recorded in history that "Nanning City Pharmacopoeia": "It can treat infectious hepatitis and sprains and fractures"; "Lingnan Herbal Medicine Records" records: "It can soothe the liver, harmonize the spleen, and heal fractures"; "National Compendium of Chinese Herbal Medicines" states: "Glechoma longituba can treat acute and chronic hepatitis, liver cirrhosis ascites, stomachache, rheumatic bone pain, and snake bites, and can be used as a refreshing drink in summer." Modern research on Glechoma longituba has shown that Glechoma longituba has antioxidant, anti-cell proliferation, anti-cancer and immunomodulatory effects. Glechoma longituba contains saponins, alkaloids, flavonoids, volatile oils and other ingredients, among which the main ingredients are soybean saponin Bb (Soyasaponin Bb, C 48 H 78 O 18 ), Abrus saponins, Schaftoside, Abrus albicans, Hypaphorine, C 14 H 18 N2O2), etc. In our in vivo and in vitro study of the anti-hepatic fibrosis activity of scutellaria baicalensis extracts, we found that total saponins of scutellaria baicalensis could significantly reduce AST and ALT in the serum of mice with chemical and immune liver damage, reduce the level of MDA in liver tissue, and increase SOD activity. Further studies through network pharmacology and molecular docking confirmed that total alkaloids and scutellaria baicalensis alkaloids have a protective effect against liver damage. KEGG pathway analysis showed that its mechanism is related to the phenylalanine metabolic pathway and the tyrosine metabolic pathway, suggesting their potential in preventing and treating liver damage and liver fibrosis.

[0006] The present invention focuses on the application of the key active components soybean saponin Bb and erythrina alkaloids (Hypaphorine, C14H18N2O2) in Aconitum and the composition in the prevention and treatment of liver injury and liver fibrosis. It is aimed at the Healthy China strategy and the major needs of the country for preventing and treating chronic liver disease and liver fibrosis, and is of great significance for the prevention and treatment of chronic diseases such as liver fibrosis.

[0007] References

[0008] 1.Zhang Chengzhong,Bu Qitao,Li Chuyan,Lu Pengfei,Liu Chang,HuangBaokang.Simultaneous determination of abrine,hypaphorine,schaftoside andsoyasaponin Bb in rat plasma by UPLC-MS / MS and its application to apharmacokinetic study after oral administration of Abrus cantoniensis Hanceextract.Biomed Chromatogr.2023;37(10):e5696.

[0009] 2.Wang M, Chen

[0010] 3. Xiao Xiao, Yao Xiangcao, Yu Yaru, Xu Chongyuan, Huang Baokang. Resource survey and pharmacognosy identification of Herba Corydalis. Journal of Pharmaceutical Practice. 2019, 4: 318-321

[0011] 4. Yao Xiangcao, Xue Jingjing, Xiao Xiao, Xu Chongyuan, Huang Baokang. Protective effects of total saponins from Aconitum carmichaelii against chemical and immune liver damage. Chinese Journal of Clinical Pharmacology, 2019, 35(18): 2071-2074.

[0012] 5. Yao Xiangcao, Xiao Xiao, Huang Baokang, Xu Zhongyuan. Molecular docking and in vitro activity study of the antiviral hepatitis B activity of Herba Corydalis. Chinese Journal of Clinical Pharmacology. 35(5):439-441,448.

[0013] 6. Xiao Xiao, Xu Zhongyuan, Yang Dejun, Huang Baokang. Comparative analysis of volatile oil and fatty acid composition of Herba Corydalis and Herba Corydalis, Journal of Pharmaceutical Practice, 2017, 35(1), 39-42. Summary of the invention

[0014] The purpose of the present invention is to provide a Chinese medicine component and composition for preventing and treating liver damage and liver fibrosis in view of the deficiencies of the prior art.

[0015] Another object of the present invention is to provide applications of the above-mentioned Chinese medicine components and compositions.

[0016] To achieve the above purpose, the technical solution adopted by the present invention is: a Chinese medicine component and composition for preventing and treating liver damage and liver fibrosis, wherein the Chinese medicine components soybean saponin Bb and erythrinaine are extracted from Chinese medicines such as Herba Corydalis and Herba Corydalis, obtained by extraction with ethanol solution or pure water, drinking water, or by necessary chemical synthesis. The composition is composed of the following raw materials in parts by mass: 35 parts of soybean saponin Bb and 3 parts of erythrinaine.

[0017] To achieve the above second purpose, the technical solution adopted by the present invention is: the use of the Chinese medicine components and compositions in the preparation of drugs or foods for preventing and treating liver damage and liver fibrosis. The soybean saponin Bb and erythrina alkaloid composition disclosed in the present invention can be in the form of medicine, or in the form of food or health food, including tablets, granules, capsules, pills, oral liquids, injections, etc., and are not limited to the above pharmaceutical dosage forms, and can also be herbal teas, brewed beverages, solid foods, etc., and are not limited to the above forms.

[0018] When the soybean saponin Bb and erythrine disclosed in the present invention are used in medicine and food, necessary excipients, fillers, diluents and other auxiliary materials that can be added to medicines or foods can be added.

[0019] The invention has the advantages that the soybean saponin Bb and erythrine prepared by using radix scutellariae as the main raw material have significant effects on preventing and treating liver damage and liver fibrosis, and can significantly reduce the elevated ALT and AST in the serum of patients with liver damage, inhibit the increase of MDA value caused by acute damage in liver tissue, increase SOD value in tissue, slow down liver tissue pathological changes, and reduce liver cell necrosis. The invention has few toxic and side effects, and can be used for preparing medicines or foods for preventing and treating liver damage and liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The structural formulas of Soyasaponin Bb and Hypaphorine are shown.

[0021] Figure 2 The results show the effects of soybean saponin Bb and erythrine on the blood biochemical indicators ALT and AST in mice with acute liver injury (Figure A shows the difference in ALT content, Figure B shows the difference in AST content, n=7). Note: Compared with the normal group, #P<0.05, ##P<0.01; compared with the model group, *P<0.05, **P<0.01.

[0022] Figure 3 The results show the effects of soybean saponin Bb and erythrine on MDA, SOD, and GSH in mice with acute liver injury (Figure A shows the difference in MDA levels, Figure B shows the difference in SOD content, and Figure C shows the difference in GSH content, n=7). Note: Compared with the normal group, #P<0.05, ##P<0.01; compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0023] Figure 4 The results show the effects of soybean saponin Bb and erythrina alkaloids on liver tissue of mice with acute liver injury caused by CCl4 (HE, ×0.5, ×40). A is the blank group (Control group); B is the model group (Model group); C is the positive control group (Positive group); D is the low-dose soybean saponin Bb group (Soy-L group); E is the high-dose soybean saponin Bb group (Soy-H group); F is the low-dose erythrina alkaloids group (Hy-L group); G is the high-dose erythrina alkaloids group (Hy-H group).

[0024] Figure 5 This is the HPLC chromatogram of the test sample of Acanthopanax salsa (peak 2: soybean saponin Bb).

[0025] Figure 6 This is the HPLC chromatogram of the test sample of Aconitum carmichaelii (erythrine).

[0026] Figure 7 The drug-time curves of the four tested components in the plasma of rats after oral administration of Aconitum scabra extract (0.3 g / kg) are shown. DETAILED DESCRIPTION

[0027] The following examples are used to illustrate specific implementation methods, which further illustrate and supplement the content of the present invention. However, the scope of the above subject matter of the present invention is not limited to the following examples. All technologies and applications realized based on the above content of the present invention belong to the scope of the present invention.

[0028] Example 1 - Preparation of Tablets

[0029] Take 3.5g of soybean saponin Bb, 0.3g of erythrine, 40g of dextrin, appropriate amount of dry starch, appropriate amount of carboxymethyl cellulose, appropriate amount of talcum powder, and appropriate amount of sucrose. Sucrose and talcum powder are used for sugar coating, mixed, granulated and tableted to obtain a total of 100 tablets. A single tablet weighs 0.50g and contains 35mg of soybean saponin Bb and 3mg of erythrine. Other requirements comply with the relevant provisions of the 2020 edition of the "Pharmacopoeia of the People's Republic of China" on tablets.

[0030] Example 2 - Preparation of capsules

[0031] Take 3.5g soybean saponin Bb, 0.3g erythrine, 40g corn starch, and an appropriate amount of cyclodextrin, mix well, and put into capsules, a total of 100 capsules. Each capsule contains 35mg soybean saponin Bb and 3mg erythrine. Other requirements must comply with the relevant provisions of the 2020 edition of the "Pharmacopoeia of the People's Republic of China" on capsules.

[0032] Example 3 - Preparation of tea

[0033] Take 1kg of Chinese herbal medicine Herba Glechomae containing soybean saponin Bb and erythrine, cut into sections, add water and boil twice, each time with 8L of pure water, combine the decoction, concentrate into extract, take 100g of dextrin, 100g of soluble starch, appropriate amount of powdered sugar, mix well, make tea blocks, and package them in batches, with a filling volume of 5.0g / bag. Other requirements meet the requirements for tea preparations.

[0034] Example 4-Effects of soybean saponin Bb and erythrine on acute liver injury

[0035] 1. Experimental Materials

[0036] 1. Experimental Animals

[0037] 56 SPF-grade C57BL / 6J mice, 8 weeks old, all male, weighing (25±3) g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., license number: SCXK(Beijing)2021-006, use license: SYXK(Beijing)2022-0013, and were kept in the animal room of the Experimental Animal Center of the Department of Pharmacy of Naval Medical University, with a temperature of 20-25°C and a humidity of 40%-60%. The light and dark cycles were alternating for 12 h each, and food and water were freely available.

[0038] 2. Drugs and reagents

[0039] The drugs and reagents used in the experiment are shown in Table 1. The structural formulas of soybean saponin Bb and erythrine are shown in Figure 1 .

[0040] Table 1. Reagents

[0041]

[0042] 3. Instruments

[0043] The experimental instruments used in the experiment are shown in Table 2.

[0044] Table 2. Experimental instruments and tools

[0045]

[0046] (II) Experimental methods

[0047] 1. Experimental grouping and modeling

[0048] After 1 week of adaptive feeding, SPF C57BL / 6J mice were randomly divided into 7 groups, 7 mice in each group, including normal group (Ctrl group), model group (Mod group), positive drug silymarin control group (Pos group, 100 mg / kg), soybean saponin Bb low-dose group (Soy-L group, 50 mg / kg), soybean saponin Bb high-dose group (Soy-H group, 100 mg / kg), erythrina alkaloid low-dose group (Hy-L group, 50 mg / kg), erythrina alkaloid high-dose group (Hy-H group, 100 mg / kg). According to the experimental settings, the drug-treated group and the positive drug group were intragastrically administered with a certain amount of 0.5% CMC-Na solution of soybean saponin Bb, erythrina alkaloid and silymarin every day, and the blank group and the model group were intragastrically administered with 0.5% CMC-Na solution every day, with an intragastrical volume of 0.1 mL / 10 g. After 3 consecutive days of administration, except for the normal group, the mice in the other groups were intraperitoneally injected with 0.2% Olive oil solution of CCl4 (10mL / kg), the normal group was intraperitoneally injected with an equal volume of olive oil. After 24 hours, the body weight was weighed, and blood was collected from the eye sockets after anesthesia. The whole blood was allowed to stand and centrifuged at 4℃ for 10min (4000r / min) to separate the serum and store it for later use. The animals were killed, and the spleen, thymus, liver and other organs were weighed after dissection. Part of the liver was fixed in 10% paraformaldehyde solution for routine hematoxylin-eosin staining (HE staining), and the rest of the liver was frozen in a -80℃ refrigerator for later use.

[0049] 2. Serum biochemical index detection

[0050] Serum was obtained after centrifugation of whole blood, and the contents of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum were detected using a fully automatic biochemical analyzer according to the instructions of the kit.

[0051] 3. Detection of liver tissue biochemical indicators

[0052] Take out the frozen mouse liver tissue and weigh it. Immediately place it in 10 times the amount of 4℃ physiological saline and homogenize it. Use a high-speed low-temperature centrifuge to remove the cell nuclei and other tissue fragments in the liver tissue homogenate to avoid affecting the determination of liver biochemical indicators. Centrifuge at 3000r / min for 10 minutes in a 4℃ low-temperature centrifuge, and aspirate the supernatant. The protein concentration in the supernatant was determined using the EasyIIProtein Quantitative Kit protein concentration determination kit, and then the xanthine oxidase method total superoxide dismutase (SOD) kit, thiobarbituric acid method malondialdehyde (MDA) kit, and reduced glutathione (GSH) kit were used to determine the activity of related indicators in the liver tissue homogenate.

[0053] 4. Liver pathological examination

[0054] Mouse liver tissue was fixed in 10% formaldehyde solution for 24 hours, trimmed, and dehydrated with 30%, 50%, 70%, 90%, 95% ethanol solution and anhydrous ethanol, then transparentized with xylene, and the transparent tissue was immersed in molten paraffin for 2 hours, then embedded in paraffin, sliced, with a thickness of 5μm, and fixed on a slide by spreading on 55℃ water surface, pasting on a slide, and drying at 60℃ for 10min. After further rehydration, HE staining, and sealing, the pathological changes of liver tissue were observed and analyzed under an optical microscope.

[0055] 5. Statistical methods

[0056] The data are The data were analyzed by one-way analysis of variance, and the t-test was used between groups. P<0.05 indicated that the difference was statistically significant, and P<0.01 indicated that the difference was very significant.

[0057] (III) Experimental results

[0058] 1. Effects of soybean saponin Bb and erythrine on serum biochemical indicators ALT and AST in mice with acute liver injury induced by CCl4

[0059] The effects of soybean saponin Bb and erythrine on serum aspartate aminotransferase and alanine aminotransferase in mice with acute liver injury induced by CCl4 are shown in Table 3. Figure 2As shown. Compared with the normal group, the ALT and AST values ​​of the model group were significantly increased (P < 0.01). Compared with the model group, the ALT value of the silymarin positive group was significantly reduced (P < 0.05), and the AST value was very significantly reduced (P < 0.01), indicating that silymarin can improve the serum indicators of mice with acute liver injury caused by CCl4, and can meet the medication requirements of the control group for this test. The low and high dose groups of soybean saponin Bb can significantly reduce the ALT and AST values ​​in the serum of mice with acute liver injury caused by CCl4 (P < 0.01); the low and high dose groups of erythrine also showed the effect of significantly reducing the ALT and AST values ​​in the serum of mice with acute liver injury (P < 0.01).

[0060] Table 3. Effects of soybean saponin Bb and erythrine on serum biochemical indicators ALT and AST in mice with acute liver injury ( n=7)

[0061]

[0062] Note: Compared with the normal group, # P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01.

[0063] 2. Effects of soybean saponin Bb and erythrine on SOD activity and GSH and MDA contents in liver tissue of mice with acute liver injury induced by CCl4

[0064] The effects of soybean saponin Bb and erythrine on MDA, SOD and GSH in liver tissue of mice with acute liver injury induced by CCl4 are shown in Table 4. Figure 3As shown. Compared with the normal group, the MDA level in the liver tissue of the model group mice increased significantly, with significant differences (P < 0.01), and the SOD value decreased, but there was no significant difference in the SOD and GSH values. Compared with the model group, the silymarin positive group, soybean saponin Bb and erythrina alkaloids can significantly reduce the MDA value in the liver tissue, among which the soybean saponin Bb high-dose group significantly reduced the lipid peroxidation byproduct MDA value (P < 0.01), and the other drug-treated groups can significantly reduce the MDA value (P < 0.001), and the effect of erythrina alkaloids on MDA shows a certain dose dependence. From this result, it can be intuitively seen that soybean saponin Bb and erythrina alkaloids can significantly inhibit the increase of MDA value in the liver of mice with acute liver injury induced by CCl4. Compared with the model group, the low and high doses of soybean saponin Bb groups can significantly increase the antioxidant SOD value (P < 0.05), and the low and high doses of erythrina alkaloids can significantly increase SOD (P < 0.01). It can be seen that soybean saponin Bb and erythrina alkaloids can significantly increase the SOD value in the liver tissue of mice with acute liver injury induced by CCl4, showing a certain antioxidant activity. The results of GSH determination are relatively complicated. The values ​​of the model group increased compared with the normal group, but there was no significant difference. Compared with the model group, the low-dose group of soybean saponin Bb and the high-dose group of erythrina alkaloids can increase the content of GSH in the liver tissue of mice with acute liver injury induced by CCl4, but there was no significant difference. Comprehensive analysis of the results of MDA, SOD and GSH determination shows that soybean saponin Bb and erythrina alkaloids can improve the antioxidant capacity of the liver and have the effect of resisting lipid peroxidation of hepatocytes.

[0065] Table 4. Effects of soybean saponin Bb and erythrine on MDA, SOD and GSH in liver tissues of acute liver injury ( n=7)

[0066]

[0067] Note: Compared with the normal group, # P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01, *** P<0.001.

[0068] 3. Pathological effects of soybean saponin Bb and erythrine on liver tissue of mice with acute liver injury induced by CCl4

[0069] The liver tissues of mice in each experimental group were taken for HE staining and observed under an optical microscope. The liver sections of mice in the normal group were smooth, the tissue and lobule structures were intact, the cells were neatly arranged, mostly in cords, there were no inflammatory changes, no inflammatory cell infiltration, and no degenerative necrotic cells. The liver lobule structure of mice in the CCl4-induced acute liver injury group was partially destroyed, the liver cords were disordered, the hepatocytes had vacuolar changes and inflammatory cell infiltration, and the boundaries of some liver tissue cells were blurred. Compared with the normal group, vacuolar hepatocytes were visible in the silymarin positive control group, but compared with the model group, the liver lobule structure was more intact, there was no obvious inflammatory cell infiltration, and the degree of pathological damage was lower, indicating that silymarin can reduce the damage of CCl4 to the liver after preventive administration as a positive control. After acute injury by CCl4, the hepatocytes in the soya saponin Bb and erythrina alkaloid groups showed mild vacuolar degeneration and inflammatory cell infiltration. However, compared with the model group, the overall liver structure was not significantly damaged, the liver tissue cells were relatively intact, there were no obvious necrotic cells, and the histopathological lesions were reduced. Compared with the soya saponin Bb group, the erythrina alkaloid group showed more obvious vacuolar changes and inflammatory cell infiltration, but no obvious hepatocyte necrosis. There was no significant difference in the effects of the two drugs on liver tissue cells at the dosage. This indicates that the preventive administration of soya saponin Bb and erythrina alkaloids in Glechoma longituba can alleviate the damage of hepatocytes to a certain extent, has a certain hepatocyte protective effect, and can reduce the liver tissue damage caused by CCl4 ( Figure 4 ).

[0070] Example 5 - Determination of the content of soybean saponin Bb and erythrine in Glechoma longituba

[0071] (I) Main experimental instruments

[0072] The experimental instruments used in this experiment are shown in Table 5.

[0073] Table 5. Experimental instruments and tools

[0074]

[0075] (II) Reagents and materials

[0076] The reagents and materials used in this experiment are listed in Table 6.

[0077] Table 6. Reagents and materials

[0078]

[0079] (III) Experimental methods

[0080] 1. Chromatographic conditions and system suitability experiments

[0081] The composition analysis was performed using a ZORBAX SB-C18 column; the mobile phase was gradient elution, with phase A being acetonitrile and phase B being 0.2% formic acid solution. The gradient elution program is shown in Table 4-3-3. The column temperature was set at 35°C. Flow rate: 1.0 mL / min. Injection volume: 10 μl. Evaporative light scattering detector conditions: drift tube temperature 105°C, gas flow rate 3.0 mL / min, gain value 2, no split.

[0082] Table 7. Liquid chromatography gradient elution program

[0083]

[0084] 2. Preparation of test solution

[0085] Accurately weigh 1.0 g of the powder of the Herba Codonopsis pilosulae sample, place it in a conical flask, add 50 mL of 70% methanol, weigh the weight, ultrasonically treat it at 40°C for 1 hour (300 W, 40 KHz), take it out, weigh it, make up for the loss, filter it, recover the solvent, add 30 mL of water to dissolve the residue, extract it 3 times with a saturated n-butanol solution (40 mL, 30 mL, 30 mL), combine the n-butanol solutions, recover the solvent, add methanol to dissolve the residue, make up to 5 mL, and filter it with a 0.22 μm microporous filter membrane to obtain the test solution.

[0086] 3. Preparation of reference solution

[0087] Accurately weigh 10.74 mg of soybean saponin Bb reference substance (batch number: wkq 22102507, purity HPLC ≥ 98%), place in a 10 mL volumetric flask, add methanol to dissolve, and dilute to the mark.

[0088] Shake well to prepare the reference solution of soybean saponin Bb. Accurately measure 0.1mL, 0.2mL, 0.4mL, 0.5mL, 0.6mL, 0.8mL and 1.0mL of the reference solution into injection vials, and accurately add methanol to make up to 1.00mL to prepare the reference solution of the standard curve.

[0089] 4. Precision experiment

[0090] Accurately measure 5 μl of soybean saponin Bb solution and inject it into liquid chromatography to determine the retention time and peak area of ​​soybean saponin Bb. The determination was repeated 5 times in parallel, and the retention time and peak area RSD% were calculated respectively.

[0091] 5. Stability test

[0092] Accurately weigh 1.0 g of JGC-001-010 powder, place it in a stoppered conical flask, add 50 mL of 70% methanol solution, prepare the test solution according to the method under the preparation of the test solution, and determine soybean saponin Bb at 0, 4, 6, 10, 16, and 24 hours, and record the retention time and peak area.

[0093] 6. Repeatability Experiment

[0094] Weigh 6 portions of 1.0 g each of the Glechoma longituba sample JGC-001-010, and prepare 6 parallel test solutions according to the preparation method under the preparation of the test solution. Determine the content of soybean saponin Bb in the test sample by HPLC, record the retention time and peak area, and calculate the content RSD%.

[0095] 7. Sample recovery experiment

[0096] Take about 0.25g of JGC-003 sample, weigh it accurately, add a certain amount of soybean saponin Bb reference substance accurately, treat it according to the test solution preparation method to obtain the test solution, prepare 6 portions of test solution for sample recovery in parallel, and inject 5μl of each portion into the high performance liquid chromatograph for determination, and calculate the content and sample recovery rate.

[0097] 8. Sample determination

[0098] 48 batches of Gastrodia elata were determined by HPLC-ELSD method, and the peak area was taken as derivative to calculate the content of soybean saponin Bb in the test samples. The content was calculated using the formula:

[0099]

[0100] 9. Effect of different chromatographic conditions on the content of the test sample

[0101] Take 1.0 g of the test samples JGC-001-010 and JGC-001-011, accurately weigh them, and place them in a stoppered conical flask. Prepare the test sample solution according to the method in this chapter. Use C18 chromatographic columns of brands such as Thermo, Agilent, and Diamonsil (for specific models, see the previous section of this chapter) to investigate the effect of the chromatographic column on the content of soybean saponin Bb; investigate the effect of column temperature at 25, 30, and 35°C; set the flow rates to 0.8, 1.0, and 1.2 mL / min to investigate the effect of the flow rate.

[0102] The content of erythrine in Aconitum was determined by referring to the above method.

[0103] IV. Experimental Results

[0104] The results are as follows Figure 5As shown in Figure 6, the content of soybean saponin Bb in scutellaria baicalensis and scutellaria baicalensis is 0.1056±0.0679% and 0.0743±0.0591% respectively. The content of erythrine in scutellaria baicalensis is 0.009% and that in scutellaria baicalensis is 0.021%.

[0105] Example 6 - Study on the kinetic characteristics of rats

[0106] 1. Experimental Materials

[0107] The experimental instruments used in the experiment are shown in Table 8.

[0108] Table 8. Experimental instruments

[0109]

[0110] The drugs and reagents used in the experiment are shown in Table 9.

[0111] Table 9. Drugs and reagents

[0112]

[0113] The experimental animals used SPF male Sprague-Dawley rats, weighing (200±10g), were purchased from Shanghai Slake Experimental Animal Co., Ltd., with a certificate number of SCXK (Shanghai) 2018-0006. They were kept in the animal room of the Experimental Animal Center of the Department of Pharmacy of the Naval Medical University, with a temperature of 20-25°C, a humidity of 40%-60%, a 12-h light-dark alternating light cycle, and free access to food and water. This animal experiment protocol was reviewed and approved by the school's Animal Ethics Committee in accordance with the regulations for the use of experimental animals.

[0114] (II) Experimental methods

[0115] 1. UPLC-MS / MS conditions

[0116] Chromatographic conditions: Shimadzu liquid chromatography system consists of SIL-30 autosampler, LC-30 binary pump and CTO-20 column oven, and the chromatographic column is Waters CORTECS T3 (2.1×100mm, 1.6μm). The column temperature is 40℃. The mobile phase is water (A) containing 0.1% formic acid and acetonitrile (B) containing 0.1% formic acid, and the flow rate is 0.6mL / min. The gradient elution program is: 0~0.6min, 15% B; 0.6~1.5min, 15~90% B; 1.5~1.51min, 90~15% B; 1.51~2.0min, 15% B.

[0117] Mass spectrometry conditions: Compound quantitative analysis was performed using a triple quadrupole mass spectrometer in electrospray ionization mode. Source parameters were set as follows: ion spray voltage, -5500 V; temperature, 500 °C; curtain gas, 50 psi; ion source gas, 55 psi. Multiple reaction monitoring (MRM) mode was used for quantitative analysis. The ion pairs corresponding to abrinine, erythrine, schaftoside, soybean saponin Bb and verapamil are m / z 219.1-188.1, m / z 247-146.1, m / z 565.2-528.9, m / z 941-615.5, and m / z 455.3-165.1, respectively, and the corresponding declustering voltages and collision energy values ​​are: abrinine 190, 35 V; erythrine 210, 30 V, schaftoside 230, 40 V; soybean saponin Bb 250, 45 V in negative ion mode; internal standard (IS) verapamil 100, 35 V in positive ion mode.

[0118] 2. Preparation of Herba Corydalis Extract

[0119] Take 500g of the powder of the herb, weigh an appropriate amount into a round-bottom flask, add 10 times the amount of 95% ethanol solution, reflux extract twice (1h each time), and recover the solvent to obtain 55g of ethanol extract. Determine the content of the main components in the extract and calculate the dosage.

[0120] 3. Preparation of stock solution and working solution

[0121] Accurately weigh four reference substances, including abrin (batch number: 111808-202003), erythrine (batch number: 112058-202001), schaftoside (batch number: 111912-202204), and soybean saponin Bb (batch number: wkq 22102507), and place them in volumetric flasks, add acetonitrile to the mark, and prepare a 1.0 mg / mL stock solution. Take equal volumes of the above stock solutions and mix them into reference substance stock solutions, and then dilute the reference substance mixed stock solutions with methanol to different concentrations of working solutions such as 500 ng / mL, 400 ng / mL, 250 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 10 ng / mL, and 5 ng / mL. The internal standard IS working solution was prepared with methanol as the solution at 0.1 μg / mL. All solutions were stored at 4°C until use and brought to room temperature before use.

[0122] 4. Calibration standards and quality control samples

[0123] Take 50 μl of working solution of different concentrations and dry it under nitrogen flow. Redissolve it with 50 μl of blank rat plasma to obtain calibration samples (500 ng / mL, 250 ng / mL, 100 ng / mL, 50 ng / mL, 25 ng / mL, 10 ng / mL and 5 ng / mL). In addition, prepare quality control (QC) samples (12.5 ng / mL, 200 ng / mL, 400 ng / mL) using the same method.

[0124] 5. Animal grouping and plasma sample content determination

[0125] Animal experiments were conducted in accordance with the "Regulations on the Administration of Experimental Animals" promulgated by the State Science and Technology Commission of the People's Republic of China. Five male SD rats were fed adaptively for one week, fasted overnight (10-14h) before administration, and returned to food 4h after administration. They were weighed before administration, and the dosage was calculated based on their body weight. The extract powder of Glechoma longituba (3g) was dissolved in an appropriate amount of 0.5% sodium carboxymethylcellulose (CMC-Na) aqueous solution to prepare a drug-containing solution with a concentration of 0.03g / mL. With reference to the clinical human dosage (2.5g / d), the dosage of 0.3g / kg was converted into a rat dosage according to the ratio of human and rat body surface area. The dosage of 0.3g / kg is equivalent to 0.27mg / kg of abrinine, 0.14mg / kg of erythrine, 3.06mg / kg of schaftoside and 6.1mg / kg of soybean saponin Bb, and the dosage is 10mL / kg. Single oral administration.

[0126] Before administration, 0.2 mL of blood was collected from the tail vein and placed in a 1.5 mL polyethylene centrifuge tube containing heparin, which was placed on ice. After administration, 0.2 mL of blood was collected from the tail vein at 0.083 h, 0.25 h, 0.5 h, 1.0 h, 2.0 h, 4.0 h, 8.0 h, 12.0 h, and 24.0 h, which was placed in a 1.5 mL polyethylene centrifuge tube containing heparin, which was placed on ice. The blood sample was immediately centrifuged at 3 000 g for 10 min to obtain a plasma sample. The plasma sample was placed on ice and stored at -20 ° C for further experiments.

[0127] Plasma samples were thawed at room temperature, 50 μl of plasma samples, standard curve samples or QC samples were placed in 1.5 mL centrifuge tubes, and 200 μl of acetonitrile solution containing internal standard IS (0.5 μg / mL) was added to the centrifuge tubes. After vortex shaking for 2.0 min and centrifugation at 14000 g for 10 min, 2 μl of the supernatant was injected into the UPLC-MS / MS system for analysis. According to the experimental conditions under the establishment and methodological investigation of the UPLC-MS / MS method, the changes in the contents of abrin, erythrine, schaftoside and soybean saponin Bb in the plasma of rats after oral administration were determined at different times, and the pharmacokinetic changes of the above four main components were analyzed.

[0128] 6. Method Validation

[0129] According to the guidelines issued by the State Food and Drug Administration and the method validation guidelines of the Chinese Pharmacopoeia (Volume 4), the established method was fully validated for specificity, linearity, accuracy, precision matrix effect, extraction recovery and stability. The entire validation experiment was carried out for three consecutive days, and each validation experiment was accompanied by calibration standards and 6 QC plasma samples.

[0130] (1) Specificity experiments and transfer effects

[0131] Specificity was evaluated by analyzing the chromatograms of blank plasma from 6 rats, blank rat plasma spiked with standards, and plasma samples after oral administration. The transfer effect was evaluated by injecting blank plasma samples at the highest concentration immediately after the calibration standard samples (500 ng / mL). The interference of the blank plasma sample should be less than 20% of the response of the lower limit of quantification (LLOQ) of the analyte and 5% of the response of the internal standard IS.

[0132] (2) Linearity and limit of quantitation

[0133] The ratio of the peak area to the IS peak area and the mass concentration is 1 / x 2 Draw a calibration curve. Evaluate the linearity through the standard curve, and the correlation coefficient r should be >0.99. Define the lowest concentration on the calibration curve with a signal-to-noise ratio >10 as the lower limit of quantitation. The relative standard deviation (RSD) of the quantitation limit sample is within 20%, and the accuracy is within the range of 80-120%.

[0134] (3) Accuracy and precision experiments

[0135] Precision is expressed as RSD, and accuracy is expressed as relative error (RE). Intra-day and inter-day precision and accuracy were evaluated by repeating the analysis of LLOQ and QC samples of 6 samples on the same day and three consecutive days. In terms of precision and accuracy, the errors should not exceed ±15% and should not exceed ±20%, respectively.

[0136] (4) Extraction recovery and matrix effect

[0137] The extraction recovery rate was evaluated by comparing the peak area ratio of the analyte extracted from the three concentrations of QC samples with the peak area obtained by dissolving the analyte in the treated blank plasma supernatant. Rat blank plasma was taken to prepare QC sample solutions with low, medium and high mass concentrations. After pretreatment, the samples were measured according to the conditions under item 2, and the peak area (A) was recorded. Six samples of each mass concentration were prepared. Another blank plasma was taken. After pretreatment, low, medium and high mass concentration reference solutions were added to the supernatant, respectively, and then measured according to the conditions under item 2. The peak area (B) was recorded and the extraction recovery rate was calculated as A / B×100%. The reference solutions of each component of the above low, medium and high mass concentrations were taken and measured according to the conditions under item 2. The peak area (C) was recorded and the matrix effect was calculated as B / C×100%.

[0138] (5) Stability test

[0139] The stability of the method was evaluated by analyzing six replicate samples of three concentrations of QC samples under four storage conditions, including short-term stability (storage at room temperature for 8 h), long-term stability (storage at -20°C for 2 weeks), three freeze-thaw cycle stability, and post-preparation stability (storage at 4°C for 12 h), with an acceptable error within ±15%.

[0140] 7. Data Analysis

[0141] Analyst 1.6 software from AB SCIEX, USA, was used for liquid chromatography mass spectrometry data acquisition and instrument control, and the blood concentration of each analyte at different time points was calculated based on the corresponding calibration curve. Phoenix WinNolin software (Certara, USA) was used for non-compartmental model analysis and calculation of pharmacokinetic parameters AUC 0-t (area under the concentration-time curve to the last detectable time point), AUC 0-∞ (the area under the concentration-time curve to infinity) and t 1 / 2 (elimination half-life), maximum plasma concentration (C max ) and the time required to reach the maximum blood concentration (T max ) is calculated from the observed values.

[0142] (III) Experimental results

[0143] The UPLC-MS / MS method was used to analyze the pharmacokinetic process of schizonepeta alkaloids, erythrine, schaftoside, soybean saponin Bb and other components in the extract of schizonepeta tenuifolia in rats. The results showed that the blood drug concentration-time curve was as follows Figure 7The pharmacokinetic parameters calculated by the non-compartmental model are shown in Table 3-2-1. After administration, the four main components were rapidly absorbed into the blood and detected at the first time point (0.083h). The lowest dose of erythrina alkaloids was detected, while the highest exposure was detected. The high exposure may be related to high permeability and good absorption rate. The concentration of erythrina alkaloids in plasma reached a peak at 2h, and the half-life was long (t 1 / 2 =9.83h) indicates that its elimination rate in the body is slow and its residence time is long (MRT=8.8h). The extract of Gastrodia elata has the highest content of soybean saponin Bb, but the lowest exposure. Soysaponin Bb can only be detected at a few time points, and the concentration reaches a peak at 2h. This may be due to the large molecular weight, high polarity, and poor absorption of soybean saponin Bb in the body. In addition, potential hydrolysis in the gastrointestinal tract and poor permeability of the intestinal epithelial membrane may also be the reasons for the low exposure.

[0144] Table 10. Pharmacokinetic parameters of active ingredients of Glechoma longituba in rat plasma ( n=6)

[0145]

[0146] Note: 1 / 2 , elimination half-life; C max , maximum plasma concentration; T max , time to peak concentration; AUC 0–t , the area of ​​the blood drug concentration-time curve when it reaches the last detectable point; AUC 0–∞ , the area under the plasma drug concentration-time curve to infinity; MRT, mean residence time; T max *, expressed as median value.

[0147] Example 7 - Preparation of soybean saponin Bb, erythrine and composition

[0148] 2kg of Gastrodia elata was crushed, sieved, and mixed with 8 times the amount of choline chloride-urea DES (molar ratio of 1:2), and subjected to ultrasonic assisted extraction, with ultrasonic power of 200-400W, extraction temperature of 60°C, and extraction time of 45min. After filtration, the filtrate was loaded onto a D101 or XAD-16 macroporous resin column, and impurities were eluted with deionized water, and then eluted with 60-80% ethanol solution. The eluate was concentrated and dried to obtain the soybean saponin Bb product, which was further purified by preparative HPLC to obtain the pure soybean saponin Bb product.

[0149] 2 kg of dried medicinal materials of Acanthopanax radiata were crushed and passed through a 50-mesh sieve. The powder was mixed with 10 times the amount of 70% ethanol aqueous solution, and ultrasonically extracted at a power of 200 W for 60 minutes. Filtered, the filtrate was concentrated under reduced pressure to obtain an extract. The extract was dissolved in water, loaded on a pretreated HPD100 macroporous resin column, and gradient eluted with water, 15% ethanol aqueous solution, and 40% ethanol aqueous solution in turn, and the 40% ethanol aqueous solution eluate was collected. The 40% ethanol aqueous solution eluate was concentrated under reduced pressure, an appropriate amount of 95% ethanol was added, and it was allowed to stand at 4°C for crystallization, filtered, and dried to obtain erythrine.

[0150] According to the contents of soybean saponins and erythrine in the herb of Acanthopanax ovata and the in vivo pharmacokinetic parameters in 6 cases of Acanthopanax ovata, 35 parts of soybean saponins and 3 parts of erythrine were taken in a mass ratio and mixed evenly to obtain a composition. Network pharmacology and molecular docking showed that the composition had a good effect in preventing and treating liver damage and liver fibrosis. The docking results are shown in Table 11.

[0151] Table 11. Main component-target molecule docking results

[0152]

Claims

1. Use of erythrinaine, soybean saponin Bb and a composition thereof in the preparation of a medicament for treating acute liver injury and liver fibrosis, wherein the composition is composed of erythrinaine and soybean saponin in a mass ratio of 3:

5.

2. The use according to claim 2, characterized in that: Soysaponin Bb, erythrine and their combination can significantly reduce the elevated ALT and AST levels in the serum of mice with acute liver injury model, slow down liver tissue lesions, and prevent and treat liver injury and fibrosis by regulating the body's immunity and antioxidant capacity.

3. The use according to claim 1 or 2, characterized in that Erythrina alkaloids, soybean saponin Bb and the composition thereof are prepared into tablets, capsules, granules, oral liquids, drop pills, pills, ointments, mixtures, patches, gels, chewable tablets, lozenges, chewing gums or teas.

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