阅读说明：本技术 一种抗肝纤维化中药复方组合物及其应用 (Anti-hepatic fibrosis traditional Chinese medicine compound composition and application thereof ) 是由 郑仕中 谭善忠 张峰 王飞虾 陈利 连娜琦 于 2021-01-28 设计创作，主要内容包括：本发明公开了一种具有抗肝纤维化作用的中药复方及其应用。该中药复方由下列重量份数的中药原料制成：黄芪10-30份,太子参10-30份,当归5-15份,女贞子5-15份,茯苓5-15份,炒白术10-30份,陈皮5-15份,酒黄芩5-15份,炙甘草3-10份。本发明根据肝纤维化的发病机理,采用辨证论治的方法,结合药理实验筛选组方,方中各药物科学配伍后,临床上对慢加急性肝衰竭肝衰竭患者有良好的保肝作用,体内对肝纤维化具有良好防治作用和效果,体外抑制活化型肝星状细胞增殖,保护肝细胞损伤的功效,具有良好的抗肝纤维化的作用,是一种非常有前景的中药复方,具有较好的市场推广价值。(The invention discloses a Chinese herbal compound with anti-hepatic fibrosis effect and application thereof. The traditional Chinese medicine compound is prepared from the following traditional Chinese medicine raw materials in parts by weight: 10-30 parts of astragalus membranaceus, 10-30 parts of radix pseudostellariae, 5-15 parts of angelica sinensis, 5-15 parts of glossy privet fruit, 5-15 parts of poria cocos, 10-30 parts of fried bighead atractylodes rhizome, 5-15 parts of dried orange peel, 5-15 parts of wine-processed scutellaria baicalensis and 3-10 parts of honey-fried licorice root. According to the pathogenesis of hepatic fibrosis, the invention adopts a method of treatment based on syndrome differentiation and combines with a pharmacological experiment to screen a formula, and after the medicines in the formula are scientifically mixed, the invention has good liver protection effect on patients with chronic and acute hepatic failure and hepatic failure clinically, has good prevention and treatment effect on hepatic fibrosis in vivo, has the efficacy of inhibiting the proliferation of activated hepatic stellate cells in vitro and protecting hepatic cell injury, has good effect of resisting hepatic fibrosis, is a traditional Chinese medicine compound with very promising prospect and has better market popularization value.)

1. The anti-hepatic fibrosis traditional Chinese medicine compound composition is characterized by being prepared from the following traditional Chinese medicine raw materials in parts by weight: 10-30 parts of astragalus membranaceus, 10-30 parts of radix pseudostellariae, 5-15 parts of angelica sinensis, 5-15 parts of glossy privet fruit, 5-15 parts of poria cocos, 10-30 parts of fried bighead atractylodes rhizome, 5-15 parts of dried orange peel, 5-15 parts of wine scutellaria baicalensis and 3-10 parts of honey-fried licorice root.

2. The traditional Chinese medicine compound composition of claim 1, which is prepared from the following traditional Chinese medicine raw materials in parts by weight: 30 parts of astragalus membranaceus, 30 parts of radix pseudostellariae, 10 parts of angelica sinensis, 10 parts of glossy privet fruit, 10 parts of poria cocos, 30 parts of fried bighead atractylodes rhizome, 10 parts of dried orange peel, 10 parts of wine-processed scutellaria baicalensis and 3 parts of honey-fried licorice root.

3. The compound traditional Chinese medicine composition of claim 1, wherein the preparation method comprises the following steps:

(1) weighing raw materials, adding water 1-10 times of the weight of the raw materials, boiling for 45-60min, and filtering to obtain a first liquid medicine; adding water 1-9 times the weight of the raw materials, decocting for 35-55min, and filtering to obtain a second medicinal liquid; adding water 1-8 times the weight of the raw materials, decocting for 30-50min, and filtering to obtain a third medicinal liquid;

(2) mixing the first liquid medicine, the second liquid medicine and the third liquid medicine prepared in the step (1), and concentrating to obtain a concentrated solution with the relative density of 1.02-1.25, namely a traditional Chinese medicine concentrated solution;

(3) freezing the traditional Chinese medicine concentrated solution prepared in the step (2) at the temperature of-10 ℃ to-50 ℃ to form a solid state, and then freezing and vacuum-drying under the vacuum of 1.3-13Pa to obtain the powdery traditional Chinese medicine compound composition.

4. The compound traditional Chinese medicine composition according to any one of claims 1 to 3, wherein the compound traditional Chinese medicine composition prepared from each raw material medicine and a pharmaceutically acceptable carrier are prepared into oral liquid, granules, capsules or tablet dosage forms.

5. The use of a compound Chinese medicinal composition according to any one of claims 1 to 3 in the preparation of a medicament for the treatment of liver fibrosis.

6. The compound traditional Chinese medicine composition of any one of claims 1 to 3 for preparing a medicine for treating liver injury of patients with chronic plus acute liver failure.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a traditional Chinese medicine compound with an anti-hepatic fibrosis effect and application thereof.

Background

Hepatic fibrosis is a common pathological process in the development of various chronic liver diseases, and the excessive proliferation and abnormal deposition of extracellular matrix components in liver tissues are caused by various reasons, so that the structural or functional abnormality of the liver is further caused, the disease condition is developed, liver cirrhosis can be formed, the functional disorder and failure of the liver are caused, and the life and health are seriously damaged. Delay and even reverse hepatic fibrosis, and has important significance for reducing the occurrence of serious complications and improving the life quality of patients.

The liver is used as a main organ for disease attack, and has the physiological characteristics that the liver is unique, the body yin uses yang, the liver stores blood mainly, the body yin nourishes liver qi, the body yang regulates qi activity mainly by dredging and clearing away the pathogenic yang, the secretion and excretion of bile can be promoted, and the transportation and transformation of the spleen and stomach can be promoted; spleen is restricted by liver, liver disease is most apt to transmit spleen, and abnormal liver and spleen can cause spleen qi deficiency, which affects normal physiological functions of spleen; the liver, blood and kidney essence are generated and nourished by food essence through the transportation and transformation of the spleen and the stomach, and simultaneously, the liver, blood and kidney essence are bred mutually, and the deficiency of liver yin and kidney yin can be further affected, so that the deficiency of liver and kidney is caused. The traditional Chinese medicine mainly treats the hepatic fibrosis by promoting blood circulation to remove blood stasis and supplementing qi and nourishing yin as auxiliary materials. However, the differentiation of liver fibrosis is well known, and there is no unified and standardized standard, and the symptoms are often compatible, and there is no medicine with obvious curative effect on liver fibrosis.

Disclosure of Invention

The invention provides a Chinese herbal compound extract with anti-hepatic fibrosis effect, a pharmaceutically acceptable capsule, tablet, granule or oral liquid dosage form medicament, a preparation method thereof and application thereof in preparing medicaments for treating hepatic fibrosis.

The invention provides a traditional Chinese medicine compound composition with an anti-hepatic fibrosis effect, which is prepared from the following traditional Chinese medicine raw materials in parts by weight: 10-30 parts of astragalus membranaceus, 10-30 parts of radix pseudostellariae, 5-15 parts of angelica sinensis, 5-15 parts of glossy privet fruit, 5-15 parts of poria cocos, 10-30 parts of fried bighead atractylodes rhizome, 5-15 parts of dried orange peel, 5-15 parts of wine-processed scutellaria baicalensis and 3-10 parts of honey-fried licorice root.

Preferably, the traditional Chinese medicine compound composition is prepared from the following traditional Chinese medicine raw materials in parts by weight: 30 parts of astragalus membranaceus, 30 parts of radix pseudostellariae, 10 parts of angelica sinensis, 10 parts of glossy privet fruit, 10 parts of poria cocos, 30 parts of fried bighead atractylodes rhizome, 10 parts of dried orange peel, 10 parts of wine-processed scutellaria baicalensis and 3 parts of honey-fried licorice root.

The invention provides a preparation method of a traditional Chinese medicine compound composition with an anti-hepatic fibrosis effect, which comprises the following steps: weighing raw material of each dose of medicinal materials of the traditional Chinese medicine compound composition, adding water, boiling, decocting, and filtering to obtain liquid medicine; repeating the above steps for three times, and mixing the filtrates to obtain crude extract; then, decocting the crude extract of the traditional Chinese medicine, and concentrating to be thick to obtain a concentrated solution of the traditional Chinese medicine; and (3) freezing and vacuum drying the traditional Chinese medicine concentrated solution to obtain a powdery traditional Chinese medicine compound extract, namely the traditional Chinese medicine compound composition.

Specifically, the preparation method of the traditional Chinese medicine compound composition comprises the following steps:

(1) weighing the medicinal materials of the Chinese medicinal compound composition, adding water, boiling, decocting, and filtering to obtain a first liquid medicine; adding water, decocting, and filtering to obtain a second medicinal liquid; adding water, decocting, and filtering to obtain a third medicinal liquid;

(2) mixing the first liquid medicine, the second liquid medicine and the third liquid medicine prepared in the step (1) to obtain a crude traditional Chinese medicine extract, and decocting and concentrating the crude traditional Chinese medicine extract to obtain a thick traditional Chinese medicine concentrated solution;

(3) and (3) taking the traditional Chinese medicine concentrated solution prepared in the step (2), freezing and drying in vacuum to obtain a powdery traditional Chinese medicine compound extract, namely the traditional Chinese medicine compound composition.

In a specific embodiment, the preparation method is as follows:

(1) weighing crude drug raw materials of each dose of medicinal materials of the traditional Chinese medicine compound composition, adding water with the amount of 1-10 times (preferably 10 times) of the weight of the raw materials, boiling for 45-60min (preferably 60min), and filtering out a first liquid medicine after decocting; adding water 1-9 times (preferably 9 times) the weight of the raw materials, decocting for 35-55min (preferably 45min), and filtering to obtain second medicinal liquid; adding water 1-8 times (preferably 8 times) the weight of the raw materials, decocting for 30-50min (preferably 40min), and filtering to obtain a third medicinal liquid;

(2) mixing the first liquid medicine, the second liquid medicine and the third liquid medicine prepared in the step (1) to obtain a crude traditional Chinese medicine extract, and decocting and concentrating the crude traditional Chinese medicine extract to obtain a concentrated solution with the relative density of 1.02-1.25, namely a concentrated traditional Chinese medicine solution;

(3) freezing the traditional Chinese medicine concentrated solution prepared in the step (2) to be solid at (-10 ℃ to-50 ℃), and then freezing and vacuum-drying under vacuum (1.3-13Pa) to obtain a powdery traditional Chinese medicine compound extract, namely the traditional Chinese medicine compound composition.

The invention also provides a pharmaceutical composition, which comprises the traditional Chinese medicine compound composition and pharmaceutically acceptable auxiliary materials, and the pharmaceutical composition can be prepared into capsules, tablets, granules or oral liquid.

The invention also provides application of the anti-hepatic fibrosis traditional Chinese medicine compound composition or the pharmaceutical composition in preparing a medicine for treating hepatic fibrosis.

The traditional Chinese medicine compound composition or the pharmaceutical composition has the effects of clinically protecting the liver function of patients with chronic and acute hepatic failure, treating the pathological process of hepatic fibrosis of mice induced by carbon tetrachloride in vivo, inhibiting the proliferation of activated hepatic stellate cells and protecting hepatic cell injury.

The invention also provides the effect of the anti-hepatic fibrosis traditional Chinese medicine compound composition in preparing liver injury for treating patients with chronic and acute hepatic failure.

The traditional Chinese medicine compound composition or the pharmaceutical composition has the effect of protecting the liver function of patients with chronic and acute liver failure clinically.

Under the guidance of the theory of traditional Chinese medicine, the invention performs treatment based on syndrome differentiation, combines the pathogenesis of hepatic fibrosis, and performs compatibility and formula on the basis of the overall treatment concept of invigorating qi and strengthening spleen, treating both symptoms and root causes, and strengthening body resistance to consolidate the constitution and eliminating pathogenic factors in traditional Chinese medicine.

The Chinese herbal compound with the anti-hepatic fibrosis effect is obtained by screening through literature retrieval and experimental verification and creative labor contribution of an inventor, and nine Chinese herbal medicines of astragalus membranaceus, radix pseudostellariae, angelica sinensis, glossy privet fruit, poria cocos, fried bighead atractylodes rhizome, dried orange peel, wine-processed scutellaria baicalensis and honey-fried licorice root are selected for composing the formula.

The invention has the beneficial effects that:

compared with the prior art, the traditional Chinese medicine compound with the anti-hepatic fibrosis effect provided by the invention has the following advantages:

(1) under the guidance of the theory of traditional Chinese medicine, the traditional Chinese medicine compound selects nine traditional Chinese medicines of astragalus, radix pseudostellariae, angelica, glossy privet fruit, tuckahoe, fried bighead atractylodes rhizome, dried orange peel, wine scutellaria baicalensis and honey-fried licorice root for composing the formula: the radix astragali is used as a monarch drug in the prescription, the radix pseudostellariae is mainly tonifying, and can tonify qi and invigorate spleen, promote the production of body fluid, help the radix astragali strengthen body resistance and tonify deficiency and improve deficiency of both qi and yin; the angelica, the salvia miltiorrhiza, the motherwort and the peach kernel are ministerial drugs, can nourish blood and activate blood, are compatible with the astragalus, can tonify qi and activate blood, and can lead tangible blood to be born in intangible qi; the rhizoma atractylodis macrocephalae, the poria cocos, the rheum officinale, the gardenia and the glossy privet fruit are adjuvant drugs, the rhizoma atractylodis macrocephalae and the poria cocos are capable of tonifying spleen and promoting diuresis, the compatible oriental wormwood is capable of clearing heat and eliminating dampness and benefiting liver and gallbladder, the radix scutellariae is less capable of clearing heat and cooling blood, the blood stasis removing effect is realized, the bleeding is prevented, the gardenia is capable of clearing heat and promoting diuresis, the glossy privet fruit is capable of nourishing liver and kidney yin essence; prepared licorice root, radix Glycyrrhizae Praeparata, is used as a guiding drug to regulate the effects of the drugs in the recipe. The whole formula has the effects of clearing heat, activating blood, promoting diuresis, removing jaundice, tonifying qi, invigorating spleen, treating both principal and secondary aspects of diseases, and strengthening body resistance, consolidating constitution and eliminating pathogenic factors. The traditional Chinese medicine compound provided by the invention has the effect of protecting the liver of a patient suffering from chronic acute hepatic failure, has the effect of resisting hepatic fibrosis, and can obviously inhibit the hepatic fibrosis progression of a mouse induced by carbon tetrachloride.

(2) The traditional Chinese medicine compound provided by the invention has an anti-hepatic fibrosis effect, and can reduce the expression of liver injury related factors in a mouse hepatic fibrosis model induced by carbon tetrachloride, wherein the liver injury related factors refer to alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), combined bilirubin (DBIL) and liver index; the traditional Chinese medicine compound provided by the invention can also relieve the carbon tetrachloride-induced structural disorder of the liver and inhibit the collagen deposition in the liver under the induction of carbon tetrachloride. The traditional Chinese medicine compound composition provided by the invention can inhibit proliferation of activated hepatic stellate cells and inhibit collagen generation in vitro; protecting liver cells from LPS + D-Gal induced damage, and down-regulating LPS + D-Gal induced liver damage factor expression, wherein the liver damage related factors refer to alanine Aminotransferase (ALT), aspartate Aminotransferase (AST) and Lactate Dehydrogenase (LDH). The traditional Chinese medicine compound provided by the invention has the effect of protecting the liver function of patients with chronic acute liver failure clinically. The traditional Chinese medicine compound composition provided by the invention has definite curative effect, makes a new breakthrough in the aspect of hepatic fibrosis treatment, and can fill the blank in the field of the development of the existing hepatic fibrosis treatment medicines in China to a certain extent.

(3) The traditional Chinese medicine compound with the anti-hepatic fibrosis effect provided by the invention has the advantages of low cost of the preparation method, environmental protection and realization of industrial mass production.

(4) The Chinese herbal compound with the anti-hepatic fibrosis effect can be prepared into various dosage forms with pharmaceutically acceptable carriers, is convenient for clinical administration, and is expected to be developed into a new anti-hepatic fibrosis medicine.

Drawings

FIG. 1 is a schematic diagram showing the effect of a Chinese herbal compound of the present invention on the proliferative activity of hepatic stellate cells and the protective effect of LPS + D-Gal induced hepatocyte damage;

a MTT is used for determining the influence of the traditional Chinese medicine compound of the experimental example on the cell viability of HSC-LX 2.

B MTT measures the influence of the traditional Chinese medicine compound of the experimental example on the hepatocyte activity under the LPS + D-Gal treatment.

C light microscope observation the effect of the traditional Chinese medicine compound of the experimental example on the shape of the liver cells under the LPS + D-Gal treatment.

FIG. 2 is a schematic diagram of the index of the compound Chinese medicine of the experimental example of the present invention that can significantly reduce the damage of LPS + D-Gal induced liver cells;

the kit A is used for determining the influence of the traditional Chinese medicine compound of the experimental example on the ALT content in the hepatic cells under the LPS + D-Gal treatment.

The kit B is used for determining the influence of the traditional Chinese medicine compound of the experimental example on the AST content of the hepatic cells under the LPS + D-Gal treatment.

And C, determining the influence of the traditional Chinese medicine compound of the experimental example on the LDH content in the hepatic cells under the LPS + D-Gal treatment by using the kit.

FIG. 3 is a schematic diagram showing the effects of different concentrations of the compound Chinese medicine of the experimental example of the present invention on the liver pathology of mouse liver fibrosis induced by carbon tetrachloride and collagen deposition. The mice were grouped as: 1 represents a normal group; 2 represents a carbon tetrachloride-induced liver fibrosis model group; 3 represents the hepatic fibrosis model and the traditional Chinese medicine compound low-dose treatment group of the experimental example; 4 represents a carbon tetrachloride-induced liver fibrosis model group; 5 represents a hepatic fibrosis model and a traditional Chinese medicine compound middle dose treatment group of the experimental example; 6 represents a carbon tetrachloride-induced liver fibrosis model group; and 3, a liver fibrosis model and a traditional Chinese medicine compound high-dose treatment group of the experimental example of the invention.

Liver photographing, HE staining, sirius red staining and Masson staining show that the traditional Chinese medicine compound of the experimental example of the invention has influence on carbon tetrachloride-induced hepatic fibrosis progression of mice.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and drawings, and the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.

The Chinese medicinal compound composition with the anti-hepatic fibrosis effect has the following specific formula and dosage of each compound:

example 1: formula 1 comprises astragalus root 20g pseudostellaria root 10g Chinese angelica root 10g ligustrum japonicum 10g poria 25g white atractylodes rhizome 10g dried orange peel 8g wine scutellaria root 8g honey-fried licorice root 5g

Example 2: formula 2 astragalus 25g radix pseudostellariae 15g angelica 12g ligustrum lucidum 10g poria 25g white atractylodes rhizome 10g dried orange peel 8g wine scutellaria 8g honey-fried licorice root 3g

Example 3: formula 3 astragalus 30g radix pseudostellariae 10g angelica 10g ligustrum lucidum 10g poria 30g bighead atractylodes rhizome 10g dried orange peel 10g wine scutellaria 10g honey-fried licorice root 3g

The preparation methods of the three formulas are implemented according to the following methods:

(1) weighing raw material medicines of each medicine of the traditional Chinese medicine compound composition, adding water with the weight 10 times of that of the raw material medicines, boiling, decocting for 60min, and filtering out a first liquid medicine; adding water 9 times the weight of the raw materials, decocting for 45min, and filtering to obtain a second medicinal liquid; adding water 8 times the weight of the raw materials, decocting for 40min, and filtering to obtain a third medicinal liquid;

(2) mixing the first liquid medicine, the second liquid medicine and the third liquid medicine prepared in the step (1) to obtain a crude traditional Chinese medicine extract, and decocting and concentrating the crude traditional Chinese medicine extract to obtain a thick traditional Chinese medicine concentrated solution;

(3) and (3) taking the traditional Chinese medicine concentrated solution prepared in the step (2), freezing and drying in vacuum to obtain a powdery traditional Chinese medicine compound extract, namely the traditional Chinese medicine compound composition.

In order to verify the preferable effect of the formula ratios of the components of the traditional Chinese medicine compound, the formula 1, the formula 2 and the formula 3 are prepared into the traditional Chinese medicine compound composition, and parallel experiments are carried out, so that the traditional Chinese medicine compound composition prepared according to the formula 3 has the best effect, and the formula ratio of the component of the formula 3 is determined to be the optimal proportioning scheme. The Chinese herbal compound proportions in the following examples are all carried out according to formula 3.

Example 4: the provided Chinese herbal compound has the effect of protecting liver clinically.

The technical method comprises the following steps:

1. 97 patients with chronic acute liver failure who were admitted to the Nanjing Hospital affiliated to Nanjing university of traditional Chinese medicine from 8 months to 2016 months in 2013 were included.

2. Grouping and administration of drugs

The patients were randomly divided into control and administration groups. Control group (conventional treatment group in western medicine): according to the 'liver failure diagnosis and treatment guideline' 2012, the patients in this group were treated with conventional western medicine only, and were not treated with Chinese herbal medicines and related Chinese patent medicines. The tablets prepared in example 3 of the present invention were administered to the administration group by water decoction, 1 dose/day, twice daily oral administration, and 1 year of treatment.

3. Indexes in serum liver injury indexes ALT, TBIL, Alb, Cr, PTA and Na are detected by using a biochemical detection kit, and an experimental operation method is according to the specification of the biochemical detection kit.

TABLE 1 comparison of laboratory examinations in two groups before treatment

TABLE 2 comparison of laboratory examination differences (+ -s) in two groups after treatment

The experimental results are shown in table 1 and table 2, and the provided traditional Chinese medicine compound has an obvious protective effect on the liver of patients with chronic and acute liver failure.

Example 5: the invention inhibits the proliferation of activated hepatic stellate cells in vitro and inhibits the hepatic cell damage induced by LPS + D-Gal.

The technical method comprises the following steps:

1. culture of cells

Human hepatic stellate cell LX2 and human hepatic cell LO2 were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody. All cells were cultured in a 37 ℃ incubator (humidity 95%, 5% CO)₂Concentration).

2. MTT method for measuring cell proliferation

The cells in log phase were digested and then differentiated according to their proliferation rate (hepatic stellate cells 2X 10)⁴1.5X 10 hepatocyte cells per well⁴One/well) to a 96-well plate, 100 μ L of medium per well. After 24h, hepatic stellate cell group was addedAt different concentrations, the control group was supplemented with an equal amount of PBS and each group was plated with 6 replicates. After the liver cell group LPS (10 mug/mL) and D-Gal (10mg/mL) are induced for 4 hours, Chinese herbal compound extracts with different concentrations are added, PBS with the same amount is added into a control group, and each group is provided with 6 multiple holes. Continuing culturing for 24h, adding 20 μ L MTT solution (5mg/ml, i.e. 0.5% MTT), continuing culturing for 4h, sucking supernatant, adding 200 μ L dimethyl sulfoxide into each well, shaking on a shaker at low speed for 10min to dissolve the crystal, measuring absorbance of each well at 490nm of an enzyme linked immunosorbent detector, setting zero-setting wells (culture medium, MTT, dimethyl sulfoxide), setting 6 wells for each group (cells, dissolution medium, culture solution, MTT, dimethyl sulfoxide), and calculating inhibition rate [ (control-blank) - (administration-blank) ]]/(control-blank). times.100%.

The results of fig. 2 show that the Chinese herbal compound of the experimental example of the invention inhibits the proliferation of hepatic stellate cells and is dose-dependent. In addition, the traditional Chinese medicine compound of the experimental example has a protective effect on hepatic cells induced by LPS + D-Gal in vitro.

Example 6: the invention protects liver cell damage.

The technical method comprises the following steps:

1. kit for detecting ALT, AST and LDH

20000 hepatocytes are inoculated into a 6-well plate, the compound of example 3 with different concentrations is added after the cells adhere to the wall, a cell culture medium is extracted, the kit detects the ALT, AST and LDH content in the culture medium, and each group of experiments is repeated for three times. The experimental operation method follows the instruction of the biochemical detection kit.

The results of FIG. 3 show that the compound Chinese medicine of the experimental example of the invention inhibits the hepatocyte from being expressed by the hepatocyte injury factor induced by LPS + D-Gal, and is dose-dependent.

Example 7: the invention relieves the mouse liver injury induced by carbon tetrachloride.

The technical method comprises the following steps:

1. carbon tetrachloride-induced mouse hepatic fibrosis model establishment

SPF grade male ICR mice (8 weeks old) weighing 20-25g were acclimatized by one week prior to the experiment.Before molding, the mice were kept under standard conditions with a room temperature of 20 + -2 deg.C, a relative humidity of 40 + -5%, and a light/dark cycle of 12 hours. Using carbon tetrachloride (CCl)₄) And olive oil [1:10(v/v)]The mixture of (2) induces hepatic fibrosis in mice by intraperitoneal injection (0.05mL/10g body weight).

2. Detection of serum ALT, AST, DBIL

The serum liver injury indexes ALT, AST and DBIL are detected by using a biochemical detection kit, and the experimental operation method is according to the specification of the biochemical detection kit. The results are shown in Table 3.

TABLE 3 influence of the Chinese herbal compound of the experimental examples of the present invention on the index of serum liver injury in carbon tetrachloride-induced mouse hepatic fibrosis model

And # P < 0.05, # P < 0.01, in the hepatic fibrosis model group compared to the normal group, and # in the treated group compared to the hepatic fibrosis model group.

The results in table 3 show that with the increase of the concentration of the compound traditional Chinese medicine in the experimental example of the invention, ALT, AST and DBIL in the serum of the mouse are obviously reduced compared with the carbon tetrachloride model group. The Chinese herbal compound of the experimental example of the invention is shown to lower liver injury indexes ALT, AST and DBIL in a carbon tetrachloride-induced mouse hepatic fibrosis model.

Example 8: the invention alleviates the liver fibrosis process in a liver fibrosis model.

The technical method comprises the following steps:

1. h & E staining

After the mice died at the dislocation of the cervical vertebrae, they were dissected, stripped of liver, removed of excess connective tissue, and placed in PBS for washing once. Fix well in 4% paraformaldehyde. Cutting tissue according to the coronal plane of the anatomical position of the mouse, placing the tissue in an embedding box, marking related information by a pencil, dehydrating, transparentizing and waxing the tissue, and then performing H & E staining.

2. Masson staining

Paraffin sections of liver tissue were fixed overnight with Bouin's solution or Zenker's solution and washed colorless with running water. Staining celestite blue for 5min, staining Mayer's hematoxylin for 5min, differentiating with 1% hydrochloric acid ethanol solution, washing with running water, staining with ponceau acid product red solution for 5-10min, washing with distilled water, treating with 1% phosphomolybdic acid water solution for 5min, directly staining with aniline blue or brilliant green for 5min, treating with 1% glacial acetic acid for 5min, dehydrating with 95% ethanol and anhydrous ethanol for several times, and sealing with neutral gum after transparent treatment with xylene. The images were observed under an optical microscope and photographed in randomly selected fields.

3. Dyeing with picric acid sirius red (sirius red)

And (4) dehydrating the paraffin sections of the liver tissues by a conventional method. And (5) dyeing the celestite blue solution for 5-10min, and washing with distilled water for 3 times. Dyeing with saturated bitter acid liquor for 15-30 min. And (3) differentiating and dehydrating by using absolute ethyl alcohol. Finally, xylene is used for transparent treatment, and neutral gum is used for sealing. The images were observed under an optical microscope and photographed in randomly selected fields.

The results in FIG. 3 show that carbon tetrachloride induced liver fibrosis model of mice has liver structural disorder, large amount of collagen deposition and obvious liver fibrosis pathological changes. With the increase of the dosage of the Chinese herbal compound, the structure of the liver is gradually recovered, the collagen deposition is reduced, and the hepatic fibrosis lesion is lightened. The Chinese herbal compound of the experimental example of the invention is shown to relieve the hepatic fibrosis process in a mouse hepatic fibrosis model induced by carbon tetrachloride.

The above embodiments are only for illustrating the technical concept and features of the present invention, and the purpose of the embodiments is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made in accordance with the spirit of the present disclosure are intended to be included within the scope of the present disclosure.