CN108671149B

CN108671149B - Traditional Chinese medicine composition for treating liver diseases and preparation thereof

Info

Publication number: CN108671149B
Application number: CN201810864158.1A
Authority: CN
Inventors: 裴坚毅
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-08-01
Filing date: 2018-08-01
Publication date: 2021-10-29
Anticipated expiration: 2038-08-01
Also published as: CN108671149A

Abstract

The invention discloses a traditional Chinese medicine composition for treating liver diseases, which comprises the following components in parts by weight: 100-150 parts of dried toad, 20-60 parts of saussurea involucrata, 30-90 parts of turtle shell, 100-200 parts of mulberry, 50-150 parts of dendrobium, 20-60 parts of chrysanthemum and 20-60 parts of liquorice. The traditional Chinese medicine composition can be used for treating liver diseases such as chronic liver disease, hepatic fibrosis, alcoholic fatty liver and liver cirrhosis, has the effect of reversing hepatic fibrosis, and is nontoxic, simple in components, convenient to prepare and low in cost.

Description

Traditional Chinese medicine composition for treating liver diseases and preparation thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a traditional Chinese medicine composition for treating liver diseases and a preparation thereof, and particularly relates to a traditional Chinese medicine composition for treating chronic liver diseases, hepatic fibrosis, liver cirrhosis, alcoholic fatty liver and the like.

Background

Hepatic Fibrosis (HF) is a pathological process in which the liver develops fibroconnective tissue proliferation and deposition under the action of pathogenic factors, and is mainly caused by the disruption of the balance of extracellular matrix (ECM) synthesis and degradation, resulting in increased ECM synthesis and decreased ECM degradation, and the basic pathological changes are the deposition of a large amount of ECM in the perisinus space and the change in composition, leading to the narrowing of the perisinus space and "capillarity", and also the wound healing response of the liver to chronic damage caused by different causes, which is called Hepatic fibrosis in mild cases.

The liver is chronically damaged and often results in the development of liver fibrosis, mainly manifested by hyperproliferation and abnormal deposition of extracellular matrix components, and hyperproliferation of Hepatic Stellate Cells (HSCs). Since HSCs are the major source cells of liver fibrosis ECM, their activation marks the onset of liver fibrosis. Hepatic fibrosis is the essential stage for various chronic liver diseases to develop into cirrhosis. Hepatic fibrosis is a compensatory reaction in the repair process after hepatic tissue injury, the ECM deposition amount is gradually increased and the range is gradually enlarged along with the progress of hepatic fibrosis, and the collagen fibrous tissue of severe patients extends into lobules, so that normal hepatic lobules are divided and damaged by fibrous connective tissue to form false lobules, namely liver cirrhosis. Liver cirrhosis is the uncompensated manifestation of liver fibrosis development, and the liver cirrhosis and the liver fibrosis are two different stages of chronic liver diseases.

Liver fibrosis is caused by long-term continuous damage of liver caused by hepatitis virus, schistosomiasis, alcohol, fatty liver, autoimmune disease, overwork, environmental pollution, drugs, poisons and the like, so that the etiology of the liver fibrosis is various and complex. Liver injury is the wound healing response of the liver to chronic damage caused by different causes, and mild cases are called liver fibrosis. Most pathological changes of hepatic fibrosis in human beings progress slowly, and some pathological changes take from months to years, with an average of about 3-5 years, from damage, inflammation, necrosis, and abnormal proliferation and deposition of extracellular matrix of liver cells. Because the liver has a strong compensatory function, even if the liver fibrosis is in an active stage, the clinical manifestations of the patient are atypical, and even if the patient has symptoms, the patient often lacks the characteristics. Many patients are found at physical examination or laparotomy for other diseases, even at autopsy.

The clinical manifestations of liver fibrosis are similar to the initial symptoms of cirrhosis, manifested as 1) fatigue and weakness, which is one of the early common symptoms of liver fibrosis; 2) often, the appetite is poor or nausea, vomiting, chronic indigestion abdominal flatulence, constipation or diarrhea, liver region dull pain and the like are accompanied, and some patients do not have obvious history of chronic liver diseases clinically and are discovered through further examination; 3) malnutrition, emaciation, anemia, and various vitamin deficiencies, such as nyctalopia and pachylosis; 4) bleeding and chronic hepatitis due to hypohepatia affecting the synthesis of prothrombin and other coagulation factors, the clinical manifestations of hepatic fibrosis often show spider nevus, epistaxis, gingival bleeding, purpura or bleeding spots on skin and mucous membrane, and women often have menorrhagia.

Hepatic fibrosis is a pathological change caused by chronic liver damage caused by various reasons, is represented by excessive and abnormal deposition of extracellular interstitial components in the liver and influences the function of the liver, and is a stage necessary for chronic liver diseases to develop into cirrhosis. The medical community now believes that liver fibrosis is likely to reverse to normal, while cirrhosis is not. The focus of current research is on molecule-to-molecule, molecule-to-cell, and cell-to-cell interactions. Although some progress has been made in diagnosis and treatment, there is still a lack of effective drugs.

However, reversing hepatic fibrosis is still a weak item in modern medicine, and no successful and satisfactory anti-fibrosis drug has been found in long-term clinical anti-fibrosis experimental studies on patients with hepatic fibrosis, liver cirrhosis and hepatocellular carcinoma at present. At present, the anti-hepatic fibrosis treatment is mainly started from the following steps: 1. removing the causes of hepatic fibrosis; 2. inhibiting HSC activation; 3. inducing and promoting apoptosis of activated HSCs; 4. inhibiting synthesis of ECM; 5. accelerating degradation of the ECM; 6. enhance the regeneration of the liver cells. Due to the extremely complex mechanism of formation and development of hepatic fibrosis, the currently discovered anti-hepatic fibrosis western medicines generally can only act on a few links, most of the anti-hepatic fibrosis western medicines are still in the experimental research stage at present, and the effectiveness and the safety of the anti-hepatic fibrosis western medicines need to be proved.

From the 70 s of the last century, scholars at home and abroad take traditional Chinese herbs as the breakthrough of anti-hepatic fibrosis, and the anti-hepatic fibrosis of traditional Chinese medicines has become a hot spot of the national research on preventing and treating hepatic fibrosis.

There is no name of hepatic fibrosis in TCM, and the chapter in Ling Shu Jing, Wu Xian is: "pathogenic factors in the liver will cause pain in the hypochondriac area. According to its main clinical manifestations, it belongs to the categories of hypochondriac pain, distension, jaundice, mass and feeling of fullness. Zhang's Yi Tong & Shi Qi Ji (Zhang's medical expert and accumulation) ' the accumulation of the pathogenic factors also means that healthy qi is deficient, and then "the pathogenic factors are recumbent". Day of Danxi Xin Fa Tongman: hypochondriac pain. The internal cause of liver fibrosis is healthy qi deficiency, the external cause is damp-heat epidemic toxin, the pathogenesis of the liver fibrosis is qi deficiency and blood stasis, and liver collateral stasis, and the liver fibrosis treatment method mainly activates blood circulation. The Chen Guo et al also consider the treatment of hepatic fibrosis to reverse the disease condition, i.e. mainly eliminate pathogenic factors (activate blood, eliminate dampness and reduce phlegm). In nearly more than 20 years, research on the traditional Chinese medicine in China in anti-hepatic fibrosis treatment shows obvious advantages and characteristics, in 2006, China promulgates the first national-level diagnosis and treatment guide of hepatic fibrosis by combining traditional Chinese medicine and western medicine, and the basic pathogenesis of hepatic fibrosis is defined as 'deficiency of both qi and yin and collateral obstruction by blood stasis'. The traditional Chinese medicine for treating hepatic fibrosis is a characteristic and an advantage of China, and has made certain progress and favorable achievement in the aspects of single traditional Chinese medicine and extract, traditional Chinese medicine prescription and compound, Chinese and western medicine combined treatment and the like in recent years. So far, the novel Chinese patent medicines such as the capsule for strengthening body resistance and removing blood stasis, the compound turtle shell liver softening tablet, the turtle shell decocted pill, the capsule for smoothing collaterals and soothing liver, the capsule for strengthening liver and the like are widely applied clinically.

Disclosure of Invention

The invention provides a traditional Chinese medicine composition for treating liver diseases and a preparation thereof, in particular to a traditional Chinese medicine composition for treating liver diseases such as chronic liver diseases, hepatic fibrosis, alcoholic fatty liver and liver cirrhosis, and the traditional Chinese medicine composition has the function of reversing the hepatic fibrosis.

The technical scheme of the invention is as follows:

a traditional Chinese medicine composition for treating liver diseases comprises the following components in parts by weight:

100-150 parts of dried toad, 20-60 parts of saussurea involucrata, 30-90 parts of turtle shell, 100-200 parts of mulberry, 50-150 parts of dendrobium, 20-60 parts of chrysanthemum and 20-60 parts of liquorice.

Preferably, the saussurea involucrate is saussurea involucrate.

Further preferably, the dried toad is 140 portions; 25-55 parts of saussurea involucrate; the turtle shell accounts for 34-84 parts; the mulberry is 110-186 parts; 55-139 parts of dendrobium; 25-56 parts of chrysanthemum; the liquorice is 25-56 parts.

Further preferably, the weight of the dried toad is 115-130 parts; 30-50 parts of saussurea involucrate; 36-78 parts of turtle shell; the mulberry is 115-174 parts; the dendrobium is 61-130 parts; 29-50 parts of chrysanthemum; 29-50 parts of liquorice.

Further preferably, the dried toad is 120 parts; 42 parts of saussurea involucrate; 69 parts of turtle shell; 117 parts of mulberry; 82 parts of dendrobium; 35 parts of chrysanthemum; the liquorice is 35 parts.

In the traditional Chinese medicine composition, the dried toad and the saussurea involucrata are used as monarch drugs, the turtle shell is used as ministerial drugs, the mulberry, the dendrobium and the chrysanthemum are used as adjuvant drugs, and the liquorice is used as conductant drugs.

The preparation method of the traditional Chinese medicine composition adopts the common methods in the field, such as decoction, pill, granule or tablet by further extraction and concentration.

The invention provides an extraction method of the traditional Chinese medicine composition, which comprises the following steps:

(1) firstly, crushing turtle shells and dried toads, decocting after warm immersion, filtering, and collecting filtrate;

(2) soaking carapax Trionycis and Bufo siccus in water, decocting, filtering, and collecting filtrate; the process of the step is carried out at least once;

(3) all filtrates were combined.

Preferably, the extraction method of the traditional Chinese medicine composition comprises the following steps:

crushing carapax Trionycis and dried Bufo siccus, soaking in warm water, decocting, filtering, and collecting filtrate;

soaking carapax Trionycis and Bufo siccus in water, decocting, filtering, and collecting filtrate;

decocting the residue with water, filtering, and collecting filtrate;

combining the three filtrates, continuously concentrating to obtain an extract with a relative density of 1.15-1.30 (60-80 ℃), and storing at low temperature of 0-4 ℃.

The invention greatly reduces the toxicity of the dried toad by selecting the extraction method, particularly the step of firstly decocting the turtle shell and the dried toad.

The invention also provides a traditional Chinese medicine preparation which is prepared from the traditional Chinese medicine composition and pharmaceutically acceptable auxiliary materials.

The traditional Chinese medicine composition disclosed by the invention is prepared from 7 traditional Chinese medicines such as dried toad, saussurea involucrata and the like, has the effects of activating blood and softening hard masses, nourishing yin and softening liver, and is used for treating hypochondrium fullness and distension or dull pain, epigastric stuffiness and anorexia, greasy taste, mental fatigue and hypodynamia, dry mouth, nasal and tooth bleeding, nausea and vomiting, or hypochondrium mass and hepatic fibrosis with the symptoms caused by blood blockage of liver and liver collaterals malnutrition.

The recipe is for hypochondriac pain and accumulation, and the disease is mainly localized in the liver and is caused by blood blocking the liver, yin-blood deficiency and liver collateral malnutrition. Liver meridian obstruction can cause hypochondriac distention and fullness; the obstruction for a long time can cause the accumulation and the formation of the food, and the food is accumulated below the hypochondrium, and the food can cause blood and qi stagnation below the rib, and transverse adverse flow of the food to the stomach, so that the gastric cavity is stuffy and anorexia, greasy in taste, and nausea and vomiting due to severe symptoms; liver heat produces burning and does not benefit kidney, and liver and kidney yin deficiency causes mental fatigue, dry mouth, yin blood deficiency, liver channel malnutrition causes dull pain, deficiency fire and collaterals, and nasal and gingival bleeding. So it is indicated for blood circulation promotion, hardness softening, yin nourishing and liver softening.

The traditional Chinese medicine composition provided by the invention comprises the following components:

the dried toads are pungent and cool in nature and enter liver meridian and mainly have the effects of breaking and dissipating the ecchymoses, so that the dried toads are called as 'eliminating lump and qi accumulation, eliminating lump and swelling' in the book Zheng 'and' being called as 'dispersing, capable of moving, capable of permeating and capable of softening' in the book Zheng Yao, and are used for treating the disease condition of the recipe caused by lump and accumulation; snow lotus flower is sweet, bitter and warm, enters liver and kidney, can dredge channels and activate blood when recorded in the handbook of Xinjiang Chinese herbal medicine, and can tonify yang and enrich blood when recorded in the handbook of Sichuan Chinese herbal medicine, so that the snow lotus flower can dredge channels and activate blood, is cooperated with dried toads to activate blood and soften hardness to treat the symptoms of obstruction and obstruction in the abdomen, can tonify nutrient and blood to treat the deficiency of yin and blood, and can prevent the cold and cool of all the medicines to prevent the cold congealing and blood stagnation, so the two medicines are monarch medicines together.

Turtle shell, turtle shell, turtle shell, root, rhizome, root, rhizome, root, rhizome, Chinese yam, rhizome, radix astragali, Chinese yam, radix astragali, radix et caulis et folium astragali, radix et caulis et folium astragali, radix astragali, Chinese medicinal herbs, radix astragali, Chinese magnoliaofficinalis, radix astragali, radix et caulis et folium astragali, radix astragali, Chinese magnoliae, Chinese medicinal herbs, radix et folium astragali, a cannivesii, radix et folium astragali, radix et caulis et folium wherein the four with the Chinese thoracis, namely, the Chinese thoracis, namely, the root, the herb, the root, the herb, the root, the herb, the root, the all used for the pine needles, the root, the.

Mulberry is sweet and cold, enters liver and kidney meridians, has the functions of nourishing blood, nourishing yin, tonifying liver and kidney, and is called as 'nourishing liver and kidney and filling blood' in the living diet book; the dendrobium is sweet and cold, enters kidney and stomach meridians, and has the effects of nourishing yin and promoting the production of body fluid; the white chrysanthemum is sweet and bitter, has the functions of nourishing the liver and improving eyesight, and can nourish the liver and kidney yin and blood to soften the liver, promote the production of body fluid and moisten the stomach and smooth stomach qi by combining the three medicines. The three ingredients are used as adjuvant drugs.

Licorice root, radix Glycyrrhizae coordinates the effects of the other drugs in the recipe, and acts as a guiding drug.

The traditional Chinese medicine composition combines various medicines, takes effects of both principal and secondary aspects, can not only activate blood and soften hardness to treat symptoms, but also nourish yin and soften liver to treat root causes, is cold and warm to be taken simultaneously, can not only cool and moisten to supplement yin and blood deficiency, but also warm and dredge to prevent stagnation, has the functions of activating blood and softening hardness, nourish yin and soften liver, enables liver collaterals to be smooth, accumulates to be scattered, nourishes yin and can remove various symptoms.

Compared with the prior art, the invention has the following beneficial effects:

firstly, the traditional Chinese medicine composition has definite curative effect on treating and reversing hepatic fibrosis, and is also proved by related pharmacodynamic animal tests;

secondly, the traditional Chinese medicine composition is proved to be non-toxic by toxicological tests;

thirdly, the traditional Chinese medicine composition has simple components, less components, low raw material cost and low energy cost required by manufacturing, thereby having low cost and convenient manufacturing;

fourthly, the traditional Chinese medicine composition can be prepared into various dosage forms, has small dosage, accurate dosage, convenient taking, carrying, storage and the like, simple preparation process and reasonable process flow.

Of course, it is not necessary for any product in which the invention is practiced to achieve all of the above-described advantages at the same time.

Detailed Description

The invention provides a Chinese medicinal composition for treating chronic liver disease, hepatic fibrosis, liver cirrhosis, alcoholic fatty liver, etc., which comprises dried toad, snow lotus herb, turtle shell, mulberry, dendrobium, chrysanthemum and liquorice, has the effects of promoting blood circulation, softening hard masses, nourishing yin and softening liver, and is used for treating hypochondrium distention or dull pain, gastric cavity stuffiness and anorexia, anorexia and greasiness, listlessness and hypodynamia, dry mouth, or nasal and dental bleeding, or nausea, or hepatic fibrosis with accumulated blocks in the hypochondrium, which are caused by blood blockage of liver and liver collaterals malnutrition.

The traditional Chinese medicine composition provided by the invention comprises 7 traditional Chinese medicines of dried toad, saussurea involucrate, turtle shell, mulberry, dendrobium, chrysanthemum and liquorice, and considering that the traditional Chinese medicine components are relatively complex, the inventor analyzes the components and the extraction method of the medicinal materials adopted in the prescription, and the traditional Chinese medicine composition is specifically as follows:

the main components of the dried toad are protein, a large amount of amino acid and vitamin, the dried toad contains trace toad venom base and resibufogenin, the components are complex, and the dried toad is always decocted by water extraction in the application process of the traditional proved recipe, so the preparation process of the invention still adopts water extraction and decoction.

The main components of the saussurea involucrate are anhydrous rutin and chlorogenic acid which are water-soluble substances.

The main components of turtle shell are gelatin, keratin, calcium phosphate, calcium carbonate and 17 amino acids, so turtle shell is best extracted with water.

Mulberry mainly contains glucose, fructose, tannin, malic acid, rutin, anthocyanin, carotene, P acid, linoleic acid, vitamin B, vitamin C, mucoid, cyanidin, calcium and the like. It is preferably extracted by decocting with water.

The main effective components of herba Dendrobii are alkaloids such as dendrobine, which are dissolved in water, and are preferably extracted by decocting with water.

The main components of licorice are glycyrrhizic acid and liquiritin, which have high solubility in water, and the process is preferably to extract the licorice by a water decoction method.

The main components of the chrysanthemum are chlorogenic acid, galuteolin, quinic acid and the like which are water-soluble substances and are extracted by a water decoction method.

In conclusion, the 7 medicinal materials in the prescription can be extracted by a method of water decoction, so that the effective ingredients can be extracted, the consistency with the original process can be kept, the stability of the product and the drug effect is facilitated, and the production and the manufacture are convenient.

In the process of medication, the formula can be prepared according to the prescription, and then the traditional Chinese medicine preparation method is used for preparing pills for taking, and the pills can also be prepared into tablets for industrial production. When the traditional Chinese medicine compound preparation is prepared, the preparation is further extracted and concentrated into extract, and the extract is prepared by granulation, tabletting and coating.

In the following exemplary description, the formulation of the Chinese medicinal composition of the present invention is further described, and the extraction process and the preparation process thereof are exemplarily described, specifically as follows:

1. formulation of

100g of dried toad; 20-60g of saussurea involucrate; 30-90g of turtle shell; mulberry 100-;

50-150g of dendrobium; 20-60g of chrysanthemum; 20-60g of liquorice.

2. Extraction process

Crushing carapax Trionycis and Bufo siccus, soaking in 10 times of water for 5 hr, decocting for 2.5 hr, and filtering to obtain filtrate; soaking carapax Trionycis and dried Bufo siccus and the rest materials including herba Saussureae Involueratae, herba Dendrobii, Mori fructus, Glycyrrhrizae radix, and flos Chrysanthemi in 8 times of water for 2.5 hr, decocting for 2.5 hr, and filtering to obtain filtrate; adding 6 times of water into the medicine residues, decocting for 2 hours, filtering, combining three extracting solutions, continuously concentrating into an extract with the relative density of 1.15-1.30 (60-80 ℃), and storing at low temperature of 0-4 ℃.

3. Preparation process

Adding appropriate amount of starch and microcrystalline cellulose into the extract, granulating, drying, pressing into 1000 tablets, and coating with film coat.

An exemplary process step is as follows:

3.1 accurately weighing 300g of starch and the extract according to the formula of the product, adding into a groove type mixer, uniformly mixing, and preparing a soft material;

3.2 adding the uniformly mixed materials into a swing granulator, and granulating (for example, 18 meshes);

3.3, distributing the materials subjected to primary granulation, putting the materials into a hot air circulation oven, and drying the materials under the condition of 50-60 t;

3.4 granulation: dispersing the agglomerated and adhered particles in the drying process to obtain particles with uniform size

Granules (18 mesh);

3.5 adding 1.9g of magnesium stearate and 5g of microcrystalline cellulose, and mixing in batches;

3.6 tabletting (0.5 g);

3.7 coating film, wherein the coating agent is hydroxypropyl methylcellulose and 5 g;

3.8 bottling;

and 3.9, warehousing after qualified inspection.

In this context, a range of values from one value to another is a general expression avoiding any recitation of all values in the range in the specification. Thus, recitation of a range of values herein is intended to encompass any value within the range and any smaller range defined by any value within the range, as if the range and smaller range were explicitly recited in the specification.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. In practice, the technical personnel according to the invention make improvements and modifications, which still belong to the protection scope of the invention.

Example 1

The formula of the traditional Chinese medicine composition in the embodiment is as follows:

120g of dried toad; 42g of saussurea involucrate; 69g of turtle shell; 117g of mulberry;

82g of dendrobium; 35g of chrysanthemum; 35g of liquorice.

Example 2

125g of dried toad; 45g of saussurea involucrate; turtle shell 71 g; 120g of mulberry;

86g of dendrobium; 40g of chrysanthemum; licorice root, radix Glycyrrhizae 40 g.

Example 3

130g of dried toad; 50g of saussurea involucrate; 76g of turtle shell; 130g of mulberry;

90g of dendrobium; 45g of chrysanthemum; licorice root, radix Glycyrrhizae 45 g.

Example 4

135g of dried toad; 55g of snow lotus herb; 80g of turtle shell; 135g of mulberry;

95g of dendrobium; 50g of chrysanthemum; licorice root, radix Glycyrrhizae 50 g.

Example 5

150g of dried toad; 60g of saussurea involucrate; turtle shell 85 g; 140g of mulberry;

100g of dendrobium; 55g of chrysanthemum; and 55g of liquorice.

According to the formula of the above 5 examples, the extraction and preparation processes are respectively adopted to prepare corresponding tablets, each tablet is 0.5g, the effective content of each tablet is 0.5g, and the recommended dosage is 0.11g/kg of medicine taken per kilogram of body weight per day. For an adult of 70kg, the recommended amounts are: three times daily, 5 tablets each time.

The treatment effect of the traditional Chinese medicine composition on a rat hepatic fibrosis model is evaluated from the following three aspects:

(1) the invention relates to a pair of Chinese medicinal compositionsDelicacy of wineEffect of the rat hepatic fibrosis model；

(2) The invention relates to a pair of Chinese medicinal compositionsBy carbon tetrachlorideThe effect of the rat liver fibrosis model;

(3) the invention relates to a pair of Chinese medicinal compositionsCaused by pig serumEffect of rat liver fibrosis model.

A first group: the invention relates to a pair of Chinese medicinal compositionsDelicacy of wineEffect of rat liver fibrosis model.

The experimental method comprises the following steps:

1. the used medicines are as follows:

experimental groups: the traditional Chinese medicine composition tablet obtained in example 1 is used, and the content of effective substances in the tablet is as follows: 0.5 g.

Positive group: the compound turtle shell liver softening tablet is provided by inner Mongolia Furui medical science and technology GmbH, approved document No: national medicine standard Z19991011, production batch number: 20140410, 0.5 g/tablet.

2. The experimental steps are as follows:

120 female SD rats with the weight of 170-.

Molding: the 120 female SD rats are randomly divided into a blank group (18 rats) and a modeling group (102 rats) according to the weight balance principle, the blank group is filled with water and fed with normal complete feed, the modeling group starts from 10 percent (volume fraction) of low-concentration alcohol, 1.5ml/100g is taken once a day, the concentration is increased by 3 to 4 percent day by day, when the concentration is 60 percent, the filling volume is reduced to 0.75ml/100g, and meanwhile, 10 percent alcohol beverage (prepared by a two-pot head and purified water) is taken as drinking water for more than 10 continuous weeks. Starting to carry out spot check from the 10 th week, sampling a blank group and a model building group, and carrying out pathological detection when the model building group is more than the blank group until experimental grouping is carried out after alcohol-induced hepatic fibrosis of rats is observed.

18 pieces before blank modeling, 3 pieces after spot check, and 15 pieces after grouping; the model building group 102 had 8 spot-checks, 27 died, and the remaining 67 were grouped into 14 model groups, 13 large/medium/small dose groups, and 14 positive groups.

The experiment adopts an alcohol-induced rat hepatic fibrosis model, the model is simple and easy to implement, the forming rate is high, but the fibrous lesion is naturally reversed after the model building is stopped, so that the intervention effect of the medicine on the hepatic fibrosis is conveniently researched, and during the grouped administration period, except for a blank group, the alcohol dose is reduced for each group of animals to continue the model building.

Wherein the blank group and the model group are intragastrically administered with purified water every day, the positive group is intragastrically administered every day, the dosage is 0.8g/kg (equivalent to 8 times of clinical dosage), the large/medium/small dosage groups of the experimental group are intragastrically administered every day by 0.96, 0.48 and 0.24g/kg (equivalent to 16, 8 and 4 times of clinical dosage), the administration is carried out once every day, the administration is carried out 7 days per week for 8 weeks continuously, the model group, the positive group and the large/medium/small dosage groups of the experimental group are modeled by 0.75ml/100g of 40 percent double-boiler during the administration period, and alcohol with the concentration of 10 percent is added into drinking water. Weighing body weight and food intake once per week, and regulating drug dosage according to body weight per week; the administration mode adopts purified water as a solvent, the experimental medicine is ground into powder and then is prepared into the required concentration by purified water.

3. Material taking and detection: after the last administration, fasting and no water supply is carried out for 12 hours, then the rat is anesthetized, and the total carotid artery is used for taking blood to measure four items of liver function and liver fiber; detecting alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), alkaline phosphatase (ALP), Total Protein (TP), Albumin (ALB), Globulin (GLB), Total Bilirubin (TBIL), Laminin (LN), Hyaluronic Acid (HA), serum type III procollagen (PC III), and type IV collagen (COI-IV) in serum;

weighing the liver and spleen and calculating the index of the liver and spleen;

taking liver tissue, and measuring the content of hydroxyproline;

liver tissues are taken for fixation, pathological sections are made, and three lobules of the liver of each rat are taken for pathological detection (two staining methods of HE and Masson are adopted).

Wherein, the liver function test is sent to a hospital clinical laboratory for testing;

four hepatic fibrosis items are measured by enzyme-linked immunoassay (ELISA), and the operation is strictly carried out according to the operation steps of the instruction;

hydroxyproline was detected using a kit.

And (4) carrying out pathological section and staining on the liver tissue, then carrying out image acquisition, and carrying out pathological scoring.

4. Statistical method

SPSS16.0 software is adopted for statistical treatment, the data of experimental animals in each group are expressed by mean plus or minus standard deviation, variance analysis is adopted among groups, t test is adopted for comparison among indexes, each group is compared with a model group, and P is less than 0.05, so that the statistical significance is achieved.

5. Results of the experiment

In the administration process, animals in the blank group and the positive group do not die, 3 animals in the model group die, 2 animals in the experiment group die in the high-dose group, and 1 animal in the experiment group and the low-dose group respectively die. Number of animals in each group at the end of experiment: blank group 15, positive group 14, model group 11, experiment group large dose group 11, experiment group medium dose group 12, experiment group small dose group 12.

The test results were as follows:

1) body weight and food intake:

the body weight parameters of each group of rats are shown in table 1 below.

TABLE 1

Effect on rat body weight

Week (week)

Blank group

Model set

Positive group

High dose group

Middle dose group

Low dose group

1

299.53±27.20

269.50±21.26^ΔΔ

272.21±19.20^ΔΔ

271.92±18.28^ΔΔ

274.77±16.05^ΔΔ

272.85±22.47^Δ-

2

306.20±29.81

272.85±22.49^ΔΔ

281.93±20.69^Δ

283.42±15.77^Δ

283.62±18.05^Δ

282.62±24.84^Δ

3

312.60±27.63

276.46±24.32^ΔΔ

287.57±22.56^Δ

290.33±22.06^Δ

290.85±22.33^Δ

290.92±26.57^Δ

4

321.53±27.40

281.85±25.62^ΔΔ

296.21±24.80^Δ

298.92±23.77^Δ

301.00±21.89^Δ

295.85±31.80^Δ

5

334.67±31.02

288.77±26.33^ΔΔ

304.29±23.62^Δ

316.50±28.59^*

310.15±16.78^*Δ

305.83±20.88^Δ

6

347.40±35.93

299.69±16.81^ΔΔ

325.79±44.52

327.58±23.82^**

321.25±18.94^**

311.17±18.46^ΔΔ

7

350.93±39.30

304.75±27.99^ΔΔ

325.43±26.22

334.92±24.63^*

328.50±27.88^*

318.42±26.49^Δ

8

353.27±30.94

306.50±21.35^ΔΔ

323.50±22.32

334.18±21.62^**

336.42±24.16^**

322.33±20.49^ΔΔ

Note: model group, administration group and blank group comparison^ΔP＜0.05，^ΔΔP is less than 0.01; the administration groups were compared with the model group^*P＜0.05^.**P＜0.01

As can be seen from Table 1 above, the body weight of the model group, the small dose group at the beginning of the administration and the test period is significantly lower than that of the blank group; the body weight of the large, medium and positive groups is also significantly lower than that of the blank group (P < 0.05, P < 0.01) 4 weeks before administration. The body weight of the large and medium dose groups was significantly higher than that of the model group (P < 0.05, P < 0.01) from the 5 th week of administration. The test result proves that the traditional Chinese medicine composition can obviously improve the weight of the rat with hepatic fibrosis. The results of the diet test in each group of rats are shown in table 2.

TABLE 2

Effect on rat food intake

Week (week)

Blank group

Model set

Positive group

High dose group

Middle dose group

Low dose group

1

18.32±0.21

17.17±0.17^ΔΔ

17.88±0.07

17.71±0.96

17.20±0.85

17.20±0.79

2

21.00±0.61

18.43±0.65^ΔΔ

20.53±1.99

19.40±1.48

20.17±1.33

19.58±0.81

3

24.53±1.16

18.93±1.27^ΔΔ

23.23±1.50

23.07±3.00

26.30±5.12

24.37±3.69

4

27.37±2.84

21.27±1.05^Δ

29.00±5.89

30.43±6.18

28.60±4.61

26.93±5.29

5

37.40±5.29

23.47±2.05^Δ

33.37±11.05

36.90±6.70^*

35.63±2.87^**

29.67±3.88

6

37.60±5.94

25.33±2.41^Δ

28.00±4.20

34.77±2.19^**

34.67±3.04^*

28.53±5.40

7

38.10±1.31

25.27±2.54^ΔΔ

33.00±7.84

36.63±0.90^**

34.77±3.00^*

32.53±3.82

8

35.10±1.31

22.40±1.64^ΔΔ

25.23±1.48

26.33±0.76^*

39.17±2.90^**

29.73±5.46

Note: model group to blank group comparison^ΔP＜0.05，^ΔΔP is less than 0.01: comparison of the drug administration group with the model group^*P＜0.05，^**P＜0.01

As can be seen from Table 2 above, the food intake at the beginning of the administration and during the test period in the model group was significantly lower than that in the blank group (P < 0.05, P < 0.01). From the 5 th week of administration, the food intake of the large and medium dose groups was significantly higher than that of the model group (P < 0.05, P < 0.01). The test result proves that the large and medium dosage groups of the traditional Chinese medicine composition can obviously improve the food intake of the rats with hepatic fibrosis.

2) Liver function

The results of the liver function tests for each group are detailed in tables 3-5.

TABLE 3

Effect on ALT and AST in rats

Group of	Dosage (g/kg)	Number of animals	ALT(U/L)	AST(U/L)	AST/ALT
						Blank group	——	15	34.20±6.10	111.60±15.79	3.43±079
Model set	——	11	53.73±15.98^ΔΔ	179.91±34.43^ΔΔ	3.56±1.01
						Positive group	0.8	14	50.14±18.66	130.71±24.71^**	2.80±0.76^*
High dose group	0.96	11	45.45±8.87	148.36±33.03^*	3.33±0.86
						Middle dose group	0.48	12	57.83±26.48	162.08±40.00	3.43±0.97
Low dose group	0.24	12	57.25±29.14	152.08±32.60	2.95±0.86

Note: model group to blank group comparison^ΔΔP is less than 0.01; the administration groups were compared with the model group^*P＜0.05，^**P＜0.01

TABLE 4

Effect on TBIL and ALP in rats

Group of	Dosage (g/kg)	Number of animals	TBIL(mmol/L)	ALP(U/L)
					Blank group	-	15	1.50±0.24	32.87±13.19
Model set	-	11	1.54±0.43	43.27±9.19^ΔΔ
					Positive group	0.8	14	1.41±0.27	40.29±16.59
High dose group	0.96	11	1.58±0.38	32.91±11.41^*
					Middle dose group	0.48	12	1.43±0.37	39.42±26.00
Low dose group	0.24	12	1.41±0.38	42.00±14.86

Note: model group to blank group comparison^ΔΔP is less than 0.01; administration group mixing mouldType group comparison^*P＜0.05

As can be seen from tables 3 and 4, ALT, AST and ALP were significantly higher in the model group than in the blank group, indicating that alcohol damages liver function of rats. The experimental group can obviously reduce AST and ALP values, and the product is proved to have certain liver protection effect.

TABLE 5

Effect on rat TP ALB GLB

Group of

Dosage (g/kg)

Number of animals

TP(g/L)

ALB(g/L)

GLB(g/L)

ALB/GLB

Blank group

——

15

64.93±3.31

28.13±1.41

36.80±2.37

0.76±0.05

Model set

——

11

64.91±3.27

28.55±2.25

36.36±2.54

0.79±0.09

Positive group

0.8

14

62.36±4.77

28.07±2.26

34.29±2.95

0.82±0.08

High dose group

0.96

11

67.91±3.99

31.72±1.95^**

36.27±3.44

0.86±0.08

Middle dose group

0.48

12

68.42±6.37

31.33±2.84^*

37.08±4.70

0.85±0.12

Low dose group

0.24

12

61.17±3.79

28.25±2.26

35.50±3.90

0.80±0.10

Note: the administration groups were compared with the model group^*P＜0.05，^**P＜0.01

Table 5 shows the results of TP, ALB, GLB, ALB/GLB tests for the blank group, model group, positive group, and each experimental group.

3) Influence on four items of hepatic fibrosis

The results of four liver fibrosis tests in each group are detailed in table 6 below.

TABLE 6

Influence on four items of hepatic fibrosis

Group of

Dosage (g/kg)

Number of animals

LN(ng/ml)

HA(ng/ml)

PCIII(ng/ml)

IV(ng/ml)

Blank group

——

15

481.55±33.64

529.83±84.47

81.70±3.46

35.89±4.38

Model set

——

11

550.30±26.35^ΔΔ

545.54±72.55

88.08±5.04^ΔΔ

33.88±2.61

Positive group

0.8

14

499.34±27.34^**

556.10±26.58

77.06±3.59^**

33.50±3.20

High dose group

0.96

11

532.08±27.03

578.82±88.04

83.22±6.37

34.13±4.56

Middle dose group

0.48

12

526.38±62.53

550.38±76.23

82.63±4.46^*

32.51±2.78

Low dose group

0.24

12

508.74±25.86^*

524.87±35.05

78.69±4.99^**

31.05±3.39

Note: comparison of blank and model groups^Δ＜0.05，^ΔΔP is less than 0.01; the administration groups were compared with the model group^*P＜0.05，^**P＜0.01

In clinical examination, if only one of four liver fibrosis indexes is increased, liver fibrosis may begin to appear in the liver, and if two or three indexes are increased, obvious liver fibrosis is indicated. In this experiment, as can be seen in table 6, the concentrations of LN and PCIII in the serum of the rat in the model group are significantly higher than those in the blank group, indicating that the hepatic fibrosis model is established. Compared with the model group, the concentration of PCI II in serum is obviously reduced in the experimental group (P is less than 0.05), and the concentration of LN and PCIII in serum is obviously reduced in the experimental group with small dosage (P is less than 0.05 and P is less than 0.01). The traditional Chinese medicine composition in the embodiment 1 of the invention is proved to have a certain treatment effect on hepatic fibers.

4) Effect on hydroxyproline in liver tissue

The results of testing the various hydroxyprolines of the experimental groups are detailed in table 7 below.

TABLE 7

Effect on hydroxyproline in liver tissue

Group of	Dosage (g/kg)	Number of animals	Hydroxyproline (μ g/mg)
				Blank group	——	15	142.48±25.29
Model set	——	11	267.70±95.11^ΔΔ
				Positive group	0.8	14	170.95±42.41^**
High dose group	0.96	11	163.90±37.14^**
				Middle dose group	0.48	12	183.40±46.53^*
Low dose group	0.24	12	190.68±33.04^**

As can be seen from Table 7, the content of hydroxyproline in the liver tissue of the rat is significantly increased compared with that of the blank group, indicating that the liver fibrosis of the rat is formed. The large/medium/small dose group and the positive group of the experimental group can obviously reduce the content of hydroxyproline (P is less than 0.05, and P is less than 0.01), and the traditional Chinese medicine composition has a certain treatment effect on hepatic fibrosis.

5) Effect on rat liver-spleen index

The results of the liver and spleen index tests of the rats in each group are detailed in the following table 8.

TABLE 8

Effect on rat liver-spleen index

Group of	Dosage form	Number of animals	Liver index	Spleen index
					Blank group	——	15	2.31±0.24	1.69±0.29
Model set	——	11	2.67±0.22^ΔΔ	1.86±0.42
					Positive group	0.8g/kg	14	2.45±0.23^*	2.21±0.94
High dose group	0.96g/kg	11	2.47±0.15^*	1.61±0.13
					Middle dose group	0.48g/kg	12	2.49±0.26^*	1.81±0.24
Low dose group	0.24g/kg	12	2.56±0.26	2.07±0.38

Note: model group to blank group comparison^ΔΔP is less than 0.01; the administration groups were compared with the model group^*P＜0.05

As can be seen from Table 8, the liver index of the rat model group is significantly higher than that of the control group (P < 0.05), indicating that liver fibrosis of the rat is formed. The liver indexes of the large and medium dose groups and the positive group of the experimental group are obviously lower than those of the model group. The experimental group large/medium/small dose groups had less effect on spleen index than the positive group.

6) Influence on pathological organization of liver disease

This section specifically evaluates the mirror images under HE and Masson staining lenses.

Liver under HE staining scope was: the liver cell nucleus is dark blue, the cytoplasm is light pink, the collagen fiber is pink slightly darker than the cytoplasm, and the fatty liver cell is vacuole with different sizes and is not colored.

The test results for each group of experiments are as follows:

blank group: the liver tissue structure is normal, the liver cells are arranged in order in a radial mode, and fat drops hardly exist in cytoplasm;

model group: normal lobular structure of the liver is damaged to form a disordered false lobular structure, a central vein disappears after deviating, liver cells in the lobules are edematous, a large amount of lipid vacuoles can be formed, meanwhile, a large amount of inflammatory cells infiltrate in a manifold area, fibroblasts are proliferated in a large amount, collagen fibers are formed, and large fibrous intervals are formed;

positive group: hepatic lobule structure is essentially normal with less steatosis surrounding the central vein;

experimental group high dose group: the liver lobules are regularly arranged, no obvious sinus cavity stenosis is seen, and little steatosis is seen;

dose groups in experimental groups: hepatic steatosis is still present around the central vein;

experimental group low dose group: there is more steatosis around the central veins of the lobules of the liver;

liver under Masson staining mirror: the liver cell nucleus presents dark blue or purple blue, the cytoplasm, muscle fiber and red cell present red, the collagen fiber presents light blue or green, and the liver cell fat changes to present vacuoles with different sizes and no coloration.

Blank group: the normal blank group has clear hepatic lobule structure, hepatic cells in the lobules are basically normal, the structure in the manifold is clear, and small bile ducts and fibrous tissue hyperplasia are not seen in the manifold area and the periphery of the manifold area;

model group: most of the manifold areas have more inflammatory cell infiltration, some of the manifold areas even have enlargement of the manifold areas, punctate necrosis, clastic necrosis, rare bridge necrosis, liver cell edema in the lobules, and formation of a large amount of lipid vacuoles, and meanwhile, the manifold areas have a large amount of inflammatory cell infiltration, and a large amount of fibroblast hyperplasia and collagen fiber formation form coarse fibrous intervals;

positive group: the hepatic lobule structure is basically normal, the periphery of the central vein has less steatosis, the infiltration of inflammatory cells is reduced, and the formation of fibroblasts and collagen fibers is obviously reduced;

experimental group high dose group: the liver lobules are arranged regularly, obvious sinus stenosis is not seen, little steatosis can be seen, inflammatory cell infiltration is obviously reduced, and the formation of fibroblasts and collagen fibers is obviously reduced;

dose groups in experimental groups: the hepatic lobules are arranged regularly, hepatic steatosis still exists around the central vein, the infiltration of inflammatory cells is less, and the formation of fibroblasts and collagen fibers is reduced;

experimental group low dose group: the liver lobules are arranged regularly basically, more fatty degeneration exists around the central veins of the liver lobules, the infiltration of inflammatory cells is reduced, and the formation of fibroblasts and collagen fibers is reduced.

Pathological scoring is carried out on three lobules of the liver of each rat according to grading standards under a Masson staining mirror, and the average value of three is used as the pathological scoring of the hepatic fibrosis of the rat. The results are shown in Table 9 below.

TABLE 9

Liver histology scoring

Group of	Dosage (g/kg)	Number of animals	Score of
				Blank group	——	15	0±0^ΔΔ
Model set	——	11	2.15±0.43^ΔΔ
				Positive group	0.8	14	1.52±0.31^**
High dose group	0.96	11	1.58±0.34^**
				Middle dose group	0.48	12	0.92±0.48^**
Low dose group	0.24	12	1.22±0.46^*

As can be seen from Table 9, the pathological score of the model group is significantly higher than that of the blank group, indicating that the hepatic fibrosis model is established. The pathological scores of the large, medium and small doses of the experimental group and the positive group are obviously lower than those of the model group (P is less than 0.05 and P is less than 0.01), and the traditional Chinese medicine composition can improve the alcoholic hepatic fibrosis tissue lesion.

The results of the experiments are summarized below:

1) weight and food intake: before administration, the body weight of rats in the model group and the administration group is obviously lower than that in the blank group (P is less than 0.05, P is less than 0.01), then the body weight and the food intake of the administration group are gradually increased, and the body weight and the food intake of the large and medium dose groups are obviously higher than those in the model group (P is less than 0.05, P is less than 0.01) from the fifth week to the end of the experiment;

2) biochemical treatment of liver function: the large dosage of the invention can obviously reduce AST and ALP values (P is less than 0.05);

3) four items of hepatic fibrosis: the dosage of the invention obviously reduces the concentration of PCIII in serum (P is less than 0.05), and the small dosage of the invention obviously reduces the concentration of LN and PCIII in serum (P is less than 0.05, P is less than 0.01);

4) hydroxyproline: the invention can reduce the content of hydroxyproline in liver tissues with large, medium and small doses (P is less than 0.05 and P is less than 0.01);

5) histopathological examination of liver: compared with a model group, the administration group of the traditional Chinese medicine composition has the advantages that liver lobules are arranged neatly, fatty degeneration is reduced, inflammatory cell infiltration is obviously reduced, fibroblast and collagen fiber formation is obviously reduced, pathological scores are obviously reduced (P is less than 0.05, P is less than 0.01), and a certain dosage relation is displayed.

The experimental results show that the weight and the food intake of the rats in the model group are obviously lower than those of the blank group, the ALT, AST and TBIL values of serum, LN and PCIII concentrations in serum and the content and the index of Hyp in liver tissues of the rats in the model group are obviously higher than those of the blank group, the liver fiber degree of the rats in the pathology inspection model group is obvious, and the comprehensive analysis of all factors indicates that the hepatic fibrosis model of the experimental rats is established.

The large dose, the medium dose and the small dose of the traditional Chinese medicine composition can obviously reduce the content of hydroxyproline in liver tissues, and the large dose and the medium dose of the traditional Chinese medicine composition can obviously reduce the AST value in the serum of a rat, obviously reduce the concentration of LN in the serum, obviously reduce the concentration of PC III in the serum of the rat, and obviously reduce the concentrations of LN and PC III in the serum of the rat. Pathological examination shows that the liver lobules of the administration group of the traditional Chinese medicine composition are arranged orderly, fatty degeneration is reduced, inflammatory cell infiltration is obviously reduced, the formation of fibroblasts and collagen fibers is obviously reduced, pathological scores are obviously reduced, and a certain dosage relation is displayed.

In conclusion, the traditional Chinese medicine composition has an obvious improvement effect on an alcoholic liver fibrosis rat model, and the model is similar to a clinical alcoholic liver fibrosis, so that the traditional Chinese medicine composition has a certain treatment effect on alcoholic liver fibrosis patients.

Second group: the influence of the traditional Chinese medicine composition on the rat hepatic fibrosis model caused by carbon tetrachloride

1. Experimental Material

Experimental animals and sources: male SD rats, 80-120g in weight, provided by the animal experimental center of the seian university medical college, animal certification number: 61001700000177, license: SCXK (shan) 2012 and 003.

Feeding conditions are as follows: the temperature is 20-24 ℃, the air is circulated, the relative humidity is 40-70%, the animals can eat freely, and the animals can be fed adaptively for more than one week before the experiment.

Experimental drugs:

positive drug: compound turtle shell liver softening tablet provided by inner mongolia fordi medical science and technology limited, approved document no: national medicine standard Z19991011, production batch number: 20140410, clinical application dose: orally administered 3 times a day, 3 granules at a time, 0.5 g/tablet.

Pharmaceutical tablets prepared according to the invention in example 1: 0.5 g/tablet.

2. Experimental methods

1) Preparation of rat hepatic fibrosis model

Dividing 100 male SD rats into blank group and modeling group 2 according to weight balance principle, 15 blank groups and the rest 85 male SD rats are modeled according to the following method:

injecting oleum Olivarum subcutaneously in blank group, and injecting carbon tetrachloride oleum Olivarum subcutaneously in molding group (40% carbon tetrachloride oil prepared from carbon tetrachloride and oleum Olivarum at ratio of 2: 3) with injection volume of 0.3ml/100g, subcutaneously injecting twice per week, doubling for the first time, and continuously injecting for more than 10 weeks at interval of 2-3 days. And (3) from the 10 th week, sampling blank groups and model building groups, wherein the model building groups are more than the blank groups, carrying out pathological detection, and carrying out experimental grouping after observing the condition that the carbon tetrachloride induces the rat hepatic fibrosis.

2) Experimental grouping and administration

15 blank groups are selected, 2 blank groups are selected, and 13 blank groups are selected; the model group is 85, 8 are spot-checked, 3 die, and the rest 74 are divided into 15 model groups, 15 large/medium dose groups, 14 small dose groups and 15 positive groups (compound turtle shell soft liver tablet group).

Purified water is given to the blank group and the model group by intragastric administration every day; the administration dosage of the large/medium/small dose group in the experimental group is 0.96 g/Kg, 0.48 g/Kg and the administration dosage of the compound turtle shell liver softening tablet group is 0.8 g/Kg. The above groups were administered 1 time daily for 7 days per week for 8 weeks. Each group of rats is raised in cages, and each group can freely take normal feed and drink distilled water at room temperature (20-25 ℃), weigh the weight and eat the food once per week, and adjust the dosage of the medicine according to the weight.

Since hepatic fibrosis of the rat can be recovered by self, model group and administration group continue modeling during administration period. During the administration period, 40% carbon tetrachloride olive oil is injected subcutaneously twice a week, and the injection volume is 0.2ml/100 g.

3) Material taking and detection:

fasting: before treatment, rats are fasted for 12 hours without water supply;

the rat is anesthetized, and the blood is taken from the common jugular vein to measure four items of liver function, hepatic fibrosis, platelet-derived growth factor and transforming growth factor beta 1;

weighing the liver and spleen, and calculating the index of the viscera;

taking liver tissue, and measuring the content of Malondialdehyde (MDA) and superoxide dismutase (SOD);

liver tissues are taken for fixation, pathological sections are made, and two kinds of staining methods, namely HE staining and Masson staining, are adopted.

4) Each detection index and detection method

4.1 liver function assay: the same as example 1;

4.2 hepatic fibrosis four items: the same as example 1;

4.3 transforming growth factor β 1 (TGF-. beta.1): TGF-beta 1 is a polypeptide capable of regulating cell growth and differentiation, and has multiple biological effects.

The determination method comprises the following steps: and (3) taking blood from the carotid artery of the rat, sealing the collected blood by using a test tube, standing for 25min at 4 ℃, setting a centrifuge at 3000r/min, working for 10min, absorbing about 1.5mL of supernatant by a liquid shifter after centrifugation is finished to obtain serum, and freezing and storing the serum in a refrigerator at-20 ℃ for later use. Serum TGF-. beta.1 was assayed by enzyme-linked immunoassay (ELISA) following the exact protocol as described.

4.4 platelet-derived growth factor (PDGF): PDGF is a basic protein stored in platelet alpha granules.

The determination method comprises the following steps: and (3) taking blood from the carotid artery of the rat, sealing the collected blood by using a test tube, standing for 25min at 4 ℃, setting a centrifuge at 3000r/min, working for 10min, absorbing about 1.5mL of supernatant by a liquid shifter after centrifugation is finished to obtain serum, and freezing and storing the serum in a refrigerator at-20 ℃ for later use. Serum PDGF was assayed by enzyme-linked immunosorbent assay (ELISA) following the protocol described in the manual.

4.5 Malondialdehyde (MDA) and superoxide dismutase (SOD), wherein SOD is the main enzyme for eliminating active oxygen in vivo, and the activity can indirectly reflect the capability of eliminating free radicals in vivo. MDA is the final product of lipid peroxide metabolism and its content may reflect the degree of tissue loss.

The determination method comprises the following steps: taking a blood-treated rat, opening an abdominal cavity, quickly taking out liver tissues, cleaning the liver tissues in physiological saline water, weighing 0.5g of the liver tissues, preparing 10% liver homogenate by using cold physiological saline, centrifuging at 3000 rpm for 10 minutes, taking supernate, and detecting the levels of MDA and SOD by using a kit.

4.6 liver histopathology

4.6.1 HE staining: the same as example 1;

4.6.2 MASSON trichromatic method: the same as example 1;

5. the statistical method comprises the following steps: the same as in example 1.

Third, experimental results

In the administration process, animals in the blank group and the large and medium dose group do not die, 3 die animals in the model group, and 1 die animal in the positive and small dose groups respectively. Number of animals in each group at the end of experiment: blank group 13, positive group 14, model group 12, large dose 15, medium dose 15, and small dose 13.

1. Effect on rat body weight and food intake

The results are shown in tables 10 and 11.

Watch 10

Weight change of rats after administration

Week (week)

Blank group

Model set

Positive group

High dose group

Middle dose group

Low dose group

1

474.00±36.22

422.93±49.29^ΔΔ

426.71±46.72^ΔΔ

425.60±52.28^ΔΔ

425.73±43.01^ΔΔ

426.93±40.19^ΔΔ

2

486.38±37.29

432.73±48.95^ΔΔ

439.64±41.92^ΔΔ

442.13±54.31^Δ

439.67±37.02^ΔΔ

438.14±38.97^ΔΔ

3

499.31±35.21

441.85±39.53^ΔΔ

456.86±41.85^ΔΔ

461.47±54.78^Δ

456.60±31.96^ΔΔ

456.50±31.41^ΔΔ

4

514.77±26.34

454.00±37.89^ΔΔ

471.57±42.73^ΔΔ

480.87±51.85^Δ

476.87±35.92^ΔΔ

481.23±43.70^Δ

5

527.62±17.73

465.00±38.42^ΔΔ

483.57±26.42^ΔΔ

505.33±38.99^*

505.20±34.81^**Δ

496.46±39.29^Δ

6

533.46±17.85

480.92±36.94^ΔΔ

498.71±20.87^ΔΔ

520.40±35.11^**

517.47±32.64^*

506.54±37.53^Δ

7

541.46±17.68

489.00±33.82^ΔΔ

506.71±18.67^ΔΔ

526.00±30.90^**

522.80±23.07^**Δ

517.62±35.75^Δ

8

541.92±16.18

504.58±28.11^ΔΔ

511.71±15.98^ΔΔ

529.07±30.19^*

525.47±19.23^*Δ

521.92±35.69

Note: comparison with blank group^ΔP＜0.05，^ΔΔP is less than 0.01; the comparison of the administration group and the model group shows that P is less than 0.05 and P is less than 0.01

As can be seen from table 10 above, the body weight of the model group, the positive group and the experimental group at the beginning of the administration and the experimental period of the small dose group is significantly lower than that of the blank group; the body weight of the large and medium dose groups is obviously lower than that of the blank group (P < 0.05, P < 0.01) 4 weeks before the administration, and the body weight of the large and medium dose groups is obviously higher than that of the model group (P < 0.05, P < 0.01) from the 5 th week of the administration. The suggestion above shows that the large and medium dosage of the invention can obviously improve the weight of the hepatic fibrosis rat.

TABLE 11

Changes in food intake after administration

Week (week)

Blank group

Model set

Positive group

High dose group

Middle dose group

Low dose group

1

19.04±0.47

16.01±0.75^ΔΔ

17.80±1.67

17.91±0.22^Δ

17.57±0.40^Δ

17.60±0.88

2

21.47±1.81

18.27±0.35^ΔΔ

19.03±0.38

19.60±1.48

18.07±0.61^Δ

18.87±0.72

3

20.25±0.92

17.40±0.64^ΔΔ

18.56±0.53

18.39±0.42^Δ

18.73±2.11

18.11±1.34

4

23.00±2.36

18.27±0.35^ΔΔ

19.03±0.38^Δ

19.60±1.48

19.20±0.66

18.87±0.72^Δ

5

25.97±0.72

20.53±0.87^ΔΔ

23.03±2.19

24.03±1.21^*

24.87±1.60^*

23.50±2.93

6

33.40±1.06

24.23±1.29^ΔΔ

27.13±3.60^Δ

30.70±3.12^*

31.10±2.43^*

26.77±4.80^Δ

7

33.93±0.93

27.70±3.12^ΔΔ

32.03±4.02

35.53±3.01^*

35.07±3.26^*

32.63±1.50

8

34.80±0.53

29.40±1.08^ΔΔ

31.50±3.75

32.17±0.55^*Δ

32.23±0.75^*ΔΔ

32.80±2.06

Note: comparison with blank group^ΔP＜0.05，^ΔΔP is less than 0.01: comparison of the drug administration group with the model group^*P＜0.05，^**P＜0.01

As can be seen from Table 11 above, the food intake at the beginning of the administration and the experimental period of the model group is significantly lower than that of the blank group (P < 0.01), the food intake of the large, medium and small dose groups and the positive group is also lower than that of the blank group 4 weeks before the administration, the significant difference exists in individual weeks (P < 0.05), and the food intake of the large and medium dose groups is significantly higher than that of the model group (P < 0.05, P < 0.01) from the 5 th week after the administration. The results show that the large and medium dosage of the invention can obviously improve the food intake of the hepatic fibrosis rats.

2. The effects on liver function

The results of liver function tests of the experimental groups are shown in tables 12-14.

TABLE 12

Group of	Dosage (g/kg)	Number of animals	ALT(U/L)	AST(U/L)	AST/ALT
						Blank group	——	13	43.62±14.21	135.15±27.84	3.28±0.80
Model set	——	12	293.67±179.13^ΔΔ	469.17±219.62^ΔΔ	1.73±0.44^ΔΔ
						Positive group	0.8	14	108.00±76.03^**	210.29±103.96^**	2.33±0.82^*
High dose group	0.96	15	99.47±71.97^**	224.00±95.90^**	2.65±1.05^**
						Middle dose group	0.48	15	81.13±43.96^**	177.27±76.42^**	2.43±0.70^**
Low dose group	0.24	13	65.08±17.87^**	163.46±43.13^**	2.58±0.72^**

Note: model group to blank group comparison^ΔΔP is less than 0.01; the administration groups were compared with the model group^**P＜0.01

Watch 13

Group of	Dosage (g/kg)	Number of animals	TBIL(mmol/L)	ALP(U/L)
					Blank group	——	13	1.48±0.39	89.62±31.16
Model set	——	12	1.38±0.37	324.75±135.79^ΔΔ
					Positive group	0.8	14	1.48±0.39	142.07±63.51^**
High dose group	0.96	15	1.38±0.34	147.93±91.40^**
					Middle dose group	0.48	15	1.49±0.36	109.60±52.28^**
Low dose group	0.24	13	1.52±0.32	110.00±32.70^**

Note: model group to blank group comparison^ΔΔP is less than 0.01: the administration groups were compared with the model group^**P＜0.01

It can be seen from tables 12 and 13 that the serum ALT, AST and ALP of the rats in the model group are significantly higher than those in the blank group (P < 0.01), which indicates that the rat hepatocytes are seriously damaged by carbon tetrachloride, and the large, medium and small dose groups in the experimental group can significantly reduce the serum ALT, AST and ALP values (P < 0.01), the reduction range is not in dose relationship, the influence of the small dose on the liver is possibly small, and the metabolic burden of the liver is aggravated when the dose is too high. The invention is proved to have the functions of protecting liver cells and reducing the damage of carbon tetrachloride to the liver cells.

TABLE 14

Group of

Dosage (g/kg)

Number of animals

TP(g/L)

ALB(g/L)

GLB(g/L)

ALB/GLB

Blank group

——

13

63.85±3.48

28.31±1.49

35.54±3.60

0.81±0.11

Model set

——

12

59.35±3.63^ΔΔ

25.67±2.77^ΔΔ

32.67±4.85

0.82±0.22

Positive group

0.8

14

61.07±2.23

27.36±1.50

33.71±1.38

0.80±0.06

High dose group

0.96

15

62.20±3.41^*

25.47±2.00

36.73±3.20

0.71±0.09

Middle dose group

0.48

15

62.00±4.36

26.07±2.52

35.93±4.03

0.74±0.12

Low dose group

0.24

13

59.31±2.95

25.62±2.02

33.69±2.78

0.77±0.12

Note: model group to blank group comparison^ΔΔP is less than 0.01: comparison of the drug administration group with the model group^*P＜0.05

As can be seen from Table 14, the concentrations of TP and ALB in the serum of the rats in the model group are obviously lower than those in the blank group (P is less than 0.01), which indicates that the generation of protein is influenced by the hepatic cells damaged by carbon tetrachloride, and the TP is increased in the large dose group of the experimental group. Indicating that the large dose of the invention can increase the total liver protein.

3. Influence on four items of hepatic fibrosis

The results of four liver fibrosis tests are shown in tables 15-16.

Watch 15

Group of	Dosage (g/kg)	Number of animals	LN(ng/ml)	HA(ng/m1)
					Blank group	——	13	386.19±46.30	175.46±20.14
Model set	——	12	506.87±55.84^ΔΔ	252.70±26.51^ΔΔ
					Her sexual group	0.8	14	421.39±35.04^**	199.38±32.39^**
High dose group	0.96	15	425.01±37.90^**	206.53±37.38^**
					Middle dose group	0.48	15	419.31±28.54^**	222.25±28.73^**
Low dose group	0.24	13	437.48±29.49^**	199.49±42.97^**

Note: model group to blank group comparison^ΔΔP is less than 0.01; comparison of the drug administration group with the model group^*P＜0.05，^**P＜0.01

TABLE 16

Group of	Dosage (g/kg)	Number of animals	PCIII(ng/m1)	COI-IV(ng/m1)
					Blank group	——	13	62.13±3.77	17.16±2.08
Model set	——	12	87.81±34.7^ΔΔ	22.78±2.51^ΔΔ
					Positive group	0.8	14	63.98±5.74^*	19.71±3.00^*
High dose group	0.96	15	67.83±12.68^*	18.66±1.72^**
					Middle dose group	0.48	15	65.56±3.99^*	18.47±1.96^**
Low dose group	0.24	13	68.01±4.10	18.33±1.79^**

In clinical examination, if only one of four liver fibrosis indexes is increased, liver fibrosis may begin to appear in the liver, and if two or three indexes are increased, obvious liver fibrosis is indicated. In the experiment, LN, HA, PCIII and COI-IV of the serum of the rat in the model group are obviously higher than those in the blank group (P is less than 0.01), which indicates that the hepatic fibrosis model of the rat is established. Compared with the model group, the large, medium and small dose groups of the experimental group obviously reduce the concentration of LN, HA and COI-IV in serum (P is less than 0.05, P is less than 0.01), and the large and medium dose groups of the experimental group obviously reduce the concentration of PCIII in serum.

4. The results of the experimental groups are shown in Table 17 for the effects on transforming growth factor beta 1 (TGF-. beta.1) and platelet-derived growth factor (PDGF).

TABLE 17

Group of	Dosage (g/kg)	Number of animals	TGF-β1(ng/ml)	PDGF(ng/ml)
					Blank group	——	13	33.91±10.42	16.95±1.42
Model set	——	12	45.44±10.56^Δ	17.39±1.60
					Positive group	0.8	14	38.48±10.43	17.02±0.89
High dose group	0.96	15	37.20±9.29	17.22±1.31
					Middle dose group	0.48	15	38.69±14.79	17.07±0.72
Low dose group	0.24	13	32.69±6.85^*	16.17±1.26

Note: comparison of blank and model groups^ΔLess than 0.05; the administration groups were compared with the model group^*P＜0.05

As can be seen from Table 17, the TGF-beta 1 concentration in the serum of the rat in the model group is obviously higher than that in the blank group (P is less than 0.05), the TGF-beta 1 concentration in the serum can be reduced by the large, medium and small dose groups of the TGF-beta 1 injection, wherein the small dose group has significant difference (P is less than 0.05).

5. Influence on SOD and MDA of liver tissue

The results of the tests of each group are shown in Table 18.

Watch 18

Group of	Dosage (g/kg)	Number of animals	SOD(U/mgprot)	MDA(nmol/mgprot)
					Blank group	——	13	68.58±12.68	2.56±0.76
Model set	——	12	44.71±9.48^ΔΔ	3.47±1.03^Δ
					Positive group	0.8	14	63.44±18.06^**	2.05±1.21^**
High dose group	0.96	15	57.83±19.92^*	2.54±0.90^*
					Middle dose group	0.48	15	60.98±11.74^**	2.40±0.95^*
Low dose group	0.24	13	61.58±12.36^**	26.5±0.51^*

Note: model group to blank group comparison^Δ＜0.05，^ΔΔP is less than 0.01; the administration groups were compared with the model group^*P＜0.05，^**P＜0.01

Compared with the blank group, the liver tissue SOD activity of the model group rat is obviously reduced (P is less than 0.01), the MDA content is obviously increased (P is less than 0.01), which shows that after the model is made by carbon tetrachloride, the capability of eliminating active oxygen in the body of each group rat is obviously reduced, the lipid peroxidation final product is obviously increased, and the liver cell damage is serious.

Compared with the model group, SOD in the positive group and the experimental group with large, medium and small doses is obviously increased (P is less than 0.05, P is less than 0.01), and MDA is obviously reduced (P is less than 0.05, P is less than 0.01). The large, medium and small dosage of the invention is proved to play a role in preventing hepatic fibrosis by inhibiting lipid peroxidation.

6. Effect on rat liver-spleen index

The results of the tests of each group are shown in Table 19.

Watch 19

Group of	Dosage (g/kg)	Number of animals	Liver index (%)	Spleen index (‰)
					Blank group	——	13	2.2343±0.22	1.3099±0.21
Model set	——	12	3.3600±0.59^ΔΔ	2.9424±0.73^ΔΔ
					Positive group	0.8	14	2.9523±0.50^**	2.1962±0.75^**
High dose group	0.96	15	2.8046±0.29^**	1.7663±0.36^**
					Middle dose group	0.48	15	2.7658±0.31^**	1.7363±0.35^**
Low dose group	0.24	13	2.7128±0.32^**	1.8254±0.73^**

Note: comparison of model groups with blank groups^ΔΔP is less than 0.01; comparison of the drug administration group with the model group^**P＜0.01

The results in Table 19 show that the liver-spleen index of the model group is obviously higher than that of the blank group, which indicates that the liver-spleen index of the rats with the hepatic fibrosis caused by carbon tetrachloride is obviously increased (P is less than 0.01), and compared with the model group and the positive group, the liver-spleen index of the rats with the large, medium and small doses in the experimental group can be reduced (P is less than 0.01).

7. Influence on pathological organization of liver disease

The mirror image under HE staining lens is: the liver cell nucleus is dark blue, the cytoplasm is light pink, the collagen fiber is pink slightly darker than the cytoplasm, and the fatty liver cell is vacuole with different sizes and is not colored.

Blank group mirroring: the hepatic lobules are complete in structure, hepatic cells are arranged regularly, hepatic plates are arranged in a radial shape by taking a central vein as a center, biliary tubules and partial blood vessels can be seen in portal areas, and steatosis, inflammation and balloon-like necrosis are not seen.

Model group mirroring: the liver normal lobular structure is destroyed to form a disordered false lobular structure, the central vein deviates or disappears, liver cells in the lobules are edematous, a large amount of lipid vacuoles are formed, thick fibrous cables and false lobules can be seen to form, the liver cells are fattened, part of the liver cells are changed like balloons, are scattered in a small amount of inflammatory cells to infiltrate, collagen fibers are formed to form thick fibrous intervals, part of liver parenchyma is widely destroyed, diffuse fibers are formed, and separated liver cell masses are regenerated in different degrees to form early cirrhosis.

Positive group: liver cellulose is small in amount and fine, and little steatosis is observed.

Experimental group high dose group: the hepatic lobules are regularly arranged, no obvious sinus cavity stenosis is seen, little steatosis is seen, and the hepatic lobule structure is normal.

Dose groups in experimental groups: hepatic steatosis still exists around the central vein, scattered in a few inflammatory cells to infiltrate, collagen fibers are formed, and large fibrous cords are seen.

Experimental group low dose group: the stem cells in the leaflets are edematous and a large number of lipid vacuoles are visible.

The mirror image under the Masson staining mirror is: the liver cell nucleus presents dark blue or purple blue, the cytoplasm, muscle fiber and red cell present red, the collagen fiber presents light blue or green, and the liver cell steatosis presents vacuoles with different sizes and is not colored.

Blank group: the hepatic lobules have a complete structure, the hepatic cells are arranged regularly, and the hepatic plates are arranged radially by taking the central vein as the center.

Model group: the terminal veins (including the central vein) are thickened, collagen fibers extend to the paranasal space, fibrosis is formed in the area of the junction and around the junction, thick fibrous cords and false lobules are formed, fat change occurs in liver cells, part of the liver cells are changed like balloons, the liver cells are scattered and infiltrated by a few inflammatory cells, and the collagen fibers are formed to form thick fibrous intervals.

Pathological scoring is carried out on three lobules of the liver of each rat according to grading standards under a Masson staining mirror, and the average value of three is used as the pathological scoring of the hepatic fibrosis of the rat. The results are shown in Table 20.

Watch 20

Group of	Dosage (g/kg)	Number of animals	Pathological scoring
				Blank group	——	13	0±0
Model set	——	12	3.26±0.66^ΔΔ
				Positive group	0.8	14	1.83±0.96^**
High dose group	0.96	15	1.81±0.63^**
				Middle dose group	0.48	15	2.29±0.73^**
Low dose group	0.24	13	1.71±0.49^**

Note: comparing the model group with the blank group, wherein delta P is less than 0.01; the administration groups were compared with the model group^**P＜0.01

As can be seen from Table 20, the pathological score of the model group was significantly higher than that of the control group, indicating that the hepatic fibrosis model was established.

The pathological scores of the large, medium and small dose groups and the positive group of the invention are obviously lower than that of the model group (P is less than 0.01), and the invention prompts that the product can improve the pathological changes of carbon tetrachloride hepatic fibrosis tissues.

The results of the experiments are summarized below:

the experiment adopts a carbon tetrachloride-induced rat hepatic fibrosis model, the model is simple and easy to implement, the molding rate is high, the hepatic fibrosis degree is relatively ideal, but the hepatic fibrosis pathological changes of the model are easy to naturally reverse after the carbon tetrachloride injection is stopped, so that in order to accurately research the treatment effect of the medicine on the hepatic fibrosis, except for a blank group, the carbon tetrachloride dosage is reduced for each group of animals, and the subcutaneous injection molding is continued.

The experimental results show that the ALT, AST, ALP, LN, HA, PCIII, COI-IV, TGF-beta 1 concentration in the serum, MDA content in the liver tissue and liver index in the serum of the model group rat are all obviously higher than those of the blank group, TP, ALB concentration and SOD content in the serum of the model group rat are all obviously lower than those of the blank group, the weight and food intake of the model group rat are obviously lower than those of the blank group, the liver injury degree of the model group rat is also very obvious through pathological examination, and the comprehensive analysis of all factors shows that the hepatic fibrosis model of the experimental rat is relatively ideal.

The large, medium and small doses of the invention can obviously reduce ALP, ALT and AST values in rat serum, and the large dose can obviously increase TP content in the rat serum; the large, medium and small dosage of the invention can obviously reduce the concentration of LN, HA and COI-IV in the serum, and the large, medium dosage can obviously reduce the concentration of PC III in the serum of a rat; the invention can obviously increase the SOD content in liver tissues and reduce the MDA content in large, medium and small doses; the invention obviously reduces the content of TGF-beta 1 in rat serum with small dosage; the invention obviously reduces the index of liver and spleen with large, medium and small dosage; the large and medium doses of the present invention significantly increased body weight and food intake in rats from the 5 th week of administration. Pathological examination shows that liver lobules of the administration group (positive group and experimental group) are arranged regularly, fatty degeneration is reduced, inflammatory cell infiltration is obviously reduced, formation of fibroblasts and collagen fibers is obviously reduced, pathological scores of the administration group are obviously reduced, and a certain dosage relation is displayed.

In conclusion, the invention has obvious improvement effect on a rat model of the carbon tetrachloride hepatic fibrosis, the animal hepatic fibrosis of the model is similar to the human hepatic fibrosis in certain aspects of morphology and pathophysiology, for example, the two hepatic fibrosis have regeneration after hepatocyte necrosis, and the physical signs and the abnormal liver function of the experimental animal are similar to those when the human chronic liver disease progresses to cirrhosis. It is suggested that it has certain therapeutic effect on hepatic fibrosis.

Third group: the influence of the traditional Chinese medicine composition on a rat hepatic fibrosis model caused by pig serum

1. Experimental Material

Experimental animals: male SD rats, weighing 120-: 61001700000286, license: SCXK (shan) 2012 and 003.

Feeding conditions are as follows: the temperature is 20-24 ℃, the air is ventilated, the relative humidity is 40-70%, the animals can freely eat and eat, and the animals can be adaptively fed for more than one week before the experiment.

The tested drugs are: the same as the first group.

Positive drug: the same as the first group.

2. Experimental methods

2.1 preparation of rat hepatic fibrosis model

100 male SD rats were divided into blank group and model building group 2 according to the weight balance principle, 15 of blank group, and the rest 85 were molded as follows.

Injecting normal saline into abdominal cavity of blank group, injecting pig serum into abdominal cavity of model group at a dosage of 0.5 ml/pig, injecting twice per week, with interval of 2-3 days, continuously for more than 5 weeks. And (3) sampling a blank group and a model building group from the 5 th week, wherein the model building group is more than the blank group, and carrying out pathological detection until experimental grouping is carried out after pig serum is observed to induce the hepatic fibrosis of the rat.

2.2 test grouping and administration

15 pieces before blank modeling, 2 pieces after spot inspection and 13 pieces after grouping; the model group is 85, 6 are spot-checked, 7 are dead, the rest 72 are divided into 14 model groups, 15 experimental groups and 15 medium dose groups respectively, 13 experimental groups and 15 positive groups respectively.

The blank group and the model group are administrated with physiological saline by intragastric administration every day, the administration dose of the large, medium and small dose groups of the experimental group is respectively 0.96, 0.48 and 0.24g/kg, the administration dose of the positive group is 0.8g/kg, and the administration doses of the groups are respectively 1 time every day and 7 days every week for 8 weeks. Each group of rats is raised in cages, and each group can freely take normal feed and drink distilled water at room temperature (20-25 ℃), weigh the weight and eat the food once per week, and adjust the dosage of the medicine according to the weight.

Because the pig serum hepatic fibrosis model can also be recovered by self, the model group and the administration group continue to inject 0.5ml of pig serum per abdominal cavity in the administration period, 2 times per week and 2-3 days apart.

2.3 taking materials and detecting

Fasting: rats were fasted for 12h before treatment.

Rats are anesthetized, and total carotid artery blood is taken to test four items of liver function, liver fibrosis, platelet derived growth factor and transforming growth factor beta 1.

Taking liver tissue, and measuring the content of Malondialdehyde (MDA), total superoxide dismutase (T-SOD), and hydroxyproline.

2.4 Each detection index and detection method

Liver function detection: same as the first set of experiments.

Four items of hepatic fibrosis: same as the first set of experiments.

Transforming growth factor beta 1 (TGF-. beta.1): as in the second set of experiments.

Platelet Derived Growth Factor (PDGF): as in the second set of experiments.

Hydroxyproline (Hyp): same as the first set of experiments.

Malondialdehyde (MDA), superoxide dismutase (SOD): as in the second set of experiments.

Liver histopathology: HE staining, Masson trichrome method: same as the first set of experiments.

2.5 statistical methods: same as the first set of experiments.

3. Results of the experiment

During the administration, the rats did not die. Number of animals in each group at the end of experiment: 13 blank groups, 14 model groups, 15 positive groups, 15 experimental group high dose groups, 15 experimental group medium dose groups and 13 experimental group low dose groups.

3.1 weight and food intake changes after administration

The body weight and food intake variability data for each group tested are detailed in tables 21 and 22.

TABLE 21

Weight change of rats after administration

Week (week)

Blank group

Model set

Positive group

High dose group

Middle dose group

Low dose group

1

393.08±28.55

367.29±27.35^Δ

374.33±23.98^Δ

370.87±26.45^Δ

375.93±24.03^Δ

373.69±29.81^Δ

2

410.69±28.50

381.79±19.73^ΔΔ

386.00±19.03^Δ

382.27±20.81^ΔΔ

394.47±21.74

394.08±28.85

3

420.54±26.90

393.14±17.40^ΔΔ

395.60±18.16^ΔΔ

394.40±20.82^ΔΔ

405.00±19.82

406.92±28.35

4

428.23±24.74

394.12±16.16^ΔΔ

409.53±28.99

405.47±22.38^Δ

413.53±22.01^*

416.69±28.72^*

5

442.15±26.57

399.57±21.53^ΔΔ

406.27±20.21^ΔΔ

420.53±28.12^*Δ

418.13±25.16^*

425.00±30.81^*

6

449.62±29.38

408.71±20.14^ΔΔ

419.00±14.92^ΔΔ

429.27±26.85^*Δ

429.60±22.82^*Δ

436.62±30.79^**

7

454.23±28.80

410.79±24.92^ΔΔ

421.27±20.24^ΔΔ

430.93±22.59^*Δ

433.13±21.56^*Δ

434.62±28.93^*

8

470.23±30.70

416.21±26.43^ΔΔ

427.27±24.16^ΔΔ

435.33±19.50^*ΔΔ

440.33±27.51^*ΔΔ

444.69±27.13^*Δ

Note: comparison with blank group^ΔP＜0.05，^ΔΔP is less than 0.01; the administration groups were compared with the model group^*P＜0.05，^**P＜0.01

As can be seen from Table 21, the individual week weights of the model group, the positive group, the experimental group, and the high dose group and the medium and low dose groups were significantly lower than those of the blank group (P < 0.05, P < 0.01). In the experimental group, the weight of the small dose group was significantly higher than that of the model group (P < 0.05, P < 0.01) from the 4 th week of administration and the large dose group from the 5 th week of administration. The traditional Chinese medicine composition disclosed by the invention is proved to be capable of obviously increasing the weight of a hepatic fibrosis rat.

TABLE 22

Changes in food intake after administration

Week (week)

Blank group

Model set

Positive group

High dose group

Middle dose group

Low dose group

1

17.37±1.04

13.60±1.54^Δ

14.67±0.76^Δ

14.77±0.50^Δ

15.57±1.58

14.90±0.85^Δ

2

18.63±1.29

15.33±0.78^Δ

17.43±1.58

17.37±1.47

17.00±1.76

17.03±1.44

3

19.67±1.01

16.87±0.93^Δ

18.47±1.78

18.87±1.60

18.70±1.60

18.50±2.07

4

25.20±1.50

19.37±0.29^ΔΔ

21.50±1.54^Δ

21.47±1.71^Δ

20.57±0.51^*Δ

21.57±1.14^*Δ

5

27.37±1.40

21.17±1.19^ΔΔ

23.57±1.00^Δ

24.03±1.33^*Δ

24.10±1.21^*Δ

23.43±0.46^*Δ

6

31.17±1.77

25.07±0.90^ΔΔ

26.87±2.38

28.90±0.17^**Δ

28.43±1.27^*

28.37±1.26^*Δ

7

37.27±1.82

27.97±0.64^ΔΔ

31.67±3.00

33.10±1.05^**

33.90±1.35^**

33.30±1.32^**

8

39.70±1.93

31.83±2.35^Δ

37.90±3.39

39.03±1.57^*

38.43±0.64^**

38.20±1.30^*

As can be seen from Table 22, the food intake at the beginning of the administration and the test period of the model group and the positive group is significantly lower than that of the blank group (P < 0.05, P < 0.01), and the food intake at the beginning of the administration and the test period of the large, medium and small dose groups is significantly lower than that of the blank group (P < 0.05, P < 0.01) at individual weeks. In the experimental group, the small dose group started from the 4 th week of administration, and the large dose group started from the 5 th week, the food intake was significantly higher than that in the model group (P < 0.05, P < 0.01). The results prove that the traditional Chinese medicine composition can obviously increase the food intake of rats with hepatic fibrosis.

3.2 Effect on liver function

The results of the liver function tests are shown in tables 23-25.

TABLE 23

Effect on ALT and AST in rats

Group of	Dosage (g/kg)	Number of animals	ALT(U/L)	AST(U/L)	AST/ALT
						Blank group	——	13	49.46±4.68	170.31±22.94	2.59±0.57
Model set	——	14	54.93±8.09^Δ	177.14±22.66	3.31±0.80^Δ
						Positive group	0.8	15	60.87±7.61	189.60±32.48	3.15±0.62
High dose group	0.96	15	69.60±12.97^**	158.67±31.78	2.32±0.46^**
						Middle dose group	0.48	15	67.73±19.23^*	169.33±38.21	2.65±0.88
Low dose group	0.24	13	57.69±10.99	179.31±35.30	3.20±0.89

Note: model group to blank group comparison^ΔP is less than 0.05, and the administration group is compared with the model group^*P＜0.05，^**P＜0.01

Watch 24

Effect on TBIL and ALP in rats

Group of	Dosage (g/kg)	Number of animals	TBIL(mmol/L)	ALP(U/L)
					Blank group	——	13	1.19±0.22	140.38±32.48
Model set	——	14	1.43±0.36	155.21±56.80
					Positive group	0.8	15	1.37±0.36	119.73±35.17
Large dose scene group	0.96	15	1.55±0.40	157.67±67.81
					Middle dose group	0.48	15	1.50±0.41	144.00±54.27
Low dose group	0.24	13	1.40±0.51	127.15±30.88

Note: model group to blank group comparison^Δ＜0.05，^ΔΔLess than 0.01; the administration groups were compared with the model group^*P＜0.05，^**P＜0.01

As can be seen from tables 23 and 24, the ALT value in serum of the model group is significantly higher than that of the blank group (P < 0.05), and the ALT values in the positive group and the large, medium and small dose groups are also significantly increased, wherein the large and medium dose groups are significantly higher than those of the model group (P < 0.05, P < 0.01), and the reason is not clear and may be related to liver burden caused by overdose. In addition, the AST and ALP of the model group have no obvious change and are probably related to the little damage of pig serum to the liver.

TABLE 25

Effect on TP, ALB and GLB in rats

Group of

Dosage (g/kg)

Number of animals

TP(g/L)

ALB(g/L)

GLB(g/L)

ALB/GLB

Blank group

——

13

56.62±3.86

25.38±1.98

31.23±3.19

0.82±0.11

Model set

——

14

56.36±3.23

22.57±2.03^ΔΔ

33.79±3.26

0.68±0.11^ΔΔ

Positive group

0.8

15

58.67±2.23^*

23.87±1.13^*

34.80±2.46

0.71±0.07

High dose group

0.96

15

58.73±1.94^*

24.60±1.18^**

34.13±1.51

0.71±0.05

Middle dose group

0.48

15

58.67±2.06^*

24.07±0.70^*

34.60±2.26

0.70±0.08

Low dose group

0.24

13

56.69±2.10

24.38±1.94^*

32.31±2.06

0.76±0.10

As shown in Table 25, the serum ALB of the model group is obviously lower than that of the blank group (P is less than 0.01), and the serum hepatic fibrosis of the pig can be presumed to influence the generation of the hepatic albumin. Compared with a model group, the ALB (P is less than 0.05, P is less than 0.01) can be obviously increased in large, medium and small dose groups of an experimental group, and the traditional Chinese medicine composition can be proved to be capable of resisting the reduction of hepatic fibrosis albumin without obviously influencing TBIL, TP and GLB.

3.3 Effect on four terms of hepatic fibrosis

The results of the tests are shown in tables 26-27.

Watch 26

Influence on four items of hepatic fibrosis

Group of	Dosage (g/kg)	Number of animals	LN(ng/ml)	HA(ng/m1)
					Blank group	——	13	472.49±71.23	343.93±22.87
Model set	——	14	633.37±70.18^ΔΔ	433.09±46.47^ΔΔ
					Positive group	0.8	15	526.79±37.37^**	373.69±39.62^**
High dose group	0.96	15	511.05±36.61^**	370.69±32.74^**
					Middle dose group	0.48	15	518.58±42.73^**	374.86±37.60^**
Low dose group	0.24	13	546.56±34.95^**	393.97±29.15^*

Watch 27

Influence on four items of hepatic fibrosis

Group of	Dosage (g/kg)	Number of animals	PCIII(ng/ml)	COI-IV(ng/ml)
					Blank group	——	13	59.60±4.28	19.45±4.14
Model set	——	14	80.20±30.91^Δ	23.66±4.47^Δ
					Positive group	0.8	15	61.98±4.29^*	20.49±2.75^*
High dose group	0.96	15	60.37±2.77^*	19.64±3.50^*
					Middle dose group	0.48	15	63.65±5.34^*	19.42±2.68^**
Low dose group	0.24	13	64.39±3.99	22.05±6.04^*

Note: model group to blank group comparison^ΔLess than 0.05; the administration groups were compared with the model group^*P＜0.05，^**P＜0.01

In the experiment, the LN, HA, PCI II and COI-IV of the rat serum are obviously increased (P is less than 0.05, P is less than 0.01) compared with the blank group in the model group; compared with the model group, the large, medium and small dose groups of the experimental group obviously reduce the concentration of LN, HA and COI-IV in the serum (P is less than 0.05, P is less than 0.01), and the large and medium dose groups obviously reduce the concentration of PCI II in the serum (P is less than 0.05). Proved by experiments, the traditional Chinese medicine composition obviously inhibits the synthesis of LN, HA, PCI II and COI-IV of hepatic fibrosis and slows down the progress of hepatic fibrosis.

3.4 Effect on transforming growth factor beta 1 (TGF-. beta.1), platelet-derived growth factor (PDGF)

The results of the tests are shown in Table 28.

Watch 28

Effect on liver tissues TGF-. beta.1, PDGF

Group of	Dosage (g/kg)	Number of animals	TGF-β1(ng/m1)	PDGF(ng/ml)
					Blank group	——	13	6.6333±0.50	8.39±0.85
Model set	——	14	6.1248±1.39	10.83±0.79^ΔΔ
					Positive group	0.8	15	6.4716±1.01	10.16±1.62^*
High dose group	0.96	15	6.1945±0.87	8.38±0.83^**
					Middle dose group	0.48	15	6.6465±1.12	7.87±0.66^**
Low dose group	0.24	13	6.6018±0.88	9.36±1.25^**

Note: model group compared to blank group Δ Δ Δ P < 0.01: comparison of the drug administration group with the model group^*P＜0.05，^**P＜0.01

As can be seen from Table 28, the PDGF content in the serum of the model group rats is higher than that in the blank group, and the PDGF content in the serum of the rats is obviously reduced in the large, medium and small dose groups of the experimental group (P < 0.01).

3.5 Effect on hepatic hydroxyproline

The results of the tests are shown in Table 29.

Watch 29

Effect on hydroxyproline

Group of	Dosage (g/kg)	Number of animals	Hyp(μg/mg)
				Blank group	——	13	258.3404±29.21
Model set	——	14	341.6095±57.12^ΔΔ
				Positive group	0.8	15	274.5437±12.07^**
High dose group	0.96	15	262.295±32.50^**
				Middle dose group	0.48	15	265.2779±53.29^**
Low dose group	0.24	13	267.6403±7.01^**

As can be seen from Table 29, the content of hydroxyproline (Hyp) in the liver tissue of rats was significantly increased (P < 0.01) compared with the blank group in the model group, suggesting hepatic fibrosis of rats. The experimental groups of large, medium and small dose groups and the positive group can obviously reduce the content of Hyp (P is less than 0.01), and the traditional Chinese medicine composition has a certain treatment effect on hepatic fibrosis.

3.6 Effect on liver tissue SOD, MDA

The results of the tests are shown in Table 30.

Watch 30

Influence on SOD and MDA of liver tissue

Group of	Dosage (g/kg)	Number of animals	SOD(U/mgprot)	MDA(mmol/mgprot)
					Blank group	——	13	151.1334±28.4818	8.8047±2.0466
Model set	——	14	107.9970±15.74^ΔΔ	16.7464±2.62^ΔΔ
					Positive group	0.8	15	135.0312±27.66^**	11.1670±2.80^**
High dose group	0.96	15	140.6750±33.76^**	12.7432±2.21^**
					Middle dose group	0.48	15	143.2360±33.30^**	11.3965±2.27^**
Low dose group	0.24	13	137.4040±15.02^**	12.3355±2.70^**

As can be seen from Table 30, in the model group, compared with the blank group, the SOD activity of liver tissue of rat is significantly reduced, the MDA content is significantly increased (P is less than 0.01), which indicates that the capability of eliminating active oxygen in vivo of the model-making rat is significantly reduced, the lipid peroxidation final product is significantly increased, and liver cells are seriously damaged. Compared with the model group, the experiment group obviously increases SOD activity (P is less than 0.01) and obviously reduces MDA content (P is less than 0.01) in large, medium and small dose groups. The traditional Chinese medicine composition of the invention is proved to play a role in preventing and treating hepatic fibrosis by inhibiting lipid peroxidation.

3.7 Effect on rat liver-spleen index

The results of the tests are shown in Table 31.

Watch 31

Index of liver and spleen

Group of	Dosage (g/kg)	Number of animals	Liver index (%)	Spleen index (‰)
					Blank group	——	13	2.6074±0.19	1.4380±0.23
Model set	——	14	2.8884±0.34^Δ	1.3840±0.34
					Positive group	0.8	15	2.7899±0.18	1.6802±0.33^*
High dose group	0.96	15	3.0245±0.28	1.6249±0.26
					Middle dose group	0.48	15	2.9216±0.23	1.6334±0.25
Low dose group	0.24	13	2.7375±0.39	1.4328±0.27

Note: model group to blank group comparison^ΔP is less than 0.05; comparison of the drug administration group with the model group^*P＜0.01

As can be seen from Table 31, the liver index of the model group was higher than that of the blank group (P < 0.05), and the liver and spleen index of the rats was not significantly affected by the large, medium and small dose groups of the experimental group.

3.8 Effect on liver pathological organization

Liver tissue under HE staining scope was: the liver cell nucleus is dark blue, the cytoplasm is light pink, the collagen fiber is pink slightly darker than the cytoplasm, and the fatty liver cell is vacuole with different sizes and is not colored.

Blank group: the structure of hepatic lobules is clear, hepatic cells in the hepatic lobules are basically normal, the hepatic cells are round, the outline is clear, the arrangement is neat, the structure of a junction area is clear, and small bile ducts and fibrous tissue hyperplasia are not found in the junction area and the periphery of the junction area;

model group: the model group shows that the hepatic lobular structure is fuzzy, hepatic cells in the lobules are edematous, the liver cells of a local focus are necrotic, a small bile duct is massively proliferated, the small bile duct radially extends into the liver parenchyma by taking a manifold area as a center, the proliferated bile duct is in a rosette shape, and the periphery of the proliferated bile duct is accompanied with fibrous tissue proliferation and inflammatory cell infiltration.

Positive group: the hepatic lobule structure is clear, local liver cells in the lobules are in punctate necrosis, small bile ducts in the liver slightly proliferate with a tract area as the center, and the periphery of the proliferated bile ducts is accompanied by slight fibrous tissue proliferation and small inflammatory cell infiltration.

Experimental group high dose group: the liver lobule structure is clear, the hyperplasia of connective tissues is reduced, the fiber interval is narrowed, part of the fiber interval is broken, and the inflammatory infiltration degree is obviously reduced;

dose groups in experimental groups: the liver lobule structure is clear, the desmoplasia is reduced, the fiber interval is narrowed, part of the fiber interval is broken, and the inflammatory infiltration degree is reduced;

experimental group low dose group: the hepatic lobule structure is clear, the small bile duct in the liver slightly proliferates by taking the region of the junction as the center, and the periphery of the proliferated bile duct is accompanied with slight fibroplasia and small inflammatory cell infiltration.

Liver tissue under Masson staining mirror is: the liver cell nucleus presents dark blue or purple blue, the cytoplasm, muscle fiber and red cell present red, the collagen fiber presents light blue or green, and the liver cell steatosis presents vacuoles with different sizes and is not colored.

Model group: the terminal veins (including the central vein) are seen to be thickened, collagen fibers extend to the paranasal space, fibrosis is formed in and around the area of the junction, thick cellulose and false lobule are formed, fat change occurs in liver cells, part of the liver cells are changed like a balloon, the liver cells are scattered and infiltrated by a few inflammatory cells, and the collagen fibers are formed to form thick fibrous intervals.

Positive group: liver cellulose is small in quantity and fine, and little steatosis can be seen;

experimental group high dose group: the liver lobules are arranged regularly, obvious sinus stenosis is not seen, little steatosis can be seen, the hyperplasia of connective tissues is reduced, the fibrous interval is narrowed, part of fibrous intervals are broken, and the inflammatory infiltration degree is obviously reduced;

dose groups in experimental groups: hepatic steatosis still exists around the central vein, the fiber interval becomes narrow, part of the fiber interval is broken, and the inflammatory infiltration degree is reduced;

experimental group low dose group: there is much fatty degeneration around the central vein of hepatic lobule, the hepatic lobule structure is clear, the small bile duct in liver slightly proliferates with the region of the collector as the center, and the periphery of the proliferated bile duct is accompanied with slight fibrous tissue proliferation and a little inflammatory cell infiltration.

Pathological scoring is carried out on three lobules of the liver of each rat according to grading standards under a Masson staining mirror, and the average value of three is used as the pathological scoring of the hepatic fibrosis of the rat. The results are shown in Table 32.

Watch 32

Liver histology scoring

Group of	Dosage (g/kg)	Number of animals	Liver pathology scoring
				Blank group	——	13	0.07±0.17
Model set	——	14	2.14±0.66^ΔΔ
				Positive group	0.8	15	1.32±1.17^*
High dose group	0.96	15	1.36±0.66^**
				Middle dose group	0.48	15	1.42±0.97^*
Low dose group	0.24	13	1.05±0.91^**

Note: model group to blank group comparison^ΔΔP is less than 0.01: the administration groups were compared with the model group^*P＜0.05，^**P＜0.01

The evaluation score of the model group is obviously higher than that of the control group, which indicates that the hepatic fibrosis model is established. The pathological scores of the large, medium and small dose groups and the positive group of the experimental group are obviously lower than that of the model group (P is less than 0.01), and the Chinese medicinal composition can improve the carbon tetrachloride hepatic fibrosis lesion.

4. Discussion of the related Art

The pig serum modeling rat does not die, has small influence on liver function of the rat, but has better liver fibrosis. The experimental results show that the serum LN, HA, PCIII, COI-IV and PDGF concentrations of the model group rats, the MDA and Hyp contents of the liver tissues and the liver indexes are all obviously higher than those of the blank group, the serum ALB concentration and the SOD content of the liver tissues of the model group rats are all obviously lower than those of the blank group, the body weight and the food intake of the model group rats are obviously lower than those of the blank group, the liver injury degree of the model group rats is also very obvious through pathological examination, and comprehensive analysis of all factors indicates that the experimental rat liver fibrosis model is established.

Similarly, the fibrosis of the pig serum hepatic fibrosis model is naturally reversed after the model is stopped, so that the intervention effect of the medicine on hepatic fibrosis is conveniently researched, and the model is still continuously made on each group of animals except a blank group in the grouping administration period.

The large, medium and small doses of the traditional Chinese medicine composition can obviously reduce the concentrations of LN, HA, COI-IV and PDGF in the serum of a rat and reduce the contents of MDA and Hyp in liver tissues, the large, medium and small doses of the traditional Chinese medicine composition can obviously reduce the concentration of PC III in the serum of the rat, and the large, medium and small doses of the traditional Chinese medicine composition can obviously improve the content of SOD in the liver tissues, and the large, medium and small doses can obviously increase the weight and food intake of the rat. Pathological examination shows that liver lobules of the administration group are arranged regularly, fatty degeneration is reduced, inflammatory cell infiltration is obviously reduced, fibroblast and collagen fiber formation is obviously reduced, pathological score is obviously reduced, and a certain dosage relation is displayed.

In conclusion, the traditional Chinese medicine composition has an obvious improvement effect on immune liver fibrosis rats, and the traditional Chinese medicine composition has a certain treatment effect on liver fibrosis patients and an effect of reversing liver fibrosis, and the reversing effect of the traditional Chinese medicine composition is equivalent to or better than that of the existing effective medicine compound turtle shell liver softening tablets.

The toxicological research of the traditional Chinese medicine composition comprises two aspects of acute toxicity and long toxicity.

In a first aspect: long poison

After SD rats of 6-7 weeks old are repeatedly gavaged with the dry powder preparation prepared according to the formula of the invention in the example 1, the toxicity reaction and the severity of animals, and the reversible degree of main toxic target organs and damage are observed, so that a reference basis is provided for clinical toxic and side effect monitoring.

The research content is as follows: 160 rats were used in total, half each in males and females, and quarantined for 7 days. Animals were grouped on day 7 of quarantine into 4 experimental groups, vehicle control group, low dose group, medium dose group, and high dose group. Groups of 40 animals were used, male and female halves. The administration is carried out by intragastric administration, the administration dosage is 100, 300 and 1000mg/kg in sequence, the administration is repeated for 6 months, and the recovery period is 1 month. During the experiment, the clinical symptoms of the animals were observed, the body weight and the food intake were measured, and hematology, blood coagulation, serum biochemistry, urine examination and ophthalmology examination were performed. Gross pathology and histopathology examinations were performed at mid-dose, end-dose and end-convalescent period.

The results of the study are as follows:

1. observation of clinical symptoms

The rats numbered F206, F316, M304, M305, M402 and M407 die, and the death of the animals is presumed to be caused by struggling during the gavage administration or the lung of the test sample caused by improper operation of the experimenter according to the comprehensive analysis of the microscopic examination results, and is related to the test sample.

The rat with the number M404 dies, no obvious abnormality is found in gross autopsy, and the microscopic examination result speculates that the M404 causes the body immunity to be reduced due to the kidney adenocarcinoma, such as the reduction of thymic cortex moderate lymphocytes and the slight reduction of spleen white marrow lymphocyte numbers, further influences the cardiopulmonary function, causes the liver hepatocytes to be moderately degenerated/necrotized, and finally the animal dies due to the exhaustion of the cardiopulmonary function. Only M404 in the experiment shows renal adenocarcinoma, and other animals do not show the lesion, so that the death of the animal is not related to the test article.

The rat numbered M412 died, no obvious abnormality was seen in gross necropsy, and microscopic examination revealed moderate dilatation of the right ventricle and moderate left atrium/right atrium of the heart, moderate dilatation of the pulmonary vessels and minimal foam-like cell aggregation, mild thymus hemorrhage, moderate congestion of the liver and liver sinuses, moderate dilatation of the renal vessels and minimal degeneration/regeneration of the renal tubules, and parathyroid gland was not seen. The reason for death in male M412 was considered to be probably due to respiratory cycle failure based on microscopic examination. Other animals did not see the above lesions and the death of this animal was not related to the test article.

Rats numbered M303 and M306 showed hair loss, which was not observed in the other dose groups, and was analyzed as an individual animal sporadic event, regardless of the test article.

The rat with the number F320 had a lump in the right forelimb, which was not observed in other animals, so that the phenomenon was sporadic in individual animals and was not related to the test article.

2. Body weight determination

During the experiment, the body weights of the animals in the low, medium and high dose groups are compared with the body weight of the animal in the vehicle control group at the same time point, and no statistical difference is seen.

3. Food intake

During the experiment, no obvious abnormal food intake is observed in the animals in the administration period and the recovery period of the low-dose group, the medium-dose group and the high-dose group of the test article.

4. Ophthalmology examination

In the middle period of administration, the end period of administration and the recovery period, the ophthalmic examination of each animal is not abnormal in the low, medium and high dose groups of the test sample.

5. Hematology examination

The animal hematology indexes of the low-dose group, the medium-dose group and the high-dose group of the test sample have no obvious abnormality related to the test sample.

6. Biochemical examination of serum

In the middle period of administration, the end period of administration and the recovery period, no obvious abnormality related to the test sample is found in the serum biochemical indexes of the low-dose group, the medium-dose group and the high-dose group of the test sample.

7. Urine examination

In the middle administration period, the end administration period and the recovery period, no obvious abnormality related to the test sample is found in the urine examination indexes of the animals in the low dose group, the medium dose group and the high dose group of the test sample.

8. Weight of visceral organs

The weight of the animal organs of the low-dose group, the medium-dose group and the high-dose group of the test sample has no obvious abnormality related to the test sample in the middle administration period, the end administration period and the recovery period.

9. Results of pathological examination

9.1 dead animals

In the experiment, 1 female animal (F206) in the low-dose group, 2 male animals (M304 and M305) in the medium-dose group, 1 female animal (F316) in the medium-dose group, and 4 male animals (M412, M402, M404 and M407) in the high-dose group die.

The microscopic examination result supposes that the death reasons of the animals F206, M304, M305, F316, M402 and M407 can be caused by struggling during the gavage administration or the lung of the test sample caused by improper operation of experimenters.

M404: according to the gross autopsy and microscopic examination results, the comprehensive analysis conjectures that M404 causes the organism immunity reduction due to the kidney adenocarcinoma, such as the reduction of thymic cortex moderate lymphocyte and the slight reduction of spleen leucocyte number, further influences the cardiopulmonary function, causes the moderate degeneration/necrosis of liver hepatocyte, and finally dies due to the exhaustion of the cardiopulmonary function. The test sample is analyzed to be related to animal individuals and not related to the test sample.

M412: death occurred on day 53 of dosing. Gross autopsy shows no obvious abnormality, microscopic examination shows moderate dilatation of the right ventricle and moderate dilatation of the left atrium/right atrium of the heart, moderate dilatation and minimal foam-like cell aggregation of the lung vessels, mild thymus hemorrhage, moderate hepatic sinus congestion of the liver, moderate dilatation of the kidney vessels and minimal degeneration/regeneration of the renal tubules, and parathyroid gland does not appear. The reason for death in male M412 was considered to be probably due to respiratory cycle failure based on microscopic examination. The test sample is analyzed to be related to animal individuals and not related to the test sample.

9.2 planned Caesarean animal

9.2.1 gross dissection results

Gross necropsy results: m207 heart can see grey-white plaque, F107 liver can see grey-white plaque, F210 liver can see swelling, F302 kidney is dark red, F407 kidney can see dark red plaque, F301 pituitary volume is enlarged, M303 brain surface can see a depression, and through comprehensive microscopic examination analysis, the animal organ abnormality is mostly considered animal accidental or background lesion, and is irrelevant to the test article. No organ abnormalities were noted in the remaining animals.

9.2.2 microscopic examination results

After the administration, the liver of the high-dose animal can be seen to be hypertrophied by hepatic cells around the central vein, the lesion frequency is 3/17, and the lesion degree is very mild; the animals in the vehicle control group and the low and medium dose groups do not have the above pathological changes; since the lesions occur in the high dose group, the incidence of the lesions is low and the degree of the lesions is low, it is not excluded that the lesions in the liver of the high dose group are associated with the administration of the test article. At the end of the recovery period, the vehicle control group and the low, medium and high dose groups did not show the pathological changes of the hypertrophy of the hepatic cells around the central vein.

At the end of the recovery period, mild necrosis was observed in liver microscopic examination of the medium 1/4 female animals, while the low and high dose groups and vehicle control groups did not see the above lesions. The incidence of lesions is low, no dose correlation exists, and mild necrosis of the liver of the female animals in the medium dose group is not considered to be related to the administration of the test article.

Histopathological changes in the other organs of the remaining groups of animals were considered sporadic or background lesions, and were not associated with the administration of the test article.

The research conclusion is that: SD rats are gavaged with the drug (dry powder) of the invention example 1, and are repeatedly administrated for 6 months, the recovery period is 1 month, long-term toxicity studies show that the drug of the invention has no toxic or side effect at low dose (100mg/kg), medium dose (300mg/kg) and high dose (1000mg/kg), and the NOVEL dose is 1000 mg/kg.

In a second aspect: acute toxicity

SD rats of 6-7 weeks old are orally taken and gavaged once with different doses of the medicament (dry powder) in the example 1, and the acute toxic reaction, the severity and the death condition of the animal caused by the test sample are observed, so that reference is provided for the safe dose setting in clinical use.

The research content is as follows: the experiment was carried out with a vehicle control group, a low dose group (2g/kg) and a high dose group (4g/kg), 10 animals per group, male and female halves. The administration volume is 20ml/kg in single intragastric administration. The animal's posture, gait, reaction state, nerve activity, appetite, fur, glasses, ear, mouth, nose, limbs, breath, feces and other clinical symptoms are observed within 2 hours after administration, and if abnormal symptoms appear, the observation is continued until the abnormal symptoms disappear 30 minutes, and the observation is carried out for 6 hours at most after administration. The observation was then continued by day 15 for clinical symptoms, activities, etc. of the animals. Body weight measurements were made on the animals at regular intervals during the experiment. Gross pathology was examined on day 15 for all animals.

The research results are as follows:

1. observation of clinical symptoms

The clinical symptom results show that no obvious abnormality is found in each group of animals within 2 hours after the administration. No significant abnormality was found in any of the groups within 14 days after the administration.

2. Results of body weight measurement

Example 1 the body weight was measured on day 1, day 2, day 3, day 7 and day 14 of the drug (dry powder) administration in each group, and no significant difference was observed compared with the vehicle control group.

3. Dissection and histopathology

All animals were necropsied 15 days after dosing and no gross pathological changes were seen in each group of animals, therefore, no histopathological examination was performed on all animals in this study.

The research conclusion is that: the above results show that the maximum tolerated dose of the drug of example 1 of the present invention (dry powder) administered by gavage to rats is 4g/kg, 53 times the dose of the drug effective in clinical practice, in terms of body surface area ratio.

Typical cases

Case 1: somebody keen, male, 49 years old, shanghai.

The patients have hepatitis B at 13 years old, and the patients are diagnosed with liver cirrhosis in the decompensation stage, esophageal varices at the bottom of the stomach, splenomegaly and ascites due to hemorrhage in 3 months in 2009 for more than 30 years. After the traditional Chinese medicine composition is taken, the liver disease condition is gradually stabilized and improved, the mental state is good, the laboratory examination is recovered to be normal from the blood routine, liver function, B ultrasonic image examination and gastroscopy, and the disease is completely cured, particularly the B ultrasonic liver ultrasonic quality image is normal and has no pathological change.

Case 2: li Jie, male, 40 years old, Xinjiang Wulu wood Qi people.

In 2016, 1 month and 5 days, patients feel uncomfortable in liver area before taking the medicine, sometimes have gingival bleeding, poor sleep and ordinary stomach and sodium, and have been diagnosed as liver cirrhosis in local hospitals. B ultrasonic examination shows that: diffuse liver disease, liver cirrhosis. Blood examination: b hepatitis E antigen quantitative examination: 2.72 to 3.37; quantitative detection of hepatitis B surface antigen: 1383 to 1428; alkaline phosphatase (alkaline phosphatase): 43.81, respectively; direct bilirubin: 3.43.

after more than ten months of treatment and observation, the patients have stable general illness state, the complexion changes from dark gray to ruddy, the spirit is exhausted to have spirit and forceful qi, the discomfort of the liver area basically disappears, the sleep is uneasy to be good, the tongue vessels are all normal, the direct bilirubin is higher than normal, and no obvious abnormality is found in other examinations.

Case 3: wang somebody, man, 62 years old.

The patient has a history of hepatitis B for decades, and has the symptoms of distending pain in the liver area, lassitude, fatigue in motion, extreme fatigue, bedridden patients with poor physical strength, and poor night sleep in the clinic, and the patient is clearly diagnosed with cirrhosis through the examination of the relevant hospitals in Shanghai. As is well known, patients with liver cirrhosis are characterized by long course of disease, poor prognosis, many complications and frequent and repeated attacks of disease.

After patients take the traditional Chinese medicine composition, the symptoms of the patients in three months show a comprehensive improvement trend (B ultrasonic + general situation), the whole set of the blood and liver functions is normal after the examination, and only the blood total bilirubin 21.7 mu mol/L is slightly higher than the direct bilirubin 7.8 mu mol/L. B ultrasonic shows that the liver is normal in shape and size, the rod surface is smooth, the envelope is complete, the intrahepatic echoes are distributed unevenly and thickened, the vein veins are deformed clearly, and no obvious abnormal blood flow signal is seen in 8mm CDPI of the intrahepatic bile duct.

Case 4: some Zhu, male, 69 years old, Shanghai.

The patient has adopted various modes once for more than thirty years due to the hemophagous liver cirrhosis, and the symptoms of the traditional Chinese and western medicine combined treatment are not obviously improved, and the patient feels mental fatigue, weakness of limbs, pain in the liver area, gingival bleeding, abdominal distension, pain and discomfort and frequent eructation in two years. Once diagnosed as hemophagous cirrhosis in Shanghai city Fengxian area central hospital, ultrasonography B showed that the portal vein was thickened and the spleen was enlarged.

After nine courses of treatment by adopting the traditional Chinese medicine composition, the subjective symptoms of the patient are obviously improved, and the same as normal.

Case 5: kangyi, woman, 42 years old, Fujian Quanzhou

The patients suffer from posthepatitic cirrhosis decompensation, esophageal venous rupture bleeding, hemorrhagic anemia, splenic hyperfunction and hypoproteinemia, and are repeatedly hospitalized for 5 times within 2014.

Under the conditions of repeated and gradually serious diseases and no medical help seeking and future preparation, the traditional Chinese medicine composition is tried out by patients, and the examination report of the traditional Chinese medicine institute in Quanzhou city before medicine taking shows that: 3.1 parts of white blood cells; 2.21 parts of red blood cells; hemoglobin 54; platelet count 57; total protein 46; albumin 28. Gastroscopy shows moderate varicosis of veins in the middle and lower parts of esophagus, portal hypertension gastropathy, B-ultrasonic diagnosis, liver parenchymal diffuse lesion, liver cirrhosis, splenomegaly accompanied with splenic vein dilatation and ascites.

After 4 months of taking the traditional Chinese medicine composition, patients are subjected to cardia peripheral vascular dissection and splenectomy, and after taking the traditional Chinese medicine composition, the relevant examination report of the second hospital affiliated to Fujian medical university shows that: total protein 76.8; albumin 39.7; globulin 37.1; b ultrasonic display: diffuse lesions of the left and right liver parenchyma, liver cirrhosis and splenic postoperative deficiency, no obvious abnormality of gallbladder and pancreas is found, and the routine examination report of blood after half a month shows that: white blood cells 5.7, red blood cell count 4.71, hemoglobin 101, platelets 301.

After 10 months of treatment, the patient is scheduled for physical examination by the working unit, and the report shows that: positive hepatitis B surface antigen, alanine aminotransferase 32.3; 4.87 parts of red blood cells; hemoglobin 103; platelets 310.

After 14 months of treatment, the physical examination reports were as follows: conventional biochemical report sheet: total protein 75.8; albumin 43; globulin 32.8, the rest normal. Blood routine: 7.0 parts of white blood cells; red blood cell count 5.36; 99 of hemoglobin; platelets 400. B, ultrasonic wave: after diffuse change of liver parenchymal echo, liver cirrhosis and splenectomy, abnormal occupation is not left.

After the patient is treated for one and a half years, the examination report of the Quanzhou traditional Chinese medicine institute shows that the blood routine has no obvious abnormal change, and the B-mode ultrasound has no obvious change compared with the previous B-mode ultrasound.

The patients begin to take the traditional Chinese medicine composition in 12 months in 2014, and take the entecavir as well as other adjuvant therapy and other medicines except for anti-hepatitis B virus therapy, so that the condition is stable, the patients recover, the mood is happy, and upper gastrointestinal hemorrhage such as hematemesis and black stool is avoided.

Case 6: wang somebody, man, 66 years old, Shanghai

The patients have been treated for schistosomiasis for more than 40 years, and the patients have been diagnosed as blood-sucking liver cirrhosis by examination of Shanghai eosino hospital and Jinshan public health center, because the patients feel distending pain in liver area, dark face, full feeling after eating and eating, lassitude and fatigue and weakness after eating for recent years.

The initial examination conditions were as follows: 1) and (3) ultrasonic detection report: substantially diffuse lesions-schistosomiasis cirrhosis; 2) total bilirubin 33.4; indirect bilirubin 28; triglyceride 1.81; low density lipoprotein 1.9.

After a patient takes the pharmaceutical composition for one month, the distending pain of the conscious liver area is improved, the stool is thin, and the spirit is good. The examination results for 5 months of treatment were: 1) ultrasonic: chronic schistosomiasis cirrhosis; 2) blood examination: total bilirubin 27; direct bilirubin 7.1; triacylglycerol 1.77; apolipoprotein a 1.09. After 8 months of treatment, the subjective symptoms of the patient are improved, the spirit is more vigorous, the distending pain in the liver area basically disappears, the abdominal distension does not feel after eating, the stool and urine are normal, the tongue fur is thin and white, the pulse is thin, the face is bright and shiny, and the examination result is as follows: 1) ultrasonic: schistosomiasis cirrhosis, portal diameter of portal vein 12.5 mm; 2) blood examination: 28.1 of total bilirubin; direct bilirubin 6.9.

The patient baseline controls are shown in Table 33 below.

Watch 33

In light of the above teachings, those skilled in the art will readily appreciate that the materials and their equivalents, the processes and their equivalents, as listed or exemplified herein, are capable of performing the invention in any of its several forms, and that the upper and lower limits of the parameters of the materials and processes, and the ranges of values between these limits are not specifically enumerated herein.

Claims

1. A traditional Chinese medicine composition for treating hepatic fibrosis is characterized by being prepared from the following components in parts by weight:

2. The Chinese medicinal composition as claimed in claim 1, wherein the saussurea involucrate is saussurea involucrate.

3. The traditional Chinese medicine composition as claimed in claim 1 or 2, wherein the amount of the Bufo siccus is 110-140 parts; 25-55 parts of saussurea involucrate; the turtle shell accounts for 34-84 parts; the mulberry is 110-186 parts; 55-139 parts of dendrobium; 25-56 parts of chrysanthemum; the liquorice is 25-56 parts.

4. The traditional Chinese medicine composition as claimed in claim 1 or 2, wherein the amount of the Bufo siccus is 130 parts by weight of 115; 30-50 parts of saussurea involucrate; 36-78 parts of turtle shell; the mulberry is 115-174 parts; the dendrobium is 61-130 parts; 29-50 parts of chrysanthemum; 29-50 parts of liquorice.

5. The traditional Chinese medicine composition according to claim 1 or 2, wherein the number of the dried toads is 120; 42 parts of saussurea involucrate; 69 parts of turtle shell; 117 parts of mulberry; 82 parts of dendrobium; 35 parts of chrysanthemum; the liquorice is 35 parts.

6. The traditional Chinese medicine composition of claim 1 or 2, which is prepared by a method comprising the following steps:

(3) all filtrates were combined.

7. A method for extracting the Chinese medicinal composition of any one of claims 1 to 6, comprising the steps of:

(3) all filtrates were combined.

8. The method for extracting the traditional Chinese medicine composition as claimed in claim 7, which comprises the following steps:

decocting the residue with water, filtering, and collecting filtrate;

and combining the three filtrates, continuously concentrating to obtain an extract with the relative density of 1.15-1.30 at the temperature of 60-80 ℃, and storing at the low temperature of 0-4 ℃.

9. A Chinese medicinal preparation, which is characterized by being prepared from the Chinese medicinal composition as claimed in any one of claims 1 to 6 and pharmaceutically acceptable auxiliary materials.