CN111617216B - Anti-hepatic fibrosis traditional Chinese medicine composition and application thereof - Google Patents

Abstract

The invention relates to an anti-hepatic fibrosis traditional Chinese medicine composition and application thereof, wherein the traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight: 10 parts of morinda officinalis, 10 parts of radix rehmanniae, 10 parts of raw astragalus membranaceus, 10 parts of herba lycopi, 9 parts of rhizoma wenyujin concinnatae and 6 parts of pericarpium citri reticulatae viride. The invention also comprises the application of the traditional Chinese medicine composition in preparing anti-hepatic fibrosis medicines. The whole formula takes kidney tonifying and qi benefiting, blood circulation activating and blood stasis removing, heat clearing and detoxifying as treatment principles, the combination of the morinda officinalis and the astragalus membranaceus in the formula can tonify the kidney yang of the innate spleen and the spleen qi of the acquired spleen, and the effects of tonifying kidney, invigorating spleen and tonifying qi are achieved together. The medicines are combined to play the effects of tonifying kidney and benefiting qi, activating blood circulation to dissipate blood stasis and clearing heat and detoxicating.

Description

Anti-hepatic fibrosis traditional Chinese medicine composition and application thereof

Technical Field

The invention relates to the technical field of traditional Chinese medicines, in particular to a traditional Chinese medicine composition for resisting hepatic fibrosis and application thereof.

Background

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

Liver fibrosis is a dynamic pathological process, and multiple links interact in multiple stages of occurrence, development and regression. The traditional Chinese medicine considers that the damp-heat toxin stasis causes chronic liver injury, and the damp-heat toxin stasis is also an initiating factor of hepatic fibrosis and is a continuous pathogenic reason. Dampness-heat toxin stagnation stagnates in the blood system of the liver collaterals, the liver collaterals are injured, the toxin causes stasis, blood stasis is not transformed and dampness and heat is generated, and the dampness-heat toxin stasis is accumulated in the liver collaterals and is the main factor for the formation and aggravation of hepatic fibrosis. The persistent existence of damp-heat and blood stasis can damage healthy qi, resulting in spleen qi deficiency, liver and kidney yin deficiency, and spleen and kidney yang deficiency, which can eventually lead to the formation of intractable symptoms such as accumulation and tympanites. The pathogenesis evolution process of chronic liver diseases (damp heat-blood stasis-stagnant heat-damp heat toxin-deficiency of qi and yin) is an important link for the formation and aggravation of hepatic fibrosis, wherein the damp heat toxin is the most key. Lingshu and Bai Bian Sheng chapter consider that the invasion of pathogenic factors into the body, internal injury, anxiety and anger, or improper diet, which results in "damp retention, blood coagulation in the interior without dispersing, body fluids with astringent and penetrating nature without removing, and accumulation with ". For example, exogenous pathogens such as "damp-heat epidemic toxin" and "poison parasitic tympantis" invade the liver or are injured by alcohol and food, which are not healed for a long time and gradually lead to dysfunction of the liver, spleen and kidney, and stagnation of qi, blood and body fluid, which leads to obstruction of channels and stagnation of qi, and then to failure of yang qi to smooth, which causes blood coagulation in the interior to be not dissipated, stagnation of body fluid infusion also occurs, and finally, phlegm-dampness, blood stasis and blood stasis are caused, and liver collateral obstruction is caused. Therefore, "blood coagulation in the interior" and "body fluid unsmooth infiltration" are the general pathogenesis of hepatic fibrosis.

The traditional Chinese medicine mainly treats the hepatic fibrosis by promoting blood circulation to remove blood stasis and supplementing qi and nourishing yin as auxiliary materials. However, the differentiation of liver fibrosis is well known, and there is no unified and standardized standard, and the symptoms are often compatible, and there is no medicine with obvious curative effect on liver fibrosis.

In order to overcome the defects, the inventor of the present invention has developed the anti-hepatic fibrosis traditional Chinese medicine composition and the application thereof based on years of clinical experience, and no report is found about the present invention.

Disclosure of Invention

The invention aims to provide a traditional Chinese medicine composition for resisting hepatic fibrosis and application thereof aiming at the defects of the prior art.

In order to achieve the purpose, the invention adopts the technical scheme that:

on one hand, the invention provides a traditional Chinese medicine composition for resisting hepatic fibrosis, which consists of the following raw material medicines in parts by weight: 0.1 to 20 portions of morinda officinalis, 0.1 to 20 portions of radix rehmanniae, 0.1 to 20 portions of raw astragalus root, 0.1 to 20 portions of herba lycopi, 0.1 to 20 portions of rhizoma wenyujin Concisa and 0.1 to 15 portions of green tangerine peel.

Preferably, the traditional Chinese medicine composition consists of the following raw material medicines in parts by weight: 5-15 parts of morinda officinalis, 5-15 parts of radix rehmanniae, 5-15 parts of raw astragalus membranaceus, 5-15 parts of herba lycopi, 5-15 parts of rhizoma wenyujin concinnatae and 5-10 parts of pericarpium citri reticulatae viride.

More preferably, the traditional Chinese medicine composition consists of the following raw material medicines in parts by weight: 10 parts of morinda officinalis, 10 parts of radix rehmanniae, 10 parts of raw astragalus membranaceus, 10 parts of herba lycopi, 9 parts of rhizoma wenyujin concisum and 6 parts of pericarpium citri reticulatae viride.

Further, the traditional Chinese medicine composition is prepared into a clinically acceptable pharmaceutical preparation according to a conventional traditional Chinese medicine preparation method.

Furthermore, the pharmaceutical preparation is granules, powder, capsules, tablets, mixture or oral liquid.

On the other hand, the invention also provides the application of the traditional Chinese medicine composition in preparing anti-hepatic fibrosis medicines.

Preferably, the invention also provides application of the traditional Chinese medicine composition in preparing medicines for reducing infiltration of inflammatory cells of liver, protecting liver cells and reducing necrosis of liver cells, inhibiting production and secretion of hepatic fibrosis cytokines, reducing collagen synthesis and accelerating collagen degradation.

On the other hand, the invention also provides an anti-hepatic fibrosis medicament, which is prepared from the traditional Chinese medicine composition and pharmaceutically acceptable conventional auxiliary agents.

Preferably, the method for preparing the medicament comprises the steps of:

(1) decocting radix Morindae officinalis, radix rehmanniae and radix astragali in water for 0.5-1.5 hr;

(2) adding herba Lycopi and rhizoma Wenyujin Concisa, decocting for 20-40 min;

(3) finally adding green tangerine peel, boiling with strong fire, and decocting with slow fire for 15 minutes;

(4) and (4) leaching out juice, and precipitating to obtain clear liquid.

Further, the invention also provides a preparation method of the medicine, which comprises the following steps:

(1) decocting radix Morindae officinalis, radix rehmanniae and radix astragali in water for 0.5-1.5 hr;

(2) adding herba Lycopi and rhizoma Wenyujin Concisa, decocting for 20-40 min;

(3) finally adding green tangerine peel, boiling with strong fire, and decocting with slow fire for 15 minutes;

(4) leaching out juice, precipitating and taking clear liquid;

(5) adding pharmaceutically acceptable conventional adjuvants.

The invention has the following therapeutic principles:

according to the etiology and pathogenesis of hepatic fibrosis and clinical characteristics, the traditional Chinese medicine considers that hepatic fibrosis belongs to the category of accumulation and the like. The pathogenesis of the disease is mainly that the disease is affected by damp-heat epidemic toxin or insect toxin, emotional disorder, drug toxicity injuring the liver, diet (wine) loss, long-term treatment is difficult to cure, viscera are weak and the like, the pathological factors are mainly related to dampness, heat, toxicity, stasis and deficiency, the disease location is mainly in the liver and relates to the spleen and the kidney.

On the basis of the academic experience of treating hepatic fibrosis by inheriting the professor 'reinforcing kidney and clearing away liver as assistance' of Wang Ling Tai in traditional Chinese medicine in Shanghai, the invention considers that the initial stage of the hepatic fibrosis is mainly the invasion of dampness, heat and toxin into liver channel, the damp-heat pathogen is accumulated in the liver and gallbladder to cause the dysfunction of catharsis, the dysfunction of the spleen and stomach due to the obstruction of the spleen and stomach causes the transportation and transformation disorder, the damp-heat toxin pathogen stays for a long time and causes the disharmony of qi and blood, the liver yin and blood consumption is damaged, the liver fails to be nourished, the qi and blood circulation is unsmooth, and stasis, phlegm and internal stagnation are generated, and the blood stasis blocks collaterals. The chronic persistent and recurrent diseases cause imbalance of yin and yang and consumption of kidney essence, so called "five zang organs damage, affecting kidney, slightly damaging kidney qi, and severely damaging kidney yang", which means "chronic diseases affecting kidney". Just as the pure plum is used, it is considered that the strong and full plum has no accumulation, but the weak people have the accumulation, which are caused by the deficiency of spleen and stomach, qi and blood damage, seven emotions and stagnation of phlegm with blood coagulation, and the people with deficiency of spleen and stomach and deficiency disorder have the accumulated diseases.

Therefore, the invention advocates that the prescription of the Baqi liver-softening granule takes the treatment principles of tonifying kidney and qi, activating blood and dissolving stasis, and clearing heat and detoxicating as treatment principles. The prescription takes radix morindae officinalis and astragalus root as monarch drugs. Morinda officinalis is sweet and pungent in flavor and slightly warm in nature, enters kidney and liver meridians, and is warm but not hot in nature, directly enters kidney meridian, and warms and tonifies kidney yang. Astragalus root, radix astragali, sweet in taste and slightly warm in nature, enters lung and spleen channels, invigorates spleen and invigorates qi, and benefits defensive qi and strengthens exterior. The combination of the two drugs can tonify both the congenital kidney yang and the acquired spleen qi, and has the effects of tonifying kidney, strengthening spleen and replenishing qi, thus being a monarch drug. The ministerial drugs are herba lycopi and rhizoma wenyujin Concisa, the herba lycopi is bitter and pungent in taste and slightly warm, enters liver and spleen channels, promotes blood circulation to remove blood stasis, induces diuresis to alleviate edema. Wenyujin Jiang is pungent, bitter and warm in flavor. It enters spleen and liver meridians, breaks blood and moves qi, and clears meridians to stop pain. Herba lycopi has the effects of promoting blood circulation to remove blood stasis and promoting diuresis, and can dispel damp evil in a body, rhizoma wenyujin concisum has the effects of promoting qi circulation to promote blood circulation to remove blood stasis, promoting stagnant qi circulation and promoting blood circulation, and the combination of the two can strengthen the effects of promoting blood circulation to remove blood stasis. The dried rehmannia root, radix rehmanniae, radix rehmanniae, and radix rehmanniae. So that Qing Pi is bitter, pungent and warm in flavor. It enters liver, gallbladder and stomach meridians, soothing liver and breaking qi, and removing food stagnation. It can soothe liver and promote qi circulation, strengthen the qi and blood circulation promoting effects of herba Lycopi and Wenyujin Concisa, and prevent qi stagnation caused by nourishing radix Morindae officinalis and radix astragali, and meanwhile, pericarpium Citri Reticulatae viride is used as channel-inducing drug to induce all the drugs to reach liver to exert drug effect. The medicines above are used together to play the effects of tonifying kidney and benefiting qi, activating blood circulation to dissipate blood stasis and clearing heat and toxic materials, the treatment of hepatic fibrosis can gradually benefit kidney yang, spleen is smooth, qi and blood are smooth, damp-heat toxin and stasis are dissipated to nourish liver yin, liver yang is used, and the condition can be slowly treated .

The invention has the advantages that:

1. the raw material medicaments and the weight part ratio of the raw material medicaments are optimized, the morinda officinalis and the astragalus membranaceus are used as monarch medicaments in the formula and are combined together to supplement the kidney yang in the first day and the spleen qi in the last day, so that the effects of tonifying the kidney, strengthening the spleen and replenishing qi are achieved, the eupatorium adenophorum and the rhizoma wenyujin concinnatae are used as ministerial medicaments, the effect of promoting blood circulation and removing blood stasis can be enhanced by combining the eupatorium adenophorum and the rhizoma wenyujin concinnatae, the rehmannia glutinosa is used as an assistant medicament, the effect of nourishing the liver and regulating the liver can be achieved, the damp-heat retention effect can be removed, the dryness-heat property of the morinda officinalis can be supplemented, the green tangerine peel can be used for soothing the liver and promoting the circulation of qi, the qi and the effects of promoting the circulation of the qi and the blood can be enhanced, the obstruction of the morinda officinalis and the astragalus membranaceus caused by the nourishing, and the green peel is used as a channel-inducing medicament to reach the liver to play the efficacy. The medicines are synergistic, and the medicines are combined to play the roles of tonifying kidney and qi, promoting blood circulation to remove blood stasis and clearing heat and toxic materials, can reduce infiltration of liver inflammatory cells, protect liver cells, reduce liver cell necrosis, inhibit the generation and secretion of hepatic fibrosis cell factors, reduce collagen synthesis and accelerate collagen degradation to play the role of resisting hepatic fibrosis.

2. The Chinese medicinal composition has wide medicinal material sources, simple preparation method, strong practicability and wide application prospect, and reduces the economic burden of patients, the pain of patients and the survival rate.

Drawings

FIG. 1 shows HE staining (x 100 fold) of liver tissue of each group of mice.

FIG. 2 shows the sirius red staining (x 40 fold) of liver tissue of each group of mice.

FIG. 3 is a Hyp comparison of liver tissues of mice in each group, Control: a normal group; model: a model group; RouGan: a baqi liver softening granule group, vs control,^**P<0.01；vs model，^##P<0.01。

FIG. 4 shows FBRS mRNA expression of liver tissue, liver and spleen mononuclear cells from various groups of mice, A: FBRS mRNA expression of liver tissues of each group of mice; b: FBRS mRNA expression of liver mononuclear cells of each group of mice; c: FBRS mRNA expression Control of splenic mononuclear cells of each group of mice: a normal group; model: a model group; RouGan: the Basil liver softening granule group vs control,^*P<0.05，^**P<0.01；vs model，^#P<0.05，^##P<0.01。

FIG. 5 shows the expression of TGF-beta 1, alpha-SMA mRNA in liver tissues of various groups of mice, A: mouse liver TGF-beta 1 of each groupmRNA expression; b: liver alpha-SMA mRNA expression of each group of mice, Control: a normal group; model: a model group; RouGan: a group of ba qi liver-softening granules; the control is carried out under the control of vs,^**P<0.01；vs model，^#P<0.05，^##P<0.01。

FIG. 6 shows the expression of mRNA of ColI, ColIII and ColIV tissues in the liver of various groups of mice, A: liver tissues of mice of each group express ColI mRNA B: liver tissue ColIIImRNA expression C of each group of mice: the ColIV mRNA expression Control of liver tissues of each group of mice: a normal group; model: a model group; RouGan: a group of ba qi liver-softening granules; the control is carried out under the control of vs,^*P<0.05，^**P<0.01；vs model，^#P<0.05，^##P<0.01。

FIG. 7 shows the mRNA expression of MMP2, MMP3, MMPP and MMP13 in the liver tissues of each group of mice, A: MMP2 mRNA expression in liver tissues of various groups of mice; b: MMP3 mRNA expression in liver tissues of various groups of mice; c: MMP9 mRNA expression in liver tissues of various groups of mice; d: MMP13 mRNA expression Control of liver tissues of mice in each group: a normal group; model: a model group; RouGan: a group of ba qi liver-softening granules; the control is carried out under the control of vs,^*P<0.05，^**P<0.01；vs model，^##P<0.01。

FIG. 8 shows the expression of TIMP1 and TIMP2 mRNA in liver tissues of mice in each group, A: TIMP1 mRNA expression in liver tissue of each group of mice; b: TIMP2 mRNA expression in liver tissue of each group of mice; control: a normal group; model: a model group; RouGan: a group of ba qi liver-softening granules; the control is carried out under the control of vs,^**P<0.01；vs model，^#P<0.05，^##P<0.01。

FIG. 9 shows the expression of FBRS and alpha-SMA proteins in liver tissues of various groups of mice, A: FBRS protein expression of each group of mouse liver tissues; b: liver tissue alpha-SMA protein expression of each group of mice, C: FBRS/GAPDH; d: α -SMA/GAPDH; control: a normal group; model: a model group; RouGan: a group of ba qi liver-softening granules; the control is carried out under the control of vs,^*P<0.05，^**P<0.01；vs model，^#P<0.05，^##P<0.01。

FIG. 10 shows the expression of TGF-beta 1, ColI proteins in liver tissues of various groups of mice, A: expressing TGF-beta 1 protein of liver tissue of each group of mice; b: liver tissue C of each group of miceol i protein expression, C: TGF-. beta.1/GAPDH; d: col I/GAPDH; control: a normal group; model: a model group; RouGan: a group of ba qi liver-softening granules; the control is carried out under the control of vs,^*P<0.05，^**P<0.01；vs model，^#P<0.05。

FIG. 11 shows the MMP13 and TIMP1 protein expression in the liver tissues of various groups of mice, A: MMP13 protein expression of liver tissues of various groups of mice; b: TIMP1 protein expression in liver tissue of mice in each group, C: MMP 13/GAPDH; d: TIMP 1/GAPDH; control: a normal group; model: a model group; RouGan: a group of ba qi liver-softening granules; the control is carried out under the control of vs,^*P<0.05，^**P<0.01；vs model，^#P<0.05，^##P<0.01。

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.

EXAMPLE 1 Chinese medicinal composition 1

The raw material medicaments are taken according to the following weight portion: 1 part of morinda officinalis, 1 part of radix rehmanniae, 1 part of raw astragalus membranaceus, 1 part of herba lycopi, 1 part of rhizoma wenyujin concisum and 1 part of pericarpium citri reticulatae viride.

EXAMPLE 2 Chinese medicinal composition (II)

The raw material medicaments are taken according to the following weight portion: 5 parts of morinda officinalis, 15 parts of radix rehmanniae, 5 parts of raw astragalus membranaceus, 15 parts of herba lycopi, 5 parts of rhizoma wenyujin concinnatae and 10 parts of pericarpium citri reticulatae viride.

EXAMPLE 3 Chinese medicinal composition (III)

The raw material medicaments are taken according to the following weight portion: 1 part of morinda officinalis, 20 parts of radix rehmanniae, 1 part of raw astragalus membranaceus, 20 parts of herba lycopi, 1 part of rhizoma wenyujin concinnatae and 20 parts of pericarpium citri reticulatae viride.

EXAMPLE 4 Chinese medicinal composition (IV)

The raw material medicaments are taken according to the following weight portion: 15 parts of morinda officinalis, 5 parts of radix rehmanniae, 15 parts of raw astragalus membranaceus, 5 parts of herba lycopi, 15 parts of rhizoma wenyujin concinnatae and 10 parts of pericarpium citri reticulatae viride.

EXAMPLE 5 Chinese medicinal composition (V)

The raw material medicaments are taken according to the following weight portion: 10 parts of morinda officinalis, 10 parts of radix rehmanniae, 10 parts of raw astragalus membranaceus, 10 parts of herba lycopi, 9 parts of rhizoma wenyujin concisum and 6 parts of pericarpium citri reticulatae viride.

Example 6 decoction

The preparation method comprises the following steps:

the Chinese medicinal composition of any one of embodiments 1 to 5 is taken and added with water to be decocted according to a conventional method. The Chinese medicinal materials are taken according to the weight part ratio, and are decocted into decoction by adding water.

Example 7 granules

The preparation method comprises the following steps:

the raw materials are taken according to the weight portions of the embodiments 1-5 respectively: decocting radix Morindae officinalis, radix rehmanniae and radix astragali in water for 0.5-1.5 hr; adding herba Lycopi and rhizoma Wenyujin Concisa, decocting for 20-40 min; finally adding green tangerine peel, boiling with strong fire, and decocting with slow fire for 15 minutes; filtering, concentrating the filtrate, and cooling to room temperature; concentrating the supernatant, adding water, stirring, standing, and concentrating the supernatant into fluid extract. Taking the clear paste, and preparing into granules.

EXAMPLE 8 preparation of tablets/capsules

The preparation method comprises the following steps:

the raw materials are taken according to the weight portions of the embodiments 1-5 respectively: decocting radix Morindae officinalis, radix rehmanniae and radix astragali in water for 0.5-1.5 hr; adding herba Lycopi and rhizoma Wenyujin Concisa, decocting for 20-40 min; finally adding green tangerine peel, boiling with strong fire, and decocting with slow fire for 15 minutes; filtering, concentrating the filtrate, and cooling to room temperature; taking supernatant, and concentrating to obtain thick extract; adding pharmaceutical adjuvants, vacuum drying, pulverizing, granulating, and making into tablet or capsule.

Example 9 animal experiments

1 materials of the experiment

1.1 Experimental animals

50 male BALB/c mice 6-8 weeks old were provided by Shanghai Sphere-BiKai laboratory animals Co., Ltd, and the animal certification was SCXK (Shanghai) 2008-0016. Animals were housed in the laboratory animal center of Shanghai university of medicine, fed with standard feed, and observed for 1 week with adaptive feeding.

1.2 Experimental drugs

The experimental Ba Qi Ruan gan Ke Li (liver softening granule for short in the following chart) is composed of Morinda officinalis 10g, dried rehmannia root 10g, raw Astragalus root 10g, herba Lycopi 10g, rhizoma Wenyujin Concisa 9g, and pericarpium Citri Reticulatae viride 6 g. The medicine is provided by Shanghai eosin hospital pharmacy, decocted by a conventional method, filtered to remove impurities, and concentrated into a stilbene liver softening concentrated solution.

2 method of experiment

2.1 mouse modeling and drug administration

50 mice were bred adaptively for 1 week and divided into a normal group, a model group and a baqi liver softening granule group according to a weight random block, wherein 10 mice were in the normal group, and 20 mice were in the other two groups. Injecting ConA into tail vein of the model-making mouse according to the weight of 12.5mg/kg mouse, 1 time/week, and continuously injecting for 10 weeks; the mice in the normal group were injected with 0.9% NaCl in the tail vein at the corresponding dose. The mice in the Basil liver softening granule group are drenched with liver softening formula liquid medicine according to the weight of 10ml/kg mice on the current modeling day, and the other two groups are drenched with normal drinking water according to the corresponding dose for 1 time/day for 10 weeks.

2.2 specimen Collection and processing

Collectively treating each group of mice the day after 10 th ConA injection of the model mice, removing eyeballs by using forceps, and taking whole blood; opening the abdominal cavity, picking the liver and spleen, weighing and recording; dividing the liver into three parts, namely cutting a liver tissue with the size of about 0.5cm multiplied by 0.5cm from the right lobe of the liver, fixing the liver tissue by 10 percent neutral formaldehyde, gradually dehydrating the liver tissue by a full-automatic dehydrator after 48 hours, and embedding and slicing the liver tissue by a paraffin embedding machine for HE and Tianlang scarlet dyeing; another two small liver tissues are taken and put into a 1.5ml EP tube to be preserved at minus 80 ℃ for measuring the Hyp content, RT-PCR and Western Blot experiments; the remaining liver tissues and intact spleen were used to isolate hepatosplenic mononuclear cells.

2.3 mouse liver function assay

Standing the whole blood of the mice for 2h at room temperature, centrifuging at 3500rpm for 15min to separate serum, taking 100 mul of serum from each mouse, placing the serum into a measuring tube, placing the measuring tube into a full-automatic biochemical analyzer, and measuring the ALT, AST and Alb content of the mice.

2.4 determination of mouse liver Hyp content

According to the specific experimental steps of the Hyp determination kit, 50mg of liver tissue is cut, gradually hydrolyzed by a hydrolysis reduction method, adsorbed by activated carbon to remove impurities, centrifuged at 3500rpm for 10min, the supernatant is taken to determine the absorbance value, and the specific content of Hyp is converted according to a formula.

2.5 liver, spleen mononuclear cell isolation

2.5.1 liver mononuclear cell isolation

1) Selecting a clean culture dish, covering a square 200-mesh wire gauze on the culture dish, placing the liver on the wire gauze, grinding, and flushing with PBS (phosphate buffer solution) while edging;

2) taking a small piece of 200-mesh nylon membrane, folding the nylon membrane into a funnel shape, placing the nylon membrane on a 15ml centrifuge tube, and filtering and transferring liver grinding fluid in a culture dish into the 15 centrifuge tube;

3) metering the volume of each tube to 15ml, balancing, and centrifuging at room temperature of 90g for 2 min;

4) taking the supernatant, transferring the supernatant to a new 15ml centrifuge tube, centrifuging the supernatant for 5min at the room temperature of 450g, and removing the supernatant;

5) resuspending with PBS and repeating the previous step;

6) re-suspending and precipitating 6ml of 40% Percoll lymphocyte separation liquid, sucking 3ml of 70% Percoll lymphocyte separation liquid by using a suction pipe, slowly adding to the bottom of the pipe, regulating the lifting speed of a centrifuge to 6g and 2g, and centrifuging at room temperature of 800g for 20 min;

7) and after the centrifugation is finished, carefully sucking the middle leucocyte layer cells, transferring the cells to a 15ml centrifuge tube, adding PBS (phosphate buffer solution), centrifuging the cells at the room temperature of 800g for 5min, and removing supernatant to obtain the precipitate, namely the liver mononuclear cells.

2.5.2 spleen mononuclear cell isolation

The 3 rd step in the liver mononuclear cell separation step is omitted, namely the spleen mononuclear cell separation step.

2.6 pathological staining of liver tissue

2.6.1 HE staining of liver tissue

Taking paraffin sections of liver tissues, baking the sections for 30-60min on a 60 ℃ baking machine, and then sequentially putting the sections into a dye vat, wherein the specific steps are as follows:

1) xylene dewaxing: xylene I for 10min and xylene II for 10 min;

2) and (3) dewatering the alcohol step by step: anhydrous ethanol I for 5min, anhydrous ethanol II for 2min, 95% ethanol for 2min, 85% ethanol for 2min, and 70% ethanol for 2 min;

3) washing with tap water for 3 times to remove ethanol;

4) staining with hematoxylin for 15min, and washing off loose color with tap water;

5) differentiating with 75% hydrochloric acid alcohol for 3s, washing the slices with tap water until the slices turn blue;

6) eosin dye liquor for 2 min;

7) dehydrating and transparent: 1min of 95% ethanol I, 1min of 95% ethanol II, 1min of anhydrous ethanol I, 1min of anhydrous ethanol II, 1min of xylene I and 1min of xylene II;

8) and (5) sealing the neutral gum.

2.6.2 hepatic tissue Tianlang scarlet staining

Taking paraffin sections of liver tissues, baking the sections for 30-60min on a 60 ℃ baking machine, and then sequentially putting the sections into a dye vat, wherein the specific steps are as follows:

1) xylene dewaxing: xylene I for 10min and xylene II for 10 min;

2) and (3) dewatering the alcohol step by step: anhydrous ethanol I for 5min, anhydrous ethanol II for 2min, 95% ethanol for 2min, 85% ethanol for 2min, and 70% ethanol for 2 min;

3) washing with tap water for 3 times to remove ethanol;

4) wiping water drops around the liver tissue with paper, dripping 1 drop of Tianlang scarlet dye solution on the liver tissue, putting the glass slide into a wet box, and placing the wet box in a 37 ℃ thermostat for 25 min;

5) differentiating with absolute ethanol for 1min, and allowing xylene to be transparent for 1 s;

6) and (5) sealing the neutral gum.

2.7RT-PCR experiments

2.7.1 Total RNA extraction

The procedure was carried out as follows, using trizol from Invitrogen, usa, according to the instructions:

1) taking out liver tissue of each group of mice from a refrigerator at-80 deg.C, shearing about 30mg with scissors, transferring into 1.5ml EP tube, adding 1ml trizol, shearing with high pressure small scissors, standing on ice for 5-10min, and centrifuging at 15000rpm at 4 deg.C for 10 min;

2) taking the supernatant, adding 0.2ml chloroform, reversing, mixing, standing at room temperature for 3min, and centrifuging at 15000rpm at 4 ℃ for 15 min;

3) taking the supernatant, adding 0.5ml of isopropanol, uniformly mixing, standing at room temperature for 10min, centrifuging at 15000rpm at 4 ℃ for 10min, and obtaining RNA (ribonucleic acid) after a small amount of white precipitate is visible at the bottom of the tube;

4) discarding the supernatant, adding 1ml of precooled 75% ethanol, washing the precipitate, and centrifuging at 10000rpm and 4 ℃ for 5 min;

5) the supernatant was discarded, air-dried for 5min, and 30. mu.l of sterilized purified water was added to dissolve the precipitate, and the next experiment was conducted directly or the precipitate was temporarily stored at-80 ℃.

2.7.2 Total RNA concentration determination

Taking 1 mul of sterilized pure water, placing the sterilized pure water in a measuring hole of an RNA concentration measuring instrument, and adjusting the instrument to zero; mu.l of each total RNA group was added to the assay wells, and the RNA concentration and the OD260/OD280 ratio were read. OD260/OD280 value is 1.8-2.1, which indicates that the RNA purity is good; when R <1.8, it indicates protein or other organic contamination in solution, and when R >2.1, it indicates that RNA has been degraded into single nucleic acid.

2.7.3 primer design and Synthesis

The gene sequence was obtained from NCBI, and primers were designed using oligo and primer5, and the designed primers were synthesized by Shanghai Biotech. Specific primer sequences are shown in Table 1.

TABLE 1 primer sequences

2.7.4 reverse transcription reaction

The following procedure was performed according to the cultures kit (# K1621):

1) adding each reactant into a centrifuge tube of RNase-free according to the following volume;

TABLE 2

2) Gently shaking, mixing the reactants, incubating at 65 ℃ for 5min, and rapidly placing on ice;

3) the following reagents were continued to be added to the above reaction species:

TABLE 3

4) Gently shaking, mixing the reactants, incubating at 25 deg.C for 5min, and then at 42 deg.C for 60 min;

5) the reaction was terminated after heating at 70 ℃ for 5 min. The obtained product is cDNA, which can be directly used for subsequent PCR amplification reaction and can also be temporarily stored at-20 ℃.

2.7.5PCR amplification reaction

1) At room temperature, the reactants were added in the following order:

TABLE 4

2) And (3) lightly and uniformly mixing the reagents to avoid generating bubbles, and carrying out amplification circulation according to the following sequence:

TABLE 5

2.7.6 analysis of results

According to 2^-△△CTMethod, relative quantification of each expression was performed using GAPDH as a reference.

2.8Western Blot experiment

2.8.1 extraction of Total protein

The liver tissues of each group were removed from the freezer at-80 deg.C, a small piece (about 30-50mg) was cut into a 1.5ml EP tube, 100. mu.l of cell lysate containing 1% PMSF was added, and the cell lysate was cut into pieces with small scissors. The sample was kept on ice for 30min to lyse the sample, and centrifuged at 14000g for 5min at 4 ℃ to obtain the supernatant.

2.8.2BCA assay for protein concentration

According to the kit specification, preparing the solution A and the solution B in the kit into the BCA working solution according to the ratio of 50:1, and preparing the working solution at present. 5mg of bovine serum albumin is weighed and dissolved in 5ml of 0.9% NaCl to prepare 1mg/ml of protein standard mother liquor, and then the mother liquor is sequentially diluted into standard products of 500 mu g/ml, 400 mu g/ml, 300 mu g/ml, 200 mu g/ml, 100 mu g/ml, 50 mu g/ml and 25 mu g/ml. And sequentially adding each standard substance into a 96-well plate according to a concentration gradient, adding 20 mu l of each standard substance into each well, diluting a sample to be detected by 50 times by using 0.9% NaCl, adding 20 mu l of the sample to be detected into each well, sequentially adding 200 mu l of BCA working solution into each well, placing the 96-well plate into a 37 ℃ incubator for incubation for 30min, moving the 96-well plate to an enzyme-labeling instrument, adjusting the wave band to 562nm, and reading. A standard curve was made from the readings and the concentration of each sample was calculated.

2.8.3 preparation of samples

The total loading mass of each sample was 30. mu.g, the total loading volume was 10. mu.l (containing 2. mu.l of loading buffer), the required volume of the sample was calculated based on the concentration of the protein sample to be measured, and the balance was made up with 0.9% NaCl.

2.8.4 glue making

Based on the measured protein molecular weight, 8%, 10% separation gel and 5% concentrated gel were prepared (formula shown in Table 6).

TABLE 6, 8%, 10% split gum and 5% concentrated gum formulations

Pouring separation gel, sealing with distilled water, pouring out distilled water after gelation, drying with filter paper, pouring concentrated gel, inserting sample comb, and avoiding air bubble.

2.8.5 protein loading and electrophoresis

Placing the glass plate in an electrophoresis tank, and adding electrophoresis liquid. The prepared sample is boiled in water bath at 100 ℃ for 5min to be denatured, and the sample is sequentially loaded, and a hole is reserved on each side of the sample to be added with a molecular weight standard. During electrophoresis, the initial voltage is 80V, and after the sample enters the separation gel, the voltage is adjusted to 110V.

2.8.6 transfer film by semi-dry method

Taking the molecular weight standard as reference, cutting off the separation gel of the required membrane transfer part, marking the upper right corner, and soaking in the membrane transfer liquid (precooling at 4 ℃) for balancing for 15-30 min. One piece of PVDF membrane and 2 pieces of filter paper with the sizes similar to those of the separation gel are cut out respectively, and the membranes are marked on the upper right corner. Soaking the membrane in methanol for 1min, and soaking the membrane and filter paper in the membrane transferring solution for 15-30 min. From the bottom to the top, the filter paper, the membrane, the separation gel and the filter paper are placed on a semi-dry membrane transfer apparatus, and a glass rod is used for removing air bubbles. Covering the cover of the electric transfer instrument, switching on the power supply, and performing electric transfer at constant voltage of 25V for 15-30min at room temperature according to the molecular weight of the transferred protein.

2.8.7 sealing

The membrane was placed on a dish, appropriate amount of blocking solution was added, placed on a shaker, and blocked for 2 hours at room temperature.

2.8.8 antibody incubation

The membrane was removed from the blocking solution and rinsed in PBST membrane wash solution for 3 min. The membrane was placed in a hybridization box, an appropriate amount of diluted primary antibody (1:1000-1:500) was added, and incubated on a shaker at 4 ℃ overnight. The membrane was taken out, rinsed in PBST membrane-washing solution for 3 times, 10min each time, another hybridization cassette was changed, secondary antibody (1:1000) was added, and incubated on a shaker at room temperature for 2 hours. The membrane was removed and the membrane was washed with PBST for 3 additional 10min each.

2.8.9 development

Opening a developing dark clamp, laying a layer of preservative film, putting the PVDF film on the preservative film with the front side facing upwards, mixing the ECL Pl mu s A liquid and the B liquid in a ratio of 1:1 according to the specification, dripping the mixture on the PVDF film, standing for 3min, sucking off the redundant liquid, covering the preservative film, removing bubbles, and moving the preservative film into a dark room for development. An X-ray film is taken out and placed on the strip, and the dark clamping cover is lightly covered to prevent the film from sliding. Pressing for 10s to 1min according to the luminous intensity of the strip, opening the cover, taking out the film, placing the film into a developing solution for development, then rinsing with clear water, transferring the film into a fixing solution for fixation, finally rinsing with clear water, and airing.

2.8.10 image analysis

Scanning the film strip into a computer by an image scanner, analyzing the strip by image analysis software, and comparing the differences of protein expression among samples by taking the ratio of the gray values of the target protein and the internal reference protein as a relative value.

3 statistical analysis

Statistical analysis is carried out by using SPSS15.0 data analysis software, the measured data are expressed by mean +/-standard deviation, the difference between groups is analyzed by adopting one-factor variance, and the LSD method is adopted for pairwise comparison. P <0.05 indicates that the difference is statistically significant.

4 results of the experiment

4.1 general conditions in groups of mice

The normal group of mice has good mental state, sensitive response, glossy hair color and regular eating and drinking; the state of the mice in the model group is more convoluted in the cage corner than the state of the mice in the model group, the response is more sluggish, and the food intake is reduced; the state of each mouse in the baqi liver-softening granule group is improved compared with that of the model group through the intervention of the baqi liver-softening granule liquid medicine. In the experimental process, no mice in the normal group died, 7 mice in the model group died, and 5 mice in the pasteur liver softening granule group died. The mortality rate of mice in the baqi liver softening granule group is reduced compared with that in the model group, but no obvious statistical difference exists (P > 0.05).

4.2 comparison of liver weights and liver indices for various groups of mice

Compared with each mouse in the normal group, the liver weight and the liver index of the liver fibrosis model mouse injected with ConA through the tail vein are obviously increased (P is less than 0.01); compared with the model group mice, the weight of the liver and the liver index of the mice in the pasteur liver softening granule group tend to be reduced, but the statistical significance is not available (P > 0.05). See table 7.

Table 7 comparison of liver weights and liver indices for each group of mice

Note: p <0.05, P <0.01 compared to normal group.

4.3 comparison of spleen weight and spleen index in mice of each group

Compared with normal mice, the liver fibrosis model mouse injected with ConA through tail vein has obviously increased spleen weight and spleen index, and the difference has statistical significance (P < 0.01); compared with the model group mice, the spleen weight and spleen index of the mice with the astragalus liver-softening granules are obviously reduced (P is less than 0.05), and the astragalus liver-softening granules have the function of improving splenomegaly. See table 8.

TABLE 8 comparison of spleen weights and spleen indices for each group

Note: p compared to normal group<0.05，**P<0.01; in comparison to the set of models,^#P<0.05。

4.4 liver function comparison of groups of mice

Compared with normal mice, the liver fibrosis model mice have significantly increased serum ALT and AST levels (P < 0.01); ALT and AST levels of the Baqi liver softening granule group are obviously improved compared with those of a model group (P is less than 0.01); there was no significant statistical difference in serum Alb levels (P >0.05) for each group of mice. See table 9.

TABLE 9 comparison of liver function in groups of mice

Note: p compared to normal group<0.01; in comparison to the set of models,^#P<0.05。

4.5 liver histopathology in groups of mice

4.5.1 general overview

The liver of each mouse in the normal group is soft, bright red in color and smooth in surface; the liver of each mouse in the model group is slightly large, the quality is slightly hard, the color is dark, scattered point-shaped or sheet-shaped necrotic regions can be seen on the surface and the section, and the surface of the liver is rugged if the liver is severe; the liver of each mouse in the baqi liver softening granule group is soft and red, and the necrotic area on the surface and the section of the liver is reduced compared with that in the model group.

4.5.2HE staining

Under the microscope, the liver lobules of each mouse in the normal group are clear and complete in structure, liver cables are radially arranged around a central vein in a cord shape to the periphery, the arrangement is neat, obvious degeneration and necrosis are avoided, hepatic blood sinuses are normal, and a large amount of inflammatory cell infiltration is not seen; most normal hepatic lobule structures of mice in the model group are damaged, hepatic cords are disorderly arranged, and the hepatic lobule structures are scattered in hepatic tissue necrosis areas with different ranges and degrees and have a large amount of inflammatory cell infiltration; compared with the model group, the liver lobule structure damage degree of each mouse of the ba qi liver softening granule group is obviously reduced, liver cords are arranged neatly, and inflammatory cell infiltration is obviously reduced. See fig. 1.

4.5.3 day Lance Red staining

Under the microscope, each mouse in the normal group only has a small amount of collagen fibers on the central vein wall and the junction area, and has no obvious collagen deposition; the normal hepatic lobule structure of each mouse in the model group is damaged, the collagen deposition is obviously increased, the fibrous interval is formed by extending along an inflammation necrotic area or a sink area, and the severe hepatic lobule can form a false lobule; compared with the model group, the pasteur astragalus liver softening granule group has obviously reduced collagen deposition and obviously improved fibrosis degree. See fig. 2.

4.6 liver tissue Hyp content of mice in each group

In hepatic fibrosis, Hyp is an important index reflecting the collagen metabolism and fibrosis degree of liver tissues. The kit method detects the expression level of Hyp of liver tissues of each group of mice, and the result shows that the Hyp content of the mice in the model group is obviously higher than that in the normal group (P is less than 0.01); compared with the model group, the content of Hyp in liver tissue of mice in the pasteur and liver softening granule group is obviously reduced (P < 0.01). See fig. 3.

4.7RT-PCR results

4.7.1 mouse liver tissue, liver and spleen mononuclear cell FBRS mRNA expression

The total RNA of liver tissues, liver and spleen mononuclear cells of all groups of mice is extracted, the expression of FBRS mRNA is detected by an RT-PCR method, and the result shows that compared with the normal group of mice, the expression quantity of the FBRS mRNA of the liver tissues, liver and spleen mononuclear cells of the mice in a liver fibrosis model group is obviously up-regulated (P <0.01), the Baqi liver softening particles have down-regulation effect on the FBRS mRNA, and compared with the model group, the Baqi liver softening particle group has significant difference (P <0.05 or P < 0.01). See fig. 4.

4.7.2 mouse liver tissue TGF-beta 1, alpha-SMA mRNA expression

RT-PCR results show that compared with a normal group, the mouse liver tissue TGF-beta 1 and alpha-SMA mRNA expression of the model group is obviously up-regulated (P is less than 0.01); compared with the model group, the expression levels of TGF-beta 1 and alpha-SMA mRNA of the baqi liver softening granule group are both obviously reduced (P is less than 0.01). See fig. 5.

4.7.3 liver tissues of each group of mice ColI, ColIII and ColIV mRNA expression

RT-PCR results show that the mRNA expression of ColI, ColIII and ColIV mRNA of mouse liver tissues in a model group is obviously up-regulated (P <0.01) compared with that in a normal group, the pasteur astragalus liver softening particles have down-regulation effect on the mRNA expression, and the pasteur astragalus liver softening particle group has significant difference (P <0.01 or P <0.05) compared with the model group. See fig. 6.

4.7.4 groups of mice liver tissue MMP2, MMP3, MMP9 and MMP13 mRNA expression

The RT-PCR result shows that the mRNA expression of mouse liver tissues MMP2, MMP3, MMP9 and MMP13 in the model group is remarkably increased (P <0.01), and the MMPmRNA in the pasteur astragalus mollissima particle group is remarkably reduced (P < 0.01). See fig. 7.

4.7.5 mouse liver tissue TIMP1 and TIMP2 mRNA expression

RT-PCR results show that compared with a normal group, the mRNA expression of the mice TIMP1 and TIMP2 in the model group is remarkably up-regulated (P <0.01), and compared with the model group, the mRNA expression of the mice TIMP1 and TIMP2 in the stilbene liver softening particle group is reduced, and the difference has statistical significance (P <0.01 or P < 0.05). See fig. 8.

4.8Western Blot results

4.8.1 expression of FBRS and alpha-SMA proteins in liver tissues of mice in each group

Extracting total proteins of liver tissues of each group of mice, and detecting the expression changes of FBRS and alpha-SMA by a Western Blot method. The results show that the expression of FBRS and alpha-SMA proteins in the liver tissues of the mice in the model group is obviously higher than that in the normal group (P <0.05 or P <0.01), and the pasteurella and daunorubicin particle group has down-regulation effect, and has obvious difference (P <0.05 or P <0.01) compared with the model group. See fig. 9.

4.8.2 liver tissue TGF-beta 1, ColI protein expression of each group of mice

The expression conditions of TGF-beta 1 and ColI proteins of liver tissues of mice in each group are detected by a Western Blot method, and the results show that compared with a normal group, the TGF-beta 1 and ColI proteins of liver tissues of mice in a model group are obviously up-regulated (P is less than 0.05 or P is less than 0.01), the TGF-beta 1 and ColI expression protein levels of mice in a balaqi liver softening granule group are obviously reduced through intervention of balaqi liver softening granule liquid medicine. See fig. 10.

4.8.3 mouse liver tissue MMP13, TIMP1 protein expression

The Western Blot result shows that the expressions of MMP13 and TIMP1 proteins in liver tissues of a model group of mice are obviously increased (P is less than 0.01), which indicates that the liver collagen metabolism dysfunction of the model group of mice is promoted, and compared with the model group, the levels of the two are obviously reduced, which indicates that the ba qi liver softening granules can improve the liver collagen metabolism. See fig. 11.

Conclusion of the experiment

The astragalus liver softening granules can play a role in resisting hepatic fibrosis by reducing infiltration of inflammatory cells of the liver, protecting liver cells and reducing necrosis of the liver cells, inhibiting the production and secretion of hepatic fibrosis cell factors, reducing collagen synthesis and accelerating collagen degradation.

Example 10 control test

1 materials of the experiment

1.1 Experimental animals

80 male BALB/c mice of 6-8 weeks old were provided by Shanghai Sphere-BiKai laboratory animals Co., Ltd, and the animal certification was SCXK (Shanghai) 2008-0016. Animals were housed in the laboratory animal center of Shanghai university of medicine, fed with standard feed, and observed for 1 week with adaptive feeding.

1.2 Experimental drugs

The first medicine is: the baqi liver-softening granule: is prepared from morinda root 10g, dried rehmannia root 10g, astragalus root 10g, eupatorium japonicum 10g, curcuma longa 9g, green tangerine orange peel 6 g.

Medicine II: is prepared from morinda root 10g, astragalus root 10g, curcuma longa 9g, green tangerine orange peel 6g through steps of disintegrating, disintegrating and disintegrating.

Medicine three: is prepared from herba Lycopi 10g and radix rehmanniae 10g by making into granule.

Medicine four: is prepared from astragalus root, matrimony vine, dried human placenta, white atractylodes rhizome, Chinese angelica root, curcuma longa and diverse wormwood herb each 10g, and is prepared into granules.

Medicine five: is prepared from rhizoma Polygoni Cuspidati, herba Artemisiae Scopariae, radix Isatidis, herba Scutellariae Barbatae, rhizoma Curculiginis, herba et Gemma Agrimoniae, herba Epimedii, radix Codonopsis, Atractylodis rhizoma, radix astragali, radix Paeoniae Rubra, radix Ginseng, Curcumae rhizoma, carapax Trionycis, fructus Aurantii Immaturus each 20g, radix Aconiti lateralis Preparata, and fructus Amomi each 9g by making into granule.

Medicine six: is prepared from morinda officinalis 21g, dried rehmannia root 22g, raw astragalus root 24g, eupatorium japonicum 19g, curcuma longa 24g and green tangerine orange peel 16 g.

2 method of experiment

2.1 mouse modeling and drug administration

80 mice were bred adaptively for 1 week and divided into normal group, model group and experiment one, two, three, four, five and six groups of 10 mice each, according to random weight block. Injecting ConA into tail vein of the model-making mouse according to the weight of 12.5mg/kg mouse, 1 time/week, and continuously injecting for 10 weeks; the mice in the normal group were injected with 0.9% NaCl in the tail vein at the corresponding dose. After the model building is successful, the mice in the first to the six groups are respectively dosed with drugs one, two, three, four, five and six according to the weight of the mice of 10ml/kg on the current day of the model building, and the model group is filled with normal drinking water according to the corresponding dose for 1 time/day for 10 weeks.

2.2 specimen Collection and processing

The same as in example 9.

2.3 mouse liver function assay

The same as in example 9.

2.4 determination of mouse liver Hyp content

The same as in example 9.

2.5 liver, spleen mononuclear cell isolation

The same as in example 9.

3 statistical analysis

Statistical analysis is carried out by using SPSS15.0 data analysis software, the measured data are expressed by mean +/-standard deviation, the difference between groups is analyzed by adopting one-factor variance, and the LSD method is adopted for pairwise comparison. P <0.05 indicates that the difference is statistically significant.

4 results of the experiment

4.1 general conditions in groups of mice

The normal group of mice has good mental state, sensitive response, glossy hair color and regular eating and drinking; the state of the mice in the model group is more convoluted in the cage corner than the state of the mice in the model group, the response is more sluggish, and the food intake is reduced; the state of each mouse in the first to the sixth groups of experiments is improved compared with that in the model group by drug intervention. In the experimental process, the mice in the normal group die, 4 mice in the model group die, 1 mouse in the drug group die, and 2, 3, 2 and 2 mice in the drug group and six mice in the drug group die respectively.

4.2 comparison of liver weights and liver indices for various groups of mice

Compared with each mouse in the normal group, the liver weight and the liver index of the liver fibrosis model mouse injected with ConA through the tail vein are obviously increased (P is less than 0.01); compared with the model group mice, the liver weight and liver index of the experimental one-six groups of mice are reduced, but the statistical significance is not achieved (P > 0.05). See table 10.

TABLE 10 comparison of liver weights and liver indices for each group of mice

Note: p <0.05, P <0.01 compared to normal group.

4.3 comparison of spleen weight and spleen index in mice of each group

Compared with normal mice, the liver fibrosis model mouse injected with ConA through tail vein has obviously increased spleen weight and spleen index, and the difference has statistical significance (P < 0.01); spleen weight and spleen index were significantly decreased in experimental one group of mice (P <0.05) and in experimental two to six groups of mice, but without significant difference, compared to model group of mice, see table 11.

TABLE 11 comparison of spleen weight and spleen index for each group

Note: p compared to normal group<0.05，**P<0.01; in comparison to the set of models,^#P<0.05。

4.4 liver function comparison of groups of mice

Compared with normal mice, the liver fibrosis model mice have significantly increased serum ALT and AST levels (P < 0.01); the ALT and AST levels of an experimental group are obviously improved compared with those of a model group (P is less than 0.01); ALT and AST levels in two to six groups of experiments are improved compared with the model group, but no significant difference exists; there was no significant statistical difference in serum Alb levels (P >0.05) for each group of mice. See table 12.

TABLE 12 comparison of liver function in groups of mice

Note: p compared to normal group<0.01; in comparison to the set of models,^#P<0.05。

5 conclusion

The degree of hepatic fibrosis of the first to the sixth groups of experiments is improved in different degrees compared with that of the model group, but the effect of the first group of experiments is obviously better than that of the second to the sixth groups of experiments, and the obvious difference exists.

It shows that even under the same treatment rules, the medicinal flavors with similar effects and the medicinal flavors with different weight proportions have obvious difference on the treatment effect.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and additions can be made without departing from the principle of the present invention, and these should also be considered as the protection scope of the present invention.

SEQUENCE LISTING

<110> affiliated eosin Hospital of medicine university in Shanghai

<120> Chinese medicinal composition for resisting hepatic fibrosis and application thereof

<130> /

<160> 26

<170> PatentIn version 3.3

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Claims (10)

1. The traditional Chinese medicine composition for resisting hepatic fibrosis is characterized by consisting of the following raw material medicines in parts by weight: 0.1 to 20 portions of morinda officinalis, 0.1 to 20 portions of radix rehmanniae, 0.1 to 20 portions of raw astragalus root, 0.1 to 20 portions of herba lycopi, 0.1 to 20 portions of rhizoma wenyujin Concisa and 0.1 to 15 portions of green tangerine peel.

2. The traditional Chinese medicine composition according to claim 1, which is prepared from the following raw material medicines in parts by weight: 5-15 parts of morinda officinalis, 5-15 parts of radix rehmanniae, 5-15 parts of raw astragalus membranaceus, 5-15 parts of herba lycopi, 5-15 parts of rhizoma wenyujin concinnatae and 5-10 parts of pericarpium citri reticulatae viride.

3. The traditional Chinese medicine composition according to claim 1, which is prepared from the following raw material medicines in parts by weight: 10 parts of morinda officinalis, 10 parts of radix rehmanniae, 10 parts of raw astragalus membranaceus, 10 parts of herba lycopi, 9 parts of rhizoma wenyujin concisum and 6 parts of pericarpium citri reticulatae viride.

4. The Chinese medicinal composition according to any one of claims 1 to 3, which is prepared into a clinically acceptable medicinal preparation according to a conventional Chinese medicinal preparation method.

5. The traditional Chinese medicine composition of claim 4, wherein the pharmaceutical preparation is a granule, a powder, a capsule, a tablet, a mixture or an oral liquid.

6. Use of the Chinese medicinal composition of any one of claims 1-3 in the preparation of a medicament for treating hepatic fibrosis.

7. Use of the composition of any one of claims 1-3 for the preparation of a medicament for reducing infiltration of inflammatory cells of the liver, protecting and reducing necrosis of liver cells, inhibiting production and secretion of cytokines that promote liver fibrosis, reducing collagen synthesis and accelerating collagen degradation.

8. An anti-hepatic fibrosis drug, which is prepared from the traditional Chinese medicine composition of any one of claims 1 to 3 and pharmaceutically acceptable conventional auxiliary agents.

9. The medicament of claim 8, wherein the preparation method comprises the steps of:

(1) decocting radix Morindae officinalis, radix rehmanniae and radix astragali in water for 0.5-1.5 hr;

(2) adding herba Lycopi and rhizoma Wenyujin Concisa, decocting for 20-40 min;

(3) finally adding green tangerine peel, boiling with strong fire, and decocting with slow fire for 15 minutes;

(4) and (4) leaching out juice, and precipitating to obtain clear liquid.

10. The method of preparing the medicament of claim 8, comprising the steps of:

(1) decocting radix Morindae officinalis, radix rehmanniae and radix astragali in water for 0.5-1.5 hr;

(2) adding herba Lycopi and rhizoma Wenyujin Concisa, decocting for 20-40 min;

(3) finally adding green tangerine peel, boiling with strong fire, and decocting with slow fire for 15 minutes;

(4) leaching out juice, precipitating and taking clear liquid;

(5) adding pharmaceutically acceptable conventional adjuvants.

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