CN110538194A

CN110538194A - Anti-hepatic fibrosis pharmaceutical composition and application thereof

Info

Publication number: CN110538194A
Application number: CN201910911084.7A
Authority: CN
Inventors: 成秋宸; 覃雯; 卓朗
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-09-25
Filing date: 2019-09-25
Publication date: 2019-12-06

Abstract

The invention discloses an anti-hepatic fibrosis pharmaceutical composition and application thereof, wherein the composition is a preparation prepared from the following raw material medicines in parts by weight: 1-5 parts of Chitosan, 2-10 parts of Genistein, 3-15 parts of EGCG and 20-100 parts of Taurine; the composition of the invention is applied to the preparation of anti-hepatic fibrosis drugs; the composition has the characteristics of improving the oxidation resistance, anti-inflammation and anti-fibrosis capability of liver cells, delaying or reversing the hepatic fibrosis process of model animals, having no obvious hepatotoxicity, stable and controllable quality, wide sources and the like through a multi-target-point acting hepatic fibrosis pathway, and has good application prospect in resisting hepatic fibrosis.

Description

Anti-hepatic fibrosis pharmaceutical composition and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to an anti-hepatic fibrosis pharmaceutical composition and application thereof.

Background

more than 10% of the world suffer from chronic liver disease, with more than 1 million patients suffering from liver fibrosis and more than 100 million patients dying from liver fibrosis each year. Hepatic fibrosis is the etiology of chronic viral hepatitis, cholestasis, drug-induced liver injury, parasitic infection, chronic alcoholic liver disease and autoimmune hepatitis, acts on the liver, is a compensation reaction in the process of repairing inflammatory injury, and is a necessary way for the progress to cirrhosis. The hepatic fibrosis is considered to have reversible stage, so the hepatic fibrosis becomes the best period of clinical medicine intervention reversion, and the hepatic fibrosis is delayed, prevented, even reversed, and the liver fibrosis has important significance for improving the liver function of a patient, delaying the formation of liver cirrhosis and complications thereof, improving the life quality of the patient and prolonging the life cycle of the patient.

So far, an ideal scheme for treating hepatic fibrosis is still lacking at home and abroad, and an anti-hepatic fibrosis drug approved by the Food and Drug Administration (FDA) is not available. Because the molecular mechanism of hepatic fibrosis is complex, multiple molecular pathways are involved, and a drug with a single target point hardly has good drug effect. Achieving multi-target treatment of liver fibrosis remains a problem that needs to be solved urgently.

Disclosure of Invention

The invention overcomes the technical problems that medicines with single target points hardly have good drug effect because the molecular mechanism of hepatic fibrosis is complex and relates to a plurality of molecular paths in the prior art, provides the application of a natural product pharmaceutical composition in treating hepatic fibrosis, improves the anti-oxidation, anti-inflammation and anti-fibrosis capability of liver cells through multi-target point action of hepatic fibrosis paths, and delays or reverses the hepatic fibrosis process, thereby achieving the purpose of treating hepatic fibrosis.

In order to solve the problems, the invention adopts the following technical scheme:

the anti-hepatic fibrosis pharmaceutical composition is a preparation prepared from the following raw material medicines in parts by weight: 1-5 parts of Chitosan, 2-10 parts of Genistein, 3-15 parts of EGCG and 20-100 parts of Taurine.

Chitosan (Chitosan), Genistein (Genistein), EGCG (gallocatechin gallate) and Taurine (Taurine) in the raw material medicines are natural products; wherein Chitosan is obtained by deacetylating active ingredients extracted from the epidermis of crustacean, Genistein is active ingredients extracted from fructus Sophorae and radix Sophorae Tonkinensis, EGCG is active ingredient extracted from folium Camelliae sinensis, and Taurin is active ingredient extracted from marine fish, and can be synthesized chemically economically; the extraction method of the raw material medicines comprises the following steps: and (3) extracting with alcohol.

In the invention, the composition is a preparation prepared from the following raw material medicines in parts by weight: 2-4 parts of Chitosan, 4-8 parts of Genistein, 6-12 parts of EGCG and 40-80 parts of Taurine.

In the invention, the composition is a preparation prepared from the following raw material medicines in parts by weight: chitosan 3 parts, Genistein 6 parts, EGCG 9 parts and Taurin 60 parts.

In the invention, the composition is prepared from raw material medicines serving as active ingredients and pharmaceutically common auxiliary materials or auxiliary ingredients, and is suitable for various formulations in clinical application; such formulations include, but are not limited to: granule, capsule, tablet, oral liquid, and injection.

In the invention, further, the composition is prepared by the following method:

S1: weighing the raw material medicines according to the parts by weight;

S2: the raw material medicines are taken as active ingredients, and are added with auxiliary materials or auxiliary ingredients commonly used in pharmacy to prepare various preparation formulations suitable for clinical application.

the invention also aims to protect the application of the pharmaceutical preparation which takes the anti-hepatic fibrosis pharmaceutical composition as an active ingredient in preparing anti-hepatic fibrosis drugs.

The pharmaceutically acceptable auxiliary materials of the present invention refer to substances contained in the dosage form in addition to the active ingredients, and include, but are not limited to, fillers (diluents), lubricants (glidants or anti-adherents), dispersants, wetting agents, binders, regulators, solubilizers, antioxidants, bacteriostats, emulsifiers, disintegrants, and the like. The binder comprises syrup, acacia, gelatin, sorbitol, tragacanth, cellulose and its derivatives (such as microcrystalline cellulose, sodium carboxymethylcellulose, ethyl cellulose or hydroxypropyl methylcellulose), gelatin slurry, syrup, starch slurry or polyvinylpyrrolidone; the filler comprises lactose, sugar powder, dextrin, starch and its derivatives, cellulose and its derivatives, inorganic calcium salt (such as calcium sulfate, calcium phosphate, calcium hydrogen phosphate, precipitated calcium carbonate, etc.), sorbitol or glycine, etc.; the lubricant comprises superfine silica gel powder, magnesium stearate, talcum powder, aluminum hydroxide, boric acid, hydrogenated vegetable oil, polyethylene glycol and the like; the disintegrating agent comprises starch and its derivatives (such as sodium carboxymethyl starch, sodium starch glycolate, pregelatinized starch, modified starch, hydroxypropyl starch, corn starch, etc.), polyvinylpyrrolidone or microcrystalline cellulose, etc.; the wetting agent comprises sodium lauryl sulfate, water or alcohol, etc.; the antioxidant comprises sodium sulfite, sodium bisulfite, sodium pyrosulfite, dibutylbenzoic acid, etc.; the bacteriostatic agent comprises 0.5% of phenol, 0.3% of cresol, 0.5% of chlorobutanol and the like; the regulator comprises hydrochloric acid, citric acid, potassium (sodium) hydroxide, sodium citrate, and buffer (including sodium dihydrogen phosphate and disodium hydrogen phosphate); the emulsifier comprises polysorbate-80, sorbitan fatty acid, pluronic F-68, lecithin, soybean lecithin, etc.; the solubilizer comprises Tween-80, bile, glycerol, etc.

the pharmaceutically acceptable auxiliary components have certain physiological activity, but the addition of the components does not change the dominance of the compounds or derivatives in the process of treating diseases, but only plays an auxiliary effect, and the auxiliary effects are only the utilization of the known activity of the components and are auxiliary treatment modes which are conventional in the field of medicine. If the auxiliary components are used in combination with the compound of the present invention, the protection scope of the present invention should still be included.

The applicant, based on many years of medical work experience combined with numerous medical tests, briefly describes the effects of the pharmaceutical components of the present application and the principle of action of the present application in treating liver fibers as follows:

Chitosan (Chitosan) as a biological material has biocompatibility, is non-toxic and non-immunogenic, can inhibit scar formation to different degrees, participates in bile acid metabolism, reduces the absorption of bile acid by organisms, relieves the damage of lipid peroxidation of liver cells to the integrity of mitochondrial membranes, protects liver cell membranes, inhibits the apoptosis of liver cells, repairs liver injury of rats, and accordingly relieves hepatic fibrosis.

Genistein has effects of resisting oxidation, inhibiting immune activation and hepatic stellate cell proliferation, reducing collagen synthesis and extracellular matrix deposition in stellate cells stimulated by TGF-beta 1 in a concentration-dependent manner, synergistically eliminating free radicals by increasing activity and expression of hepatic antioxidant enzyme, reducing expression of alpha-SMA, blocking expression of fibroblast growth factor PDGF, and resisting fibrosis in vitro.

EGCG (gallocatechin gallate) reduces oxidative stress by removing ROS and LPO in HSC cells, inhibits the growth of HSC by inhibiting a Rho signal pathway, can inhibit the generation of collagen and the activity of collagenase to inhibit the synthesis of collagen, and can reduce the expression of MMPs (MMPs), thereby inhibiting hepatic fibrosis.

taurine (Taurine) is a sulfur-containing beta-amino acid, is one of the main components of the rare Chinese medicinal bezoar, is a self-anti-injury substance widely existing in vivo, and can promote bile excretion, inhibit hepatic stellate cells of rats in vitro, inhibit proliferation and collagen synthesis of fibroblasts, and inhibit deposition of extracellular matrix of liver cells of rats so as to relieve the degree of hepatic fibrosis. Research shows that taurine reduces the resistance of blood vessels in the liver by regulating NO signal conduction path, thereby regulating the blood microcirculation of the liver, improving the liver function and inhibiting the liver fibrosis.

Due to the adoption of the technical scheme, the invention at least comprises the following beneficial effects:

(1) The pharmaceutical composition is cut in from the main pathological mechanism of fibrosis, and four natural products of Chitosan (Chitosan), Genistein (Genistein), EGCG (gallocatechin gallate) and Taurine (Taurine) are selected for combination, so as to achieve the purpose of treating hepatic fibrosis by multiple routes and multiple targets; wherein Chitosan has the functions of reducing cholesterol, inhibiting bacteria, preventing and controlling hypertension and good biocompatibility. Genistein has estrogen-like effect of soybean isoflavone, and has effects of resisting oxidation, resisting tumor, inhibiting bacteria, reducing blood lipid, and resisting hepatic fibrosis. EGCG is the most effective active component in green tea catechin extract, is a compound containing multiple phenolic hydroxyl groups, has multiple drug effects of resisting virus, oxidation, arteriosclerosis, thrombosis, angiogenesis, bacteria, inflammation and tumor, and has oxidation resistance higher than that of ascorbic acid. Taurine is an amino acid converted from sulfur-containing amino acid, and has the drug effects of resisting inflammation, reducing blood pressure, reducing blood sugar, resisting arrhythmia, resisting bacteria, enhancing immunity, benefiting gallbladder, strengthening liver and the like; research proves that Chitosan, Genistein, EGCG and Taurine are prepared into a compound, and the compound can inhibit the hyperplasia of lipid storage cells, reduce the synthesis of collagen, promote liver repair, regulate the blood microcirculation of liver and achieve the aim of resisting hepatic fibrosis by the synergistic action of the medicines in the aspects of anti-inflammation, anti-necrosis, liver cell protection and the like.

(2) The invention has the uniqueness that the liver fibrosis pathway is acted by multiple targets, the anti-oxidation, anti-inflammation and anti-fibrosis capability of liver cells is improved, the liver fibrosis process of model animals is delayed or reversed, the liver fibrosis is free from obvious hepatotoxicity, the quality is stable and controllable, the sources are wide, and the like, and the liver fibrosis resistant liver cancer.

(3) When the pharmaceutical composition is applied to the process of treating hepatic fibrosis, the pharmaceutical composition can be compounded with other medicines or medicine carriers to prepare preparations such as granules, capsules, tablets, oral liquid, injection and the like, the proportion of the components forming the preparation has a synergistic effect, the content of active ingredients is high, the use and carrying are convenient, the oral administration and absorption are rapid, the bioavailability is high, and the prevention and treatment effect is obvious through animal test verification.

Drawings

FIG. 1 is a graph showing the effect of CGET on the pathological structure of liver tissue and the degree of liver fibrosis of a liver fibrosis rat through HE staining of liver tissue sections of seven groups of rats;

FIG. 2 shows the effect of CGET on pathological structure of liver tissue and degree of liver fibrosis in rats by Sirius red staining of liver tissue sections of seven groups of rats.

In the figure, a is a control group, B is a model group, C is a drug low-dose group a, D is a drug low-dose group B, E is a drug low-dose group C, F is a drug medium-dose group, and G is a drug high-dose group.

The specific figure analysis is shown in an experimental method.

Detailed Description

The present invention will be further described with reference to examples and tests.

example 1

the embodiment provides an anti-hepatic fibrosis pharmaceutical composition, which is a preparation prepared from the following raw material medicines in parts by weight: chitosan 1 part, Genistein 2 parts, EGCG 3 parts and Taurin 20 parts.

The preparation method comprises the following steps:

S1: weighing the raw material medicines according to the parts by weight;

The embodiment also provides an application of the pharmaceutical preparation taking the anti-hepatic fibrosis pharmaceutical composition as an active ingredient in preparing an anti-hepatic fibrosis medicament.

Example 2

The embodiment provides an anti-hepatic fibrosis pharmaceutical composition, which is a preparation prepared from the following raw material medicines in parts by weight: chitosan 3 parts, Genistein 6 parts, EGCG 9 parts and Taurin 60 parts.

The preparation method comprises the following steps:

s1: weighing the raw material medicines according to the parts by weight;

Example 3

The embodiment provides an anti-hepatic fibrosis pharmaceutical composition, which is a preparation prepared from the following raw material medicines in parts by weight: 5 parts of Chitosan, 10 parts of Genistein, 15 parts of EGCG and 100 parts of Taurine.

The preparation method comprises the following steps:

s1: weighing the raw material medicines according to the parts by weight;

The following experiments prove that the pharmaceutical composition (hereinafter referred to as CGET medicament) has the effect of treating hepatic fibrosis diseases.

1. Experimental Material

Experimental animals: the 6-week-old SPF SD rat is male, has the physical quality of 180-: SCXK myzus 2009-: SYXK Ostrin 2014-. The test bench is placed in an environment with the humidity of 50-70 percent and the temperature of 25 +/-1 ℃ and 12 hours of illumination, and is adapted to a week in a laboratory for carrying out the test.

Chemical reagents: CCl4, saline;

the detection kit comprises: total Bilirubin (TBIL), serum alkaline phosphatase (ALP), alanine Aminotransferase (ALT), and aspartate Aminotransferase (AST) were purchased from Abbott, Inc. of USA and assigned to the department of the Guilin medical school, Hospital laboratory.

2. Experimental methods

2.1 hepatic fibrosis modeling and drug administration treatment of rats

Randomly divided into 7 groups, i.e., control group, model group (CCl4), drug low dose A group (1.25, 2.5, 3.75, 25 mg/Kg. d), drug low dose B group (2.5, 5, 7.5, 50 mg/Kg. d), drug low dose C group (5, 10, 15, 100 mg/Kg. d), drug medium dose group (10, 20, 30, 200 mg/Kg. d), drug high dose group (20, 40, 60, 400 mg/Kg. d);

except for normal group, which was given physiological saline of equal volume, the other 6 groups of rats were orally gavaged with 40% carbon tetrachloride (dissolved in corn oil) for 8 weeks continuously, 2 times per week, to replicate the hepatic fibrosis model of the rats. Gavage started the next day after molding, where the drug low dose group a was given to Chitosan: 1.25 mg/Kg. d, Genistein: 2.5 mg/Kg. d, EGCG: 3.75 mg/Kg. d, Taurine: 25 mg/Kg. d; drug low dose group B administration Chitosan: 2.5 mg/Kg. d, Genistein: 5 mg/Kg. d, EGCG: 7.5 mg/Kg. d, Taurine: 50mg/Kg d; drug low dose group C administration Chitosan: 5 mg/Kg. d, Genistein: 10 mg/Kg. d, EGCG: 15 mg/Kg. d, Taurine: 100mg/Kg d; mid-drug dose groups were given to Chitosan: 10 mg/Kg. d, Genistein: 20 mg/Kg. d, EGCG: 30 mg/Kg. d, Taurine: 200mg/Kg d; drug high dose groups were given to Chitosan: 20 mg/Kg. d, Genistein: 40 mg/Kg. d, EGCG: 60 mg/Kg. d, Taurine: 400mg/Kg d; the model group and the blank group were simultaneously administered with an equal volume of physiological saline for 8 consecutive weeks.

2.2 serum liver function assay

24 hours after the last administration and physiological saline, chloral hydrate anesthetizes the rats, then the rats are sacrificed after abdominal aorta blood drawing, serum is separated, and the separated serum is placed in a refrigerator at the temperature of minus 80 ℃ for biochemical index analysis. Taking rat serum, respectively detecting ALT, AST and ALP levels of each group with alanine Aminotransferase (ALT), aspartate Aminotransferase (AST) and serum alkaline phosphatase (ALP) biochemical kit, and averaging.

2.3 liver spleen index and collagen area percentage detection

a. Calculating liver and spleen indices

Anesthetizing the rat 24 hours after the last administration of the normal saline, weighing, killing the rat, completely stripping the liver and the spleen, respectively washing the liver and the spleen in cold normal saline, and weighing; respectively calculating the liver index and the spleen index of each group, and taking the average value;

Above, liver index is liver wet mass/body mass × 100%; spleen index is spleen wet mass/body mass x 100%;

b. Calculate percent collagen area

Taking the dead right liver leaf of the rat in the mode, fixing the right liver leaf in 4% paraformaldehyde solution, embedding paraffin, staining a section in a Sirius red, scanning the section, calculating the area percentage of collagen by Aperio ImageScope12.3, and averaging;

the percentage of collagen area is defined as the area of collagen staining in the section/area of liver tissue in the section x 100%.

2.4 histopathological Observation of the liver

a part of liver tissue was fixed with 4% paraformaldehyde, cut into about 3mm × 5mm × 5mm, embedded with paraffin, and sliced continuously at 4 μm. Dewaxing with xylene, gradient dewatering with ethanol, and rinsing with tap water and distilled water to hydrate. HE dyeing, washing, dehydrating and sealing transparent sheets; the tissue structure was observed under an optical microscope.

3. Results of the experiment

note: statistical treatment: data are presented as mean ± standard deviation, results are analyzed using variance, significance is tested using t test in SPSS19.0, and significance is different with P < 0.05.

3.1 serum liver function assay

serum ALT, AST and ALP levels in seven groups of rats are shown in table 1:

TABLE 1 measurement of ALP, ALT and AST contents (x. + -. s) in the serum of each rat group

Note: p <0.05 in comparison to control

# compared to model group, P <0.05

As can be seen from Table 1, the ALP, ALT and AST levels in the model group are all significantly increased compared with the blank group, and the difference has statistical significance (P < 0.05); ALP decreased with increasing amounts of CGET drug. In ALT and AST indexes, too large and too small drug amount is not obvious in reduction. The results show that the ALP value of CCL4 model rats reduced by CGET drug under the dosage of drug low-dose group C, drug medium-dose group and drug high-dose group is statistically significant (P is less than 0.05). All five groups of drugs reduced the ALT index in rats and were statistically significant compared to CCl 4. The CGET drug has statistical significance in reducing the AST value of CCL4 model rats compared with the model group under the drug low-dose C group and the drug medium-dose group (P is less than 0.05); the CGET medicament can reduce the serum ALT, AST and ALP levels of rats and is helpful for reducing liver injury of the rats.

3.2 liver spleen index and collagen area percentage detection

The Liver index (Liver index), Spleen index (spleenindex) and collagen area percentage of the seven groups of rats are shown in table 2:

TABLE 2 liver-spleen index and collagen area percentage (x. + -.s) of rats in each group

Note: p <0.05 in comparison to control

# compared to model group, P <0.05

The liver index in a CCl4 rat model is mainly reduced, the liver index is increased after CGET is added, and the difference is not statistically significant (P is more than 0.05) when a drug low-dose group C, a drug medium-dose group and a drug high-dose group are compared with a control group; wherein the difference between the drug low dose group C and the drug high dose group is statistically significant (P < 0.05);

In a CCl4 rat model, spleen indexes are mainly increased, after CGET is added, the spleen indexes are increased, and compared with a drug low-dose group A, a drug low-dose group B and a drug low-dose group C, the difference has no statistical significance (P is more than 0.05);

The area of fibrosis in the stained area was calculated according to the whole section scanning software, the collagen area in the model group was significantly increased, the difference was statistically significant (P <0.05) compared to the control group, the collagen area in all CGET groups was decreased, and the difference was statistically significant (P <0.05) compared to the model group, as shown in table 2.

3.3 histopathological Observation of the liver

As shown in figure 1, HE staining observation of liver tissues of each group shows that liver lobules of the group A have complete structure and no cell degeneration and inflammatory cell infiltration; B. c, hepatic lobule structural disorder, collagen hyperplasia form false lobule, a large number of hepatic cells denaturize, the neutrophil infiltrates; D-G has slightly disordered hepatic lobule structures, hepatic cell degeneration is obviously reduced compared with a model group, collagen hyperplasia has no pseudolobule, hepatic cell degeneration is caused, and the number of hepatic lobule structures is reduced compared with B, C groups, and a small amount of inflammatory cell infiltration is caused.

As shown in figure 2, Sirius red staining observation is carried out on each group of liver tissues, the lobular structure of the A liver is complete, and no collagen hyperplasia exists; B. c, hepatic lobule structural disorder, a large amount of collagen (red) is proliferated to form pseudolobules and the collagen area is large; the D-G hepatic lobule structure is slightly disordered, and the collagen proliferation area is obviously reduced compared with the B, C group.

According to the comprehensive analysis of FIGS. 1-2, it can be seen that: the CGET drug administration group (C-G group) has the advantages of obviously reduced hepatocyte degeneration, little fibroplasia, reduced inflammatory cell infiltration and no obvious false lobule formation. The CGET medicament has obvious improvement effect on hepatic fibrosis. And the improvement effect is not obvious due to the low dosage of the group C.

The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.

Claims

1. The anti-hepatic fibrosis pharmaceutical composition is characterized by being a preparation prepared from the following raw material medicines in parts by weight: 1-5 parts of Chitosan, 2-10 parts of Genistein, 3-15 parts of EGCG and 20-100 parts of Taurine.

2. The anti-hepatic fibrosis pharmaceutical composition of claim 1, wherein the composition is prepared from the following raw materials in parts by weight: 2-4 parts of Chitosan, 4-8 parts of Genistein, 6-12 parts of EGCG and 40-80 parts of Taurine.

3. The anti-hepatic fibrosis pharmaceutical composition of claim 1, wherein the composition is prepared from the following raw materials in parts by weight: chitosan 3 parts, Genistein 6 parts, EGCG 9 parts and Taurin 60 parts.

4. The anti-hepatic fibrosis pharmaceutical composition of claim 1, wherein the composition is prepared from raw material drugs as active ingredients and pharmaceutically common adjuvants or auxiliary ingredients, and is in various dosage forms suitable for clinical application.

5. The anti-hepatic fibrosis pharmaceutical composition according to claim 1, wherein the composition is prepared by the following method:

s1: weighing the raw material medicines according to the parts by weight;

6. Use of the pharmaceutical composition according to any one of claims 1-5 as an active ingredient for the preparation of a medicament for treating liver fibrosis.