CN115337312A - Cholic acid complex and preparation method and application thereof - Google Patents

Abstract

The cholic acid compound provided by the invention can effectively improve liver echo of a model mouse, relieve pathological damage of the liver, reduce NAS (non-additive stratum corneum) score, reduce liver lipid deposition and relieve inflammatory reaction of serum and the liver, thereby having a remarkable treatment effect on non-alcoholic fatty liver diseases. The invention overcomes the technical problem that a single compound cannot play a comprehensive role in aiming at complex diseases by selecting the specific cholic acid type to be matched with the specific cholic acid dosage, realizes effective treatment on the non-alcoholic fatty liver disease, provides data support for clinical medication and lays a foundation for further research and development of the non-alcoholic fatty liver disease.

Description

Cholic acid complex and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a cholic acid compound and a preparation method and application thereof.

Background

Non-alcoholic fatty liver disease (NAFLD) is a disease characterized by lipodegeneration and lipopexia of parenchymal liver cells, NAFLD is suffered by 25% of people in the world at present, and non-alcoholic steatohepatitis (NASH) is a subtype of NAFLD, is characterized by inflammatory injury of liver cells and is an important rate-limiting link in the process of converting simple fatty liver into cirrhosis and liver cancer. However, current international treatment approaches for NASH are limited and therapeutic drugs are still blank, and several drugs for NASH are failed in clinical trials, including obeticholic acid (OCA) which is an agonist of Farnesoid X Receptor (FXR) that is endowed with great promise. The reason for failure of these drugs is that most of them are single compounds that cannot fully act on complex diseases and often cause some adverse reactions (e.g. skin pruritus).

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

An object of the present invention is to provide a cholic acid complex, which solves at least one of the problems of the prior art.

The second object of the present invention is to provide the use of the bile acid complex.

The present invention also provides a method for preparing the cholic acid complex.

The fourth object of the present invention is to provide a drug comprising the above-mentioned bile acid complex.

The fifth purpose of the invention is to provide the application of the medicine.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the present invention provides a cholic acid complex comprising: 27-45 parts of tauroursodeoxycholic acid and 15-25 parts of taurochenodeoxycholic acid; and at least one of taurocholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, and taurolithocholic acid.

Further, the bile acid complex comprises: 30-40 parts of tauroursodeoxycholic acid and 16-22 parts of taurochenodeoxycholic acid; and at least one of 7 to 20 parts of taurocholic acid, 0.1 to 1 part of cholic acid, 0.05 to 1 part of ursodeoxycholic acid, 0.05 to 0.5 part of chenodeoxycholic acid, 0.05 to 0.5 part of deoxycholic acid and 0.05 to 0.5 part of taurocholic acid.

Further, the bile acid complex comprises: 30-37 parts of tauroursodeoxycholic acid and 17-21 parts of taurochenodeoxycholic acid; and at least one of 8 to 15 parts of taurocholic acid, 0.3 to 0.6 part of cholic acid, 0.09 to 0.4 part of ursodeoxycholic acid, 0.1 to 0.3 part of chenodeoxycholic acid, 0.08 to 0.2 part of deoxycholic acid and 0.08 to 0.2 part of taurocholic acid.

The invention also provides application of the cholic acid complex in preparing a medicament for treating non-alcoholic fatty liver disease.

Further, the non-alcoholic fatty liver disease includes non-alcoholic steatohepatitis.

The invention also provides a preparation method of the cholic acid complex, which comprises the following steps: and uniformly mixing the tauroursodeoxycholic acid and the taurochenodeoxycholic acid, and at least one of taurocholic acid, cholic acid, ursodesoxycholic acid, chenodeoxycholic acid, deoxycholic acid and taurochenolithocholic acid in a formula amount to obtain the cholic acid compound.

The present invention also provides another preparation method of the cholic acid complex described above, which comprises: performing biotransformation on the poultry bile or the poultry bile powder by using hydroxysteroid dehydrogenase, and then performing alcohol extraction, concentration and drying to prepare the cholic acid compound;

wherein the hydroxysteroid dehydrogenase comprises a 7 α -hydroxysteroid dehydrogenase and/or a 7 β -hydroxysteroid dehydrogenase.

In addition, the invention also provides a medicine for treating the non-alcoholic fatty liver disease, which comprises the cholic acid compound and pharmaceutically acceptable auxiliary materials.

Further, the dosage form of the medicament comprises an oral preparation or an injection preparation.

Further, the effective administration dose of the medicament is 39-312 mg/kg, and preferably 156mg/kg.

Further, the non-alcoholic fatty liver disease includes non-alcoholic steatohepatitis.

The invention also provides the application of the medicine for treating the non-alcoholic fatty liver disease in preparing products for treating the non-alcoholic fatty liver disease;

preferably, the non-alcoholic fatty liver disease includes non-alcoholic steatohepatitis.

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

through a large number of experiments, the inventor of the invention discovers that the cholic acid compound provided by the invention can effectively improve the liver echo of a model mouse, relieve pathological damage of the liver, reduce NAS (non-aqueous ammonia storage) scores, reduce liver lipid deposition and relieve the inflammatory reaction of serum and the liver, thereby having a remarkable treatment effect on non-alcoholic fatty liver diseases. The invention overcomes the technical problem that a single compound cannot play a comprehensive role in aiming at complex diseases by selecting the specific cholic acid type to be matched with the specific cholic acid dosage, realizes effective treatment on the non-alcoholic fatty liver disease, provides data support for clinical medication and lays a foundation for further research and development of the non-alcoholic fatty liver disease.

The active ingredient of the medicine for treating the non-alcoholic fatty liver disease provided by the invention is the cholic acid compound provided by the invention, so that the medicine can also play an obvious role in preventing and/or treating the non-alcoholic fatty liver disease based on the beneficial effects of the cholic acid compound, and is safe, non-toxic and small in side effect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph showing the results of the weight change trend of 8 weeks of molding according to the experimental example of the present invention;

FIG. 2 is a graph showing the results of the weight change trend of 8 weeks after administration according to the experimental example of the present invention;

FIG. 3 is an ultrasonic image of a molded 8-week liver provided by an experimental example of the present invention;

FIG. 4 is a comparison graph of abdominal ultrasound liver echoes after 4 weeks of dosing according to an experimental example of the present invention;

FIG. 5 is a statistical graph of abdominal ultrasound liver echo scores 4 weeks after administration according to an experimental example of the present invention;

FIG. 6 is a comparison graph of abdominal ultrasound liver echoes after 8 weeks of dosing according to an experimental example of the present invention;

FIG. 7 is a statistical graph of abdominal ultrasound liver echo scores after 8 weeks of administration according to an example of the present invention;

FIG. 8 is a diagram of the morphology of the liver in the experimental example of the present invention after 8 weeks of modeling;

FIG. 9 is a graph of liver coefficient results 8 weeks after molding according to the experimental example of the present invention;

FIG. 10 is a diagram of the morphology of the liver in the experimental example of the present invention after 8 weeks of administration;

FIG. 11 is a graph showing the results of liver coefficients 8 weeks after administration in accordance with the experimental example of the present invention;

FIG. 12 is a graph (x 200) showing HE pathological staining of mouse liver after 8 weeks of molding according to the experimental example of the present invention;

FIG. 13 is a graph of HE pathological staining of mouse liver at 8 weeks post-dose (X200) in accordance with the experimental examples of the present invention;

FIG. 14 is a graph of HE pathological staining of mouse liver at 8 weeks post dose (X400) in accordance with the experimental examples of the present invention;

FIG. 15 is a graph of mouse liver oil red O staining after 8 weeks of molding according to the experimental example of the present invention;

FIG. 16 is a staining pattern of mouse liver oil red O at 8 weeks after administration (X200) in accordance with the experimental example of the present invention;

FIG. 17 is a graph showing the results of the levels of TC, ALT, and AST in the serum of mice 8 weeks after molding according to the experimental example of the present invention;

FIG. 18 is a graph showing the results of the test example of the present invention on the levels of TC, ALT and AST in the serum of mice after 4 weeks of administration.

Detailed Description

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" and other forms is not limiting.

Generally, the nomenclature used, and the techniques thereof, in connection with the cell and tissue cultures, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly employed in the art.

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

According to one aspect of the present invention, there is provided a bile acid complex comprising: tauroursodeoxycholic acid and taurochenodeoxycholic acid; and at least one of taurocholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, and taurolicholic acid.

It should be noted that the main active ingredients of the cholic acid complex provided by the present invention are tauroursodeoxycholic acid and taurochenodeoxycholic acid, and besides the main active ingredients, at least one of taurocholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid and taurochenolithocholic acid is also included as a complex active ingredient. For example, taurocholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, or taurocholic acid; or selecting combination of taurocholic acid and cholic acid, or selecting combination of deoxycholic acid and taurocholic acid, or selecting combination of taurocholic acid and ursodeoxycholic acid, or other combination composed of two kinds of matched active ingredients; or selecting taurocholic acid, cholic acid and ursodeoxycholic acid, or ursodeoxycholic acid, chenodeoxycholic acid and deoxycholic acid, or taurocholic acid, deoxycholic acid and taurolicholic acid, or other combinations of three matched active ingredients; or selecting four combinations of taurocholic acid, cholic acid, ursodeoxycholic acid and chenodeoxycholic acid, or four combinations of ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid and taurocholic acid, or other combinations composed of four matching active ingredients; or selecting combination of taurocholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid and deoxycholic acid, or other combination of five kinds of active ingredients; in addition, taurocholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid and taurolicholic acid can also be selected as the active ingredients.

Wherein, the content of tauroursodeoxycholic acid (TUDCA) is 27-45 parts, such as but not limited to 27 parts, 30 parts, 32 parts, 35 parts, 38 parts, 40 parts, 42 parts or 45 parts; the content of taurochenodeoxycholic acid (TCDCA) is 15 to 25 parts, and may be, for example, but not limited to, 15 parts, 18 parts, 20 parts, 22 parts, or 25 parts. In addition, the content of the main active ingredients can be limited by the dosage ratio, for example, the content ratio of tauroursodeoxycholic acid to taurochenodeoxycholic acid can be 1.3-2.0.

Through a large number of experiments, the inventor of the invention discovers that the cholic acid compound provided by the invention can effectively improve the liver echo of a model mouse, relieve pathological damage of the liver, reduce NAS (non-aqueous ammonia storage) scores, reduce liver lipid deposition and relieve the inflammatory reaction of serum and the liver, thereby having a remarkable treatment effect on non-alcoholic fatty liver diseases. The invention overcomes the technical problem that a single compound cannot play a comprehensive role in aiming at complex diseases by selecting the specific cholic acid type to be matched with the specific cholic acid dosage, realizes effective treatment on the non-alcoholic fatty liver disease, provides data support for clinical medication and lays a foundation for further research and development of the non-alcoholic fatty liver disease.

By adjusting and optimizing the dosage of the active ingredients of the cholic acid complex provided by the invention, the cholic acid complex preferably comprises: 30-40 parts of tauroursodeoxycholic acid and 16-22 parts of taurochenodeoxycholic acid; and at least one of 7 to 20 parts of taurocholic acid, 0.1 to 1 part of cholic acid, 0.05 to 1 part of ursodeoxycholic acid, 0.05 to 0.5 part of chenodeoxycholic acid, 0.05 to 0.5 part of deoxycholic acid and 0.05 to 0.5 part of taurocholic acid. The optimized cholic acid compound has more remarkable treatment effect on the non-alcoholic fatty liver disease.

For alternative co-actives, the amount of taurocholic acid (TCA) used for each component may be, for example, but is not limited to, 7 parts, 8 parts, 10 parts, 12 parts, 15 parts, 18 parts, or 20 parts; the content of Cholic Acid (CA) may be, for example, but not limited to, 0.1 part, 0.2 part, 0.5 part, 0.8 part or 1 part; the content of ursodeoxycholic acid (UDCA) may be, for example, but not limited to, 0.05 parts, 0.08 parts, 0.1 parts, 0.2 parts, 0.3 parts, 0.4 parts, 0.5 parts, 0.6 parts, 0.7 parts, 0.8 parts, 0.9 parts, or 1 part; the content of chenodeoxycholic acid (CDCA) may be, for example, but not limited to, 0.05 parts, 0.08 parts, 0.1 parts, 0.2 parts, or 0.5 parts; the content of deoxycholic acid (DCA) may be, for example, but not limited to, 0.05 parts, 0.08 parts, 0.1 parts, 0.2 parts, 0.3 parts, 0.4 parts, or 0.5 parts; the content of taurolicholic acid (TLCA) can be, for example, but is not limited to, 0.05 parts, 0.08 parts, 0.1 parts, 0.2 parts, 0.3 parts, 0.4 parts, or 0.5 parts.

On the basis, the invention further optimizes the formula of the cholic acid complex, and the cholic acid complex as a preferred embodiment comprises: 30-37 parts of tauroursodeoxycholic acid and 17-21 parts of taurochenodeoxycholic acid; and at least one of 8 to 15 parts of taurocholic acid, 0.3 to 0.6 part of cholic acid, 0.09 to 0.4 part of ursodeoxycholic acid, 0.1 to 0.3 part of chenodeoxycholic acid, 0.08 to 0.2 part of deoxycholic acid and 0.08 to 0.2 part of taurocholic acid.

According to a second aspect of the present invention there is provided the use of a bile acid complex as described above in the manufacture of a medicament for the treatment of non-alcoholic fatty liver disease. Particularly, the cholic acid complex provided by the invention has a particularly remarkable effect in the treatment of nonalcoholic steatohepatitis.

According to a third aspect of the present invention, there is provided a method for preparing the cholic acid complex described above, comprising: uniformly mixing tauroursodeoxycholic acid and taurochenodeoxycholic acid, and at least one of taurocholic acid, cholic acid, ursodesoxycholic acid, chenodeoxycholic acid, deoxycholic acid and taurocholic acid to obtain the cholic acid compound.

The preparation method of the cholic acid compound provided by the invention has the advantages of simple process and convenience in operation, does not need specific technical personnel or expensive equipment, and can effectively save the cost.

In addition, the present invention also provides another preparation method of the cholic acid complex, comprising:

performing biotransformation on the poultry bile or the poultry bile powder by using hydroxysteroid dehydrogenase, and then performing alcohol extraction, concentration and drying to prepare the cholic acid compound;

wherein the hydroxysteroid dehydrogenase comprises a 7 alpha-hydroxysteroid dehydrogenase and/or a 7 beta-hydroxysteroid dehydrogenase.

The preparation method of the cholic acid compound provided by the invention has the advantages of simple process, convenience in operation, environment friendliness and capability of effectively saving cost on the basis of the characteristic of recycling of the alcohol solvent.

When the bile acid complex is prepared using this method, the bile acid complex further comprises at least one of cholesterol, a bile pigment, an amino acid, a polypeptide, a protein, a metal element, preferably all of cholesterol, a bile pigment, an amino acid, a polypeptide, a protein, and a metal element.

When the cholic acid complex comprises the above substances, preferably, the mass percentage of tauroursodeoxycholic acid, taurodeoxycholic acid, taurocholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid and taurolithocholic acid in the cholic acid complex is 60% to 85%, and for example, may be, but is not limited to, 60%, 65%, 70%, 75%, 80% or 85%.

In addition, according to the application, the invention also provides a medicine for treating the non-alcoholic fatty liver disease, and the medicine comprises the cholic acid compound and pharmaceutically acceptable auxiliary materials.

The active ingredient of the medicine for treating the non-alcoholic fatty liver disease provided by the invention is the cholic acid compound provided by the invention, so that the medicine can also play an obvious role in preventing and/or treating the non-alcoholic fatty liver disease based on the beneficial effect of the cholic acid compound, and is safe, non-toxic and small in side effect.

The pharmaceutically acceptable auxiliary materials refer to excipients and additives used in the production of medicines and the preparation of prescriptions, and refer to substances which are reasonably evaluated in the aspect of safety and contained in pharmaceutical preparations besides active ingredients. The same pharmaceutic adjuvant can be used for pharmaceutic preparations of different administration routes and has different functions and purposes. The pharmaceutically acceptable auxiliary materials added in the medicine provided by the invention can play roles in forming, serving as a carrier or improving the stability, and also has important functions of solubilization, dissolution assistance or sustained and controlled release and the like.

Typical but non-limiting pharmaceutically acceptable excipients include: one or more of solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, antioxidants, chelating agents, permeation enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, or release retardants.

In a preferred embodiment, the dosage form of the medicament comprises an oral formulation or an injectable formulation.

When administered orally, the above-mentioned drugs may be formulated into any orally acceptable formulation form, for example, but not limited to, tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions or oral emulsions.

Among these, carriers for tablets generally include lactose and corn starch, and additionally, lubricating agents such as magnesium stearate may be added. Diluents used in capsules generally include lactose and dried corn starch. Oral suspensions are generally prepared by mixing the active ingredient with suitable emulsifying and suspending agents.

Optionally, some sweetener, aromatic or colorant may be added into the above oral preparation.

When the medicine is administered in the form of injection, the medicine can be prepared into any preparation form acceptable for injection, such as, but not limited to, injection solution or powder injection.

Among the vehicles and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. In addition, the sterilized fixed oil may also be employed as a solvent or suspending medium, such as a monoglyceride or diglyceride.

In a preferred embodiment, the effective dose of the drug is 39-312 mg/kg, such as, but not limited to, 39mg/kg, 78mg/kg, 156mg/kg or 312mg/kg, and when the dose is 156mg/kg, the dose can be effectively controlled based on the efficacy of the drug.

The invention also provides the application of the medicine for treating the non-alcoholic fatty liver disease in preparing products for treating the non-alcoholic fatty liver disease;

preferably, the non-alcoholic fatty liver disease comprises non-alcoholic steatohepatitis.

The invention is further illustrated by the following examples. The materials in the examples are prepared according to known methods or are directly commercially available, unless otherwise specified.

Example 1

The present embodiment provides a cholic acid complex comprising: 27 parts of tauroursodeoxycholic acid, 25 parts of taurochenodeoxycholic acid, 7 parts of taurocholic acid, 1 part of cholic acid, 0.05 part of ursodeoxycholic acid, 1 part of chenodeoxycholic acid, 0.05 part of deoxycholic acid and 0.5 part of taurochenolithocholic acid.

Example 2

The present embodiment provides a cholic acid complex comprising: 45 parts of tauroursodeoxycholic acid, 15 parts of taurochenodeoxycholic acid, 20 parts of taurocholic acid, 0.1 part of cholic acid, 0.5 part of ursodeoxycholic acid, 0.1 part of chenodeoxycholic acid, 0.5 part of deoxycholic acid and 0.05 part of taurochenolithocholic acid.

Example 3

The present embodiment provides a cholic acid complex comprising: 40 parts of tauroursodeoxycholic acid, 22 parts of taurochenodeoxycholic acid, 10 parts of taurocholic acid, 0.6 part of cholic acid, 0.09 part of ursodeoxycholic acid, 0.3 part of chenodeoxycholic acid, 0.08 part of deoxycholic acid and 0.2 part of taurochenolithocholic acid.

Example 4

The present embodiment provides a cholic acid complex comprising: 30 parts of tauroursodeoxycholic acid, 21 parts of taurochenodeoxycholic acid, 8 parts of taurocholic acid, 0.6 part of cholic acid, 0.09 part of ursodeoxycholic acid, 0.3 part of chenodeoxycholic acid, 0.08 part of deoxycholic acid and 0.2 part of taurolithocholic acid.

Example 5

The present embodiment provides a bile acid complex comprising: 37 parts of tauroursodeoxycholic acid, 17 parts of taurochenodeoxycholic acid, 15 parts of taurocholic acid, 0.3 part of cholic acid, 0.4 part of ursodeoxycholic acid, 0.1 part of chenodeoxycholic acid, 0.2 part of deoxycholic acid and 0.08 part of taurolithocholic acid.

Example 6

The present embodiment provides a bile acid complex comprising: 32 parts of tauroursodeoxycholic acid, 18 parts of taurochenodeoxycholic acid, 11 parts of taurocholic acid, 0.4 part of cholic acid, 0.2 part of ursodeoxycholic acid, 0.2 part of chenodeoxycholic acid, 0.1 part of deoxycholic acid and 0.1 part of taurolithocholic acid.

Example 7

The present embodiment provides a bile acid complex comprising: 35 parts of tauroursodeoxycholic acid, 20 parts of taurochenodeoxycholic acid, 13 parts of taurocholic acid, 0.5 part of cholic acid, 0.3 part of ursodeoxycholic acid, 0.22 part of chenodeoxycholic acid, 0.15 part of deoxycholic acid and 0.15 part of taurochenolithocholic acid.

Example 8

The present embodiment provides a bile acid complex comprising: 32 parts of tauroursodeoxycholic acid, 18 parts of taurochenodeoxycholic acid and 11 parts of taurocholic acid.

Example 9

The present embodiment provides a cholic acid complex comprising: 32 parts of tauroursodeoxycholic acid, 18 parts of taurodeoxycholic acid, 0.2 part of ursodesoxycholic acid and 0.2 part of chenodeoxycholic acid.

Example 10

The present embodiment provides a cholic acid complex comprising: 32 parts of tauroursodeoxycholic acid, 18 parts of taurochenodeoxycholic acid, 0.4 part of cholic acid, 0.1 part of deoxycholic acid and 0.1 part of taurocholic acid.

Example 11

The present embodiment provides a bile acid complex comprising: 32 parts of tauroursodeoxycholic acid, 18 parts of taurochenodeoxycholic acid, 11 parts of taurocholic acid, 0.2 part of ursodeoxycholic acid, 0.2 part of chenodeoxycholic acid and 0.1 part of taurolithocholic acid.

Example 12

The present example provides a cholic acid complex, which is prepared by the following steps:

the 7 alpha-hydroxysteroid dehydrogenase and the 7 beta-hydroxysteroid dehydrogenase were mixed with chicken chook, and the mixture was mixed uniformly, taking enzyme cells as an example, 7 alpha enzyme cells: adjusting the pH value to 6.5-9, reacting at room temperature overnight, adding ethanol to a final concentration of 80 +/-5%, precipitating with ethanol at 16 ℃ for more than 4h, filtering or centrifuging to collect clear liquid, and concentrating and drying in vacuum to obtain powder at the temperature of lower than 80 ℃.

Preparing chicken bile: removing impurities from fel gallus Domesticus, filtering, collecting juice, adding ethanol to final concentration of 80 + -5%, precipitating with ethanol at 16 deg.C for more than 4 hr, filtering or centrifuging to collect clear liquid, and concentrating; adding weak polar solvent ethyl acetate into the concentrated clear paste for extraction for 3 times, wherein the volume ratio of the two is 1.

Comparative example 1

This comparative example provides a cholic acid complex comprising: 30 parts of tauroursodeoxycholic acid, 30 parts of taurochenodeoxycholic acid, 8 parts of taurocholic acid, 2 parts of cholic acid, 0.01 part of ursodeoxycholic acid, 2 parts of chenodeoxycholic acid, 0.01 part of deoxycholic acid and 1 part of taurochenolithocholic acid.

Comparative example 2

This comparative example provides a cholic acid complex, which differs from example 4 in that ursodeoxycholic acid is replaced with lithocholic acid.

Comparative example 3

The comparative example provides a cholic acid complex, which consists of the following components in parts by weight: 37.2 parts of cholic acid, 29.4 parts of tauroursodeoxycholic acid, 25.4 parts of taurochenodeoxycholic acid and 4 parts of taurocholic acid.

The cholic acid complexes of examples 1 to 11 and comparative examples 1 to 3 were prepared by uniformly mixing the components in the prescribed amounts.

Examples of the experiments

Main apparatus and reagents:

1. model building, animal grouping and administration

Healthy male SPF grade C57BL/6 mice, aged 8-10 weeks, were used and purchased from: chinese institute for food and drug certification (daxing), license number: SCXK (Jing) 2017-0005; after the acceptance of the receiving personnel is qualified, the animals are raised in animal houses of the institute of basic theory of traditional Chinese medicine of the academy of traditional Chinese medicine, and the license numbers are as follows: SYXK (Jing) 2021-0017. 4-5 animals per cage, relative humidity range of animal house 50-60%; the temperature is 22 ℃ to 25 ℃, and 12h/12h simulates day-night alternation of light and shade.

After 1 week of acclimatization, western diet feeding was simulated by high-fat diet and high-sugar drinking water (containing 21.1% fat,41% sucrose, and 1.25% Cholesterol and a high sugar solution (23.1 g/L d-fraction and 18.9g/L d-glucose)) for 16 weeks after acclimatization of the normal diet, and before 8 weeks, 1-time intraperitoneal injection of carbon tetrachloride (0.2 μ L (0.32 μ g/g) was performed, and after successful molding, the normal diet, the model group, each dose group of the bile acid complex (39 mg/kg;78mg/kg;156mg/kg;312 mg/kg) provided in example 4, examples 1-3, 5-12, and comparative examples 1-3 groups (156 mg/kg), drain bear gall powder (78 mg/kg) control group, positive pyridone group (30 mg/kg), specific administration of the glitazone group, and specific administration of the CMC 1-8 week as control group, and the normal diet was administered continuously for each week after molding.

TABLE 1 dosage form

Index detection:

1. general index

Carefully observing and recording the mental state, fur change, activity condition, drinking water and food intake of each group of mice every day; and the body weight of each mouse was measured weekly and the blood glucose was recorded monthly.

2. General index of liver

Weighing the mice after 8 weeks of modeling and 8 weeks of drug administration, killing the eyeballs after blood sampling, quickly splitting the abdominal cavity, picking the liver of the mice, weighing the mice on a culture dish, and calculating the liver coefficient (the weight of the liver accounts for the percentage of the total weight); weighing, placing in normal saline to remove residual blood, and carrying out macroscopic observation and photographing on the liver after the filter paper absorbs water. Preparing the liver midlobes into paraffin sections; freezing and slicing the papilla leaves; the rest liver tissue is put into liquid nitrogen to be frozen rapidly and then is frozen and stored at minus 80 ℃.

HE staining

Mice livers were subjected to HE staining and NAFLD Activity Scoring (NAS) 8 weeks after dosing for 8 weeks, respectively, of molding.

1. Paraffin section dewaxing to water: sequentially placing the slices into xylene I20 min-xylene II 20 min-absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5min-75% alcohol 5min, and washing with tap water.

2. Hematoxylin staining: staining the slices with hematoxylin staining solution for 3-5min, washing with tap water, differentiating with differentiation solution, washing with tap water, returning blue to blue with blue returning solution, and washing with running water.

3. Eosin staining: the slices are dehydrated for 5min respectively by 85 percent and 95 percent gradient alcohol, and are dyed for 5min in eosin dye solution.

4. Dewatering and sealing: and sequentially placing the slices into absolute ethyl alcohol I for 5min, absolute ethyl alcohol II for 5min, absolute ethyl alcohol III for 5min, xylene I for 5min and xylene II for 5min, and sealing the slices with neutral gum.

5. Microscopic examination and image acquisition.

6. Semi-quantitative scoring criteria (NAS score)

NAS integration (0 to 8 points): (1) adiposis of liver cells: 0min (< 5%); 1 part (5-33%); 2 min (34-66%); score 3 (> 66%). (2) Intralobular inflammation (20-fold mirror count necrotic foci): 0min, none; 1 point (< 2); 2 min (2-4); 3 min (> 4). (3) Ballooning of hepatocytes: 0min, none; 1 minute, rare; 2 fen, mostly seen.

NAS is a semi-quantitative scoring system rather than a diagnostic program, NAS < 3 points can exclude NASH, NAS > 4 points can diagnose NASH, and between the two, NASH is possible. NAFL is prescribed in those who do not have endoplasmic inflammation, ballooning and fibrosis but have hepatic steatosis > 33%, and those who have not reached such a degree of steatosis are simply called hepatocellular steatosis.

4. Oil red O dyeing

Mouse livers were stained with oil red O at 8 weeks post-molding and 8 weeks post-dose, respectively.

1. Fresh frozen sections were fixed: and (3) rewarming and drying the frozen slices, fixing in a fixing solution for 15min, washing with tap water and drying in the air.

2. Oil red dyeing: the slices are soaked in oil red dye solution for 8-10min (covered and protected from light).

3. Background differentiation: taking out the slices, standing for 3s, sequentially immersing in 60% isopropanol in two cylinders for differentiation for 3s and 5s respectively. The slices were sequentially immersed in 2 cylinders of pure water for 10 seconds each.

4. Hematoxylin staining: taking out the slices, standing for 3s, immersing in hematoxylin for counterstaining for 3-5min, and 3-jar soaking with pure water for 5s, 10s and 30s respectively. Differentiation is carried out for 2-8s by using a differentiation solution (60% alcohol as a solvent), each 10s by using 2 cylinders of distilled water, each 1s by using a blue returning solution, the slice is slightly immersed into 2 cylinders of tap water for immersion, each 5s and each 10s, and the staining effect is examined under a microscope.

5. Sealing: and (5) sealing the glycerol gelatin sealing agent.

6. Microscopic examination and image acquisition and analysis.

5. Mouse liver ultrasonic detection

Liver ultrasonography was performed at 8 weeks (n = 4), 4 weeks (n = 6), and 8 weeks (n = 6) after the molding, respectively. Before detection, fasting is carried out at night, after abdominal cavity injection anesthesia is carried out on tribromoethanol on the next day, hair removal is carried out on the upper abdomen 3cm below the xiphoid process by using a hair removal paste, the abdomen is fully exposed, the supine position is fixed, sufficient couplant is smeared to enable a probe to be fully contacted with the abdominal wall, abdominal liver ultrasonic examination is carried out by using a Vevo 2100 small animal ultrasonic instrument, an MS400 probe is used, the frequency of the probe is 30MHZ, and liver sections (three marked sections are selected) under B Model are respectively intercepted under the same conditions (the same depth, the same width and the same gain value are fixed) to carry out grey value evaluation.

The mice in each group are randomly selected for liver ultrasonic detection, and the liver injury condition is evaluated through the liver and kidney echo contrast intensity. A hepatic echo higher than a renal echo represents severe liver damage (-1 point); a liver echo slightly above a kidney echo represents a minor liver injury (+ 1 point); liver echoes were comparable to kidney echoes, and weaker than kidney echoes represented no apparent damage to the liver (+ 2 points).

6. Serum biochemical index detection

The mouse orbital blood was collected (about 100 μ L) at 8 weeks (n = 4), 4 weeks (n = 6), and 8 weeks (n = 6) after molding, and after standing at room temperature for 4 hours, the supernatant was aspirated after centrifugation at 3500rpm at 4 ℃ for 15 min. The liver function indexes are measured and respectively detected according to the operation of a reagent specification: alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), alkaline phosphatase (ALP), direct Bilirubin (DBIL), and Total Bilirubin (TBIL); blood lipids TC, TG, HDL, LDL. Inflammatory factors interleukin 1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha).

7. Biochemical index detection of liver

Taking mouse liver at 8 weeks (n = 6) of administration, preparing homogenate, and detecting inflammatory factors TNF-alpha and IL-1 beta by biochemical index using Hitachi 7600 full-automatic biochemical analyzer; TGF-beta, a fibrosis-related indicator; lipid-related indices TC, TG, LDL-C.

8. Statistical analysis

Data is represented by calculation

Data were analyzed for one-way analysis of variance (ANOVA) using GraphPad Prism 8 software, post-test using Sidak test; when P is present<When the concentration is 0.05, the significant difference is judged between the two groups.

2. Results of the experiment

1. Effect of cholic acid Complex on status and weight of NASH model mouse

The body weight of the 8-week-old mice was shown in fig. 1, and the body weight of the model group was slightly reduced compared to that of the normal group, but there was no significant difference.

The body weights of the mice after 8 weeks of administration are shown in FIG. 2 (A: example 4 cholic acid complex group A; B: example 4 cholic acid complex group B; C: example 4 cholic acid complex group C; D: example 4 cholic acid complex group D), and compared with the normal group, the body weights of the mice in the model group were significantly reduced (P < 0.05) at 1 week (experiment 9 week) after administration, but no significant difference was observed between 2-8 weeks after administration and the normal group; mice were reduced to different extents in each dose group of cholic acid complex and in the bear bile powder-draining group 1 week after administration compared with the model group, wherein the weight reductions in the two groups of cholic acid complex C, D of example 4 were significant (P < 0.05P- < -0.01) and had significant statistical differences; example 4 bile acid complex D dose group significantly decreased body weight 2 weeks after dosing (P < 0.05); at week 4 post-dose C, both groups D significantly reduced body weight (P < 0.01P < -0.05; the positive pioglitazone group showed different increases in weight average compared with the model group at weeks 1, 2, 3, 5 and 7 after administration, but had no statistical difference, while the decrease at weeks 4, 6 and 8 was probably related to the anesthesia of the animals by ultrasonic examination at the corresponding time points.

2. Influence of cholic acid complex on liver ultrasonic index of NASH model mouse

After 8 weeks of modeling, randomly extracting 4 small animal livers from the normal group and the model group for ultrasonic detection, and intercepting three representative fixed sections of livers in a Bmodel mode. As shown in fig. 3, liver echoes were diffusely enhanced in thin dots in the model group compared to the normal group.

After 4 weeks of administration, the results are shown in FIG. 4 (A: example 4 bile acid complex group A; B: example 4 bile acid complex group B; C: example 4 bile acid complex group C; D: example 4 bile acid complex group D), FIG. 5 (1control 2Model 3 drainage bear bile powder group; 4 pioglitazone group; A: example 4 bile acid complex group A; B: example 4 bile acid complex group B; C: example 4 bile acid complex group C; D: example 4 bile acid complex group D): each administration group has the function of reducing abnormal liver echoes to a certain extent, and the evaluation of the ultrasonic echo contrast intensity of the liver and the kidney shows that: the positive drug (pioglitazone) had the best efficacy with a significant difference (P < 0.01) compared to the model group, and then each group was improved to a different extent, wherein the cholic acid complex D and C of example 4 were more significant in the dose group, but not statistically different.

For 8 weeks, as shown in FIG. 6 (A: example 4 bile acid complex group A; B: example 4 bile acid complex group B; C: example 4 bile acid complex group C; D: example 4 bile acid complex group D), FIG. 7 (1control 2model 3 drainage bear bile powder group; 4 pioglitazone group; A: example 4 bile acid complex group A; B: example 4 bile acid complex group B; C: example 4 bile acid complex group C; D: example 4 bile acid complex group D), each administration group had some efficacy as compared with the model group, as can be derived from the liver and kidney ultrasound echo comparative intensity score: example 4 bile acid complex C, B dose group was able to significantly improve liver echo (P < 0.01. From the 4-and 8-week results of dosing: the pioglitazone group had significant efficacy in 4 weeks of administration, but the efficacy was not significant after 8 weeks of administration.

3. Influence of cholic acid complex on liver gross and liver coefficient of NASH model mouse

After 8 weeks of model building, 4 eyeballs of mice in the normal group and the model group are randomly selected for blood sampling, and then the liver of the mice is photographed as shown in figure 8.

The results show that the liver tissues of the mice in the normal control group are bright red, smooth and compact in surface; the model group, slightly whitish in color, appeared as many tiny milky white particles on the liver surface and the liver appeared coarse.

The liver coefficients after 8 weeks of molding are shown in fig. 9: the liver coefficient was significantly increased in the model group compared to the normal group (P < 0.001), indicating the possible presence of edema, hyperemia or hyperplastic hypertrophy of the liver.

The liver macrostructure results after 8 weeks of administration are shown in FIG. 10 (A: group A of the bile acid complex of example 4; B: group B of the bile acid complex of example 4; C: group C of the bile acid complex of example 4; D: group D of the bile acid complex of example 4), and the liver tissues of the normal group of mice are bright red, smooth and glossy in surface and dense in texture; the liver tissue of the model group turns white, the liver tissue has white reticular texture, and the tail-shaped leaf morphological abnormality is suspected to be wrapped by lipid; compared with the model group, the color, texture and lipid encapsulation of the liver in each administration group are improved to some extent. In example 4, the cholic acid compound C and D in the dose group has the advantages that the liver tissue color is obviously reddish compared with the model group, the surface texture is lightened, the improvement is most obvious, and the cholic acid compound C and D are superior to the drainage bear gall powder group and the positive medicine pioglitazone group.

The results of liver coefficients after 8 weeks of administration are shown in FIG. 11 (1Control 2Model 3 drainage bear bile powder group; 4 pioglitazone group; A: example 4 bile acid complex group A; B: example 4 bile acid complex group B; C: example 4 bile acid complex group C; D: example 4 bile acid complex group D), where the liver coefficients of the model group were increased but not significantly different from those of the normal group, and were decreased from those of the model group at 4 weeks; compared with the model group, the liver coefficient of the cholic acid complex A, C and D dose group in example 4 is reduced, wherein the reduction of the C and D dose group is most obvious and has no statistical significance.

4. Effect of cholic acid complexes on liver histopathology in NASH model mice

Serum ALT is normal and does not mean that there is no inflammatory injury to the liver tissue, nor is elevated ALT necessarily NASH. Liver biopsy is still the gold standard for diagnosing NASH to date. Carrying out HE staining on the liver tissues of the mice after 8 weeks of modeling, and observing the pathological change of the liver of the mice in the NASH model, wherein as shown in figure 12, the liver tissue capsule of a normal group is formed by elastic fiber-rich compact connective tissues with uniform thickness, the liver lobules are obviously divided and regularly arranged, the center of the liver lobules is a central vein, the periphery of the liver lobules is hepatic cells and hepatic blood sinuses which are approximately radially arranged, and the hepatic cells are round and full; the liver plates are regularly and tidily arranged, and liver sinuses are not obviously expanded or extruded; no obvious abnormality in the portal area between adjacent hepatic lobules; no significant inflammatory changes were seen. A great deal of hepatic cell steatosis is widely seen around the central vein in the tissue of the model group, and round vacuoles (1) with different sizes are seen in cytoplasm; connective tissue hyperplasia (2) is visible around a large number of central veins, and lymphocyte infiltration (3) is rare; a small amount of hepatocyte ballooning degeneration is seen locally, cell swelling, nuclear centering, cytosolic vacuolation (4).

According to a NASH semi-quantitative scoring system, the scoring of each group at 8 weeks of model building is shown in a table 2, the scoring of hepatic steatosis, lobular endophthalmitis and NAS total score of rats in a NASH model group are obviously higher than those in a normal group (P < 0.05P < -0.01; balloon-like changes were not significantly different. Wherein the total scores of the normal group NAS are 0 point, the total scores of the model group NAS are more than or equal to 4 points, the NASH can be definitely diagnosed, and the NASH modeling success is shown.

TABLE 2 NASH Pathology Scale after 8 weeks of modeling (n = 4)

After 8 weeks of administration, as shown in FIG. 13 (A: example 4 bile acid complex A group; B: example 4 bile acid complex B group; C: example 4 bile acid complex C group; D: example 4 bile acid complex D group), FIG. 14 (A: example 4 bile acid complex A group; B: example 4 bile acid complex B group; C: example 4 bile acid complex C group; D: example 4 bile acid complex D group), the model group showed massive steatosis in hepatocytes, round vacuoles of varying sizes visible in cytoplasm, small ballooning degeneration in hepatocytes, inflammatory cell multifocal infiltrations visible in and around lobules and veins, and few nuclear inclusions. All drugs had some improvement in steatosis, with the best improvement in the bile acid complex C dose group of example 4 (P < 0.05). The scores for each group are shown in table 3, according to the NASH semi-quantitative scoring system.

The hepatic steatosis, the intralobular inflammation score and the NAS total score of rats in the NASH model group are all obviously higher than those of a normal group (P <0.0001; balloon-like changes were not significantly different. Wherein example 4 bile acid complex C has a significant statistical difference in improved steatosis and NAS total score compared to the model group (P < 0.05P < -0.01; the total NAS component of the bear gall powder drainage group is obviously lower than that of the model group (P < 0.05). The effect of the pioglitazone group was not evident after 8 weeks of administration.

Table 3 NAS pathology score table after 8 weeks of dosing (n = 10)

Effect of 5 cholic acid Complex on liver lipid aggregation in NASH model mice

The oil red O fat staining method is one of the commonly used methods for displaying the fat content in tissues, and oil red O is a fat-soluble dye which can be highly dissolved in fat and can specifically stain neutral fats such as triglyceride in tissues.

The results of oil-red O staining of mouse liver tissue cryosections 8 weeks after molding showed (fig. 15) that there were a large number of red lipid droplets (black arrows) in the liver tissue and a large amount of triglycerides accumulated.

After 8 weeks of administration, as shown in FIG. 16 (A: example 4 bile acid complex group A; B: example 4 bile acid complex group B; C: example 4 bile acid complex group C; D: example 4 bile acid complex group D), liver tissues of the model group mice showed a large number of red lipid droplets distributed diffusely, compared with the normal group; compared with the model group, the liver lipid droplets of mice of each administration group are improved to a certain extent, wherein the B and C dose groups are improved most obviously.

6. Influence of cholic acid complex on serum biochemical indexes of NASH model mice

Serum biochemical indicators are one of the criteria commonly used in clinical assisted diagnosis of NASH. Alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), alkaline phosphatase (ALP) to characterize the extent of liver damage; direct Bilirubin (DBIL) and Total Bilirubin (TBIL) reflect hepatocyte metabolism; the blood lipids TC, TG and LDL are closely related to the lipid metabolism of the liver.

After 8 weeks of modeling, an enzyme labeling instrument is used, the content of TC, ALT and AST in serum is detected according to the specification of a biochemical kit, the result is shown in figure 17, compared with a normal group, the liver lipid metabolism and liver injury of a model group mouse are obvious (P is less than 0.0001), but the indexes of a certain mouse in the model group are obviously abnormal (ear tags 15 and 57), the mouse is judged to be a non-modeled mouse, and therefore the mouse is removed before a later-stage efficacy test.

After 4 weeks of administration, the serum contents of TC, ALT and AST were again measured, and the results are shown in FIG. 18 (A: group A of the cholic acid complex of example 4; group B of the cholic acid complex of example 4; group C: group C of the cholic acid complex of example 4; group D of the cholic acid complex of example 4; group 1Control 2model 3 drainage bear bile powder; group 4 pioglitazone), wherein the serum contents of TC in the mice of the normal group and the model group are obviously increased compared with the serum contents in the mice of the 4 weeks before, but no statistical difference exists between the serum contents of TC, ALT and AST; the TC content in the serum of each administration group is not obviously different from that of the model group. Compared with the normal group, the serum liver function index ALT and AST level of the model group mice is obviously increased (P is less than 0.05); compared with the model group, the cholic acid complex A, C and pioglitazone group in the example 4 can obviously reduce ALT and AST levels (P < 0.05); example 4 cholic acid complex B and drained bear bile powder group were able to significantly reduce AST levels (P < 0.05) without significant improvement of ALT.

After 8 weeks of administration, biochemical indicators of serum were measured using a fully automatic biochemical analyzer, and the results are shown in table 4.

TABLE 4 Biochemical index content level in serum 8 weeks after administration

In the aspect of liver injury indexes, compared with a normal group, the ALT and AST levels of the model group are increased to different degrees, but no statistical difference exists, and compared with the model group, the ALT and AST levels of each administration group are not obviously changed and have no statistical difference.

In the aspect of bile metabolism indexes, compared with a normal group, the serum ALP level of the model group mice is obviously increased (P < 0.05); the cholic acid complex dose groups showed a different reduction in ALP levels compared to the model, but were not statistically significant. The serum TBIL levels in the model group mice were significantly increased compared to the normal group (P < 0.05), and the TBIL levels were decreased to different degrees in each dose group of the bile acid complex and the positive drug group compared to the model group, wherein the bile acid complex C group was statistically different from the pioglitazone group in example 4 (P < 0.05P < -0.01. The model group had no significant change in DBIL compared to the normal group, but the example 4 bile acid complexes a, C, pioglitazone group all significantly reduced DBIL levels compared to the model group (P < 0.05P-were-0.01P-were-0.0001.

In terms of lipid metabolism index, the serum Total Cholesterol (TC) level of the model group mice is remarkably increased (P < 0.05) compared with that of the normal group, and the TC is not reduced in each administration group. The mean levels of TG and LDL in the serum of each administration group did not decrease, and there was no significant difference.

In the aspect of inflammation indexes, compared with a normal group, the IL-1 beta and TNF-alpha levels of mice in a model group are obviously increased (P < 0.0001); each dosing group had significantly reduced IL-1 β levels compared to the model group (P <0.0001; TNF- α levels were reduced in each administration group compared to the model group, with significant differences in each group except the bear gall powder-draining group (P < 0.0001P-coverall 0.0001. There was no difference between the respective administration groups.

7. Influence of cholic acid complex on biochemical indexes of liver of NASH model mouse

After 8 weeks of administration, biochemical index measurements of liver homogenate were performed using a fully automated biochemical analyzer, and the results are shown in table 5.

TABLE 5 Biochemical index content levels in liver 8 weeks after dosing

In the aspect of lipid metabolism, compared with a normal group, the liver tissues of the model group of mice have obviously increased levels of TC, TG and LDL (P < 0.0001); the TC levels were reduced in each of the dosing groups compared to the model group, with the significant differences between the bile acid complex C (P < 0.0001), D (P < 0.001) and the bear gall powder draining group (P < 0.001) of example 4; the TG level was reduced in each administration group compared to the model group, with the significant difference (P < 0.05) between the cholic acid complex C group and the bear bile powder-draining group of example 4; LDL levels were reduced in each of the administered groups compared to the model group, with the significant differences between the bile acid complex C (P < 0.0001), D (P < 0.001) and bear bile powder-draining group (P < 0.001) of example 4. It is worth mentioning that in the lipid metabolism index, the decrease of TC and LDL-C in the cholic acid complex C dose group of example 4 is significantly better than that in the pioglitazone group (P < 0.05P < -0.01; wherein the dose group of cholic acid complex C in example 4 has a significantly better LDL-C lowering effect than the dose group of cholic acid complex A in example 4 (P < 0.05).

In terms of inflammation, both TNF-alpha (P < 0.0001) and transforming growth factor beta (TGF-beta) (P < 0.001) levels were significantly elevated in the model group compared to the normal group. TNF-alpha levels were reduced in each of the groups compared to the model group, and were statistically different in each of the other groups except for the cholic acid complex B group of example 4 (P < 0.05). TGF-beta levels were reduced in each of the administered groups compared to the model group, with significant differences (P < 0.01) between the bile acid complex A, B and the drained bear bile powder group of example 4. There was no tendency or difference in the comparison of the control group, model group and model group, and the administration group for inflammatory factors such as IL-1. Beta.

The experiment results show that the cholic acid compound provided by the invention or the cholic acid compound prepared by applying the preparation method provided by the invention can effectively improve the liver echo of a model mouse, relieve the pathological damage of the liver, reduce the NAS score, reduce the lipid deposition of the liver and relieve the inflammatory reaction of serum and the liver, thereby having a remarkable treatment effect on the non-alcoholic steatohepatitis. The experimental result provides data support for clinical medication of the cholic acid compound, and lays a foundation for further research and development of the cholic acid compound.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. A bile acid complex, comprising: 27-45 parts of tauroursodeoxycholic acid and 15-25 parts of taurochenodeoxycholic acid; and at least one of taurocholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, and taurolicholic acid.

2. A bile acid complex as claimed in claim 1 comprising: 30-40 parts of tauroursodeoxycholic acid and 16-22 parts of taurochenodeoxycholic acid; and at least one of 7 to 20 parts of taurocholic acid, 0.1 to 1 part of cholic acid, 0.05 to 1 part of ursodeoxycholic acid, 0.05 to 0.5 part of chenodeoxycholic acid, 0.05 to 0.5 part of deoxycholic acid and 0.05 to 0.5 part of taurocholic acid.

3. A bile acid complex as claimed in claim 2 comprising: 30-37 parts of tauroursodeoxycholic acid and 17-21 parts of taurochenodeoxycholic acid; and at least one of 8 to 15 parts of taurocholic acid, 0.3 to 0.6 part of cholic acid, 0.09 to 0.4 part of ursodeoxycholic acid, 0.1 to 0.3 part of chenodeoxycholic acid, 0.08 to 0.2 part of deoxycholic acid and 0.08 to 0.2 part of taurocholic acid.

4. Use of a bile acid complex as claimed in any of claims 1 to 3 for the manufacture of a medicament for the treatment of non-alcoholic fatty liver disease.

5. The use according to claim 4, wherein the non-alcoholic fatty liver disease comprises non-alcoholic steatohepatitis.

6. A method for preparing a bile acid complex according to any one of claims 1-3, characterised in that the method comprises: uniformly mixing tauroursodeoxycholic acid and taurochenodeoxycholic acid, and at least one of taurocholic acid, cholic acid, ursodesoxycholic acid, chenodeoxycholic acid, deoxycholic acid and taurocholic acid to obtain the cholic acid compound.

7. A method of preparing a bile acid complex as claimed in any of claims 1 to 3, which method comprises: performing biotransformation on the poultry bile or the poultry bile powder by using hydroxysteroid dehydrogenase, and then performing alcohol extraction, concentration and drying to prepare the cholic acid compound;

wherein the hydroxysteroid dehydrogenase comprises a 7 alpha-hydroxysteroid dehydrogenase and/or a 7 beta-hydroxysteroid dehydrogenase.

8. A medicament for the treatment of non-alcoholic fatty liver disease comprising a bile acid complex of any of claims 1 to 3 and a pharmaceutically acceptable excipient.

9. The medicament of claim 8, wherein the dosage form of the medicament comprises an oral preparation or an injection preparation;

preferably, the effective administration dose of the medicament is 39-312 mg/kg, preferably 156mg/kg;

preferably, the non-alcoholic fatty liver disease comprises non-alcoholic steatohepatitis.

10. Use of the medicament for the treatment of nonalcoholic fatty liver disease according to claim 8 or 9 for the preparation of a product for the treatment of nonalcoholic fatty liver disease;

preferably, the non-alcoholic fatty liver disease includes non-alcoholic steatohepatitis.

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