Cholic acid complex, its preparation method and application

Abstract

The present invention provides a cholic acid complex, a preparation method and an application thereof, relating to the technical field of medicine. The cholic acid complex provided by the present invention can effectively improve the liver echo of model mice, reduce liver pathological damage and NAS score, reduce liver lipid deposition, and alleviate the inflammatory responses in serum and liver, thereby having a significant therapeutic effect on non-alcoholic fatty liver disease. By selecting specific types of cholic acid in combination with specific dosages of cholic acid, the present invention overcomes the technical problem that a single compound cannot play a comprehensive role in treating complex diseases, realizes the effective treatment of non-alcoholic fatty liver disease, provides data support for clinical medication, and also lays a foundation for its further research and development.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and in particular to a bile acid complex and its preparation method and application. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a group of diseases characterized by fatty degeneration and fat accumulation in hepatic parenchymal cells. Currently, 25% of the world's population suffers from NAFLD. Non-alcoholic steatohepatitis (NASH) is a subtype of NAFLD, which is characterized by inflammatory damage of hepatocytes and is an important rate-limiting link in the transformation process from simple fatty liver to liver cirrhosis and liver cancer. However, currently, the treatment methods for NASH in the world are very limited, and there are even no treatment drugs. Multiple drugs for treating NASH have failed in clinical trials, including obeticholic acid (OCA), a farnesoid X receptor (FXR) agonist that was given great hope. Analyzing the reasons for the failure of these drugs, most of them are that a single compound cannot play a comprehensive role in treating complex diseases and often cause some adverse reactions (such as skin itching, etc.).

[0003] In view of this, the present invention is specifically proposed. Summary of the Invention

[0004] One of the purposes of the present invention is to provide a bile acid complex to solve at least one of the technical problems existing in the prior art.

[0005] Another purpose of the present invention is to provide the application of the above-mentioned bile acid complex.

[0006] Another purpose of the present invention is to provide the preparation method of the above-mentioned bile acid complex.

[0007] Another purpose of the present invention is to provide a drug containing the above-mentioned bile acid complex.

[0008] Another purpose of the present invention is to provide the application of the above-mentioned drug.

[0009] In order to achieve the above purposes of the present invention, the following technical solutions are specifically adopted:

[0010] The present invention provides a bile acid complex, including: 27-45 parts of tauroursodeoxycholic acid and 15-25 parts of taurochenodeoxycholic acid; and at least one of taurine cholate, cholate, ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, and tauroursodeoxycholic acid.

[0011] Further, the bile acid complex includes: 30-40 parts of tauroursodeoxycholic acid and 16-22 parts of taurochenodeoxycholic acid; and at least one of 7-20 parts of taurocholic acid, 0.1-1 part of cholic acid, 0.05-1 part of ursodeoxycholic acid, 0.05-0.5 part of chenodeoxycholic acid, 0.05-0.5 part of deoxycholic acid, and 0.05-0.5 part of tauroursodeoxycholic acid.

[0012] Further, the bile acid complex includes: 30-37 parts of tauroursodeoxycholic acid and 17-21 parts of taurochenodeoxycholic acid; and at least one of 8-15 parts of taurocholic acid, 0.3-0.6 part of cholic acid, 0.09-0.4 part of ursodeoxycholic acid, 0.1-0.3 part of chenodeoxycholic acid, 0.08-0.2 part of deoxycholic acid, and 0.08-0.2 part of tauroursodeoxycholic acid.

[0013] The present invention also provides the use of the above-mentioned bile acid complex in the preparation of a drug for treating non-alcoholic fatty liver disease.

[0014] Further, the non-alcoholic fatty liver disease includes non-alcoholic steatohepatitis.

[0015] The present invention also provides a preparation method of the above-mentioned bile acid complex, and the preparation method includes: mixing the formula amounts of tauroursodeoxycholic acid and taurochenodeoxycholic acid, and at least one of taurocholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, and tauroursodeoxycholic acid evenly to obtain the bile acid complex.

[0016] The present invention also provides another preparation method of the above-mentioned bile acid complex, and the preparation method includes: subjecting avian bile or avian bile powder to biotransformation by hydroxysteroid dehydrogenase, and then performing alcohol extraction, concentration and drying to prepare the bile acid complex;

[0017] Wherein, the hydroxysteroid dehydrogenase includes 7α-hydroxysteroid dehydrogenase and / or 7β-hydroxysteroid dehydrogenase.

[0018] In addition, the present invention also provides a drug for treating non-alcoholic fatty liver disease, which includes the above-mentioned bile acid complex and pharmaceutically acceptable excipients.

[0019] Further, the dosage form of the drug includes an oral preparation or an injection preparation.

[0020] Further, the effective dosage of the drug is 39-312 mg / kg, preferably 156 mg / kg.

[0021] Further, the non-alcoholic fatty liver disease includes non-alcoholic steatohepatitis.

[0022] The present invention also provides an application of the above-mentioned drug for treating non-alcoholic fatty liver disease in the preparation of a product for treating non-alcoholic fatty liver disease;

[0023] Preferably, the non-alcoholic fatty liver disease includes non-alcoholic steatohepatitis.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The inventors of the present invention have found through a large number of experiments that the bile acid complex provided by the present invention can effectively improve the liver echo of model mice, reduce liver pathological damage and NAS score, reduce liver lipid deposition, and alleviate the inflammatory response in serum and liver, thus having a significant therapeutic effect on non-alcoholic fatty liver disease. By selecting specific types of bile acids and specific dosages of bile acids in combination, the present invention overcomes the technical problem that a single compound cannot play a comprehensive role in treating complex diseases, realizes the effective treatment of non-alcoholic fatty liver disease, provides data support for clinical medication, and also lays a foundation for its further research and development.

[0026] The drug for treating non-alcoholic fatty liver disease provided by the present invention has the bile acid complex provided by the present invention as its active ingredient. Therefore, based on the beneficial effects of the bile acid complex, the drug can also play an obvious preventive and / or therapeutic role in non-alcoholic fatty liver disease, and is safe, non-toxic, and has few side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0028] Figure 1 It is a result graph of the body weight change trend during 8 weeks of modeling provided by the experimental examples of the present invention;

[0029] Figure 2 It is a result graph of the body weight change trend during 8 weeks of drug administration provided by the experimental examples of the present invention;

[0030] Figure 3 It is a liver ultrasound image during 8 weeks of modeling provided by the experimental examples of the present invention;

[0031] Figure 4 It is a comparison graph of liver echo by abdominal ultrasound 4 weeks after drug administration provided by the experimental examples of the present invention;

[0032] Figure 5 It is a statistical graph of liver echo score by abdominal ultrasound 4 weeks after drug administration provided by the experimental examples of the present invention;

[0033] Figure 6 Abdominal ultrasound liver echo comparison chart after 8 weeks of drug administration provided by the experimental example of the present invention;

[0034] Figure 7 Abdominal ultrasound liver echo score statistical chart after 8 weeks of drug administration provided by the experimental example of the present invention;

[0035] Figure 8 Liver gross morphology chart after 8 weeks of modeling provided by the experimental example of the present invention;

[0036] Figure 9 Liver coefficient result chart after 8 weeks of modeling provided by the experimental example of the present invention;

[0037] Figure 10 Liver gross morphology chart after 8 weeks of drug administration provided by the experimental example of the present invention;

[0038] Figure 11 Liver coefficient result chart after 8 weeks of drug administration provided by the experimental example of the present invention;

[0039] Figure 12 Mouse liver HE pathological staining chart (×200) after 8 weeks of modeling provided by the experimental example of the present invention;

[0040] Figure 13 Mouse liver HE pathological staining chart (×200) after 8 weeks of drug administration provided by the experimental example of the present invention;

[0041] Figure 14 Mouse liver HE pathological staining chart (×400) after 8 weeks of drug administration provided by the experimental example of the present invention;

[0042] Figure 15 Mouse liver oil red O staining chart after 8 weeks of modeling provided by the experimental example of the present invention;

[0043] Figure 16 Mouse liver oil red O staining chart (×200) after 8 weeks of drug administration provided by the experimental example of the present invention;

[0044] Figure 17 Results chart of TC, ALT, and AST content levels in mouse serum after 8 weeks of modeling provided by the experimental example of the present invention;

[0045] Figure 18 Results chart of TC, ALT, and AST content levels in mouse serum after 4 weeks of drug administration provided by the experimental example of the present invention. Detailed implementation method

[0046] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings as commonly understood by one of ordinary skill in the art. The meanings and ranges of the terms should be clear. However, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or extrinsic definition. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms is non-restrictive.

[0047] Generally, the nomenclature and the techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein are those that are well known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods that are well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art or as described herein. The nomenclature, as well as the laboratory procedures and techniques used in connection with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry as described herein, are those that are well known and commonly used in the art.

[0048] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by one of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0049] According to one aspect of the present invention, there is provided a cholic acid complex, comprising: tauroursodeoxycholic acid and taurochenodeoxycholic acid; and at least one of taurine cholate, cholate, ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, and tauroursodeoxycholic acid.

[0050] It should be noted that the main active ingredients of the cholic acid complex provided by the present invention are tauroursodeoxycholic acid and tauroursodeoxycholic acid. In addition to the main active ingredients, it also includes at least one of taurocholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, and tauroursodeoxycholic acid as a combined active ingredient. For example, taurocholic acid, or cholic acid, or ursodeoxycholic acid, or chenodeoxycholic acid, or deoxycholic acid, or tauroursodeoxycholic acid can be selected; the combination of taurocholic acid and cholic acid can also be selected, or the combination of deoxycholic acid and tauroursodeoxycholic acid can be selected, or the combination of taurocholic acid and ursodeoxycholic acid can be selected, or other combinations composed of two combined active ingredients; the combination of taurocholic acid, cholic acid, and ursodeoxycholic acid can also be selected, or the combination of ursodeoxycholic acid, chenodeoxycholic acid, and deoxycholic acid can be selected, or the combination of taurocholic acid, deoxycholic acid, and tauroursodeoxycholic acid can be selected, or other combinations composed of three combined active ingredients; the combination of taurocholic acid, cholic acid, ursodeoxycholic acid, and chenodeoxycholic acid can also be selected, or the combination of ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, and tauroursodeoxycholic acid can be selected, or other combinations composed of four combined active ingredients; the combination of taurocholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, and deoxycholic acid can also be selected, or other combinations composed of five combined active ingredients; in addition, taurocholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, and tauroursodeoxycholic acid can all be selected as combined active ingredients.

[0051] Among them, the content of tauroursodeoxycholic acid (TUDCA) is 27-45 parts, for example, it can be, but is not limited to, 27 parts, 30 parts, 32 parts, 35 parts, 38 parts, 40 parts, 42 parts, or 45 parts; the content of tauroursodeoxycholic acid (TCDCA) is 15-25 parts, for example, it can be, but is not limited to, 15 parts, 18 parts, 20 parts, 22 parts, or 25 parts. In addition, the content of the above main active ingredients can also be limited by the dosage ratio. For example, the content ratio of tauroursodeoxycholic acid to tauroursodeoxycholic acid can be 1.3-2.0:1.

[0052] The inventor of the present invention found through a large number of experiments that the cholic acid complex provided by the present invention can effectively improve the liver echo of model mice, reduce liver pathological damage and NAS score, reduce liver lipid deposition, and reduce the inflammatory response in serum and liver, thus having a significant therapeutic effect on non-alcoholic fatty liver disease. By selecting specific cholic acid types in combination with specific cholic acid dosages, the present invention overcomes the technical problem that a single compound cannot play a comprehensive role in treating complex diseases, realizes the effective treatment of non-alcoholic fatty liver disease, provides data support for clinical medication, and also lays a foundation for its further research and development.

[0053] By adjusting and optimizing the dosage of the active ingredients of the cholate complex provided by the present invention, it is preferred that the cholate complex includes: 30-40 parts of tauroursodeoxycholic acid and 16-22 parts of taurochenodeoxycholic acid; and at least one of 7-20 parts of taurine cholate, 0.1-1 part of cholate, 0.05-1 part of ursodeoxycholic acid, 0.05-0.5 part of chenodeoxycholic acid, 0.05-0.5 part of deoxycholic acid, and 0.05-0.5 part of tauroursodeoxycholic acid. The optimized cholate complex has a more significant therapeutic effect on non-alcoholic fatty liver disease.

[0054] For the optional co-active ingredients, the dosage of each component, for example, the content of taurine cholate (TCA) can be, but is not limited to, 7 parts, 8 parts, 10 parts, 12 parts, 15 parts, 18 parts, or 20 parts; the content of cholate (CA) can be, but is not limited to, 0.1 part, 0.2 part, 0.5 part, 0.8 part, or 1 part; the content of ursodeoxycholic acid (UDCA) can be, but is not limited to, 0.05 part, 0.08 part, 0.1 part, 0.2 part, 0.3 part, 0.4 part, 0.5 part, 0.6 part, 0.7 part, 0.8 part, 0.9 part, or 1 part; the content of chenodeoxycholic acid (CDCA) can be, but is not limited to, 0.05 part, 0.08 part, 0.1 part, 0.2 part, or 0.5 part; the content of deoxycholic acid (DCA) can be, but is not limited to, 0.05 part, 0.08 part, 0.1 part, 0.2 part, 0.3 part, 0.4 part, or 0.5 part; the content of tauroursodeoxycholic acid (TLCA) can be, but is not limited to, 0.05 part, 0.08 part, 0.1 part, 0.2 part, 0.3 part, 0.4 part, or 0.5 part.

[0055] On this basis, the present invention further optimizes the formulation of the cholate complex. The cholate complex as a preferred embodiment includes: 30-37 parts of tauroursodeoxycholic acid and 17-21 parts of taurochenodeoxycholic acid; and at least one of 8-15 parts of taurine cholate, 0.3-0.6 part of cholate, 0.09-0.4 part of ursodeoxycholic acid, 0.1-0.3 part of chenodeoxycholic acid, 0.08-0.2 part of deoxycholic acid, and 0.08-0.2 part of tauroursodeoxycholic acid.

[0056] According to the second aspect of the present invention, there is provided the use of the above-mentioned cholate complex in the preparation of a drug for treating non-alcoholic fatty liver disease. In particular, the cholate complex provided by the present invention has a particularly significant effect in the treatment of non-alcoholic steatohepatitis.

[0057] According to the third aspect of the present invention, there is provided a method for preparing the above-mentioned cholate complex, including: mixing evenly the formula amounts of tauroursodeoxycholic acid and taurochenodeoxycholic acid, and at least one of taurine cholate, cholate, ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, and tauroursodeoxycholic acid to obtain the cholate complex.

[0058] The preparation method of the cholic acid complex provided by the present invention has a simple process, is convenient to operate, and does not require specific technicians or expensive equipment, which can effectively save costs.

[0059] In addition, the present invention also provides another preparation method of the above cholic acid complex, including:

[0060] After the biological transformation of avian bile or avian bile powder by hydroxysteroid dehydrogenase, the cholic acid complex is prepared by alcohol extraction, concentration and drying;

[0061] Among them, the hydroxysteroid dehydrogenase includes 7α-hydroxysteroid dehydrogenase and / or 7β-hydroxysteroid dehydrogenase.

[0062] The preparation method of the cholic acid complex provided by the present invention has a simple process and is convenient to operate. Based on the characteristic that the alcohol solvent can be recycled, it also has the advantages of environmental protection and can effectively save costs.

[0063] When using this method to prepare the cholic acid complex, the cholic acid complex further includes at least one of cholesterol, bile pigment, amino acid, polypeptide, protein, and metal element, preferably including all of cholesterol, bile pigment, amino acid, polypeptide, protein, and metal element.

[0064] When the cholic acid complex includes the above substances, preferably, the mass percentages of tauroursodeoxycholic acid, taurochenodeoxycholic acid, taurocholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, and tauroursodeoxycholic acid in the cholic acid complex are 60% - 85%, for example, it can be, but is not limited to, 60%, 65%, 70%, 75%, 80%, or 85%.

[0065] In addition, according to the above application, the present invention also provides a drug for treating non-alcoholic fatty liver disease, and the drug includes the above cholic acid complex and pharmaceutically acceptable excipients.

[0066] The drug for treating non-alcoholic fatty liver disease provided by the present invention has the active ingredient as the cholic acid complex provided by the present invention. Therefore, based on the beneficial effects of the cholic acid complex, the drug can also play an obvious preventive and / or therapeutic role on non-alcoholic fatty liver disease, and at the same time, it is safe, non-toxic, and has few side effects.

[0067] Among them, pharmaceutically acceptable excipients refer to excipients and additives used in the production of drugs and the preparation of prescriptions. They refer to substances that, except for the active ingredients, have been reasonably evaluated in terms of safety and are included in pharmaceutical preparations. The same pharmaceutical excipient can be used in pharmaceutical preparations for different administration routes and has different functions and uses. The pharmaceutically acceptable excipients added to the drugs provided by the present invention can play the roles of shaping, acting as a carrier, or improving stability. In addition, they also have important functions such as solubilization, cosolubilization, or sustained and controlled release.

[0068] Typical but non-limiting pharmaceutically acceptable excipients include: one or more of solvents, propellants, solubilizers, cosolubilizers, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, or release retardants.

[0069] In a preferred embodiment, the dosage form of the drug includes oral preparations or injection preparations.

[0070] When administered orally, the above drugs can be made into any orally acceptable preparation form, for example, but not limited to, tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions, or oral emulsions.

[0071] Among them, the carriers used for tablets generally include lactose and corn starch. In addition, lubricants such as magnesium stearate can also be added. The diluents used for capsules generally include lactose and dry corn starch. Oral suspensions usually mix the active ingredient with suitable emulsifiers and suspending agents.

[0072] Optionally, some sweeteners, fragrances, or colorants can also be added to the above oral preparation forms.

[0073] When administered by injection, the above drugs can be made into any injection-acceptable preparation form, for example, but not limited to, injection solutions or lyophilized powders.

[0074] Among them, the carriers and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterilized non-volatile oils can also be used as solvents or suspension media, such as monoglycerides or diglycerides.

[0075] In a preferred embodiment, the effective administration dose of the drug is 39-312 mg / kg, for example, it can be, but is not limited to, 39 mg / kg, 78 mg / kg, 156 mg / kg or 312 mg / kg. When the administration dose is 156 mg / kg, on the basis of ensuring the curative effect, the dosage can be effectively controlled.

[0076] The present invention also provides an application of the above-mentioned drug for treating non-alcoholic fatty liver disease in the preparation of a product for treating non-alcoholic fatty liver disease;

[0077] Preferably, the non-alcoholic fatty liver disease includes non-alcoholic steatohepatitis.

[0078] The present invention will be further described below through examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0079] Example 1

[0080] This example provides a bile acid complex, including: 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 taurolithocholic acid.

[0081] Example 2

[0082] This example provides a bile acid complex, including: 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 taurolithocholic acid.

[0083] Example 3

[0084] This example provides a bile acid complex, including: 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 taurolithocholic acid.

[0085] Example 4

[0086] This example provides a bile acid complex, including: 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.

[0087] Example 5

[0088] This example provides a cholic 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.

[0089] Example 6

[0090] This example provides a cholic 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.

[0091] Example 7

[0092] This example provides a cholic 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 taurolithocholic acid.

[0093] Example 8

[0094] This example provides a cholic acid complex, comprising: 32 parts of tauroursodeoxycholic acid, 18 parts of taurochenodeoxycholic acid, and 11 parts of taurocholic acid.

[0095] Example 9

[0096] This example provides a cholic acid complex, comprising: 32 parts of tauroursodeoxycholic acid, 18 parts of taurochenodeoxycholic acid, 0.2 part of ursodeoxycholic acid, and 0.2 part of chenodeoxycholic acid.

[0097] Example 10

[0098] This example 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 taurolithocholic acid.

[0099] Example 11

[0100] This example provides a cholic 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.

[0101] Example 12

[0102] This example provides a cholic acid complex, which is prepared by the following method:

[0103] Mix 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase evenly with chicken bile extract. Taking the enzyme cells as an example, the ratio of 7α enzyme cells to 7β enzyme cells is 1:1 - 1:5. Adjust the pH to 6.5 - 9, react overnight at room temperature, add ethanol to a final concentration of 80 ± 5%, precipitate with alcohol at a temperature below 16°C for more than 4 hours, filter or centrifuge to collect the supernatant, and concentrate and dry it into powder under vacuum at a temperature below 80°C.

[0104] Preparation of chicken bile: Remove impurities from chicken bile, filter, take the juice, add ethanol to a final concentration of 80 ± 5%, precipitate with alcohol at a temperature below 16°C for more than 4 hours, filter or centrifuge to collect the supernatant, and concentrate; Add ethyl acetate, a weakly polar solvent, to the concentrated extract and extract it 3 times. The volume ratio of the two is 1:2. Collect the aqueous phase and concentrate it into chicken bile extract for standby.

[0105] Comparative Example 1

[0106] This comparative example provides a bile acid complex, including: 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 tauroursodeoxycholic acid.

[0107] Comparative Example 2

[0108] This comparative example provides a bile acid complex. The difference from Example 4 is that ursodeoxycholic acid is replaced with lithocholic acid.

[0109] Comparative Example 3

[0110] This comparative example provides a bile acid complex, which is composed 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.

[0111] The preparation methods of the bile acid complexes in the above Examples 1 - 11 and Comparative Examples 1 - 3 are all obtained by mixing the components in the formula amounts evenly.

[0112] Experimental Example

[0113] Main instruments and reagents:

[0114]

[0115]

[0116]

[0117] I. Model establishment, animal grouping, and drug administration

[0118] Healthy male SPF C57BL / 6 mice, 8 - 10 weeks old, were purchased from the National Institutes for Food and Drug Control (Daxing), with the license number: SCXK(Beijing)2017 - 0005. After being accepted by the receiving personnel, the animals were raised in the animal house of the Institute of Basic Theory of Traditional Chinese Medicine, China Academy of Chinese Medical Sciences, with the license number: SYXK(Beijing)2021 - 0017. 4 - 5 mice were housed in each cage, and the relative humidity range in the animal house was 50 - 60%; the temperature was 22°C to 25°C, and the light - dark cycle was simulated with 12h / 12h light - dark alternation.

[0119] With normal diet and water, after 1 - week adaptive feeding, except for the normal group, the mice were fed a Western diet by high - fat feed and high - sugar drinking water (containing 21.1% fat, 41% sucrose, and 1.25% cholesterol and a high - sugar solution (23.1g / L d - fructose and 18.9g / L d - glucose)) for 16 weeks. In the first 8 weeks, carbon tetrachloride (0.2μL (0.32μg / g)) was intraperitoneally injected once a week. After successful modeling, the mice were randomly divided into groups: normal group, model group, each dose group of the bile acid complex provided in Example 4 (39mg / kg; 78mg / kg; 156mg / kg; 312mg / kg), groups of Examples 1 - 3, 5 - 12 and Comparative Examples 1 - 3 (156mg / kg), the control group of drained bear bile powder (78mg / kg), and the positive drug pioglitazone group (30mg / kg). The specific grouping and dosing doses are shown in Table 1 below. The normal group and the model group were given CMC - Na as a control, and each dosing group was continuously dosed for 8 weeks.

[0120] Table 1 Dosing Dose Table

[0121]

[0122]

[0123] Index Detection:

[0124] 1. General Index

[0125] Every day, carefully observe and record the mental state, fur changes, activity status, water intake and food intake of the mice in each group; and measure the body weight of each mouse every week and record the blood glucose every month.

[0126] 2. Gross Liver Index

[0127] After 8 weeks of modeling and 8 weeks of drug administration, the mice were weighed, sacrificed after blood collection from the eyeballs, the abdominal cavity was quickly dissected, the livers of the mice were removed, placed on a petri dish and weighed, and the liver coefficient (the percentage of liver weight to total body weight) was calculated; after weighing, the residual blood was removed with physiological saline, and after the water was blotted dry with filter paper, macroscopic observation and photography of the liver were performed. The middle lobe of the liver was made into paraffin sections; the papillary lobe was made into frozen sections; the remaining liver tissues were quickly frozen in liquid nitrogen and stored at -80 °C.

[0128] 3. HE staining

[0129] After 8 weeks of modeling and 8 weeks of drug administration, HE staining and NAFLD Activity Score (NAS) scoring were performed on the livers of the mice.

[0130] 1. Dewaxing and hydrating paraffin sections: The sections were successively placed in xylene I for 20 min - xylene II for 20 min - absolute ethanol I for 5 min - absolute ethanol II for 5 min - 75% alcohol for 5 min, and washed with tap water.

[0131] 2. Hematoxylin staining: The sections were stained with hematoxylin solution for 3 - 5 min, washed with tap water, differentiated with the differentiating solution, washed with tap water, blued with the bluing solution, and rinsed with running water.

[0132] 3. Eosin staining: The sections were dehydrated in gradient alcohols of 85% and 95% for 5 min each, and stained with eosin solution for 5 min.

[0133] 4. Dehydration and mounting: The sections were successively placed in absolute ethanol I for 5 min - absolute ethanol II for 5 min - absolute ethanol III for 5 min - xylene I for 5 min - xylene II for 5 min for clearing, and mounted with neutral balsam.

[0134] 5. Microscopic examination and image acquisition.

[0135] 6. Semi - quantitative scoring criteria (NAS score)

[0136] NAS score (0 - 8 points): ① Hepatic steatosis: 0 points (<5%); 1 point (5% - 33%); 2 points (34% - 66%); 3 points (>66%). ② Lobular inflammation (counting necrosis foci under 20 - fold magnification): 0 points, none; 1 point (<2); 2 points (2 - 4); 3 points (>4). ③ Ballooning degeneration of hepatocytes: 0 points, none; 1 point, rare; 2 points, common.

[0137] NAS is a semi - quantitative scoring system rather than a diagnostic procedure. NAS < 3 points can exclude NASH, NAS > 4 points can diagnose NASH, and those between the two are possible NASH. It is stipulated that those with hepatic steatosis > 33% without lobular inflammation, ballooning degeneration, and fibrosis are NAFL, and those with steatosis less than this degree are only called hepatocyte steatosis.

[0138] 4. Oil Red O Staining

[0139] The livers of mice were subjected to Oil Red O staining at 8 weeks after modeling and 8 weeks after drug administration, respectively.

[0140] 1. Fixation of fresh frozen sections: Rewarm and dry the frozen sections, fix them in the fixing solution for 15 min, wash with tap water, and air dry.

[0141] 2. Oil Red staining: Immerse the sections in the Oil Red staining solution for 8 - 10 min (cover to avoid light).

[0142] 3. Background differentiation: Take out the sections, immerse them in two tanks of 60% isopropanol for differentiation successively after staying for 3 s, for 3 s and 5 s respectively. Immerse the sections in two tanks of pure water for washing successively, for 10 s each.

[0143] 4. Hematoxylin staining: Take out the sections, immerse them in hematoxylin for counterstaining for 3 - 5 min after staying for 3 s, wash with three tanks of pure water, for 5 s, 10 s, and 30 s respectively. Differentiate with the differentiating solution (using 60% alcohol as the solvent) for 2 - 8 s, wash with two tanks of distilled water for 10 s each, blue with the bluing solution for 1 s, gently immerse the sections in two tanks of tap water for washing, for 5 s and 10 s respectively, and examine the staining effect under the microscope.

[0144] 5. Sealing the sections: Seal the sections with glycerin gelatin mounting medium.

[0145] 6. Microscopic examination and image acquisition and analysis.

[0146] 5. Ultrasonic detection of mouse liver

[0147] Liver ultrasonic detection was performed at 8 weeks after modeling (n = 4), 4 weeks after drug administration (n = 6), and 8 weeks after drug administration (n = 6) respectively. Overnight fasting was performed before the detection. The next day, after anesthesia by intraperitoneal injection of tribromoethanol, depilate the upper abdomen 3 cm below the xiphoid process with depilatory cream, fully expose the abdomen, fix in the supine position, apply sufficient coupling agent to make the probe fully contact with the abdominal wall, use a Vevo2100 small animal ultrasonic instrument for abdominal liver ultrasonic examination, use an MS400 probe, the probe frequency is 30 MHZ, and intercept the liver sections under B Model (select three landmark sections) for gray value evaluation under the same conditions (fix the same depth, width, and gain value).

[0148] Randomly selected mice from each group were subjected to liver ultrasonic detection, and the liver injury status was evaluated by comparing the echo intensity between the liver and the kidney. Higher liver echo than kidney echo indicates severe liver injury (-1 point); slightly higher liver echo than kidney echo indicates mild liver injury (+1 point); similar liver echo to kidney echo or weaker liver echo than kidney echo indicates no obvious liver injury (+2 points).

[0149] 6. Detection of serum biochemical indexes

[0150] At 8 weeks (n = 4) after modeling, 4 weeks (n = 6) of administration, and 8 weeks (n = 6) of administration, orbital blood was collected from mice (about 100 μL). After standing at room temperature for 4 h, the upper serum was aspirated after centrifugation at 4 °C and 3500 rpm for 15 min. According to the reagent instruction manual, the liver function indexes: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), direct bilirubin (DBIL), total bilirubin (TBIL); blood lipids TC, TG, HDL, LDL; inflammatory factors interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α) were detected respectively.

[0151] 7. Detection of liver biochemical indexes

[0152] At 8 weeks of administration (n = 6), the livers of mice were taken, homogenates were prepared, and a Hitachi 7600 automatic biochemical analyzer was used to detect the biochemical indexes inflammatory factors TNF-α, IL-1β; fibrosis-related index TGF-β; lipid-related indexes TC, TG, LDL-C.

[0153] 8. Statistical analysis

[0154] Data calculation is expressed as Using GraphPad Prism 8 software, one-way analysis of variance (ANOVA) was performed on the data, and the Sidak test was used for post hoc test; when P < 0.05, significant differences between the two groups were determined.

[0155] II. Experimental results

[0156] 1. Effects of bile acid complex on the animal state and body weight of NASH model mice

[0157] The body weight of mice at 8 weeks after modeling was as Figure 1 shown. The body weight of the model group decreased slightly compared with the normal group, but there was no significant difference.

[0158] The body weight of mice after grouping at 8 weeks of administration was as Figure 2(A: Group A of cholic acid complex in Example 4; B: Group B of cholic acid complex in Example 4; C: Group C of cholic acid complex in Example 4; D: Group D of cholic acid complex in Example 4). Compared with the normal group, the body weight of the mice in the model group decreased significantly at 1 week after administration (the 9th week of the experiment) (P<0.05), but there was no significant difference compared with the normal group from 2 to 8 weeks after administration. Compared with the model group, the body weights of the mice in each dose group of cholic acid complex and the group of drained bear bile powder decreased to varying degrees at 1 week after administration. Among them, the body weights of Group C and Group D of the cholic acid complex in Example 4 decreased significantly (P<0.05; P<0.01), and there were significant statistical differences. The body weight of Group D of the cholic acid complex in Example 4 decreased significantly at 2 weeks after administration (P<0.05). The body weights of Group C and Group D decreased significantly at the 4th week after administration (P<0.01; P<0.05). The body weights of the mice in the positive drug pioglitazone group increased to varying degrees compared with the model group at the 1st, 2nd, 3rd, 5th, and 7th weeks after administration, but there was no statistical difference. However, it decreased at the 4th, 6th, and 8th weeks, which may be related to the anesthesia of the animals during ultrasonic detection at the corresponding time points.

[0159] 2. Effects of cholic acid complex on liver ultrasonic indexes of NASH model mice

[0160] After 8 weeks of modeling, 4 mice were randomly selected from the normal group and the model group for small animal liver ultrasonic detection, and three representative fixed sections of the liver were intercepted in the Bmodel mode. As Figure 3 shown, compared with the normal group, the liver echo in the model group showed diffuse fine dot-like enhancement.

[0161] After 4 weeks of administration, the results were as Figure 4 (A: Group A of cholic acid complex in Example 4; B: Group B of cholic acid complex in Example 4; C: Group C of cholic acid complex in Example 4; D: Group D of cholic acid complex in Example 4), Figure 5 (1Control; 2Model; 3Group of drained bear bile powder; 4Pioglitazone group; A: Group A of cholic acid complex in Example 4; B: Group B of cholic acid complex in Example 4; C: Group C of cholic acid complex in Example 4; D: Group D of cholic acid complex in Example 4) shown: Each administration group had a certain effect on reducing abnormal liver echo. It could be seen from the comparison intensity score of liver and kidney ultrasonic echoes that the positive drug (pioglitazone) had the best efficacy, with a significant difference compared with the model group (P<0.01). Secondly, each administration group had varying degrees of improvement. Among them, the dose groups of Group D and Group C of the cholic acid complex in Example 4 were more obvious, but there was no statistical difference.

[0162] After 8 weeks of administration, as Figure 6 (A: Group A of cholic acid complex in Example 4; B: Group B of cholic acid complex in Example 4; C: Group C of cholic acid complex in Example 4; D: Group D of cholic acid complex in Example 4), Figure 7(1 Control; 2 Model; 3 Group of Drainage Ursodeoxycholic Acid Powder; 4 Pioglitazone Group; A: Group of Cholic Acid Complex A in Example 4; B: Group of Cholic Acid Complex B in Example 4; C: Group of Cholic Acid Complex C in Example 4; D: Group of Cholic Acid Complex D in Example 4) As shown, compared with the model group, each administration group had certain pharmacodynamic effects. It can be concluded from the comparison intensity score of liver and kidney ultrasound echoes that the groups of Cholic Acid Complex C and B in Example 4 at certain doses could significantly improve liver echoes (P<0.01; P<0.05). From the results at 4 weeks and 8 weeks of administration: The pioglitazone group had significant curative effects during the 4-week administration, but the pharmacodynamic effects were not obvious after 8 weeks of administration.

[0163] 3. Effects of Cholic Acid Complex on Gross Appearance and Liver Index of NASH Model Mice

[0164] After 8 weeks of modeling, 4 mice were randomly selected from the normal group and the model group. After blood collection from the eyeballs, the livers of the mice were photographed as Figure 8 .

[0165] As shown in the results, the liver tissues of the mice in the normal control group were bright red in color, with smooth, lubricated and dense surfaces; in the model group, the color was slightly pale, and many tiny milky white particles appeared on the liver surface and the liver appearance was rough.

[0166] The liver index after 8 weeks of modeling was as Figure 9 shown: Compared with the normal group, the liver index of the model group increased significantly (P<0.001), indicating that there might be edema, congestion or hyperplasia and hypertrophy in the liver.

[0167] The gross liver results after 8 weeks of administration were as Figure 10 (A: Group of Cholic Acid Complex A in Example 4; B: Group of Cholic Acid Complex B in Example 4; C: Group of Cholic Acid Complex C in Example 4; D: Group of Cholic Acid Complex D in Example 4) shown. The liver tissues of the mice in the normal group were bright red in color, with smooth, lubricated and shiny surfaces and dense textures; the liver tissues in the model group were pale in color, and white reticular textures appeared in the liver tissues, and the caudate lobe had an abnormal shape and was suspected to be wrapped by lipids; compared with the model group, the color, texture and lipid wrapping phenomenon of the liver in each administration group were improved to a certain extent. Among them, the liver tissues of the groups of Cholic Acid Complex C and D in Example 4 were significantly redder in color than those in the model group, and the surface texture became lighter, with the most obvious improvement, being superior to the group of Drainage Ursodeoxycholic Acid Powder and the positive drug pioglitazone group.

[0168] The liver index results after 8 weeks of administration were as Figure 11(1 Control; 2 Model; 3 Group of drained bear bile powder; 4 Pioglitazone group; A: Group of cholic acid complex A in Example 4; B: Group of cholic acid complex B in Example 4; C: Group of cholic acid complex C in Example 4; D: Group of cholic acid complex D in Example 4) As shown, compared with the normal group, the liver coefficient of the model group increased, but there was no significant difference. Compared with the liver coefficient at 4 weeks after modeling, the value decreased. Compared with the model group, the liver coefficients of the groups of cholic acid complexes A, C, and D in Example 4 showed a downward trend, and the decreases in the C and D dose groups were the most obvious, but there was also no statistical significance.

[0169] 4. Effect of cholic acid complex on liver histopathology of NASH model mice

[0170] Normal serum ALT does not mean no liver tissue inflammatory damage, and increased ALT does not necessarily mean NASH. Liver biopsy is still the gold standard for diagnosing NASH. After 8 weeks of modeling, HE staining of mouse liver tissue was performed to observe the pathological changes of the liver in NASH model mice, such as Figure 12 As shown, the capsule of the liver tissue in the normal group was composed of dense connective tissue rich in elastic fibers with uniform thickness. The boundaries of the hepatic lobules were obvious and arranged regularly. The central vein was in the center of the hepatic lobule, and the surrounding hepatocytes and hepatic sinusoids were arranged in a roughly radial pattern. The hepatocytes were round and plump; the hepatic plates were arranged regularly and neatly, and the hepatic sinusoids were not significantly dilated or compressed; there were no obvious abnormalities in the portal areas between adjacent hepatic lobules; no obvious inflammatory changes were seen. In the model group, a large number of hepatocytes showed fatty degeneration around the central vein, and round vacuoles of different sizes could be seen in the cytoplasm (1); a large amount of connective tissue hyperplasia was seen around the central vein (2), and lymphocyte infiltration was rarely seen (3); local ballooning degeneration of a small number of hepatocytes was seen, with cell swelling, nuclei in the center, and cytoplasmic vacuolization (4).

[0171] According to the NASH semi - quantitative scoring system, the scores of each group at 8 weeks after modeling are shown in Table 2. The scores of hepatic steatosis, intra - lobular inflammation, and the total NAS score of the NASH model group rats were significantly higher than those of the normal group (P < 0.05; P < 0.01; P < 0.0001); there was no obvious difference in ballooning degeneration. Among them, the total NAS score of the normal group was 0 points, and the total NAS score of the model group was ≥4 points, which could be clearly diagnosed as NASH, indicating that the NASH modeling was successful.

[0172] Table 2 NASH pathological score table after 8 weeks of modeling (n = 4)

[0173]

[0174] After 8 weeks of drug administration, as Figure 13 (A: Group of cholic acid complex A in Example 4; B: Group of cholic acid complex B in Example 4; C: Group of cholic acid complex C in Example 4; D: Group of cholic acid complex D in Example 4), Figure 14(Group A of cholic acid complex in Example 4; Group B of cholic acid complex in Example 4; Group C of cholic acid complex in Example 4; Group D of cholic acid complex in Example 4) As shown, in the model group, a large number of hepatocytes showed fatty degeneration, and round vacuoles of different sizes could be seen in the cytoplasm. A small number of hepatocytes showed ballooning degeneration. Small focal infiltrations of inflammatory cells could be seen in the lobules and around the veins, and intranuclear inclusions were rarely seen. All drugs had a certain improvement on fatty degeneration, among which the improvement in the cholic acid complex C dose group of Example 4 was the most obvious (P<0.05). According to the NASH semi-quantitative scoring system, the scores of each group are shown in Table 3.

[0175] The scores of hepatic steatosis, lobular inflammation and total NAS in the NASH model group of rats were all significantly higher than those in the normal group (P<0.0001; P<0.001; P<0.0001); there was no significant difference in ballooning degeneration. Among them, compared with the model group, cholic acid complex C in Example 4 had significant statistical differences in improving fatty degeneration and total NAS (P<0.05; P<0.01); the total NAS score of the group of drained bear bile powder was significantly lower than that of the model group (P<0.05). The efficacy of the pioglitazone group was not obvious after 8 weeks of administration.

[0176] Table 3 NAS pathological score table after 8 weeks of administration (n = 10)

[0177]

[0178]

[0179] Effect of cholic acid complex on liver lipid aggregation in NASH model mice

[0180] Oil red O fat staining method is one of the common methods to show the fat content in tissues. Oil red O is a fat-soluble dye, which can be highly dissolved in fat and can specifically stain neutral fats such as triglycerides in tissues.

[0181] The results of oil red O staining of frozen sections of mouse liver tissues after 8 weeks of modeling showed ( Figure 15 ) that there were a large number of red lipid droplets (black arrows) in the liver tissues, and a large amount of triglyceride accumulation.

[0182] After 8 weeks of administration, see Figure 16 (Group A of cholic acid complex in Example 4; Group B of cholic acid complex in Example 4; Group C of cholic acid complex in Example 4; Group D of cholic acid complex in Example 4). Compared with the normal group, a large number of red lipid droplets were diffusely distributed in the liver tissues of the model group mice; compared with the model group, the lipid droplets in the liver of each administration group mice had a certain improvement, among which the B and C dose groups had the most obvious improvement.

[0183] 6. Effect of cholic acid complex on serum biochemical indexes in NASH model mice

[0184] Serum biochemical indexes are one of the commonly used clinical criteria for the auxiliary diagnosis of NASH. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) are used to characterize the degree of liver injury; direct bilirubin (DBIL) and total bilirubin (TBIL) reflect hepatocyte metabolism; blood lipids TC, TG, and LDL are closely related to liver lipid metabolism.

[0185] After 8 weeks of modeling, an enzyme-linked immunosorbent assay (ELISA) reader was used to detect the contents of TC, ALT, and AST in the serum according to the instructions of the biochemical reagent kit. The results are as Figure 17 shown. Compared with the normal group, the liver lipid metabolism and liver injury in the model group of mice were significantly different (P<0.0001). However, there were individual mice in the model group with significantly abnormal indexes (ear tags 15 and 57), which were judged as non-modeled mice and were therefore excluded before the subsequent efficacy tests.

[0186] After 4 weeks of drug administration, the contents of TC, ALT, and AST in the serum were detected again. The results are as Figure 18 (A: Group A of cholic acid complex in Example 4; B: Group B of cholic acid complex in Example 4; C: Group C of cholic acid complex in Example 4; D: Group D of cholic acid complex in Example 4; 1 Control; 2 Model; 3 Group of drained bear bile powder; 4 Pioglitazone group) shown. The content of TC in the serum of the normal group and the model group of mice increased significantly compared with that 4 weeks ago, but there was no statistical difference between the two groups. There was also no significant difference in the content of TC in the serum of each drug administration group compared with the model group. Compared with the normal group, the levels of ALT and AST, the liver function indexes in the serum of the model group of mice, increased significantly (P<0.05). Compared with the model group, the levels of ALT and AST in Group A and C of the cholic acid complex in Example 4 and the pioglitazone group could be significantly reduced (P<0.05). The cholic acid complex B in Example 4 and the group of drained bear bile powder could significantly reduce the level of AST (P<0.05), but had no obvious improvement effect on ALT.

[0187] After 8 weeks of drug administration, a fully automatic biochemical analyzer was used to detect the biochemical indexes of the serum. The results are shown in Table 4.

[0188] Table 4 Contents and levels of biochemical indexes in the serum after 8 weeks of drug administration

[0189]

[0190]

[0191] In terms of liver injury indexes, compared with the normal group, the levels of ALT and AST in the model group increased to varying degrees, but there was no statistical difference. Compared with the model group, the levels of ALT and AST in each drug administration group did not change significantly and there was no statistical difference.

[0192] In terms of bile metabolism indicators, compared with the normal group, the serum ALP level of the mice in the model group was significantly increased (P<0.05); compared with the model group, the ALP level was reduced to varying degrees in each dose group of the bile acid complex, but there was no statistical significance. Compared with the normal group, the serum TBIL level of the mice in the model group was significantly increased (P<0.05). Compared with the model group, the TBIL level was reduced to varying degrees in each dose group of the bile acid complex and the positive drug group. Among them, there were statistical differences between the bile acid complex C group of Example 4 and the pioglitazone group (P<0.05; P<0.01). Compared with the normal group, there was no obvious change in DBIL in the model group, but the DBIL levels in the bile acid complex A, C, and pioglitazone groups of Example 4 were significantly reduced compared with the model group (P<0.05; P<0.01; P<0.0001).

[0193] In terms of lipid metabolism indicators, compared with the normal group, the serum total cholesterol (TC) level of the mice in the model group was significantly increased (P<0.05), while there was no tendency for each administration group to reduce TC. There was no tendency for the TG and LDL levels in the serum of each administration group to decrease, and there was no significant difference.

[0194] In terms of inflammatory indicators, compared with the normal group, the levels of IL-1β and TNF-α in the mice in the model group were significantly increased (P<0.0001); compared with the model group, the IL-1β levels in each administration group were significantly reduced (P<0.0001; P<0.0001; P<0.01; P<0.001; P<0.05; P<0.001); compared with the model group, the TNF-α levels in each administration group were reduced, and there were significant differences in the other groups except the drained bear bile powder group (P<0.0001; P<0.0001; P<0.01; P<0.01; P<0.05). There was no difference between each administration group.

[0195] 7. Effects of Bile Acid Complex on Liver Biochemical Indexes of NASH Model Mice

[0196] After 8 weeks of administration, a fully automatic biochemical analyzer was used to detect the biochemical indexes of liver tissue homogenate, and the results are shown in Table 5.

[0197] Table 5 Content Levels of Biochemical Indexes in the Liver after 8 Weeks of Administration

[0198]

[0199]

[0200] In terms of lipid metabolism, compared with the normal group, the levels of TC, TG, and LDL in the liver tissues of mice in the model group were significantly increased (P<0.0001); compared with the model group, the TC levels in each administration group were decreased, among which there were significant differences in Example 4 cholate complex C (P<0.0001), Group D (P<0.001), and the drained bear bile powder group (P<0.001); compared with the model group, the TG levels in each administration group were decreased, among which there were significant differences in Example 4 cholate complex C group and the drained bear bile powder group (P<0.05); compared with the model group, the LDL levels in each administration group were decreased, among which there were significant differences in Example 4 cholate complex C (P<0.0001), Group D (P<0.001), and the drained bear bile powder group (P<0.001). It is worth mentioning that among the lipid metabolism indexes, the reduction of TC and LDL-C in the dose group of Example 4 cholate complex C was significantly better than that of the pioglitazone group (P<0.05; P<0.01); among them, the reduction effect of LDL-C in the dose group of Example 4 cholate complex C was significantly better than that of the dose group of Example 4 cholate complex A (P<0.05).

[0201] In terms of inflammation, compared with the normal group, the levels of TNF-α (P<0.0001) and transforming growth factor β (TGF-β) (P<0.001) in the mice of the model group were significantly increased. Compared with the model group, the TNF-α levels in each administration group were decreased, and there were statistical differences in all the administration groups except Example 4 cholate complex B group (P<0.05). Compared with the model group, the TGF-β levels in each administration group were decreased, among which there were significant differences in Example 4 cholate complex A, B groups and the drained bear bile powder group (P<0.01). There was no trend and difference in the comparison of inflammatory factors such as IL-1β between the control group, the model group, and the model group and the administration group.

[0202] The above experimental results show that the cholate complex provided by the present invention or the cholate complex prepared by using the preparation method provided by the present invention can effectively improve the liver echo of model mice, reduce liver pathological damage and NAS score, reduce liver lipid deposition, and alleviate the inflammatory reaction in serum and liver, thus having a significant therapeutic effect on non-alcoholic steatohepatitis. The experimental results provide data support for the clinical use of cholate complex and lay a foundation for its further research and development.

[0203] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cholic acid complex, characterized in that, it comprises: 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; The cholic acid complex is prepared by the following steps: The preparation method includes: mixing the formula amounts of tauroursodeoxycholic acid, taurochenodeoxycholic acid, taurocholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, and taurolithocholic acid evenly to obtain the cholic acid complex.

2. Use of the cholic acid complex according to claim 1 in the preparation of a drug for treating non-alcoholic fatty liver disease.

3. According to the use described in claim 2, characterized in that, the non-alcoholic fatty liver disease includes non-alcoholic steatohepatitis.

4. A drug for treating non-alcoholic fatty liver disease, characterized in that, it comprises the cholic acid complex according to claim 1 and pharmaceutically acceptable excipients.

5. According to the drug described in claim 4, characterized in that, the dosage form of the drug includes oral preparations or injection preparations.

6. According to the drug described in any one of claims 4 to 5, characterized in that, the non-alcoholic fatty liver disease includes non-alcoholic steatohepatitis.

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