CN110538170A

CN110538170A - Application of macamides compound or salt thereof in preparation of medicine for preventing or treating hepatic fibrosis diseases

Info

Publication number: CN110538170A
Application number: CN201911007619.4A
Authority: CN
Inventors: 谢伟全; 刘涛; 涂剑; 张素君; 尹辉; 周芊芊; 李子涵
Original assignee: University of South China
Current assignee: University of South China
Priority date: 2019-10-22
Filing date: 2019-10-22
Publication date: 2019-12-06
Anticipated expiration: 2039-10-22
Also published as: CN110538170B

Abstract

The application discloses application of macamides or salts thereof in preparing medicines for preventing or treating hepatic fibrosis diseases. The invention discovers that the macamides compound has obvious anti-hepatic fibrosis effect for the first time, thereby discovering the new application of the macamides compound in preparing medicines for preventing hepatic fibrosis diseases or treating hepatic fibrosis diseases.

Description

Application of macamides compound or salt thereof in preparation of medicine for preventing or treating hepatic fibrosis diseases

Technical Field

The application relates to the technical field of medicines, in particular to application of macamides compounds or salts thereof in preparing medicines for preventing or treating hepatic fibrosis diseases.

Background

liver Fibrosis (Liver Fibrosis) is a chronic Liver disease caused by various factors, such as inflammatory reaction, oxidative stress, autophagy, cytokines, anticoagulation, etc., and its formation is also a result of the interweaving of various factors. The nature of liver fibrosis is that collagen-based extracellular matrix (ECM) is synthesized in much greater amounts than it is degraded, resulting in massive ECM deposition. Numerous studies have demonstrated that activation of Hepatic Stellate Cells (HSCs) plays a crucial role in the development of Hepatic fibrosis. In physiological states, ECM is synthesized primarily by HSCs; when liver tissue is injured, HSC activation becomes Myofibroblast (MFB) secreting synthetic ECM, which is excessively deposited in the matrix of liver cells, eventually causing liver fibrosis.

The number of liver disease patients in China is large, 2-3% of fibrosis patients can develop liver cirrhosis annually, the number of patients who die of liver cirrhosis, liver cancer and other diseases is up to 15-30 ten thousand, and the number of patients who die of liver disease accounts for about 45% of the number of patients who die of liver disease worldwide. By 2015, epidemiological investigation results showed that about 2.57 million persons of Hepatitis B Virus (HBV) carriers worldwide die from HBV-induced cirrhosis of the liver in 88.7 million persons. Cirrhosis causes 120 deaths worldwide each year, with a mortality rate greater than 5 major cancers, and about 81 million deaths worldwide in 2015 due to liver cancer. The liver disease is the most heavily burdened in China worldwide, about 3 hundred million people are affected, and the investigation result in 2015 shows that the liver cancer causes about 42.2 million people to die in China.

However, the existing antiviral drugs for treating hepatitis such as lamivudine, interferon, adefovir dipivoxil, sofosbuvir and the like cannot effectively reverse hepatic fibrosis of patients, and a specific hepatic fibrosis resistant drug aiming at a single link or target point cannot be developed clinically. Therefore, the vigorous research and development of anti-hepatic fibrosis drugs is of great significance for delaying and even reversing hepatic fibrosis.

Maca (Lepdium meyenii, Maca), an annual or biennial herb in south America, belongs to the genus Lepidium (L.) of the family Brassicaceae (Cruciferae). Native to the 4000-4500-meter elevation of Peru Andes mountain, and the maca has a genome suitable for high-altitude areas. It is also known as Peru ginseng because of its rich health-care action. The maca components mainly comprise primary metabolites and secondary metabolites, wherein the primary metabolites are nutrient components such as polysaccharide, protein, amino acid, trace components and the like, and the secondary metabolites are functional components which are chemical substances with main drug effects of maca and comprise macamide, glucosinolate, benzyl isothiocyanate, carboline alkaloid, pyridine alkaloid, imidazole alkaloid, macaene and the like. In studies of shore inflammation, liu billows and the like, after careful combing and analysis of more than 30 maca characteristic compounds found, the characteristic active ingredient for the efficacy of maca was confirmed to be macamide, i.e., the benzamides in maca are the characteristic active ingredients in maca. Maca amide is regarded as a characteristic component of maca, is praised as a natural active nutrient by scientists, and is the focus of the current maca research. Macamides have been reported to have a wide range of medicinal values, such as improving sexual function, inhibiting the activity of fatty acid hydrolase, protecting nerves, resisting fatigue, inhibiting carcinogenesis, resisting tumors, improving osteoporosis, and the like. However, reports about anti-hepatic fibrosis of macamide are not found at present.

disclosure of Invention

In order to solve the technical problems, the invention aims to provide the application of the maca amide compounds or the salts thereof in preparing the medicines for preventing or treating hepatic fibrosis diseases; the invention discovers that the macamides compound has obvious anti-hepatic fibrosis effect for the first time, thereby discovering the new application of the macamides compound in preparing medicines for preventing hepatic fibrosis diseases or treating hepatic fibrosis diseases.

The technical scheme provided by the invention is as follows:

Application of macamides or salts thereof in preparing medicines for preventing or treating hepatic fibrosis diseases.

Preferably, the structural general formula of the macamides is shown as the formula (I):

Wherein R1 is saturated or unsaturated straight-chain alkane or saturated or unsaturated straight-chain alkane containing keto; r2 is H or methoxy; .

Preferably, the macamides are in particular N-benzyl-hexadecylamide, N-benzyl-octadecyl amide, N-m-methoxy-benzyl-octadecyl amide, N-benzyl-linolenamide, N-m-methoxy-benzyl-linolenamide, benzyl-linoleamide, N-m-methoxy-benzyl-linoleamide, N-benzyl-docosahexaenoic acid amide, N-benzyl-eicosapentaenoic acid amide, N-benzyl-stearidonamide, N-m-methoxy-benzyl-docosahexaenoic acid amide, N-m-methoxy-benzyl-eicosapentanoic acid amide, N-m-methoxy-benzyl-octadecatetraenoic acid amide, Any one or more of N-benzyl-5-carbonyl-octadecadienamide, N-benzyl-octadecadienamide and N-benzyl-octadecadienamide.

Preferably, the maca amide compound is N-benzyl hexadecamamide.

Preferably, the maca amide compounds are extracted from maca or obtained by artificial synthesis.

Preferably, the hepatic fibrosis disease is a hepatic fibrosis disease caused by any one or more of chronic viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, toxin, drug, autoimmune liver disease, hepatic congestion and genetic metabolic disease.

Preferably, the medicament is any one of oral preparation, injection preparation and sublingual buccal preparation.

Preferably, the oral preparation is any one of tablets and capsules; the injection preparation is any one of liquid injection and powder injection.

Preferably, the content of the macamides per unit of the preparation is 20mg to 500 mg.

the invention carries out two-level research on the anti-hepatic fibrosis effect of the maca amide compounds on cells and animals, and the result shows that the maca amide compounds can inhibit the high expression of alpha-smooth muscle actin (alpha-SMA) and type I Collagen (Collagen I, COL-I) in hepatic fibrosis tissues in the hepatic fibrosis process by down-regulating the expression of the alpha-smooth muscle actin (alpha-SMA) and the type I Collagen (Collagen I, COL-I) in the hepatic fibrosis tissues, further reverse the hepatic fibrosis pathological process induced by left common bile duct ligation (LMBDL), and have good antagonistic effect on the activation of hepatic stellate cells. The invention discovers that the maca amide compounds have obvious anti-hepatic fibrosis effect for the first time, thereby discovering the new application of the maca amide compounds in preparing medicines for preventing hepatic fibrosis diseases or treating hepatic fibrosis diseases, and having small toxic and side effects, safety and effectiveness.

Drawings

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

FIG. 1 is a graph showing the observation results of the liver morphology of mice in the normal control group, the sham-operated group, and the LMBDL group plus physiological saline and macamide prevention group in example 1 of the present invention;

FIG. 2 shows the results of HE staining and Masson staining of liver in mice of normal control group, sham group, LMBDL group plus physiological saline and macamide prevention group in example 2 of the present invention;

FIG. 3 is a development view showing the induction result of TGF-1 on human hepatic fibrosis stellate cell LX-2 in example 4 of the present invention;

FIG. 4 is a graph showing the protein concentration of the result of the induction of TGF-1 to human hepatic fibrosis stellate cell LX-2 in example 4 of the present invention;

FIG. 5 is a development of the effect of macamides on the expression of fibrin α -SMA in human hepatic fibrosis stellate cells LX-2 cells in example 5 of the invention;

FIG. 6 is a graph of the protein concentration of macamides in example 5 of the invention on the effect of expression of the fibrotic protein α -SMA in human hepatic fibrosis stellate cells LX-2 cells;

FIG. 7 is a development of the effect of macamides on the expression of the fibrotic protein Collagen I in human hepatic fibrosis stellate cells LX-2 cells in example 5 of the present invention.

Detailed Description

in order to make those skilled in the art better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. 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 application.

it will be understood that when an element is referred to as being "fixed" or "disposed" on another element, it can be directly on the other element or be indirectly disposed on the other element; when an element is referred to as being "connected to" another element, it can be directly connected to the other element or be indirectly connected to the other element.

It will be understood that the terms "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like, refer to an orientation or positional relationship illustrated in the drawings for convenience in describing the present application and to simplify description, and do not indicate or imply that the referenced device or element must have a particular orientation, be constructed and operated in a particular orientation, and thus should not be construed as limiting the present application.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present application, the meaning of a plurality of or a plurality of is two or more unless specifically limited otherwise.

It should be understood that the structures, ratios, sizes, and the like shown in the drawings are only used for matching the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the practical limit conditions of the present application, so that the modifications of the structures, the changes of the ratio relationships, or the adjustment of the sizes, do not have the technical essence, and the modifications, the changes of the ratio relationships, or the adjustment of the sizes, are all within the scope of the technical contents disclosed in the present application without affecting the efficacy and the achievable purpose of the present application.

The embodiments of the present application are written in a progressive manner.

As shown in fig. 1 to fig. 7, the embodiment of the present invention provides an application of maca amide compounds or salts thereof in preparing a medicament for preventing or treating liver fibrosis diseases.

Preferably, the maca amide compound is N-benzyl hexadecamamide.

In the application, the maca amide compounds refer to amide compounds obtained by extracting and separating maca, and the structural general formula of the maca amide compounds is shown as a formula (I), wherein R1 is H or methoxyl; r2 is a saturated or unsaturated linear alkane or a saturated or unsaturated linear alkane containing a ketone group, and specific compounds are listed. The maca amide compounds can also be obtained by artificial synthesis. The extraction method can be conventional method such as extracting with petroleum ether, ethyl acetate, and n-butanol, and separating and purifying with column; the artificial synthesis adopts the synthesis method commonly used in the field.

The hepatic fibrosis disease can be hepatic fibrosis disease caused by any factor, and specifically comprises hepatic fibrosis disease caused by any one or more of chronic viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, toxin, medicament, autoimmune liver disease, hepatic congestion and hereditary metabolic disease.

The application of the maca amide compounds or the salts thereof in preparing the medicines for preventing or treating hepatic fibrosis diseases can be prepared into medicines with different dosage forms, such as oral preparations, injection preparations, sublingual buccal preparations and the like, by adding related auxiliary materials or administration carriers.

In the present application, the content of the macamides per unit of the preparation is from 20mg to 500mg, preferably from 20mg to 300mg, more preferably from 30 to 200mg, more preferably from 50 to 100 mg.

Example 1 left median Choledocholithiasis (LMBDL) -induced liver fibrosis in mice

Experimental materials: C57L/6 mice, 3% sodium pentobarbital solution, surgical instruments.

The experimental method comprises the following steps:

LMBDL group: male C57BL/6 mice of 12 weeks old are anesthetized by intraperitoneal injection with 3% pentobarbital sodium solution, the skin and muscle layers of the abdominal cavity are cut, common bile duct is dissociated, the left common bile duct is ligated, and the abdominal cavity is sutured.

The sham operation group: male C57BL/6 mice of 12 weeks old were anesthetized by intraperitoneal injection using 3% sodium pentobarbital solution, the skin and muscle layers of the abdominal cavity were dissected, the common bile duct was dissociated, and the abdominal cavity was sutured.

Prevention group: mice were treated at a rate of 0.25mg/10g body weight per mouse per 20g mouse per mouse, 0.5mg was prepared as 20. mu.l of N-benzylhexadecanamide (NBH) solution of formula (I) (12.5mg powder, dissolved in 0.5ml DMSO), dissolved in 180. mu.l of physiological saline, and injected intraperitoneally. After 24 hours, LMBDL surgery was performed.

The structural formula of the N-benzyl hexadecamamide is as follows:

The experimental results are as follows: after about 10 days, ALT and AST were detected to be significantly increased, and the general view of the liver of the LMBDL group, the sham operation group, the normal control group and the treatment group is shown in FIG. 1.

Example 2HE Staining and Sirius Red Staining (Sirius Red Staining) Effect of macamides on hepatic fibrosis in mice induced by common bile duct ligation

Experimental materials: camera, tissue section preparation, microscope.

the experimental method comprises the following steps: fixing mouse liver tissue with paraformaldehyde, dehydrating, transparentizing, soaking in wax, embedding in paraffin with embedding machine, solidifying wax block, and storing in refrigerator at 4 deg.C. The wax blocks were then made into paraffin sections for HE staining and sirius red staining.

HE staining: dewaxing by dimethylbenzene I and dimethylbenzene II for 10 minutes respectively; putting the slices into alcohol solutions of 100%, 95%, 85%, 75% and the like for 10 minutes respectively in sequence, washing the slices for 5 minutes by tap water, and washing the slices for 5 minutes by 1 multiplied by PBS; after 30 minutes of warm bath at 237 ℃ with 0.3% H2O, washing with 1 XPBS for 5 minutes again; dyeing: hematoxylin is soaked for 2 minutes, the water is washed for 10 minutes, 1% hydrochloric acid ethanol is differentiated for 5 seconds, the water is washed for 10 minutes, eosin is dyed for 2 minutes, and the water is washed for 10 minutes; slicing in 75%, 85%, 95%, 100% alcohol solutions for 5min, 1/2 xylene for 5min, and xylene I and II for 5min, and sealing with gum.

Masson staining: paraffin sections were dewaxed to water, chromated or demerculated (formaldehyde fixed tissue this step may be omitted). Sequentially washing with tap water and distilled water, staining with Regaud hematoxylin staining solution or Weigart hematoxylin staining solution for 5-10min, and washing with water thoroughly, if over-staining can be differentiated by hydrochloric acid and ethanol. Washing with distilled water, soaking and washing with Masson's Lichun acid reddening solution for 5-10min, 2% glacial acetic acid water solution, differentiating with 1% phosphomolybdic acid water solution for 3-5min, directly dyeing with aniline blue or light green solution for 5min, soaking and washing with 0.2% glacial acetic acid water solution for a while, 95% alcohol, anhydrous alcohol, xylene transparent, and neutral gum.

the experimental results are as follows: the HE staining result shows that the liver structure of the normal liver tissue of the sham operation group is clear, the hepatic lobule structure is normal, the hepatic cell cord is clear, and the hepatic cell is complete; the LMBDL group has serious liver tissue damage and obvious fibrous tissue hyperplasia; the macamide can obviously reduce the damage of liver tissues and the proliferation degree of fibrous tissues in a group prevented by macamide. Masson staining showed: the BDL group red collagen fibers gradually increase in thickness and length, and the formation of false lobules can be partially seen, which indicates the successful establishment of a mouse hepatic fibrosis model; collagen fibers of the macamide prevention group are obviously less than those of the control group, and fiber interval staining is relatively shallow, so that macamide can obviously relieve hepatic fibrosis, as shown in fig. 2.

example 3 Effect of macamides on choledocholithiasis-induced liver fibrosis mice glutamic-pyruvic transaminase (ALT) and glutamic-oxaloacetic transaminase (AST).

Experimental materials: macamide, surgical instruments, a high-speed centrifuge, a biochemical detector and the like.

The experimental method comprises the following steps: mice in a sham operation group, an LMBDL group, a macamide prevention group and a normal control group are subjected to orbital bleeding, centrifuged (4000rpm for 5min), and supernatant is taken and sent to a hospital laboratory to detect the contents of serum glutamic-pyruvic transaminase (ALT) and glutamic-oxalacetic transaminase (AST) by using a biochemical detector.

the experimental results are as follows: compared with a normal control group, hepatic fibrosis mice ALT and AST induced by common bile duct ligation are obviously increased, and macamide can obviously relieve the phenomenon, as shown in the following table 1:

table 1: effect of macamides on glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase in serum of mice

Example 4 Induction of TGF beta-1 on human hepatic fibrosis stellate cell LX-2 cell

Experimental materials: human LX-2 cell strain, phage-free high-grade low-toxin fetal calf serum, alpha-SMA antibody, Human TGF beta-1, high-sugar culture medium, pancreatin, cell lysate, PMSF, cell curette, PBS liquid, BCA protein quantitative kit, deionized water, skimmed milk powder and the like.

The experimental method comprises the following steps: cells were inoculated in six-well plates, and when the cell density reached a certain level, TGF-1 was added at different concentrations, and divided into control groups (0 for TGF-1), 1ng/ml, 10ng/ml, and 100 ng/ml. Stimulating for 24h, and then carrying out Western blot detection. Washing cells with precooled PBS for 3-4 times, then sucking residual PBS liquid with filter paper, adding 80 μ L RIPA and 100mol/L PMSF 0.8 μ L (100:1) ice in a six-well plate for 30 minutes, scraping protein with a cell curette, sucking out protein in an EP tube, performing ultrasonic treatment at 10000rpm for 30 minutes, and centrifuging at 4 ℃; a96-well plate is filled with 3. mu.l of protein sample, 17. mu.l of deionized water, and 200. mu.l of BCA solution (50:1) per well, and two blank controls are provided, in FIG. 3, GAPDH and Tublin are two reference proteins for immunoblotting, and the blank control refers to a well to which no protein sample is added. Incubate at 37 ℃ for 30 minutes. Measuring OD value (570nm) by enzyme-linked immunoassay, and calculating protein concentration according to the OD value; boiling protein, loading, gel electrophoresis, transferring a membrane, sealing, sequentially incubating a primary antibody (an antibody aiming at the alpha-SMA protein) and a corresponding secondary antibody (an antibody aiming at the primary antibody), washing the membrane and developing.

The experimental results are as follows: with increasing TGF β -1 concentration, increased expression of α -SMA implies increased degree of liver fibrosis, as shown in figures 3-4.

Example 5 Effect of macamides on the expression of the fibrotic proteins alpha-SMA, Collagen I in human hepatic fibrosis stellate cell LX-2 cells.

experimental materials: macamide, a 0.22 mu M sterile filter membrane, a Human LX-2 cell line, phage-free low-toxin high-grade fetal bovine serum, an alpha-SMA antibody, Human TGF beta-1, a high-sugar culture medium, pancreatin, cell lysate, PMSF, a cell curette, PBS liquid, a BCA protein quantification kit, deionized water, skimmed milk powder and the like.

The experimental method comprises the following steps: inoculating cells with a six-hole plate, adding 10ng/ml TGF beta-1 to stimulate for 24 hours when the cell density reaches a certain value, and adding macamides into different holes until the final concentration is respectively as follows: protein is extracted after 24 hours of 0 mu mol/l, 10 mu mol/l, 100 mu mol/l and 1000 mu mol/l and Western blot detection is carried out. Washing cells with precooled PBS for 3-4 times, then sucking residual PBS liquid with filter paper, adding 80 mu L of RIPA and 100mol/L PMSF 0.8 mu L (100:1) ice in a six-well plate for 30 minutes, scraping protein with a cell curette, sucking out the protein in an EP tube, performing ultrasonic treatment at 10000rpm for 30 minutes, and centrifuging at 4 ℃; a96-well plate was filled with 3. mu.l protein sample, 17. mu.l deionized water, and 200. mu.l BCA solution (50:1) per well, and two blank controls were set. Incubate at 37 ℃ for 30 minutes. Measuring OD value (570nm) by enzyme-linked immunoassay, calculating protein concentration according to OD value, boiling protein, loading, performing gel electrophoresis, transferring membrane, sealing, sequentially incubating primary antibody and corresponding secondary antibody, washing membrane, and developing.

The experimental results are as follows: along with the increase of the concentration of the macamides, the expression of hepatic fibrosis marker proteins alpha-SMA and Collagen I in the LX-2 cells is gradually reduced, which indicates that the macamides have the effect of reversing hepatic fibrosis, and is shown in figures 5-7.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of macamides or salts thereof in preparing medicines for preventing or treating hepatic fibrosis diseases.

2. The use according to claim 1, characterized in that the general structural formula of the macamides is represented by formula (I):

Wherein R1 is saturated or unsaturated straight-chain alkane or saturated or unsaturated straight-chain alkane containing keto; r2 is H or methoxy.

3. Use according to claim 2, characterized in that said macamides are in particular N-benzyl-hexadecanamide, N-benzyl-octadecanamide, N-m-methoxy-benzyl-octadecanamide, N-benzyl-linolenamide, N-m-methoxy-benzyl-linolenamide, benzyl-linoleamide, N-m-methoxy-benzyl-linoleamide, N-benzyl-docosahexaenoic acid amide, N-benzyl-eicosapentaenoic acid amide, N-benzyl-stearidonamide, N-m-methoxy-benzyl-docosahexaenoic acid amide, N-m-methoxy-benzyl-eicosapentaenoic acid amide, N-m-methoxy-benzyl-linolenic acid amide, N-m-methoxy-linolenic acid amide, N-benzyl-linolenic acid amide, N-m-methoxy-benzyl-linolenic acid amide, Any one or more of N-m-methoxy-benzyl-octadecatetraenoic acid amide, N-benzyl-5-carbonyl-octadecadienamide, N-benzyl-octadecadienamide and N-benzyl-octadecadienamide.

4. Use according to claim 3, characterized in that said macamides are in particular N-benzylhexadecamamide.

5. Use according to any one of claims 1 to 4, characterized in that said macamides are obtained by extraction from maca or by artificial synthesis.

6. the use of claim 1, wherein the hepatic fibrosis disease is a hepatic fibrosis disease caused by any one or more of chronic viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, toxin, drug, autoimmune liver disease, hepatic congestion, and inherited metabolic disease.

7. The use according to claim 1, wherein the medicament is any one of an oral preparation, an injectable preparation, and a sublingual buccal preparation.

8. The use according to claim 7, wherein the oral preparation is any one of a tablet and a capsule; the injection preparation is any one of liquid injection and powder injection.

9. Use according to claim 7, characterized in that the content of macamides per unit of preparation is between 20mg and 500 mg.