阅读说明：本技术 甘肃蚤缀的用途 (Application of Gansu flea patch ) 是由 崔玉磊 沈娜 辛化伟 王振 李春雷 于 2020-06-09 设计创作，主要内容包括：本发明公开了一种抗肝纤维化提取物及其用途,提供了甘肃蚤缀或其提取物在制备预防和/或治疗肝纤维化疾病的产品的用途。本发明通过动物实验说明甘肃蚤缀或其提取物具有确切的抗肝纤维化作用,因此可用来制备预防和/或治疗肝纤维化疾病的产品。(The invention discloses an anti-hepatic fibrosis extract and application thereof, and provides application of Gansu flea conjugate or extract thereof in preparing products for preventing and/or treating hepatic fibrosis diseases. Animal experiments show that Gansu flea conjugate or extract thereof has exact anti-hepatic fibrosis effect, so that the Gansu flea conjugate or extract thereof can be used for preparing products for preventing and/or treating hepatic fibrosis diseases.)

1. Use of Gansu Siphonius or extract thereof in the preparation of a product for preventing and/or treating liver fibrosis.

2. Use according to claim 1, characterized in that: the extract is selected from ethanol extract or ethyl acetate extract of Gansu flea; further, the ethyl acetate extract is a product obtained by using ethyl acetate to extract the ethanol extract of Gansu flea as a raw material.

3. Use according to claim 1, characterized in that: the extract is prepared by the following method:

extracting Gansu flea with ethyl acetate, subjecting the ethyl acetate to reverse phase silica gel chromatography, eluting with 60% methanol-water, eluting with 80% methanol-water, and collecting 80% methanol-water eluate.

4. Use according to claim 1, characterized in that: the extract is prepared by the following method:

taking an ethyl acetate extract of Gansu flea, performing reverse phase silica gel chromatography, eluting with 50-60% methanol-water, eluting with 75-95% methanol-water, and collecting 75-95% methanol-water eluate;

preferably, the ethyl acetate extract of Gansu flea is subjected to reversed phase silica gel chromatography, eluting with 60% methanol-water, eluting with 80% methanol-water, and collecting 80% methanol-water eluate.

5. Use according to claim 3 or 4, characterized in that: degreasing before ethyl acetate extraction; further, the degreasing is carried out by using petroleum ether, diethyl ether, pentane or hexane.

6. Use according to any one of claims 3 to 5, characterized in that: the similarity of the chromatogram of the extract measured by HPLC and figure 6 is more than 90%; the chromatographic conditions were as follows:

stationary phase: octadecyl bond and silica gel

Mobile phase: the mobile phase A is 0.2% formic acid water solution, the mobile phase B is acetonitrile solution, and the gradient elution procedure is as follows: 13-46% of B, 0-90 min.

7. Use according to claim 6, characterized in that: the detection wavelength of HPLC was 330 nm.

8. Use according to claim 1, characterized in that: the liver fibrosis is caused by viral liver disease, chemical liver injury, non-alcoholic fatty liver disease, fatty liver or autoimmune liver disease.

9. Use according to claim 1, characterized in that: the liver fibrosis is caused by chemical liver injury.

10. Use according to claim 1, characterized in that: the product is selected from oral, injection or external preparation.

Technical Field

The invention relates to the technical field of prevention and treatment of hepatic fibrosis, in particular to an anti-hepatic fibrosis extract and application thereof.

Background

Hepatic fibrosis is a disease in which extracellular matrix (ECM) and connective tissue in the liver are abnormally accumulated and proliferated to destroy normal structure and physiological function, and is a necessary way for various chronic liver diseases (such as chronic viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, autoimmune liver disease, and the like) to progress to cirrhosis and liver cancer. By 2019, clinical researches on hepatic fibrosis are reported, but no cure is provided so far, and for patients with advanced hepatic fibrosis (cirrhosis or liver cancer), liver transplantation is the final life-saving straw, but limited liver resources and immune rejection after transplantation are still huge problems faced by liver transplantation.

There have been international clinical reports of etiological treatment cases, such as the antiviral drug tenofovir disoproxil, with effects of reversing liver fibrosis and cirrhosis, but the study sample size is relatively small, controls are lacking, and secondary liver biopsy patients are few. Some Chinese patent medicines in China show a certain anti-hepatic fibrosis effect and are used for clinical treatment, such as Anluo chemical fiber pills, compound turtle shell liver softening tablets, capsules for strengthening body resistance and removing blood stasis and the like, but the problems of difficult quality control, unclear action mechanism, less standardized clinical research and the like of traditional Chinese medicine products exist. Most drugs (such as polyene phosphatidyl choline, obeticholic acid, silymarin, glycyrrhizic acid preparations, glucocorticoid and the like) show a certain anti-hepatic fibrosis effect in animal experiments, but specific action mechanisms of the drugs are unknown and are accompanied by certain side effects and drug resistance, so that further intensive research needs to be carried out and alternative therapeutic drugs need to be searched.

Disclosure of Invention

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

use of Gansu Siphonius or extract thereof in the preparation of a product for preventing and/or treating liver fibrosis.

Gansu Siphonium chinense (Arenaria kansuensis Maxim.) also called Arenaria kansuensis, the Tibetan medicine is named as "Zhonggabao", the Tibetan language is the "Renzhouxiancao which is the essence of the spiritual qi of the Tiandi, and has a long medicinal history, and the Tibetan medicine records that the Sichenkia kansuensis Maxim has the effects of clearing lung heat, relieving cough, reducing blood pressure, invigorating stomach, assisting digestion and the like and treating ulcer, swelling, cancer, scrofula and the like of the stomach and intestine. Modern pharmacological studies show that the medicine has the curative effects of bacteriostasis, antioxidation, anti-inflammation, immunoregulation, antivirus, liver protection and the like.

The extract used in the invention is separated from the ethanol extract of Gansu flea; further, it is separated from the ethyl acetate extract.

Furthermore, the ethyl acetate extract is a product obtained by using ethyl acetate to extract the raw material of the ethanol extract of Gansu flea.

Further, the extract is prepared by the following method:

extracting the ethanol extract of Gansu flea with ethyl acetate, performing reversed phase silica gel chromatography on the ethyl acetate part, eluting with 50-60% methanol-water, eluting with 75-95% methanol-water, and collecting the 75-95% methanol-water eluate.

Further, subjecting the ethyl acetate extract of Gansu flea to reversed phase silica gel chromatography, eluting with 60% methanol-water, eluting with 80% methanol-water, and collecting 80% methanol-water eluate.

Further, the extract is prepared by the following method:

collecting Gansu flea-decorated ethyl acetate extract, performing reverse phase silica gel chromatography, eluting with 60% methanol-water, eluting with 80% methanol-water, and collecting 80% methanol-water eluate.

The stationary phase of the reversed phase silica gel chromatography is selected from octadecyl bond, silica gel or similar materials.

Further, before ethyl acetate extraction, degreasing treatment is carried out; further, the degreasing is carried out by using petroleum ether, diethyl ether, pentane or hexane. Pentane and hexane are the major components of petroleum ether.

Further, the similarity of the chromatogram of the extract measured by HPLC and figure 6 is more than 90%. In view of the differences in the production areas, cultivation areas, growth environments and the like of natural plants, the types and contents of chemical components of different plants in the same natural plant are different, which inevitably results in the differences of the components in the extract. Therefore, when the extract is defined by the HPLC chromatogram, the extract chromatogram having a similarity of 90% or more to fig. 6 of the present invention can be determined to be the same as the extract of the present invention.

The chromatographic conditions for the above assay were as follows:

stationary phase: octadecyl bond and silica gel

Mobile phase: the mobile phase A is 0.2% formic acid water solution, the mobile phase B is acetonitrile solution, and the gradient elution procedure is as follows: 13-46% of B, 0-90 min.

In the invention, the detection wavelength can be adjusted according to the actual detection condition, most of the existing detectors can simultaneously carry out full-wave-band detection, and the atlas under the required detection wavelength can be randomly checked and called as required after the detection is finished. The invention finds that the optimal detection wavelength may be 330nm, and the detection wavelength which can be adjusted or manually set can be in the range of 320-340 nm based on the conditions of instrument, operation error and the like. Of course, it is not excluded that chromatograms of the components contained in the exact reaction extract can be obtained at other wavelengths as well.

The hepatic fibrosis is caused by viral liver disease, chemical liver injury, non-alcoholic fatty liver disease, fatty liver or autoimmune liver disease.

Further, the liver fibrosis is caused by chemical liver injury.

The product is selected from oral, injection or external preparation. The Gansu flea or the extract thereof and the pharmaceutic adjuvant can be prepared into injections, inhalants, tablets, capsules, pills and the like.

The invention has the beneficial effects that:

the invention verifies that the flavonolignan-rich component in the Gansu flea-decorated ethyl acetate extract has the effect of resisting hepatic fibrosis on a mouse model of hepatic fibrosis, and the result shows that the ethyl acetate extract can obviously relieve the damage degree to liver cells, has the effect of obviously improving the collagen deposition of liver tissues and the formation of liver fibers, can obviously inhibit the expression of hepatic fibrosis related proteins TGF-beta 1 and alpha-SMA, shows that the flavonolignan which is the effective component of the ethyl acetate extract can inhibit the progress of the hepatic fibrosis, and has the obvious effect of resisting the hepatic fibrosis.

Drawings

FIG. 1 shows HPLC analysis of the ethyl acetate fraction of Gansu flea, the impurity fraction (AS1) and the target fraction (AS 2). The chromatogram of the ethyl acetate part in the graph A, the chromatogram of the impurity part AS1 in the graph B, and the chromatogram of the target part AS2 in the graph C.

FIG. 2 is a photograph of representative liver and hematoxylin-eosin stained tissue section (20X) of Gansu flea conjugate extract target site AS2 in mice.

FIG. 3 is a Masson staining picture (20X) of a representative liver tissue section of mice with the Gansu flea conjugate extract target site AS 2.

FIG. 4 is a graph of the effect of Dolichthys gansuensis extract target site AS2 on TGF- β 1 expression in mouse liver tissue.

FIG. 5 is a graph of the effect of Gansu flea conjugate extract target site AS2 on the expression of α -SMA in mouse liver tissue.

FIG. 6 is a HPLC analysis of the Gansu flea target site AS 2.

Detailed Description

The application of the flea euphorbia in combination with flavone lignin in anti-hepatic fibrosis is described in detail in the following embodiments. It is to be understood that these examples are illustrative of the present invention, but not limiting the scope of the invention.