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CN-121971539-A - Preparation method and application of theabrownin-based chemical liver injury resistant drug

CN121971539ACN 121971539 ACN121971539 ACN 121971539ACN-121971539-A

Abstract

The invention relates to the technical field of biological medicine, and discloses a preparation method and application of a chemical liver injury resistant medicament based on theabrownin, wherein the preparation method comprises the steps of extracting the theabrownin with the molecular weight of 1kDa to 100kDa from post-fermented tea; establishing a model of acute liver injury of mice induced by carbon tetrachloride and carrying out preventive administration, evaluating in-vivo efficacy through serum biochemical, histopathological and immunohistochemical indexes, constructing an in-vitro macrophage inflammation model, and analyzing a molecular mechanism by utilizing flow cytometry and an immunoblotting technology. Experiments prove that theabrownin can clear active oxygen in cells, inhibit macrophage polarization to pro-inflammatory phenotype, and remarkably reduce the release of inflammatory factors such as IL-6 and the like by reducing the expression of phosphorylated NF-kappa Bp65 and blocking nuclear translocation thereof. The invention solves the problem of lack of molecular mechanism analysis in the prior art, and is suitable for preparing oral or injection medicaments for treating various chemical poison toxic hepatitis.

Inventors

  • DIAO HONGYAN
  • CAI RUI
  • XIAO ZIXUAN

Assignees

  • 浙江大学医学院附属第一医院(浙江省第一医院)

Dates

Publication Date
20260505
Application Date
20260128

Claims (10)

  1. 1. The preparation method of the chemical liver injury resistant medicament based on theabrownin is characterized by comprising the following steps of: S100, preparing and controlling the quality of a test drug theabrownin, wherein the test drug theabrownin is a water-soluble brown pigment compound extracted from post-fermented tea of puer tea or black tea, the molecular weight distribution range of the test drug theabrownin is between 1kDa and 100kDa, and the main components of the test drug theabrownin comprise polyphenol oxidized polymers, polysaccharides, proteins and the compounds thereof; Step 200, constructing an acute liver injury model of an animal induced by carbon tetrachloride and performing drug administration intervention, wherein the step 200 adopts a C57BL/6 mouse or an ICR mouse as an experimental animal, and the step 200 comprises a preventive drug administration stage, an acute liver injury modeling stage and a sample collection stage; step S300, in-vivo pharmacodynamics evaluation based on multi-dimension indexes, wherein the step S300 comprises serum biochemical index detection, histopathological evaluation and immunohistochemistry and inflammatory factor detection; Step S400, constructing an in-vitro macrophage inflammation model and verifying a cell level mechanism, wherein in the step S400, a mouse mononuclear macrophage leukemia cell or a phorbol ester induced differentiated human monocyte is adopted as an in-vitro cell model, and lipopolysaccharide is adopted as a stimulus source to induce the in-vitro cell model to polarize towards an M1 type pro-inflammatory phenotype; And step S500, analyzing a molecular mechanism based on a signal path, wherein the step S500 comprises protein extraction, immunoblotting and key signal protein detection, and the key signal protein detection aims at key proteins in a nuclear factor kappa B path.
  2. 2. The preparation method of the chemical liver injury resistant medicament based on theabrownin according to claim 1, which is characterized by further comprising the step of taking the test drug theabrownin as the only active ingredient after S500 is completed, or combining the test drug theabrownin with pharmaceutically acceptable auxiliary materials, and preparing tablets, capsules, granules, oral liquid, dripping pill oral preparation, freeze-dried powder injection or small water injection, wherein the pharmaceutically acceptable auxiliary materials comprise disintegrating agents, adhesives, lubricants and diluents.
  3. 3. The method for preparing the chemical liver injury resistant drug based on theabrownin according to claim 1, wherein in the step S100, the drug theabrownin to be tested comprises a crude extract prepared by a water extraction and alcohol precipitation method or a refined component purified by macroporous resin adsorption chromatography and gel column chromatography, wherein the drug theabrownin to be tested is dark brown powder, and the drug theabrownin to be tested is calculated to have the purity of more than 90% by a normalization method under the conditions that the detection wavelength is 278nm, the mobile phase is methanol-0.1% phosphoric acid aqueous solution with the volume ratio of 20:80, and the chromatographic column is C18 column.
  4. 4. The method for preparing a chemical liver injury resistant drug based on theabrownin according to claim 1, wherein in the step S200, the test animals are continuously subjected to the gastric lavage treatment of the test drug in the preventive administration period of 7 to 21 days, the administration dose of 500 to 1000mg/kg body weight, the test animals are subjected to the intraperitoneal injection of carbon tetrachloride in the last administration period of 8 to 12 hours, and the carbon tetrachloride is dissolved in olive oil or corn oil plant oil carrier at a concentration of 0.1 to 5% and the injection dose of 1 to 10mL/kg body weight.
  5. 5. The method for preparing a theabrownin-based chemical liver injury resistant drug according to claim 1, wherein in the step S300, the serum biochemical index detection is used for evaluating the integrity and damage degree of liver cell membranes by detecting the activity units of alanine aminotransferase and aspartate aminotransferase in serum, and the histopathological evaluation is used for observing liver lobule structure by hematoxylin-eosin staining technique, and observing whether to reduce balloon-like degeneration of liver cells, reduce area of punctate or sheet necrosis area, and maintain normal morphology of liver blood sinuses.
  6. 6. The method for preparing the theabrownin-based chemical liver injury resistant medicament according to claim 1, wherein in the step S300, the immunohistochemistry and inflammatory factor detection is used for detecting the expression level of pro-inflammatory factors in liver tissues by using a real-time fluorescent quantitative PCR technology or an enzyme-linked immunosorbent assay ELISA technology, wherein the pro-inflammatory factors comprise interleukin-6, interleukin-1 beta and tumor necrosis factor-alpha, and the immunohistochemistry and inflammatory factor detection is used for quantifying the infiltration amount of macrophages in the liver tissues by using immunofluorescence staining of F4/80 specific surface markers.
  7. 7. The method according to claim 1, wherein in the step S400, the step S400 comprises a cell morphology analysis and a cell oxidative stress level detection, wherein the cell morphology analysis observes whether the in vitro cell model has an activated form of pseudopodia extension and enlargement of cell bodies under the lipopolysaccharide stimulation, and whether the test drug theabrownin treatment can maintain the in vitro cell model in a circular resting form.
  8. 8. The method for preparing a theabrownin-based chemical liver injury resistant drug according to claim 7, wherein the cell oxidative stress level detection uses a dihydroethidium or a C11-BODIPY fluorescent probe to label active oxygen or lipid peroxide in the in-vitro cell model, fluorescence intensity is detected by flow cytometry, and the test drug theabrownin reduces the average fluorescence intensity of the active oxygen in the in-vitro cell model at a cell level.
  9. 9. The method for preparing the chemical liver injury resistant drug based on theabrownin according to claim 1, wherein in the step S500, the key signal protein detection comprises detection of NF-kappa B p subunit, phosphorylated NF-kappa B p, ikappa B alpha and phosphorylated forms thereof, and the test drug theabrownin reduces the relative expression quantity of the phosphorylated NF-kappa B p65 protein and inhibits translocation of the NF-kappa B p subunit from cytoplasm to nucleus so as to block transcription initiation of downstream inflammatory factors.
  10. 10. The use of a method for the preparation of a theabrownin-based anti-chemical liver injury drug according to any one of claims 1 to 9, wherein the chemical toxicant toxic hepatitis covers chemical liver injury characterized by oxidative stress and macrophage-mediated inflammatory reaction, and the chemical toxicant comprises carbon tetrachloride, alcohol, acetaminophen, and heavy metals.

Description

Preparation method and application of theabrownin-based chemical liver injury resistant drug Technical Field The invention relates to the technical field of biological medicines, in particular to a preparation method and application of a chemical-resistant liver injury drug based on theabrownin. Background Liver is an important organ for metabolism, detoxification and synthesis of human body, and its dysfunction is closely related to various liver diseases, including viral hepatitis, alcoholic liver disease, drug or toxic liver injury, autoimmune hepatitis, etc. These diseases have serious threats to human health and have become a global public health problem. Among them, liver injury caused by chemical poison is often used for liver disease pathology research and drug screening due to clear pathogenesis and mature model. Carbon tetrachloride (CCl 4) is a typical hepatotoxic substance, and after being metabolized by cytochrome P450 enzyme in the liver, forms highly active intermediates such as trichloromethyl radical (CCl 3), and further causes lipid peroxidation of hepatic cell membranes, damages membrane structural integrity, induces hepatic cell necrosis, and accompanies remarkable oxidative stress and inflammatory reaction. At present, the prevention and treatment strategies for chemical liver injury such as CCl4 mainly focus on the aspects of antioxidation, anti-inflammation, lipid peroxidation inhibition, liver cell membrane stability maintenance and the like. Although a certain liver protecting medicine can be selected clinically, most medicines still have single acting target points, adverse reactions possibly caused by long-term use or different curative effects on different types of liver injury. Therefore, the development of liver protecting components with multiple targets, high efficiency and low toxicity from natural products has become one of the important directions for the development of new drugs. Theabrownin is a water-soluble brown pigment polymer formed by deep oxidative polymerization of tea polyphenols in tea (especially dark tea, puer tea, etc.). Studies have shown that theabrownin has various biological activities such as antioxidation, lipid metabolism regulation, intestinal microecology improvement, etc. However, the current research on theabrownin is mostly focused on the field of metabolic related diseases, and the application of the theabrownin in liver protection is still limited, especially for acute and severe toxic hepatitis induced by CCl4, whether the theabrownin has definite relieving or treating effect or not, and the specific curative effect and action mechanism of the theabrownin have not been systematically revealed and effectively confirmed in the prior art. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a preparation method and application of a theabrownin-based chemical liver injury resistant medicament, and solves the problems in the background art. The preparation method and the application of the chemical-resistant liver injury medicine based on theabrownin comprise the complete technical flow from raw material preparation, quality control to multi-level medicine effect verification. The method firstly relates to the preparation and quality control of the test drug theabrownin, wherein the test drug theabrownin is a water-soluble brown pigment compound extracted from post-fermented tea of Pu' er tea or dark tea. In the aspect of material basis, the molecular weight distribution range of the theabrownin of the tested drug is controlled between 1kDa and 100kDa, and the main chemical components of the theabrownin comprise polyphenol oxidized polymer, polysaccharide, protein and a compound thereof. In the preparation process, the crude extract is obtained by a water extraction and alcohol precipitation method, or the refined component is further obtained by macroporous resin adsorption chromatography and gel column chromatography. For the purity detection of the test drug theabrownin, a High Performance Liquid Chromatography (HPLC) method is adopted, the detection wavelength is 278nm, the mobile phase is methanol-0.1% phosphoric acid aqueous solution with the volume ratio of 20:80, and the peak area ratio is calculated by a normalization method, so that the purity of the test drug theabrownin is ensured to be more than 90%. The method further comprises the steps of constructing a model of carbon tetrachloride-induced acute liver injury of the animal and intervening in dosing. C57BL/6 mice or ICR mice are selected, and the pathological process of chemical liver injury is simulated through the combination of a preventive administration stage and an acute liver injury modeling stage. Wherein, the modeling agent carbon tetrachloride is dissolved in the vegetable oil carrier, and the liver is induced to generate trichloromethyl free radical by the intraperitoneal injection way, thereby triggering lipid peroxidation and inflammator