CN111012781A

CN111012781A - Application of demethylenetetrahydroberberine hydrochloride in preparation of medicine for preventing or treating liver injury

Info

Publication number: CN111012781A
Application number: CN202010016431.2A
Authority: CN
Inventors: 张玉彬; 朱倩倩; 张元强; 闻婧
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-01-08
Filing date: 2020-01-08
Publication date: 2020-04-17

Abstract

The invention relates to the field of biological medicines, and in particular relates to application of demethylenetetrahydroberberine hydrochloride in preparation of a medicine for preventing and/or treating thioacetamide-induced liver injury. The demethylenetetrahydroberberine hydrochloride medicine has better solubility and medicine absorptivity, has good anti-inflammatory and antioxidant activities, has obvious effects on the treatment of thioacetamide induced acute mild and severe degree and chronic liver injury, can achieve the treatment purpose under a lower oral administration dosage compared with the traditional liver protection medicine, can improve the compliance of patients, and achieves the treatment effect of diseases.

Description

Application of demethylenetetrahydroberberine hydrochloride in preparation of medicine for preventing or treating liver injury

Technical Field

The invention relates to the field of biological medicines, and in particular relates to application of demethylenetetrahydroberberine hydrochloride in preparation of a medicine for preventing and/or treating thioacetamide-induced liver injury.

Background

The liver is the most important metabolic organ in the human body and participates in the metabolism of various substances such as saccharides, lipids, vitamins and the like. There are also many drug metabolizing enzymes in the liver and metabolic elimination of almost all drugs and exogenous chemicals must be accompanied by the liver. Excessive use or abuse of drugs, and inadvertent ingestion of hepatotoxic substances, can result in acute, chronic, or explosive damage to the liver. And the acute liver failure has the characteristics of fast onset, multiple complications and high fatality rate, and has great threat to human health, severe liver injury is faced, liver transplantation is the only effective treatment means at present, but organ transplantation has great risk and the medical technical problems such as immunological rejection and the like which are difficult to solve.

Chronic liver disease is a very common disease, and Hepatic Fibrosis (HF) is a consequence of chronic liver injury, which refers to a pathological change characterized by excessive deposition of cell matrix (ECM) in the liver due to liver injury predisposing factors, such as liver viral disease, alcoholic hepatitis, biliary tract disease, hemochromatosis or liver disease, which results in the persistent necrosis of liver cells, secondary liver inflammation or persistent inflammatory response during post-injury repair process, resulting in abnormal proliferation of fibrous connective tissue in the liver. When the fibrous tissue continuously grows, once invading the interior of the liver tissue, the structure of the normal liver tissue is damaged, a plurality of nodules surrounded by the fibrous tissue are formed, finally, the texture of the liver is hardened, and the liver cirrhosis is formed, and a plurality of complications caused by the liver cirrhosis can cause life risks to patients. At present, no specific and effective method exists for treating hepatic fibrosis clinically, and the treatment scheme mainly aims at preventing or relieving liver injury so as to block or even reverse the liver fibrosis process. The existing commonly used drugs for treating hepatic fibrosis are various liver-protecting drugs or antiviral drugs, such as silymarin, obeticholic acid, glycyrrhizic acid, interferon and the like.

At present, few research reports on the pharmacological activity of the demethylenetetrahydroberberine hydrochloride are reported, and the laboratory researches the effective action of the demethylenetetrahydroberberine hydrochloride on fatty liver injury and proves that the demethylenetetrahydroberberine hydrochloride has lower toxicity and higher efficient pharmaceutical activity compared with the berberine hydrochloride and the demethyleneberberine hydrochloride.

Demethylenetetrahydroberberine hydrochloride, represented by formula (I)

Demethylenetetrahydroberine hydrochloride (formula I) is named as Demethyenetetrahydroberine in English, CAS number is 47346-21-4, academic name is 6H-Dibenzoa, g ] quinolizine-2,3-diol,5,8,13,13 a-tetrahydroxy-9, 10-dimethyloxy; 6H-dibenzo [ a, g ] quinolizine-2,3-diol,5,8,13,13a-tetrahydro-9, 10-dimethoxy. The invention patent refers to it as DMTHB for short. The structural formula in the formula (I) is demethylenetetrahydroberberine hydrochloride, which is a reduction product of demethyleneberberine hydrochloride and has a molecular formula: C19H21NO4, molecular weight: 327.2, the demethylenetetrahydroberberine hydrochloride can form various salts with inorganic or organic acids, in molecular form such as chloride, sulfate, phosphate, bromide, iodide, citrate, fumarate, maleate, malate and succinate.

The invention content is as follows:

in order to make up for the deficiency of the prior art in the aspect, the invention aims to provide a new application of the demethylenetetrahydroberberine hydrochloride.

The method comprises the following specific steps:

according to the invention, the prevention and treatment effects of the demethylenetetrahydroberberine hydrochloride (DMTHB) on thioacetamide-induced liver injury are observed by establishing thioacetamide-induced acute mild and severe and chronic liver injury animal models. Research results show that the hydrochloric acid Demethylenetetrahydroberberine (DMTHB) has preventive, protective and therapeutic effects on preparing drugs for preventing and/or treating thioacetamide-induced liver injury.

Specifically, the method comprises the following steps:

the invention compares the hepatostellate cytotoxicity of the demethylenetetrahydroberberine hydrochloride and the demethyleneberberine hydrochloride by an MTT method, and the research result is as follows: the demethylenetetrahydroberberine hydrochloride and the demethyleneberberine hydrochloride are respectively used for incubating hepatic stellate cells (LX2) for 24 hours, when the concentration of the demethylenetetrahydroberberine hydrochloride reaches 400 mu M, the hepatic stellate cells are remarkably inhibited, and when the concentration of the demethyleneberberine hydrochloride reaches 200 mu M, the hepatic stellate cells are already remarkably inhibited. The research result shows that the toxicity of the hydrochloric acid demethylenetetrahydroberberine is lower than that of the hydrochloric acid demethyleneberberine.

The invention detects the prevention and treatment effects of DMTHB on H2O 2-induced L02 cell injury at the cell level through an ROS fluorescent probe. The experiment adopts an ROS fluorescent probe method, and the cell level detection DMTHB probes the treatment effect of H2O2 induced liver injury simulating liver injury. Normal hepatocyte cell lines (L02) were inoculated into 6-well plates at cell numbers of 4X 104/cm2, and cultured for 24 hours while the cells were in the vigorous growth phase. The administration group is respectively administered with 40 mu mol/l BBR, DMB and DMTHB for treatment, meanwhile, each hole of the model group and the administration treatment group is kept at 2mMH2O2, after 1 hour of damage induction, ROS probe is added, cell nucleus is stained by DAPI, and the experiment result is observed by detecting fluorescence intensity through a fluorescence microscope. The experimental result shows that DMTHB has the best effect of eliminating ROS generated by liver cell injury under the same concentration.

According to the invention, by establishing an animal model of thioacetamide induced acute mild liver injury, under the condition of keeping the intragastric administration dosage of 50mg/kg, 150mg/kg and 300mg/kg, the prevention and treatment effects of different dosages of demethylenetetrahydroberberine hydrochloride (DMTHB) on thioacetamide induced acute liver injury are observed. Furthermore, the ICR experimental mouse is used as a research object to establish an animal model of thioacetamide induced acute mild liver injury, and the pharmacodynamic action of the demethylenetetrahydroberberine hydrochloride with different doses on the injury model is researched. The experimental mice are divided into a control group, a model group, a silymarin positive drug group of 150mg/kg and a demethylenetetrahydroberberine administration group of 50mg/kg, 150mg/kg and 300mg/kg, and are subjected to intragastric administration for 5 days, and meanwhile, the control group and the model group are respectively subjected to intragastric administration for 5 days by using excipients with the same dosage. Mice in the model group and the administration group were intraperitoneally injected with thioacetamide at a dose of 150mg/kg at the fourth day of gavage, and mice in the control group were injected with the same dose of vehicle for two days. After the last injection, fasting was not followed for 18 hours, after which the mice were sacrificed and liver biochemical markers were examined. After the second injection, the mouse status was observed. The mice in the model group are listened, the hair is sparse and dark, while the mice in the administration group are obviously more active, and the hair is dense and bright. Experimental result analysis shows that the hydrochloric acid demethylenetetrahydroberberine administration group can obviously reduce ALT and AST elevation caused by mouse thioacetamide induced acute liver injury, wherein the 50mg/kg dosage effect is the best. The result shows that the hydrochloric acid demethylenetetrahydroberberine has good effects of resisting thioacetamide induced acute mild liver injury and protecting liver function.

The invention establishes an thioacetamide induced acute severe liver injury animal model by taking an ICR experimental mouse as a research object, and researches and compares the preventive efficacy of silymarin, berberine hydrochloride, demethyleneberberine hydrochloride and demethylenetetrahydroberberine hydrochloride on the acute severe liver injury model. The experimental mouse is divided into a control group, a model group, a 50mg/kg silymarin positive drug group, a 50mg/kg berberine hydrochloride control group, a 50mg/kg demethyleneberberine hydrochloride control group and a demethylenetetrahydroberberine administration group with doses of 12.5mg/kg, 25mg/kg and 50mg/kg, and is perfused for 4 days, and the control group and the model group are separately perfused for 4 days by excipients with the same dose. Mice of the model group, the positive drug group, the BBR control group, the DMB control group, and the DMTHB administration group were intraperitoneally injected with thioacetamide at a dose of 300mg/kg on the third day of gavage, while the control group was injected with the same dose of vehicle, and were fasted for 18 hours after injection, after which the mice were sacrificed. The experimental result shows that after the high-dose thioacetamide is injected, the mortality rate of the mice in the model group is 50%, the mortality rate of the mice in the positive drug group is 50%, the mortality rate of the mice in the BBR group is 37.5%, the mortality rate of the mice in the DMTB group is 37.5%, the mortality rate of the mice in the low-dose DMTHB group is 25%, the mortality rate of the mice in the medium-dose DMTHB group is 12.5%, and the mortality rate of the mice in the high-dose DMTHB group is 0. Experimental result analysis shows that the demethylenetetrahydroberberine hydrochloride can effectively relieve severe hepatitis symptoms of mice, and under the same dosage and in the same administration mode, the treatment effect is superior to silymarin, berberine hydrochloride and demethyleneberberine hydrochloride, wherein the effect of 50mg/kg of DMTHB is the best. According to the detection of biochemical indexes of the liver, the result also shows that DMTHB can obviously reduce the elevation of ALT and AST caused by the acute liver injury induced by thioacetamide of mice. Research shows that the hydrochloric acid demethylenetetrahydroberberine has good effects of resisting thioacetamide induced acute severe liver injury and protecting liver function.

The invention detects and establishes the cell level H by a method of ROS fluorescent probe-DCFH-DA₂O₂Induced L02 cell injury model. Since thioacetamide cannot be directly absorbed by cells in vitro and the damage to the liver in vivo is partially related to the generation of ROS, H commonly adopted by researchers in daily experiments is selected during in vitro cell experiments₂O₂Induction of ROS production by hepatocytes mimics thioacetamide-induced liver damage. In the experiment, normal liver cell line (L02) was inoculated into 6-well plates at a cell number of 4X 104/cm2, cultured for 24 hours, and used at 2mM H when the cells were in the vigorous growth phase₂O₂Inducing for 1h to generate a large amount of ROS, and causing damage to liver cells.

The invention establishes an thioacetamide induced chronic liver injury animal model by taking an ICR experimental mouse as a research object, and researches the prevention and treatment effects of chronic liver fibrosis models of the demethylenetetrahydroberberine hydrochloride with different dosages. The experimental mice are divided into a control group, a model group, a silymarin positive drug group and a decamethylene tetrahydroberberine administration group with dosage of 50mg/kg, 150mg/kg and 300mg/kg, and are subjected to intragastric administration for 8 weeks, and meanwhile, the control group and the model group are respectively subjected to intragastric administration with excipient with the same dosage for 8 weeks. On the fourth day of gavage, mice of the model group, the positive drug group and the DMTHB administration group are injected with thioacetamide in an intraperitoneal mode at the dose of 100mg/kg once every other day, meanwhile, the control group is injected with excipient with the same dose, the dose of the thioacetamide is adjusted to 200mg/kg after the mice are adapted after one week of injection, the injection is performed once every other day, and the injection is continued for seven weeks. Mice were fasted for 18 hours after the last injection, after which they were sacrificed and the biochemical indices of the liver were examined. The experimental result shows that after the thioacetamide is injected for a long time, the mortality rate of the mice in the model group is 37.5 percent, the mortality rate of the mice in the positive medicine group is 37.5 percent, the mortality rate of the mice in the low-dose DMTHB group is 25 percent, the mortality rate of the mice in the medium-dose DMTHB group is 12.5 percent, and the mortality rate of the mice in the high-dose DMTHB group is 12.5 percent. Experimental result analysis shows that the demethylenetetrahydroberberine hydrochloride can effectively relieve chronic hepatic fibrosis symptoms of mice, wherein the DMTHB of 50mg/kg has the best effect. The results of biochemical index detection of the liver also show that DMTHB can obviously reduce the rising effects of ALT, AST and hydroxyproline caused by acute liver injury induced by thioacetamide in mice. Research shows that the hydrochloric acid demethylenetetrahydroberberine has good effects of resisting thioacetamide induced chronic liver injury and protecting liver function.

Advantageous effects

1. Thioacetamide (TAA; C)₂H₅NS) is an organic sulfur bactericide, can cause liver injury of experimental animals, and can cause the continuous injury state of the liver after being taken for a long time, and further can develop fibrosis and cirrhosis. Studies have demonstrated that thioacetamide-S-oxide, a potential metabolite of TAA, can alter cell permeability, Ca²⁺The absorption, in turn, amplifies the nucleoli and causes inhibition of mitochondrial activity, ultimately leading to liver necrosis. Damaged hepatocytes produce a variety of proinflammatory cytokines, generate Reactive Oxygen Species (ROS) in different tissues, and then activate HSCs to secrete more cytokines through the above-mentioned mechanism and induce various liver diseases such as hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. Research by Shiyingying et al shows that TAA-induced liver injury in mice has high success rate and is not easy to reverse, and the model is very similar to the process of liver injury and liver fibrosis of human in morphology, hemodynamics and biochemical metabolic change. (Shiyingying, Tanyanying, Tang Da Xuan, et al. shallow speaking about research progress of thioacetamide induced hepatic fibrosis animal model [ J]In addition, the thioacetamide is used for molding by an intraperitoneal injection mode, and the administration mode has good repeatability, strong controllability and easy quantification, so the invention adopts the thioacetamide for molding.

The laboratory reports the prevention and treatment effects of demethyleneberberine hydrochloride and berberine hydrochloride on thioacetamide-induced liver injury. However, the demethyleneberberine hydrochloride and berberine hydrochloride are in the form of quaternary ammonium salt, so that the solubility of the medicine is extremely low, and the absorption and utilization rate of the medicine is not high. The hydrochloric acid demethylenetetrahydroberberine is a reduction structure of demethyleneberberine, does not have a quaternary ammonium salt structure, and obviously improves the drug solubility and the drug absorption compared with the former two. The administration group of the demethylenetetrahydroberberine hydrochloride can obviously reduce ALT and AST elevation caused by liver injury induced by thioacetamide of mice no matter the dosage is high or low, and shows the treatment of the liver injury and the protection of liver function by the demethylenetetrahydroberberine hydrochloride. The drug effect of berberine hydrochloride, demethyleneberberine hydrochloride and demethylenetetrahydroberberine hydrochloride are compared in the acute severe liver injury model. The experimental results show that after a large amount of thioacetamide is injected in a short period, the death rate of the mice in the model group is 50%, the death rate of the mice in the positive drug group is 50%, the death rate of the mice in the BBR group of 50mg/kg is 37.5%, the death rate of the mice in the DMHB group of 50mg/kg is 37.5%, the death rate of the mice in the DMTHB group of 12.5mg/kg is 25%, the death rate of the mice in the DMTHB group of 25mg/kg is 12.5%, and the death rate of the mice in the DMTHB group of 50mg/kg is 0. The result shows that the demethylenetetrahydroberberine hydrochloride can play a role in quickly relieving the liver injury of a mouse under the action of a large amount of thioacetamide under a lower dosage and protecting the life of the mouse, and the drug effect is better. The results of biochemical index detection of liver also show that the pharmacological action of DMTHB in reducing ALT and AST increase caused by acute severe liver injury induced by thioacetamide in mice is also more obvious than that of BBR and DMB. According to HE staining analysis, obvious cell necrosis phenomenon appears in the liver of a mouse thioacetamide-induced liver injury model group, while the administration group is improved. The hydrochloric acid demethylenetetrahydroberberine administration group has the most obvious effect on relieving the above hepatic cell diseases, and can restore the hepatic cell diseases to a level close to normal.

In conclusion, the demethylenetetrahydroberberine hydrochloride administration group can remarkably reduce the ALT and AST increasing effects caused by thioacetamide-induced liver injury of mice, and the improvement effects of the berberine hydrochloride administration group and the demethyleneberberine hydrochloride administration group on the ALT and AST increasing effects are not as remarkable. The result shows that the effects of the demethylenetetrahydroberberine hydrochloride on resisting the liver injury of the thioacetamide and protecting the liver function are obviously superior to those of a berberine hydrochloride administration group and the demethyleneberberine hydrochloride. Because the absorption of the medicine is enhanced, the anti-inflammatory activity and the antioxidant activity are enhanced, the administration dosage and the administration frequency can be reduced in the administration treatment process, the treatment process is optimized, and the treatment effect of patients is enhanced.

2. The hydrochloric acid demethylenetetrahydroberberine is a reduction product of hydrochloric acid demethyleneberberine, and compared with the hydrochloric acid berberine and the hydrochloric acid demethyleneberberine, the hydrochloric acid demethylenetetrahydroberberine has better water solubility, and the invention finds that the hydrochloric acid demethylenetetrahydroberberine has lower toxicity, better anti-inflammatory activity and better antioxidant activity than the hydrochloric acid berberine and the hydrochloric acid demethyleneberberine.

3. The invention adopts the mode of intragastric administration, and the absorption problems of the demethyleneberberine hydrochloride and the berberine hydrochloride generally need to adopt a large-dose oral administration mode, and the product greatly reduces the effective dose, thereby improving the compliance of patients from the aspect of administration.

Drawings

FIG. 1 shows that demethylenetetrahydroberberine hydrochloride and demethyleneberberine hydrochloride inhibit the synthesis of H in L02 cell₂O₂Induction of ROS production

FIG. 2 Thioacetamide induced pathological morphological change of liver tissue of mice with acute mild liver injury

FIG. 3 is a diagram of pathological morphological changes of liver tissues of mice with acute severe liver injury induced by thioacetamide

FIG. 4 photograph of liver of mice with severe acute liver injury induced by thioacetamide

FIG. 5 analysis of cytotoxicity of demethylenetetrahydroberberine hydrochloride on hepatic stellate cell LX2

FIG. 6 the pathological morphological change of liver tissue of mice with chronic liver injury induced by thioacetamide

FIG. 7 Thioacetamide induced chronic liver injury mouse liver tissue Massoin staining

FIG. 8 Thioacetamide induced chronic liver injury mouse liver tissue α -sma

FIG. 9 photograph of liver of mice with thioacetamide-induced chronic liver injury

Detailed Description

The following examples may assist those skilled in the art in a more complete understanding of the present invention, but are not intended to limit the invention in any way.

Term(s) for

DMTHB: hydrochloric acid demethylenetetrahydroberberine

4H-DMB: hydrochloric acid demethylenetetrahydroberberine

BBR: berberine hydrochloride

DMB: hydrochloric acid demethyleneberberine

ALT/AST: glutamic-pyruvic transaminase/glutamic-oxalacetic transaminase

MDA: malondialdehyde

GSH: glutathione

HYP: hydroxyproline

IP: abdominal injection

IG: gavage stomach

Example 1 cell-level antioxidant Activity of Desmethylenetetrahydroberberine hydrochloride (DMTHB)

The method comprises the following steps: the ROS probe method is utilized to detect the ROS eliminating capacity of the demethylenetetrahydroberberine hydrochloride at the cellular level. We first pre-incubated normal hepatocytes (L02) with 40. mu.M of demethyleneberberine hydrochloride, demethylenetetrahydroberberine hydrochloride, respectively, for 4 h. Then adding hydrogen peroxide H into the culture dish₂O₂The liver cell line (L02) was stimulated for 1h (1mM) to produce large amounts of ROS, which resulted in cell damage. The ROS scavenging effect is observed under a fluorescence microscope by utilizing the principle that a fluorescence probe DCFH-DA (50 mu M) is combined with ROS to generate fluorescence.

As a result: fluorescence microscopy showed that DMTHB at various concentrations significantly inhibited ROS production by peroxide in L02 cells, as shown in fig. 1.

Example 2 protective Effect of Desmethylenetetrahydroberberine hydrochloride (DMTHB) on Thioacetamide induced acute mild liver injury

The method comprises the following steps: male ICR mice, weighing 22-24g, were randomly divided into 6 groups of 8 mice each. Each group is normal control group, model group, Silymarin (SILY) positive drug group (150mg/kg), demethylenetetrahydroberberine hydrochloride low dose group (50mg/kg), demethylenetetrahydroberberine hydrochloride medium dose group (150mg/kg), and demethylenetetrahydroberberine hydrochloride high dose group (300 mg/kg). The administration group was continuously gavage for 5 days, while the control group and the model group were administered the same dose of vehicle for 5 days. Mice in the model group and the administration group were intraperitoneally injected with thioacetamide at a dose of 150mg/kg at the fourth day of gavage, and mice in the control group were injected with the same dose of vehicle for two days. After the last injection, the mice were sacrificed after fasting for 18 hours without water deprivation, and the liver tissue of the same site was taken from each mouse and ground in physiological saline on ice to prepare a 10% liver homogenate. Lipid peroxide MDA levels in liver tissue were determined according to kit requirements.

As a result: TABLE 1 Demethylenetetrahydroberberine hydrochloride antioxidant action on thioacetamide-induced acute mild liver injury liver

(n＝8，mean±variance，#p<0.05、##p<0.01、###p<0.001、####p<0.0001VScontrol，*p<0.05、**p<0.01、***p<0.001、****p<0.0001VSmodel)

Example 3 protective Effect of Demethylenetetrahydroberberine hydrochloride (DMTHB) on Thioacetamide induced acute mild liver injury pathologic Change

The method comprises the following steps: male ICR mice, weighing 22-24g, were randomly divided into 6 groups of 8 mice each. Each group is normal control group, model group, Silymarin (SILY) positive drug group (150mg/kg), demethylenetetrahydroberberine hydrochloride low dose group (50mg/kg), demethylenetetrahydroberberine hydrochloride medium dose group (150mg/kg), and demethylenetetrahydroberberine hydrochloride high dose group (300 mg/kg). The administration group was continuously gavage for 5 days, while the control group and the model group were administered the same dose of vehicle for 5 days. Mice in the model group and the administration group were intraperitoneally injected with thioacetamide at a dose of 150mg/kg at the fourth day of gavage, and mice in the control group were injected with the same dose of vehicle for two days. After the last injection, the mice are sacrificed after fasting for 18 hours without water prohibition, and different mice take liver tissues of the same part, are fixed by 10 percent paraformaldehyde, adopt an HE staining method and then use a microscope for pathological examination.

As a result: the liver pathology research result shows that the liver morphology of the control group is normal, the liver tissue structure of the model group is pathologically changed, partial liver cell focal necrosis and inflammatory cell infiltration occur, and the liver cell boundary begins to be fuzzy. The silymarin positive drug group (150mg/kg), the demethylenetetrahydroberberine hydrochloride low dose group (50mg/kg), the demethylenetetrahydroberberine hydrochloride medium dose group (150mg/kg) and the demethylenetetrahydroberberine hydrochloride high dose group (300mg/kg) all can relieve the liver cell pathological changes and enable the liver cell pathological changes to be recovered to be close to normal levels, as shown in figure 2.

Example 4 protective Effect of Demethylenetetrahydroberberine hydrochloride (DMTHB) on thioacetamide induced acute mild liver function

The method comprises the following steps: male ICR mice, weighing 22-24g, were selected for this experiment and randomly divided into 6 groups of 5 mice each. Each group is normal control group, model group, Silymarin (SILY) positive drug group (150mg/kg), demethylenetetrahydroberberine hydrochloride low dose group (50mg/kg), demethylenetetrahydroberberine hydrochloride medium dose group (150mg/kg), and demethylenetetrahydroberberine hydrochloride high dose group (300 mg/kg). The administration group was continuously gavage for 5 days, while the control group and the model group were administered the same dose of vehicle for 5 days. Mice in the model group and the administration group were intraperitoneally injected with thioacetamide at a dose of 150mg/kg at the fourth day of gavage, and mice in the control group were injected with the same dose of vehicle for two days. After the last injection, fasting is carried out for 18 hours without water prohibition, then the mice are killed, eyeballs are picked up for blood sampling, the blood is kept still for 30min at room temperature, centrifugation is carried out at 3000rpm/min at 4 ℃, serum is separated conventionally, and ALT and AST in the serum are analyzed and measured by adopting a kit of Nanjing Jiangguan company.

As a result: TABLE 2 Desmethylenetetrahydroberberine hydrochloride (DMTHB) on thioacetamide induced acute mild liver injury mice ALT, AST level changes

(n＝8，mean±variance，#p<0.05、##p<0.01、###p<0.001、####p<0.0001VScontrol，*p<0.05、**p<0.01、***p<0.001VS model)

Example 5 protective Effect of Demethylenetetrahydroberberine hydrochloride (DMTHB) on Thioacetamide induced acute severe liver injury pathological changes

The method comprises the following steps: male ICR mice, weighing 22-24g, were randomly divided into 5 groups of 8 mice each. The control group, the model group, the silymarin positive drug group of 50mg/kg, the berberine hydrochloride comparison group of 50mg/kg, the demethyleneberberine hydrochloride comparison group of 50mg/kg and the demethylenetetrahydroberberine administration group of 12.5mg/kg, 25mg/kg and 50mg/kg are respectively used for gastric lavage for 4 days, and the control group and the model group are respectively used for gastric lavage for 4 days by giving excipients with the same dose. And (3) injecting thioacetamide into the abdominal cavity of mice of a model group, a positive medicine group, a BBR (BBR) comparison group, a DMB comparison group and a DMTHB (dimethyl-tetrahydrofolate) administration group at a dose of 300mg/kg on the third day of intragastric administration, injecting an excipient at the same dose into the control group, fasting the mice for 18 hours without water prohibition after injection, killing the mice, taking liver tissues of the same part of different mice, fixing the different mice by using 10% paraformaldehyde, and performing pathological examination by using a microscope by adopting an HE (Hehe staining) method.

As a result: the liver pathology research result shows that the liver morphology of the normal control group is not abnormal, most liver cell focal necrosis appears in the model group, a large amount of inflammatory cells infiltrate, the boundary of the liver cells is uneven, and the structure of the liver tissue is obviously damaged in the whole view. The silymarin positive drug group (50mg/kg), the berberine hydrochloride group (50mg/kg), the demethyleneberberine hydrochloride group (50mg/kg), the demethylenetetrahydroberberine hydrochloride low dose group (12.5mg/kg), the demethylenetetrahydroberberine hydrochloride medium dose group (25mg/kg) and the demethylenetetrahydroberberine hydrochloride high dose group (50mg/kg) can all relieve the above liver cell pathological changes, wherein each demethylenetetrahydroberberine hydrochloride group has the optimal effect, as shown in figure 3.

Example 6 protective Effect of Demethylenetetrahydroberberine hydrochloride (DMTHB) on Thioacetamide induced acute severe liver injury

The method comprises the following steps: male ICR mice, weighing 22-24g, were randomly divided into 5 groups of 8 mice each. The control group, the model group, the silymarin positive drug group of 50mg/kg, the berberine hydrochloride comparison group of 50mg/kg, the demethyleneberberine hydrochloride comparison group of 50mg/kg and the demethylenetetrahydroberberine administration group of 12.5mg/kg, 25mg/kg and 50mg/kg are respectively used for gastric lavage for 4 days, and the control group and the model group are respectively used for gastric lavage for 4 days by giving excipients with the same dose. On the third day of gavage, mice of a model group, a positive medicine group, a BBR (brain-brain receptor) comparison group, a DMTHB (DMTHB) comparison group and a DMTHB administration group are injected with thioacetamide in an intraperitoneal mode at a dose of 300mg/kg, meanwhile, the control group is injected with excipient with the same dose, the mice are fasted for 18 hours after injection, then the mice are killed, eyeballs are picked up for blood taking, after standing for 30 minutes at room temperature, the temperature is 4 ℃ and 3000rpm are centrifuged for 10 minutes, serum is separated conventionally, and ALT and AST in the serum are analyzed and determined by adopting a kit of Nanjing.

As a result: TABLE 3 reduction of mortality in mice with severe acute liver injury induced by thioacetamide by demethylenetetrahydroberberine hydrochloride

TABLE 4 influence of demethylenetetrahydroberberine hydrochloride on the treatment of thioacetamide induced liver index, serum ALT and AST in mice with severe acute liver injury

(n＝8，mean±variance，#p<0.05、##p<0.01、###p<0.001、####p<0.0001VScontrol，*p<0.05、**p<0.01、***p<0.001VSmodel)

The experimental result shows that after a large amount of thioacetamide is injected in a short time, the mortality rate of the mice in the model group is 50%, the mortality rate of the mice in the positive drug group is 50%, the mortality rate of the mice in the BBR group is 37.5%, the mortality rate of the mice in the DMTB group is 37.5%, the mortality rate of the mice in the low-dose DMTHB group is 25%, the mortality rate of the mice in the medium-dose DMTHB group is 12.5%, and the mortality rate of the mice in the high-dose DMTHB group is 0. Experimental result analysis shows that the demethylenetetrahydroberberine hydrochloride can play a role in quickly relieving liver injury of mice under a large amount of thioacetamide under a lower dose than berberine hydrochloride and demethyleneberberine hydrochloride, and can better protect the lives of the mice. The detection result of biochemical indexes of the liver also shows that the DMTHB can effectively reduce the elevation of ALT and AST caused by acute severe liver injury induced by thioacetamide of mice under lower dosage, and the result also shows that the DMTHB has more significant difference in improving the biochemical indexes than BBR and DMB by applying statistical analysis. Research shows that the demethylenetetrahydroberberine hydrochloride has better effect of resisting thioacetamide induced acute severe liver injury than berberine hydrochloride and demethyleneberberine hydrochloride, and the effective dose is lower.

Example 7 Demethylenetetrahydroberberine hydrochloride toxicity test on hepatic stellate cells

The method comprises the following steps: the experiment adopts an MTT cell viability measuring method to detect the toxic effect of the demethylenetetrahydroberberine hydrochloride on hepatic stellate cells at a cell level. Inoculating hepatic stellate cell line (LX2) into 96-well plate according to cell number 4 × 104/cm2, culturing for 18 hr, adding demethyleneberberine hydrochloride and demethylenetetrahydroberberine hydrochloride with different concentrations when cell growth is observed in vigorous growth stage, and administering for 24 hr. Then the culture medium is discarded, the serum-free culture medium containing MTT (0.5mg/ml) is substituted, the culture medium is continuously incubated for 4 hours and then taken out, the culture medium is completely sucked out by using a syringe, 150 mu l of DMSO is added into each hole, the culture medium and the blue-violet crystalline formazan are shaken on a shaking table for 30min to fully dissolve the blue-violet crystalline formazan, and the absorbance is measured at 490 nm.

The results are shown in FIG. 5, and the toxicity of demethylenetetrahydroberberine hydrochloride to hepatic stellate cells is lower than that of demethyleneberberine hydrochloride

Example 7 protective Effect of Demethylenetetrahydroberberine hydrochloride on Thioacetamide-induced chronic hepatic injury

The method comprises the following steps: male ICR mice, weighing 22-24g, were randomized into 6 groups of 8 mice each. The groups are respectively a control group, a model group, a silymarin positive medicine group and a decamethylene tetrahydroberberine administration group with dosage of 50mg/kg, 150mg/kg and 300mg/kg, and are intragastrically administered for 8 weeks, and simultaneously the control group and the model group are respectively intragastrically administered with excipient with the same dosage for 8 weeks. On the fourth day of gavage, mice of the model group, the positive drug group and the DMTHB administration group are injected with thioacetamide in an intraperitoneal mode at the dose of 100mg/kg once every other day, meanwhile, the control group is injected with excipient with the same dose, the dose of the thioacetamide is adjusted to 200mg/kg after the mice are adapted after one week of injection, the injection is performed once every other day, and the injection is continued for seven weeks. After the last injection, the mice are fasted for 18 hours without water prohibition, then the mice are killed, eyeballs are picked up for blood sampling, the blood is kept still for 30min at room temperature, the centrifugation is carried out at 3000rpm/min at 4 ℃, the serum is separated conventionally, and ALT and AST in the serum are analyzed and measured by adopting a kit of Nanjing Jiangguan company. Homogenating liver tissues of the same part of different mice, and detecting the change of the hydroxyproline level in the liver by adopting a kit of Nanjing construction company.

As a result: TABLE 5 Demethylenetetrahydroberberine hydrochloride to reduce mortality in mice with thioacetamide-induced chronic liver injury

TABLE 6 reduction of thioacetamide-induced chronic hepatic injury mouse serum ALT, AST and hydroxyproline levels by demethylenetetrahydroberberine hydrochloride

The experimental result shows that after the thioacetamide is injected for a long time, the mortality rate of the mice in the model group is 37.5 percent, the mortality rate of the mice in the positive medicine group is 37.5 percent, the mortality rate of the mice in the low-dose DMTHB group is 25 percent, the mortality rate of the mice in the medium-dose DMTHB group is 12.5 percent, and the mortality rate of the mice in the high-dose DMTHB group is 12.5 percent. Experimental result analysis shows that the demethylenetetrahydroberberine hydrochloride can effectively relieve chronic hepatic fibrosis symptoms of mice. The results of biochemical index detection of the liver also show that DMTHB can obviously reduce the elevation of ALT, AST and hydroxyproline caused by the slow liver injury induced by thioacetamide of mice. Research shows that the hydrochloric acid demethylenetetrahydroberberine has good effects of resisting thioacetamide induced chronic liver injury, inhibiting the liver fibrosis process and protecting the liver function.

Because liver fibrosis is a long-term liver injury process, previous researches of the invention show that the demethylenetetrahydroberberine hydrochloride has superior effects in preventing and treating acute liver injury compared with berberine hydrochloride and demethyleneberberine hydrochloride, so that a comparison group of the demethylenetetrahydroberberine hydrochloride and the demethyleneberberine hydrochloride is not added in the experiment, whether the DMTHB is suitable for being taken as a long-term medicament is only confirmed, and experimental results prove that the DMTHB also has excellent liver protection effect on patients who are contacted with liver injury factors for a long time.

Example 8 protective Effect of Demethylenetetrahydroberberine hydrochloride on Thioacetamide-induced pathological changes in chronic hepatic injury

The method comprises the following steps of randomly dividing male ICR mice into 6 groups with weight of 22-24g, randomly dividing the mice into 8 groups, wherein each group comprises a control group, a model group, a silymarin positive drug group, a demethylenetetrahydroberberine administration group with dosage of 50mg/kg, 150mg/kg and 300mg/kg, performing intragastric administration for 8 weeks, simultaneously administering excipients with the same dosage to the control group and the model group, performing intragastric administration for 8 weeks, starting to perform intraabdominal injection on the mice of the model group, the positive drug group and the DMTHB administration group on the fourth day of intragastric administration with thioacetamide with dosage of 100mg/kg, performing intraperitoneal injection once every other day, simultaneously injecting excipients with the same dosage to the control group, performing injection for one week until the mice are adapted, then adjusting the dosage of the thioacetamide to 200mg/kg, performing injection once every other day, continuously injecting for seven weeks, fasting the mice for 18 hours after the last injection, then killing the mice, taking liver tissues of the same parts from different mice, using HE staining and Massion staining after being fixed by using an immunohistochemical index to detect liver fibrosis by a microscope, and performing pathological liver fibrosis examination by using a α -sma microscope.

The results are that as shown in fig. 6, the liver tissue structure of the normal group mice is clear and complete, the liver cells are arranged in a rope shape around the central vein, the region of the junction is free from inflammatory cell infiltration, the central interstitium is free from fibrous tissue proliferation, the liver tissue structure of the model group mice is incomplete, the collagenous fibers are obviously proliferated, a plurality of false lobules are generated, and more inflammatory cells are infiltrated, the liver tissue structure of each dosage group of the hydrochloric acid demethylenetetrahydroberberine administration group is clear, the collagenous fibers are slightly proliferated, no obvious false lobules are observed, and the inflammatory cell infiltration is obviously reduced, as shown in fig. 7, only a small amount of light blue fibers which are thin and short are seen at the edge of the blood vessel are normal, and no fibrous proliferation occurs at other parts, the hepatic collagens of the model group mice are distributed more, the fibers extend from the central vein to the peripheral area, and the fibers are obviously thicker compared with the normal group, and the fibrosepta and obvious false lobules are formed, while the symptoms of the administration group are obviously reduced, the hepatic fibers are only the region of the junction and the hepatic parenchyma, the liver fibers are not formed, the fibers, the fiber septa and the normal group α - α, and the liver tissue structure of the normal group has no obvious effect of inhibiting the chronic liver fibrosis, even has no collagen growth, and no yellow tissue damage, and no effect of inhibiting effect of the chronic liver fibrosis is observed in the central vein inhibiting the central vein, and the chronic liver fibrosis.

Claims

1. Application of demethylenetetrahydroberberine hydrochloride in preparing medicine for preventing or treating liver injury is provided.