Application of biochanin A in preparation of medicine for treating APAP-induced acute liver injury

Abstract

The invention relates to an application of biochanin A in preparation of a medicine for treating APAP-induced acute liver injury. Animal experiments prove that the natural plant compound biochanin A can alleviate acetaminophen (APAP)-induced acute liver injury and has the beneficial effect of effectively reducing the acetaminophen-induced acute liver injury, which is mainly shown in that the biochanin A can obviously inhibit acetaminophen-induced hepatic cell focal necrosis, and the biochanin A can be used for treating acute liver injury induced by acetaminophen, so that the biochanin A can be used for treating acute liver injury induced by acetaminophen. The expression level of cell factors in the liver is reduced, the hepatotoxicity of the acetaminophen is relieved, and the compound can be used for preparing the medicine for treating the acute liver injury caused by the acetaminophen.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to the application of the natural plant compound biochanin A in the preparation of a drug for treating APAP-induced acute liver injury. Background Art

[0002] Acetaminophen (APAP), as an antipyretic and analgesic drug, is widely used clinically for treating colds, fevers, pain, etc. Overdose of APAP can cause dose-related hepatocyte necrosis, and the resulting drug-induced liver injury is the main cause of acute liver failure in Western developed countries. This disease progresses rapidly and has a high mortality rate. Compared with acute liver injury caused by other factors, APAP drug-induced liver injury has a longer cure cycle and is more likely to develop into chronic liver injury. With the abuse of APAP drugs, the incidence of drug-induced liver injury (DILI) has gradually increased, seriously threatening people's lives and health. At present, N-acetyl-L-cysteine (NAC) is the only clinically approved antidote for drug-induced liver injury, but it can cause side effects such as bronchospasm, nausea, vomiting, etc., and the therapeutic effect has limitations. Therefore, finding a safe, effective and less side-effect alternative drug for treating APAP-induced acute liver injury is still a clinical problem that needs to be solved urgently.

[0003] Biochanin A (Bio A) is a light yellow powder, soluble in organic solvents such as ethanol and ether, with a melting point of 210-213 °C, and the molecular formula is C 16 H 12 O5, and the chemical structure is shown in the following formula (Ⅰ):

[0004]

[0005] It is reported that biochanin A, as an isoflavone compound, is an O-methylated isoflavone compound and is widely present in some edible plants such as soybeans, red clover, alfalfa, peanuts, and chickpeas, especially with the highest content in red clover. The molecular structure of biochanin A is similar to that of animal estrogen, and it can competitively bind to estrogen receptors to exert estrogen-like effects. It is an important phytoestrogen and has various beneficial effects on the health of humans and animals. It can be used for the prevention and treatment of diseases such as osteoporosis and menopausal syndrome. In addition, biochanin A also has various pharmacological effects such as anti-tumor, anti-inflammatory, antibacterial, hypoglycemic, antioxidant, and neuroprotective effects. Chinese Patent CN108938712A discloses the application of chickpea extract in the preparation of drugs for treating androgen-dependent diseases. The 5α-reductase inhibitor screened from chickpeas has good application prospects in the treatment and prevention of androgen-dependent diseases such as benign prostatic hyperplasia and prostate cancer. However, there is still no relevant report on whether biochanin A can be used to treat APAP-induced liver injury symptoms. The present invention mainly explores the effect of biochanin A on APAP-induced acute liver injury and the mechanism of alleviating APAP-induced acute liver injury symptoms. Summary of the Invention

[0006] The purpose of the present invention is to provide a new use of biochanin A in the medical field. The present invention provides a new application of a natural plant compound in the preparation of a drug for treating acetaminophen-induced acute liver injury.

[0007] The present invention provides an application of a natural plant compound, biochanin A, in the preparation of a drug for treating acetaminophen-induced acute liver injury. The natural plant compound biochanin A (Biochanin A, Bio A) is a natural phytoestrogen, an isoflavone derived from leguminous plants such as chickpeas and red clover, and has estrogen-like effects. The molecular formula is C 16 H 12 O5, and the chemical structure is shown in the following formula (Ⅰ):

[0008]

[0009] According to some embodiments of the present invention, the natural plant compound biochanin A can alleviate acetaminophen-induced acute liver injury symptoms.

[0010] According to some embodiments of the present invention, the natural plant compound biochanin A can reduce the serum ALT and AST levels of acetaminophen-induced acute liver injury.

[0011] According to some embodiments of the present invention, the natural plant compound biochanin A can alleviate the pathological morphological changes of the liver tissue of mice induced by acetaminophen.

[0012] According to some embodiments of the present invention, the natural plant compound biochanin A can inhibit acetaminophen-induced hepatocyte necrosis.

[0013] According to some embodiments of the present invention, the compound biochanin A can reduce acetaminophen hepatotoxicity.

[0014] According to some embodiments of the present invention, the natural plant compound biochanin A can inhibit the production of inflammatory factors in the lungs of mice induced by acetaminophen. The inflammatory factors include but are not limited to TNF-α, Ccl-2, and Ccl-7.

[0015] Therefore, the present invention proposes that the natural plant compound biochanin A can be applied to the preparation of drugs for treating acetaminophen-induced acute liver injury.

[0016] A drug for treating acetaminophen-induced acute liver injury, comprising the natural plant compound biochanin A and a pharmaceutically acceptable carrier, excipient, adjuvant, or a combination thereof.

[0017] Use of the natural plant compound biochanin A or the pharmaceutical composition in the preparation of a drug, wherein the drug can be used for preventing, treating, or reducing acetaminophen-induced acute liver injury.

[0018] The acetaminophen-induced acute liver injury described in the present invention is a condition caused by an oxidative stress response triggered by toxic intermediates (such as NAPQI) generated during the liver metabolism of APAP, resulting in hepatocyte necrosis, inflammatory response, and liver function impairment. Through the above technical solutions, the present application first applies the natural plant compound biochanin A to the prevention and treatment of APAP-induced acute liver injury. Experimental results prove that biochanin A can significantly reduce APAP-induced liver injury and alleviate hepatocyte necrosis and inflammatory response. Taking the treatment of a mouse model with acetaminophen combined with biochanin A as an example, the present invention proves that the compound biochanin A can significantly improve the condition of acetaminophen-induced acute liver injury, and discloses a new use of the compound biochanin A in the prevention and treatment of acetaminophen-induced acute liver injury.

[0019] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0020] In this invention, a mouse model of acute liver injury induced by paracetamol was prepared and treated with the compound biochanin A. By detecting and systematically statistically analyzing the levels of serum ALT and AST in mice, the pathological morphological changes of liver tissues, and the expression levels of liver inflammatory factors, it was found that biochanin A could reduce the levels of serum ALT and AST in acute liver injury induced by paracetamol, alleviate the pathological morphological changes of liver tissues in APAP model mice, inhibit the focal necrosis of hepatocytes induced by paracetamol, and inhibit the production of pulmonary inflammatory factors (such as TNF-α, Ccl-2, and Ccl-7) in mice induced by paracetamol. This invention reveals that the phytoestrogen biochanin A has a protective effect on APAP-induced acute liver injury and relieves the symptoms of acute liver injury in mice induced by paracetamol by exerting an anti-inflammatory effect. It can be used as a potential new drug for the treatment of acute liver injury induced by paracetamol. In addition, this invention uses the plant-derived natural compound biochanin A to treat acute liver injury induced by paracetamol, which is safe, non-toxic, and has no side effects, facilitating clinical application and promotion. Brief Description of the Drawings

[0021] Figure 1 It is a statistical result chart of serum transaminases in mice of the acute liver injury model induced by APAP protected by biochanin A. The meanings of the annotation symbols are as follows: The data were analyzed by one-way analysis of variance (One-way ANOVA), and * indicates that the difference is statistically significant, p < 0.05.

[0022] Figure 2 It is a pathological result chart of liver tissue injury in mice of the acute liver injury model induced by APAP protected by biochanin A, including the HE staining chart of the morphological changes of liver tissues and the quantitative statistical chart of the necrosis area. The meanings of the annotation symbols are as follows: The data were analyzed by one-way analysis of variance (One-way ANOVA), and * indicates that the difference is statistically significant, p < 0.05. Scale bar: 100μm.

[0023] Figure 3 It is a statistical result chart of the mRNA expression levels of liver inflammatory factors in mice of the acute liver injury model induced by APAP inhibited by biochanin A. The meanings of the annotation symbols are as follows: The data were analyzed by one-way analysis of variance (One-way ANOVA), * indicates that the difference is statistically significant, p < 0.05, and ** indicates that the difference is statistically significant, p ≤ 0.01. Detailed Embodiments

[0024] Example 1

[0025] Preparation of Animal Model

[0026] 1.1 Experimental Animals

[0027] Male C57BL / 6J mice aged 6 - 8 weeks with a body weight of 20 - 22 g were selected for the experiment and purchased from Beijing Spebefu Laboratory Animal Technology Co., Ltd.

[0028] 1.2 Reagents and Instruments

[0029] Biochanin A (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.); sesame oil (purchased from Yuanye Biotechnology Co., Ltd.); phosphate buffer (purchased from Shanghai Gibco Biotechnology Co., Ltd.); paracetamol (purchased from Shanghai Macklin Biochemical Technology Co., Ltd.); ALT kit (purchased from Nanjing Jiancheng Bioengineering Institute); AST kit (purchased from Nanjing Jiancheng Bioengineering Institute).

[0030] 1.3 Preparation Method of Animal Model

[0031] Twenty-five C57BL / 6J mice were randomly divided into 4 groups, namely the control group (7 mice), the paracetamol group (APAP, 7 mice), the biochanin A group (7 mice), and the paracetamol combined with biochanin A group (7 mice). The administration dose of APAP was 300 mg / kg, and the administration dose of biochanin A was 50 mg / kg. In the combined group, biochanin A was dissolved in sesame oil and dissolved by ultrasound, and APAP powder was dissolved in PBS solution and heated at 60 °C to fully dissolve it. Biochanin A and APAP were simultaneously administered to mice by gavage, and samples were taken 24 h later. All mice were kept under conditions of 12 h light and 12 h darkness, with sufficient supply of water and food, and the room temperature was maintained at 22 - 24 °C.

[0032] Example 2 Biochanin A can reduce the serum ALT and AST levels in APAP-induced acute liver injury

[0033] Biochemical detection of serum ALT and AST levels

[0034] 2.1 Experimental Method

[0035] (1) The mice were sacrificed, and blood was collected in a centrifuge tube containing ethylenediaminetetraacetic acid (EDTA) anticoagulant, centrifuged (13000 RPM, 15 min, 4 °C), and the supernatant was aspirated. Serum transaminase was measured according to the ALT and AST kits.

[0036] (2) The serum sample was diluted 5 times with PBS, and 10 μL of the diluted serum was taken for detection.

[0037] (3) According to the detection instructions, 250 μL of the mixture of reagent 1 and reagent 2 (reagent 1: reagent 2 = 4:1) was added to each serum sample.

[0038] (4) Use a multifunctional microplate reader to read the absorbance at a wavelength of 340 nm at different incubation time points, and calculate the ALT and AST values of the samples according to the formula in the instruction manual. The data was analyzed using a t-test.

[0039] 2.2 Experimental results

[0040] Figure 1 are the serum ALT and AST levels of each group of mice. Compared with the control group, treatment of mice with APAP led to a significant increase in serum ALT and AST levels. Treatment of APAP mice with biochanin A could significantly reduce the levels of ALT and AST, and there were statistical differences between groups. The above results indicate that biochanin A can alleviate the symptoms of APAP-induced acute liver injury in mice and reduce the serum ALT and AST levels induced by APAP.

[0041] Example 3 Biochanin A can inhibit APAP-induced hepatocyte necrosis

[0042] Preparation and staining of pathological paraffin sections

[0043] 3.1 Reagents and instruments

[0044] HE staining solution (purchased from Beijing Leagene Biotechnology Co., Ltd.), absolute ethanol (purchased from Guangdong Guanghua Sci-Tech Co., Ltd.), xylene (purchased from Guangdong Guanghua Sci-Tech Co., Ltd.), paraffin (purchased from Leica), 4% paraformaldehyde (purchased from Beijing Leagene Biotechnology Co., Ltd.), paraffin slicer (purchased from Leica).

[0045] 3.2 Experimental methods

[0046] (1) Dehydration of samples and preparation of paraffin sections

[0047] Sacrifice the mice and collect the livers, place them in 4% paraformaldehyde solution, and transfer them to 70% absolute ethanol for standby after fixation for 48 h. The dehydration, clearing, and wax infiltration processes are as follows: 80% ethanol for 2 h - 90% ethanol for 2 h - 95% ethanol for 10 h - 100% ethanol for 0.5 h - 100% ethanol for 0.5 h - 100% ethanol for 0.5 h - xylene for 0.5 h - xylene for 0.5 h - xylene for 0.5 h - paraffin for 0.5 h - paraffin for 0.5 h - paraffin for 1 h. After the above steps are completed, paraffin embedding is carried out to prepare paraffin sections, which are air-dried for standby.

[0048] (2) HE staining

[0049] The paraffin sections were baked in an incubator at 60 °C for 1.5 h, and HE staining was completed according to the following procedure: xylene for 3 min - xylene for 3 min - xylene for 3 min - absolute ethanol for 2 min - absolute ethanol for 2 min - 95% ethanol for 1 min - 90% ethanol for 1 min - 80% ethanol for 1 min - draining for 30 s - hematoxylin staining solution for 10 min - ultrapure water for 5 s - 1% hydrochloric acid alcohol for 10 s - tap water for 10 min - eosin solution for 3 min - 80% ethanol for 10 s - 90% ethanol for 3 s - 95% ethanol for 2 min - absolute ethanol for 3 min - absolute ethanol for 3 min - absolute ethanol for 3 min - xylene for 3 min - xylene for 3 min - xylene for 3 min. Sealing with neutral resin. Six fields of view were randomly photographed for each HE section under an optical microscope, and the necrotic area was statistically analyzed using the t-test.

[0050] 3.3 Experimental results

[0051] Figure 2 The typical images of HE staining of liver sections of mice in each group and the quantitative statistical results of the necrotic area are shown. The APAP treatment group showed significant liver pathological changes, including extensive necrosis and disappearance of the outline, and the necrotic area (within the dotted line box) was approximately 30%. The biochanin A treatment group significantly inhibited the liver pathological changes, and the necrotic area decreased to less than 20%, and the difference between groups was statistically significant. The above results indicate that biochanin A can alleviate the liver pathological changes induced by APAP and inhibit hepatocyte necrosis. Scale bar: 100 μm.

[0052] Example 4 Biochanin A can inhibit the production of inflammatory factors in the lungs of mice induced by APPA

[0053] Detection of the expression level of liver inflammatory factors

[0054] 4.1 Reagents and instruments

[0055] TRIZOL lysis solution (purchased from Invitrogen), isopropanol (purchased from Guangdong Guanghua Sci-Tech Co., Ltd.), chloroform (purchased from Guangdong Guanghua Sci-Tech Co., Ltd.), absolute ethanol (purchased from Guangdong Guanghua Sci-Tech Co., Ltd.), DEPC water (purchased from Sigma), SYBR Green fluorescent dye (purchased from Toyobo), ReverTra Ace qPCR RT Kit (purchased from Toyobo), PCR instrument (purchased from Eppendorf), fluorescence quantitative PCR instrument (purchased from Thermo).

[0056] 4.2 Experimental method

[0057] (1) RNA extraction from liver tissues

[0058] a. Take about 20 mg of liver tissue in a 1.5 mL centrifuge tube, add 500 μL of TRIzol lysis solution, and homogenize and lyse on ice by ultrasonic treatment.

[0059] b. Add 100 μL of chloroform to the homogenate mixture, invert it up and down 15 times, let it stand for 2 min, and centrifuge (12000 RCF, 4 °C, 15 min).

[0060] c. Pipette 200 μL of the supernatant into a new 1.5 mL centrifuge tube, add 200 μL of isopropanol, invert it up and down 30 times, let it stand for 8 min, and centrifuge (12000 RCF, 4 °C, 10 min). Discard the liquid and retain the RNA precipitate.

[0061] d. Add 1 mL of 80% absolute ethanol (prepared with DEPC water) to each centrifuge tube containing the retained RNA precipitate, vortex thoroughly, centrifuge (7500 RCF, 4 °C, 5 min), discard the liquid and retain the RNA precipitate, centrifuge (7500 RCF, 4 °C, 1 min), aspirate and discard the remaining liquid, and open the centrifuge tube to air dry for 8 - 10 min.

[0062] e. Add 50 - 100 μL of DEPC water to dissolve the RNA precipitate for standby.

[0063] (2) Reverse transcription reaction

[0064] a. Take 1 μL of the RNA solution into a PCR tube, add 6 μL of RNase-free water, mix well, and perform PCR amplification denaturation under the conditions of 65 °C for 5 min.

[0065] b. Take out the PCR tube and place it on ice. Add 0.5 μL of Enzyme Mix, 2 μL of RTBuffer (5X), and 0.5 μL of Primer Mix to the mixture solution, mix well, and perform PCR reverse transcription amplification under the conditions of heating at 37 °C for 15 min and heating at 98 °C for 5 min.

[0066] c. Add 190 μL of ultrapure water, mix well, and set aside for standby.

[0067] (3) Real-time fluorescence quantitative PCR (Quantitative Real-time PCR, qPCR)

[0068] a. Add 5 μL of the cDNA template, 6 μL of SYBR Green, and 1 μL of the 10 μM target gene primer to a 96-well PCR plate, and mix well.

[0069] b. Real-time PCR amplification cycle program and reaction conditions - pre-incubate at 95°C for 10 min, incubate at 50°C for 2 min. Use a two-step amplification step, set to incubate at 95°C for 15 s, incubate at 60°C for 1 min, and set 40 cycles. The melting curve uses the program of incubating at 95°C for 15 s, incubating at 60°C for 1 min, and incubating at 97°C for 1 s. Finally, incubate at 37°C for 30 s for cooling.

[0070] c. Read the CT value and calculate the relative expression level of the target gene.

[0071] 4.3 Experimental results

[0072] Figure 3 The mRNA expression levels of inflammatory factors TNF-α, Ccl-2, and Ccl-7 in liver tissues of each group were shown. Compared with the control group, APAP intervention significantly increased the levels of cytokines in the livers of mice, while the biochanin A treatment group significantly inhibited the expression of cytokines in the liver, and there were statistical differences among groups. The above results indicate that biochanin A can inhibit the production of inflammatory factors TNF-α, Ccl-2, and Ccl-7 in the lungs of APPA-induced mice, and relieve the symptoms of acute liver injury induced by acetaminophen by exerting an anti-inflammatory effect.

Claims

1. Use of compound biochanin A in the preparation of a medicament for treating acetaminophen-induced acute liver injury, characterized in that, Biochanin A is a compound represented by formula (I) or a pharmaceutically acceptable salt 2. The application according to claim 1, wherein The compound biochanin A is a natural phytoestrogen.

3. The application according to claim 1, characterized in that The compound biochanin A can relieve the symptoms of acetaminophen-induced acute liver injury.

4. The application according to claim 1, wherein The compound biochanin A can reduce the serum ALT and AST levels of acetaminophen-induced acute liver injury.

5. The application according to claim 1, characterized in that The compound biochanin A can alleviate the pathological morphological changes of acetaminophen-induced mouse liver tissue.

6. The application according to claim 1, characterized in that The compound biochanin A can inhibit acetaminophen-induced hepatocyte necrosis.

7. The application according to claim 1, wherein The compound biochanin A can reduce acetaminophen hepatotoxicity.

8. The application according to claim 1, wherein The compound biochanin A can inhibit the production of inflammatory factors induced by acetaminophen in mouse lungs.

9. The application according to claim 8, wherein The inflammatory factors include TNF-α, Ccl-2 and Ccl-7.

10. A drug for treating acetaminophen-induced acute liver injury, characterized in that, It includes the compound biochanin A described in claim 1 and a pharmaceutically acceptable carrier, excipient, adjuvant or their combination.

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