Traditional Chinese medicine compound composition for treating drug-induced liver injury as well as preparation method and application of traditional Chinese medicine compound composition

Abstract

The invention provides a traditional Chinese medicine compound composition for treating drug-induced liver injury as well as a preparation method and application of the traditional Chinese medicine compound composition. The composition is prepared from the following components in parts by mass: 15 parts of rhizoma alismatis, 15 parts of cortex moutan, 20 parts of radix paeoniae rubra, 15 parts of rhizoma polygoni cuspidati, 15 parts of herba siegesbeckiae and 10 parts of radix curcumae. According to the pathogenesis of the drug-induced liver injury, syndrome differentiation and treatment are adopted to obtain the composition, the curative effect of the composition and the preparation thereof is determined through experiments, and the unique effect of traditional Chinese medicine in treating the drug-induced liver injury is achieved.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a Chinese medicine compound composition, in particular to a Chinese medicine compound composition for treating drug-induced liver injury, and a preparation method and application thereof. Background Art

[0002] Drug-induced liver injury (DILI) refers to liver injury induced by various prescription or over-the-counter chemical drugs, biological agents, traditional Chinese medicines, natural medicines, health products, dietary supplements and their metabolites and even excipients. For the vast majority of drugs, the probability of a single drug causing liver injury is not high in the general population, but due to the large number of drugs and the common use of drugs in combination, DILI is relatively common overall and is increasingly common in clinical practice, and may lead to serious adverse prognosis outcomes.

[0003] 1. Clinical manifestations of drug-induced liver injury: DILI can be asymptomatic liver function abnormalities or acute or chronic liver injury. Clinically, it can manifest as non-specific symptoms such as nausea, fatigue, abdominal pain, or specific liver injury symptoms such as jaundice, itching, hepatic encephalopathy, and ascites. In addition, patients with specific hepatotoxicity may also experience fever, rash, eosinophilia, and even DRESS syndrome. Most are self-limited and rarely develop into chronic.

[0004] 2. Clinical classification of drug-induced liver injury: The 2015 version of the guidelines divides DILI into intrinsic and idiosyncratic types based on the pathogenesis, among which iDILI is further divided into immune iDILI and genetic iDILI; the 2023 version of the guidelines adds indirect DILI on this basis, that is, drugs indirectly cause liver damage by changing the patient's original liver disease or immune status, and the incubation period is often several months. Common drugs that cause indirect DILI are anti-tumor drugs, glucocorticoids, and biological agents.

[0005] 3. Biochemical diagnostic criteria: The 2023 version of the guidelines adopts the biochemical criteria for acute DILI developed by the International Serious Adverse Event Consortium (iSAEC), namely, alanine aminotransferase (ALT) ≥ 5× upper limit of normal (ULN) or alkaline phosphatase (ALP) ≥ 2× ULN [especially accompanied by elevated gammaglutamyltransferase (GGT) and excluding elevated ALP levels caused by bone disease] or ALT ≥ 3× ULN and total bilirubin (TBil) ≥ 2× ULN. Those that do not meet the above threshold standards and the causal relationship is assessed as drug-induced are called drug-induced liver biochemical abnormalities.

[0006] 4. Western medicine treatment:

[0007] 4.1 Discontinuation and elimination of related drugs

[0008] When liver damage occurs, suspected and related drugs should be discontinued in time, and the drugs in the body should be cleared and excreted immediately. The latest study by Fisher et al. shows that once DILI is diagnosed, liver damage-related drugs should be discontinued in time, drugs belonging to the same biochemical family should be avoided as much as possible, and treatment should be started as early as possible. This plays a vital role in the prognosis of DILI. Most patients can recover after stopping the drug, a few develop chronic diseases, and very few progress to liver failure. Drugs can be removed from the body through methods such as vomiting, gastric lavage, catharsis, diuretics, and hemodialysis or perfusion, peritoneal dialysis, plasma exchange, etc. when necessary.

[0009] 4.2 General treatment

[0010] Rest in bed, avoid physical activities, eat a light diet, appropriately supplement high-protein and easily digestible foods, supplement various vitamins and trace elements, and pay attention to maintaining water, electrolytes and acid-base balance to enhance drug excretion. Avoid using drugs that cause liver damage again or conducting provocative tests. Closely monitor liver function, coagulation and other indicators.

[0011] 4.3 Drug Application

[0012] 4.3.1 Application of detoxifying drugs: Acetylcysteine, tiopronin, glutathione, and glucuronolactone have a detoxifying effect on the liver because they contain sulfhydryl groups that can bind to toxic products. Tiopronin is hydrolyzed by amidase in the human body to generate glycine-based aliphatic amino groups, which are related to the synthesis of purine nucleotides in the human body and can promote liver cell regeneration. Reduced glutathione is a tripeptide composed of glutamic acid, cysteine, and glycine. The sulfhydryl groups it contains can protect liver cell membranes, promote metabolism, detoxification, excrete bile, and scavenge free radicals, enhance the stability of liver cell membranes, and play a liver-protecting role. At the same time, it can also reduce the impact of oxidative stress on liver histology, participate in carbohydrate metabolism and tricarboxylic acid cycle in the body, and enhance the liver's oxidation, reduction, and hydrolysis capabilities.

[0013] 4.3.2 Application of drugs for liver protection, gallbladder promotion and jaundice relief

[0014] When patients show signs of liver function impairment such as elevated transaminases or decreased albumin, cholestasis, etc., liver-protecting and anti-jaundice drugs may be used as appropriate. Optional therapeutic drugs include antioxidants (promoting the removal of reactive metabolites), precursors of protective substances, intervention agents that prevent the occurrence of damage, or repair agents for membrane damage. For mild to moderate hepatocellular injury and mixed DILI, silymarin can be used for mild inflammation, and bicyclol can be used for severe inflammation. Studies have shown that S-adenosylmethionine can improve the efficacy of cholestatic DILI. Diammonium glycyrrhizinate enteric-coated capsules and magnesium isoglycyrrhizinate are the fourth-generation preparations of glycyrrhizic acid. Currently, my country's CFDA has approved the treatment of acute DILI. It is used to treat acute hepatocellular or mixed DILI with a sharp increase in ALT, and can effectively reduce inflammatory cell infiltration and hepatocyte degeneration and necrosis. Ursodeoxycholic acid has the effects of stabilizing cell membranes, immunomodulation, and mitochondrial protection, which can relieve patients' fatigue and itching, and is used for cholestatic DILI.

[0015] 4.3.3 Application of glucocorticoids

[0016] It is mainly used for drug reactions mediated by immune responses, but not all allergic reactions are sensitive to glucocorticoids. Studies have shown that early treatment of DILI patients with a tendency to severe disease with low-dose glucocorticoids can protect liver cell membranes, enhance liver detoxification function, and quickly control the progression of the disease, relieve symptoms, and reduce the incidence of severe hepatitis.

[0017] The general principles of drug selection and combination are: ① Patients with milder conditions can usually choose one anti-inflammatory and hepatoprotective drug; ② If combined medication is required, drugs with different anti-inflammatory and hepatoprotective mechanisms should be selected for combined treatment based on the characteristics of the pathogenesis of DILI caused by different drugs, but the combination of drugs should usually not exceed 2 drugs.

[0018] 4.4 Surgical treatment

[0019] ① Artificial liver support therapy has achieved remarkable results in the treatment of drug-induced liver disease, and is even better than the treatment of acute viral liver failure. Its purpose is to timely reduce toxic drugs and toxic metabolites in the body, reduce inflammatory mediators, alleviate the "inflammatory storm", and improve the success rate of treatment. ② Liver transplantation is considered to be the most effective treatment for drug-induced liver failure. Liver transplantation can also be considered when severe drug-induced liver disease leads to liver failure, severe cholestasis, and chronic liver damage progressing to cirrhosis.

[0020] 5. Treatment with Traditional Chinese Medicine: Through analysis, it was found that the early stage of DILI is mainly characterized by excess symptoms such as liver depression and spleen deficiency, internal accumulation of dampness and heat, and internal stasis of toxins, while the middle and late stages are mainly characterized by deficiency symptoms such as deficiency of both yin and yang, and insufficient qi and blood. Therefore, clinical medication is often used to coordinate the liver and spleen, with the main treatment methods being to soothe the liver and strengthen the spleen, detoxify and eliminate dampness, and promote qi and blood circulation.

[0021] Traditional Chinese medicine has its unique therapeutic effect in treating liver damage. Therefore, it is necessary to continue to research and develop traditional Chinese medicine compound prescriptions with good therapeutic effect on liver damage and low adverse reactions based on existing technologies. Summary of the invention

[0022] The purpose of the present invention is to provide a Chinese medicine compound composition with a scientific and reasonable ratio and good effect in treating drug-induced liver injury, so as to give full play to the unique role of traditional Chinese medicine in treating drug-induced liver injury. Another purpose of the present invention is to provide a preparation method of the Chinese medicine compound composition and its application.

[0023] To achieve the above object, the present invention provides the following technical solutions:

[0024] In a first aspect, the present invention provides a Chinese herbal compound composition for treating drug-induced liver injury, comprising, by mass, 15 parts of Alisma orientalis, 15 parts of Moutan bark, 20 parts of Red Peony Root, 15 parts of Polygonum cuspidatum, 15 parts of Siegesbeckia herb, and 10 parts of Curcuma aromatica.

[0025] In a second aspect, the present invention also provides a traditional Chinese medicine preparation for treating drug-induced liver injury, wherein the preparation comprises an extract of the traditional Chinese medicine compound composition as described above.

[0026] In a third aspect, the present invention also discloses a method for preparing a Chinese medicine preparation for treating drug-induced liver injury, the method comprising adding water to the raw materials of the Chinese medicine compound composition as described above, decocting to obtain an extract, and concentrating to obtain a decoction preparation.

[0027] Preferably, the amount of water added is 10 times the weight of the Chinese medicine raw material.

[0028] More preferably, the extraction process is: adding water to the raw materials of the Chinese medicine compound composition and boiling for 1 to 3 times, each time for 0.5 to 1 hour, filtering and combining the extracts, concentrating by rotary evaporation, and finally obtaining a Chinese medicine concentrate.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] According to the pathogenesis of drug-induced liver injury, the present invention adopts syndrome differentiation and treatment, and screens the composition of the Chinese medicine compound composition through clinical screening. The optimal formula is: 15g of Rhizoma Alismatis, 15g of Cortex Moutan, 20g of Radix Paeoniae Rubra, 15g of Polygonum Cuspidati, 15g of Herba Siegesbeckiae, and 10g of Radix Curcumae. Professor Yang Jin, a famous Chinese medicine doctor in Jiangsu Province and an instructor of the academic experience inheritance work of famous old Chinese medicine experts in the country, has achieved good clinical effects in clinical treatment of drug-induced liver injury with the composition of the present invention. Professor Yang believes that the pathogenesis of drug-induced liver injury is stasis and heat entering the collaterals, and turbidity and toxicity stagnation, so he created a Chinese medicine compound preparation with the main effects of cooling blood, unblocking collaterals and removing turbidity, blood stasis, clearing heat and unblocking collaterals, cooling blood and dispersing blood, and removing turbidity and detoxification.

[0031] The experimental results show that the Chinese medicine compound composition provided by the present invention can improve mouse liver damage to varying degrees from different aspects. Specifically, the Chinese medicine compound composition provided by the present invention can significantly reduce AST and ALT levels, significantly increase SOD and GSH-Px content, reduce ROS generation, reduce MDA levels in the liver, and play a good preventive and therapeutic role in acetaminophen-induced drug-induced liver damage in mice by improving liver tissue pathological damage, improving oxidative stress, improving liver microcirculation, and reducing damage to organelles such as the endoplasmic reticulum. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The liver injury index determination in the embodiment of the present invention is shown, wherein (A) liver coefficient; (B) ALT; (C) AST, and the statistical significance on the figure is: compared with the control group, ### P<0.0001; compared with the model group, **P<0.01, ***P<0.0001;

[0033] Figure 2 The liver damage oxidation index determination is shown, including (A) SOD; (B) MDA; (C) ROS; (D) GSH-Px. The statistical significance of the figure is: compared with the control group, ## P<0.0001; compared with the model group, *P<0.05, **P<0.0001;

[0034] Figure 3The pathological morphological changes of liver tissues of mice in each group are shown (photos at 100X and 200X), in which the hollow arrows indicate the hemorrhagic necrotic areas in the liver, and the solid arrows indicate the normal hepatocytes; in the control group, under the microscope, the normal liver lobules were slightly polygonal with clear structures; under the microscope, the liver cells of APAP group mice were degenerated and necrotic, the outlines of liver cells in the necrotic areas were still preserved, nuclear fragments were visible, and very few neutrophils were seen at the edges; in the positive drug (silymarin) group and the three groups of Chinese medicine compound compositions ZL, ZM, and ZH, the liver tissue structures were clear, and no obvious inflammatory cell infiltration and fibrous tissue hyperplasia were observed in the liver lobules and portal areas. DETAILED DESCRIPTION

[0035] According to traditional Chinese medicine, the liver stores blood and is responsible for regulating qi. Drug toxins enter the liver with blood and are regulated by the liver. If the innate endowment is abnormal and the liver is damaged, drug toxins accumulate in the liver, or drug toxins damage the liver, causing qi stagnation. The ribs are where the liver meridian is located, so there will be pain in the ribs. If the liver is depressed and the spleen is deficient, the spleen will not function properly, so there will be poor appetite and nausea. If the spleen is deficient and water and dampness are not transported, dampness blocks heat and steams on the skin, there will be rashes and itchy skin. If the liver and gallbladder are fumigated, bile does not flow through the normal path and overflows the skin, there will be jaundice. If blood stasis is resolved over time, there will be accumulation. The disease is located in the liver and is closely related to the spleen, gallbladder, and stomach. Over time, it affects the kidneys. The key pathogenesis is liver depression and spleen deficiency, dampness and heat, and qi stagnation and blood stasis. Dr. Yang believes that drug-induced liver injury belongs to the category of flank pain in traditional Chinese medicine. Dr. Yang believes that the pathogenesis of drug-induced liver injury is the entry of blood stasis and heat into the collaterals, and the stagnation of turbid toxins. The treatment method should be to cool the blood, dredge the collaterals and resolve turbidity, so as to remove blood stasis, clear away heat and dredge the collaterals, cool the blood and disperse the blood, resolve turbidity and detoxify.

[0036] The square solution rule of the present invention is:

[0037] In the formula, Polygonum cuspidatum can break up blood retention, promote dampness and reduce jaundice, while Paeonia lactiflora can cool blood, eliminate turbidity, and relieve blood stasis. The treatment of the disease must seek the root cause. Liver damage is caused by blood stasis and heat entering the collaterals, and turbid toxins stagnate. The two can break up the yin knots and expel turbid toxins, and they are both the main medicines.

[0038] Cortex Moutan removes stagnation and soothes the five internal organs. Its root property is downward, and it can drain the blood in the middle and lower parts of the body. Curcuma has a light smell but a strong taste, and it can cure blood stasis and relieve qi. The combination of the two herbs enhances the effects of Polygonum cuspidatum and Red Peony Root in cooling blood, activating blood circulation, removing blood stasis and relieving pain, and they are both assistant herbs.

[0039] The dampness and heat are expelled from the urine, and the formula is guided by the situation. The rhizoma alismatis in the formula clears heat and removes turbidity, dries the earth and removes dampness, promotes diuresis and relieves stranguria. Once the dampness and heat are removed, the clear qi will rise. The herb Siegesbeckiae removes rheumatism, relieves heat and toxicity, and clears the joints, and serves as an adjuvant. All the medicines work together to promote blood circulation, clear the meridians, and remove turbidity and toxicity.

[0040] Drug splitting explanation:

[0041] 1. "Changsha Yaojie" says: Alisma is salty and slightly cold, and enters the foot Shaoyin kidney meridian and foot Taiyang bladder meridian. It dries the earth and drains dampness, promotes diuresis and relieves stranguria, eliminates dizziness caused by drinking, treats dryness and thirst caused by dampness, and is effective for treating gas and water swelling, choking and nausea. The active ingredients of Alisma have also been reported to have certain anti-inflammatory and antioxidant pharmacological effects. Among them, 24-acetyl alisma alcohol A is a natural triterpenoid compound isolated from Alisma, which was found to reduce the expression of ROS and inflammatory factors by regulating the AMPK / mTOR pathway. Alisma alcohol A has been reported to inhibit the production of inflammatory cytokines by blocking the activation of NF-κB. 23-acetyl alisma alcohol C can reduce the expression of inflammatory factors such as TNF-α, IL-6 and IL-1β in serum by interfering with the NF-κB signaling pathway. 23-acetyl alisma alcohol B can reduce the levels of IL-12 and IFN-γ in serum to improve inflammatory response, and can reduce oxidative stress damage by activating Nrf2. According to the literature, 23-acetyl alisomol B, 24-acetyl alisomol A, 23-acetyl alisomol C, alisomol A and alisomol C are high in ApL. 23-acetyl alisomol B and 23-acetyl alisomol C are prescribed ingredients in the pharmacopoeia. The study tested the anti-inflammatory and antioxidant activities of 23-acetyl alisomol B, 24-acetyl alisomol A and 23-acetyl alisomol C. The results showed that the above three key chemical components have significant anti-inflammatory and antioxidant activities. Therefore, it is speculated that 23-acetyl alisomol B, 24-acetyl alisomol A and 23-acetyl alisomol C may be the key pharmacological material basis for ApL to exert its effects.

[0042] 2. The "Collection of Notes on the Classic of Materia Medica" states: Cortex Moutan tastes pungent, bitter, cold, slightly cold, and non-toxic. It is mainly used to treat cold and heat, stroke, spasm, convulsions, epilepsy, evil spirits, remove hard masses, blood stasis in the stomach and intestines, calm the five internal organs, and treat carbuncle. It can remove seasonal qi, headache, guest heat, five kinds of fatigue, fatigue, head and waist pain, wind lockjaw, and epilepsy. Studies have shown that TGM can reduce the increase of serum ALT and AST in the injury group; promote the increase of serum protein content and liver glycogen synthesis in the injury group, which shows that TGM enhances the ability of liver cells to resist damage from the perspective of liver metabolic function; it can also reduce the content of MDA in the liver of the injury group and increase the activity of serum and liver GSH-Px. GSH-Px can oxidize H2O2 and MDA into water and non-toxic alcohol, and is an important part of the body's antioxidant system, indicating that TGM has a strong ability to scavenge free radical products and resist oxidative damage.

[0043] 3. According to Shennong's Herbal Classic, red peony root is bitter and flat, and can cure abdominal pain caused by evil qi, remove blood stasis, break up hard masses, treat cold and heat, hernia, relieve pain, promote urination, and invigorate qi. Studies have shown that total glycosides of red peony root can induce and enhance the activity of liver drug fermentation, which is beneficial for clearing the damage of certain foreign bodies to liver cells. This may be one of the mechanisms for its liver-protecting effect of reducing jaundice and enzymes.

[0044] 4. "Yuzhou Yaojie" says: "Herba Siegesbeckiae is bitter in taste and cold in nature. It enters the liver meridian of the foot, stops numbness, stretches cramps, clears joints, and expels rheumatism, sores, carbuncles, and swellings. It is good for oral administration and application. Studies have shown that HS extract can not only effectively scavenge free radicals and protect cell membranes, but also enhance the activity of antioxidant enzymes SOD and CAT, improve the ability to scavenge free radicals, and reduce the content of lipid peroxidation products MDA, thereby achieving the effect of protecting the body.

[0045] 5. The "Compendium of Materia Medica" states: Polygonum cuspidatum is slightly warm. It is mainly used to promote menstrual flow and break up blood retention. The "Treatise on Materia Medica" states: It dispels wind, eliminates dampness, breaks up blood stasis, and promotes menstruation. It is used to treat rheumatic pain in the muscles and bones, damp-heat jaundice, stranguria and leucorrhea, amenorrhea in women, postpartum lochia retention, accumulation of masses, and hemorrhoidal bleeding. It is also used for injuries from falls, burns, and ulcers and tinea. Studies have shown that PD can reduce mitochondrial damage in hepatocytes, inhibit the collapse of mitochondrial membrane potential and the opening of mPTP, and improve the mitochondrial function of hepatocytes. In addition, PD can inhibit oxidative stress in hepatocytes induced by severe shock and maintain the stability of hepatocyte lysosomes. Its mechanism of action is related to PD activating the SIRT1-SOD2 pathway.

[0046] 6. The "Explanation of Bencao Jing" says: Curcuma is cold in nature, cold in qi, bitter in taste, non-toxic, and can cure blood stasis, lower qi, promote tissue regeneration and stop bleeding, break bad blood, hematuria, and wounds. Curcuma has a cold qi, and is endowed with the water qi of Tiandong, and enters the foot Shaoyin kidney meridian and the hand Taiyang cold water small intestine meridian. It tastes bitter and non-toxic, and has the two flavors of earth gold and fire, and enters the hand Taiyin lung meridian and the hand Shaoyin heart meridian. The smell is more descending than ascending, and it is yin. There are many kinds of chemical components in warm turmeric, mainly sesquiterpenes, monoterpenes, diterpenes, curcumin, polysaccharides, sterols, alkaloids, resins, polypeptides, flavonoids, etc. The pharmacological effects of warm turmeric mainly include anti-tumor, anti-inflammatory and analgesic, anti-viral, cardiovascular and cerebrovascular protection, nervous system effects, kidney protection, antioxidant, pregnancy termination and anti-early pregnancy, liver protection, cytotoxicity, anti-depression, and anti-diabetic effects. Curcuma oil, curcumene, turmericdiol, curcuma cyclodiene, and diterpenoids are the main anti-inflammatory and analgesic active ingredients in Curcuma. Volatile oils and curcuminoids are the main antiviral active ingredients in Curcuma. Curcuma oil is the main antioxidant active ingredient in Curcuma. Sesquiterpenoids in Curcuma have anti-lipid peroxidation effects. Curcuma alcohol extract has antioxidant stress activation and has a protective effect on endothelial damage. Curcuma raw products achieve liver protection by blocking the TGF-TGF / Smad signaling pathway and regulating liver fibrosis by upregulating the MMP-2 / TIMP-1 ratio.

[0047] The following will be combined with the specific embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] In the description of the present invention, it should be noted that the terms "upper", "lower", "inner", "outer", "front end", "rear end", "two ends", "one end", "the other end" and the like indicating the orientation or positional relationship are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0049] Example 1 Preparation of a Chinese herbal compound composition for treating drug-induced liver injury

[0050] Select medicinal materials according to the following mass proportions: 15g of Alisma orientalis, 15g of moutan bark, 20g of red peony root, 15g of polygonum cuspidatum, 15g of siegetable herb, and 10g of curcuma.

[0051] Take the raw materials of the Chinese medicine composition according to the dosage, add water in an amount 10 times the weight of the medicinal materials, decoct and extract 1 to 3 times, each time for 0.5 hour, filter, combine the extracts, concentrate by rotary evaporation, and obtain a water decoction.

[0052] Example 2 Observation of the therapeutic effect on mice with drug-induced liver injury

[0053] 1. Drugs and reagents

[0054] Experimental drug: decoction of the Chinese medicine compound prepared in Example 1

[0055] Experimental reagents: acetaminophen (APAP), purchased from Shanghai Yuanye Biotechnology Co., Ltd., batch number D30HS205008; silymarin capsules, purchased from Jiangsu Provincial Hospital of Traditional Chinese Medicine, approval number HJ20181067. Alanine aminotransferase assay kit (ALT) test, item number is Guoxie injection 20162401795. Aspartate aminotransferase assay kit (AST) test, item number is Guoxie injection 20162401792. ELISA kits: all purchased from Nanjing Lapuda Biotechnology Co., Ltd. (Nanjing Lapuda Biotechnology Co., Ltd). Total superoxide dismutase (SOD) test, item number is SH001, batch number is 20231212. Malondialdehyde (MDA) test, item number is SH002, batch number is 20231212. Glutathione peroxidase (GSH-Px) detection, the item number is SH004, the batch number is 20231212. Reactive oxygen species (ROS) determination detection, the item number is SH026, the batch number is 20231212. Isoflurane, purchased from Beijing Yi Ze Jia Co., Ltd., the batch number is 21012801. Anhydrous ethanol, purchased from Nanjing Chemical Reagent, the item number is 211129859K. Xylene, purchased from Nanjing Chemical Reagent, the item number is 211208731F. Hematoxylin, purchased from Regen's Reagent, the item number is DH0006. Eosin, purchased from Regen's Reagent, the item number is DH0051. Masson's trichrome staining solution kit, purchased from Regen's Reagent, the item number is DC0032.

[0056] 2. Experimental instruments

[0057]

[0058]

[0059] 3. Experimental animals

[0060] Sixty SPF male Babl / c mice, weighing 18–22 g, were purchased from Qinglongshan Animal Breeding Farm with production license number: SCXK (Zhejiang) 2019-0002.

[0061] 4. Experimental methods

[0062] 4.1. Preparation of main reagents

[0063] (1) Preparation of water decoction of Chinese medicine compound composition

[0064] Two groups of 180g of the Chinese medicine compound composition were fully soaked in 1000mL of pure water for 2 hours, boiled over high heat, then simmered for 1 hour, and filtered; 800mL of pure water was added for the second decoction, boiled over high heat, then simmered for 1 hour, and filtered. The two decoctions were combined and concentrated in a Chinese medicine rotary evaporator. The dosage for mice was obtained according to the calculation formula for the body surface area of ​​humans and animals, and the Chinese medicine compound was concentrated into 77ml of high-dose concentrate, that is, the concentration of the liquid was 2.34g / mL. Then the water decoction of the Chinese medicine compound composition was diluted to 1.17g / mL and 0.585g / mL, which were the liquid concentrations corresponding to the high, medium and low doses of the Chinese medicine compound, and placed in a refrigerator at 4°C for storage.

[0065] (2) Preparation of positive drug silymarin group

[0066] Use a precision electronic balance to weigh 140 mg of silymarin and dissolve it in 28 mL of normal saline to prepare a 50 mg / kg solution. Vortex mix and prepare it before use.

[0067] (3) Preparation of APAP modeling drug

[0068] Use a precision electronic balance to weigh 400 mg of APAP powder and dissolve it in 10 mL of hot saline. Prepare a solution at a dosage of 400 mg / kg. Vortex to mix and prepare it before use.

[0069] 4.2 Establishment of a mouse model of drug-induced liver injury

[0070] Sixty healthy Babl / c male mice (seven weeks old, 18-22g) were adaptively fed for 7 days and then randomly divided into 6 groups, with 10 mice in each group, namely control group (Control), positive drug silybin group (Silybin: 50mg / kg), model group (APAP: 400mg / kg), low concentration group of the combination (ZL: 5.85g / kg), medium concentration group of the combination (ZM: 11.7g / kg), and high concentration group of the combination (ZH: 23.4g / kg). The low, medium and high groups of the combination and the positive drug silybin group were all given continuous gavage (0.1 ml / 10 g) for 7 days, and the control group and the model group were given normal saline (0.1 ml / 10 g) by continuous gavage for 7 days. One hour after the administration on the 7th day, the mice in the normal control group were intraperitoneally injected with normal saline (0.1 ml / 10 g), and the mice in the other 5 groups were all intraperitoneally injected with 400 mg / kg of APAP (dissolved in hot normal saline) to induce drug-induced liver injury.

[0071] 4.3. General monitoring of mice

[0072] Starting from the first day of the experiment, the weight of the mice was recorded once a day, and the general condition and appearance of the mice, such as their mental state, activity, hair color, diet and excretion, were observed, and any abnormalities were recorded.

[0073] 4.4 Determination of biochemical indices such as transaminase in mice

[0074] Mice were anesthetized by inhalation of isoflurane gas, and then blood was collected by removing the eyeballs. After standing at room temperature for 20 minutes, the blood was centrifuged at 3500 rpm and 4°C for 10 minutes, and the serum was placed in a 4°C refrigerator for testing. The serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and other biochemical indicators were analyzed by an automatic biochemical analyzer.

[0075] 4.5. Determination of mouse liver index

[0076] After the mice were killed, the livers were removed, rinsed in PBS solution, and excess water was absorbed on filter paper. The livers were weighed, and the ratio of liver weight to body weight was calculated to obtain the liver index.

[0077] 4.6. ELISA to detect oxidative stress indicators in mice

[0078] The activities of serum oxidative stress indicators ROS, MDA, SOD, and GSH-Px were detected by ELISA. The specific operation method was carried out according to the instructions of the ELISA kit.

[0079] 4.7 Histopathological analysis of mouse liver

[0080] After the determination of mouse liver index, 3 mouse livers were taken from each group and fixed in 4% paraformaldehyde solution, followed by dehydration, embedding, slicing, staining, and observation. The specific experimental steps are as follows:

[0081] (1) Dehydration: After fixation for 48 h, the mouse liver was placed in a tissue dehydrator for gradient alcohol dehydration. The dehydration steps were 50%, 70%, 85%, and 95% alcohol concentrations, each for 1.5 h. 100% alcohol dehydration was performed for 30 min twice. The sample was immersed in 100% alcohol and xylene in a 1:1 ratio for 30 min to prevent incomplete dehydration. Finally, xylene was used for transparency for 30 min three times.

[0082] (2) Wax immersion and embedding: The liver tissue was immersed in a 1:1 mixture of xylene and paraffin for 2 h, and then immersed in 100% paraffin for 4 h to fully replace the xylene inside the liver tissue with paraffin. The wax-immersed liver tissue was then placed in an embedding box with the heart tissue located in the center of the mold. The heart tissue was completely embedded with hot wax and placed in a freezer until the wax block solidified.

[0083] (3) Sectioning: After simple trimming of the embedded wax block, it is sliced ​​longitudinally and continuously on a microtome with a thickness of 2.5 μm. The sections are placed in warm water, and an adhesive glass slide is inserted into the water surface at an angle to pick up the sections, which are then baked at 50°C for 30 min.

[0084] (4) Dewaxing and rehydration: The baked sections were first soaked in xylene for 30 min twice, then soaked in 100%, 95%, 90%, 80%, 70%, and 50% alcohol gradient for 10 min, and then soaked in distilled water for 10 min.

[0085] (5) H&E staining: After dewaxing, the slices were dried for 10 minutes, immersed in hematoxylin solution for 8 minutes, rinsed with running water for 3 minutes, soaked in 1% hydrochloric acid alcohol solution three times for differentiation, rinsed with running water for 3 minutes, blued with 0.6% ammonia solution, and finally rinsed with running water for 3 minutes. Then, the slices were soaked in eosin solution for 3 minutes, and rinsed with running water for 3 minutes. Finally, the slices were immersed in 70%, 90%, 95%, and 100% alcohol for 5 minutes each, and then sealed. After the slices were dried, they were sealed with neutral gum, covered with coverslips, and examined under a microscope for pathological analysis.

[0086] 4.8 Statistical analysis

[0087] All data are expressed as mean ± standard deviation Description, Graphpad Prism 8.0 was used for statistical analysis, and the data were subjected to one-way ANOVA test and independent sample T test. P value < 0.05 was considered statistically significant, and P value < 0.01 was considered statistically significant.

[0088] 5. Experimental results

[0089] 5.1. Effect of Chinese herbal medicine on the general condition and liver index of mice with APAP-induced liver injury

[0090] By observing and recording the general condition and weight of the mice during the experimental period, it was found that the mice in the blank control (Control) group were in good mental state, moved freely, had shiny hair, and ate and drank well; the mice in the liver injury model (Model) group were listless after modeling, had messy hair, weakened grip, and slightly decreased food and water intake; compared with the model group, the conditions of the mice in each treatment group improved to varying degrees.

[0091] Monitoring and recording of mouse liver coefficient, the results are shown in Figure 1A. It was found that compared with the control group, the liver index of the model group mice was significantly increased (P < 0.0001), indicating that the model can cause liver damage after modeling. After intervention with the low, medium and high dose groups of the Chinese herbal compound composition, the liver index of the mice was significantly reduced compared with the model. The effect of the Chinese herbal compound was dose-dependent, indicating that the Chinese herbal compound composition can effectively improve liver damage caused by APAP.

[0092] 5.2 Effects of Chinese herbal medicine combination on APAP-induced liver injury markers ALT and AST in mice

[0093] The ELISA results of ALT and AST are shown in Figure 1 B. Figure 1 C. Compared with the control group, the ALT and AST indexes of mice in the model group were significantly increased, indicating the presence of liver damage. Compared with the model group, the Chinese medicine compound composition can effectively reduce the ALT and AST indexes and alleviate liver damage. The high-dose group has a more significant effect on reducing the ALT and AST indexes.

[0094] 5.3 Effects of Chinese herbal medicine compound on APAP-induced oxidative stress in mice

[0095] The results of SOD are visible Figure 2 A, compared with the control group, the SOD index of mice in the model group was significantly reduced, indicating the presence of liver damage. Compared with the model group, the SOD index of the positive drug and the Chinese medicine compound group was increased, indicating that the positive drug and the Chinese medicine compound have therapeutic effects on drug-induced liver damage; the results of MDA can be seen Figure 2 B, Compared with the control group, the MDA index of mice in the model group increased significantly, which can indirectly reflect the increased degree of peroxidation damage in liver tissue, indicating the presence of liver damage. Compared with the model group, the MDA of the positive drug and Chinese medicine compound groups were both reduced, indicating that both positive drugs and Chinese medicine compound have therapeutic effects on drug-induced liver injury.

[0096] The results of ROS are visible Figure 2 C. Compared with the control group, the ROS index of mice in the model group was significantly reduced, indicating that the redox balance was broken and oxidative stress was generated, indicating the presence of liver damage. Compared with the model group, the ROS of the positive drug and the Chinese medicine compound group were both increased, indicating that the positive drug and the Chinese medicine compound had a therapeutic effect on drug-induced liver injury, and the data of the Chinese medicine compound group were closer to the control group, indicating that the effect of the Chinese medicine compound group was better than that of the positive drug group; the results of GSH-PX can be seen Figure 2 D. Compared with the control group, the GSH-PX index of mice in the model group was significantly decreased, indicating that GSH was consumed in large quantities, resulting in oxidative stress, suggesting the presence of liver damage. Compared with the model group, the GSH-PX index of mice in the positive drug and Chinese herbal compound groups was increased, indicating that both positive drugs and Chinese herbal compound have therapeutic effects on drug-induced liver injury.

[0097] 5.4 Effects of Chinese herbal medicine combination on APAP-induced liver pathology in mice

[0098] HE staining results are shown in Figure 3 , it was found that the normal liver lobules in the control group were slightly polygonal and had a clear structure under the microscope. The hepatocytes were arranged radially around the central vein, and the portal area was between the hepatic lobules, where interlobular arteries, veins and interlobular bile ducts were visible. No obvious inflammatory cell infiltration or fibrous tissue hyperplasia was observed in the hepatic lobules and portal areas. Under the microscope in the model group, degeneration and necrosis of hepatocytes were observed in the APAP group mice, with the outlines of hepatocytes in the necrotic area still preserved and nuclear fragments visible. Very few neutrophils were seen at the edge. This shows that APAP can cause obvious pathological damage to the liver tissue of mice. Compared with the model group, the liver tissue structure was clear in the positive drug (silymarin) group, with no obvious degeneration and necrosis of hepatocytes, a small number of small foci of inflammatory cell infiltration in the liver tissue, and no other obvious lesions.

[0099] The liver tissue structures of the three groups of Chinese herbal compound compositions ZL, ZM and ZH were clear, with hepatocytes arranged radially around the central vein. The portal areas were between the liver lobules, and interlobular arteries, veins and interlobular bile ducts were visible. No obvious inflammatory cell infiltration and fibrous tissue hyperplasia were observed in the liver lobules and portal areas.

[0100] 6. Experimental conclusion

[0101] The results of animal experiments showed that both the positive drug and the Chinese herbal compound combination can significantly improve the liver damage in mice, and the effect of the Chinese herbal compound combination is better than that of the positive drug group. The effect of the Chinese herbal compound is dose-dependent, among which the high-concentration Chinese herbal compound group has the best effect, which is better than the data of the control group, proving that the combination has a significant effect in treating drug-induced liver injury.

[0102] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

[0103] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified and replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A Chinese medicinal compound composition for treating drug-induced liver injury, characterized in that: The composition comprises 15 parts of oriental rhizome, 15 parts of moutan bark, 20 parts of red peony root, 15 parts of knotweed, 15 parts of siegetable herb, and 10 parts of curcuma by weight.

2. A Chinese medicine preparation for treating drug-induced liver injury, characterized in that: The preparation comprises the extract of the Chinese medicine compound composition as claimed in claim 1.

3. A method for preparing a traditional Chinese medicine preparation for treating drug-induced liver injury, characterized in that: Add water to the raw materials of the Chinese medicine compound composition as claimed in claim 1, boil them and obtain an extract, which is then concentrated to obtain a water decoction preparation.

4. The method for preparing the Chinese medicine preparation for treating drug-induced liver injury according to claim 3, characterized in that: The amount of water added is 10 times the weight of the Chinese medicine raw materials.

5. The method for preparing the Chinese medicine preparation for treating drug-induced liver injury according to claim 3, characterized in that: The extraction process is as follows: water is added to the raw materials of the Chinese medicine compound composition and boiled for 1 to 3 times, each time for 0.5 to 1 hour, the extracts are combined after filtration, and concentrated by rotary evaporation to finally obtain the Chinese medicine concentrate.

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