Application of kummerowia striata in preparation of medicine for preventing and / or treating liver injury

Abstract

The invention discloses application of kummerowia striata in a medicine for preventing and / or treating liver injury, and belongs to the field of traditional Chinese medicines. The research shows that the Thunb. Schindl or the extract of the Thunb. Schindl can obviously and rapidly reduce transaminase and improve liver function indexes, has the capability of resisting oxidative stress level, protects the histological form of the liver, can comprehensively condition, is mild and safe, and can prevent and / or treat liver injury, so that the Thunb. Schindl or the extract of the Thunb. Schindl has a protective effect on diseases caused by the liver injury, and has a good application prospect. The compound is expected to become a novel treatment medicine in the aspect of liver injury diseases.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine, and particularly relates to application of cloverleaf herbaceous grass in preparing medicines for preventing and / or treating liver damage. Background Art

[0002] The liver is the largest organ in the human body and plays a major role in regulating the physiological functions of the human body. Drug abuse, alcoholism, and long-term exposure to toxic environments can cause damage to the liver. Drug-induced liver injury (DILI) refers to a disease caused by abnormal liver function caused by drugs or their metabolites or a hypersensitivity reaction of the liver to drugs and metabolites. Drug-induced liver injury is one of the common and serious adverse drug reactions. In severe cases, it can cause cirrhosis, liver cancer, acute liver failure, and even death. Its incidence rate is increasing year by year. It is often the result of multiple mechanisms acting successively or together. So far, there is still a lack of simple, objective, and specific diagnostic indicators and effective treatment methods.

[0003] Acetaminophen (APAP) is a widely used over-the-counter analgesic and antipyretic drug that is safe and effective at therapeutic doses. However, excessive use of APAP can lead to acute liver damage, the extent of which is positively correlated with the dose and can develop into acute liver failure. In Western countries, APAP overdose is the main cause of liver failure.

[0004] According to the 2017 US FDA statistics, APAP causes 46% of acute liver failure, and no less than 500 people die from overdose of APAP each year. In my country, there are hundreds of Western medicines or compound Chinese medicines containing acetaminophen, especially some medicines for treating colds. These medicines are almost all over-the-counter drugs, widely available in the market and easy to buy. Therefore, patients often suffer from liver damage due to taking multiple APAP-containing drugs at the same time. Therefore, studying the detoxification of APAP is of great significance to my country.

[0005] With the rapid development of medical technology, many studies have been conducted on drugs for the treatment of liver damage, mainly including Chinese herbal medicine, biological drugs, chemical drugs and trace elements. However, some studies have shown that some drugs for treating liver damage will inevitably produce biological toxicity. For example, N-acetylcysteine, a commonly used drug for the treatment of drug-induced liver damage in clinical practice, requires large doses of intravenous injection and is contraindicated for asthma patients. Clinically, therapeutic doses can cause systemic anaphylactic reactions, and side effects such as bronchial spasm, bleeding, nausea and vomiting, diarrhea or constipation may occur. It can be seen that the prevention and treatment of liver damage is still a serious problem.

[0006] Traditional Chinese medicine is a treasure of traditional Chinese medicine. Many of them have good therapeutic effects on diseases and do not produce very obvious toxic side effects. Therefore, it is of great practical significance to research and develop drugs for the clinical treatment of liver injury from traditional Chinese medicine. Summary of the invention

[0007] The purpose of the present application is to overcome at least one shortcoming of the prior art and to provide the use of Herba Trifoliatae in drugs for preventing and / or treating liver damage.

[0008] The technical solution adopted in this application is:

[0009] Application of Kummerowia striata (Thunb.) Schindl or an extract thereof in the preparation of medicines or health products for preventing and / or treating liver damage, diseases caused by liver damage, or diseases related to liver damage.

[0010] Three-leaf herbaceous grass, also known as chicken eye grass and herbaceous grass, is the dried whole herb of the leguminous plant Three-leaf herbaceous grass. It is mainly produced in Guangdong, Guangxi, Fujian and other places. It was first included as a food in the "Compendium of Materia Medica for Famine Relief". Three-leaf herbaceous grass is cool in nature and sweet in taste. It has the effects of cooling blood and detoxifying, clearing away heat and relieving exterior symptoms, dispersing blood stasis and eliminating accumulation. It can be used to treat sore throat, colds, sores, swelling and pain, dysentery and other symptoms.

[0011] The Latin name of three-leaf herbaceous grass is: Kummerowia striata (Thunb.) Schindl.

[0012] In some embodiments, the Herba Lycopodiellae extract is a water extract, an alcohol extract, or a water-alcohol extract of Herba Lycopodiellae.

[0013] In some embodiments, the liver injury includes drug-induced liver injury, chemical liver injury, and alcoholic liver injury.

[0014] In some preferred embodiments, the liver injury is drug-induced liver injury, specifically drug-induced liver injury caused by overdose of acetaminophen (APAP).

[0015] In some embodiments, the liver injury is acute liver injury or chronic liver injury.

[0016] In some preferred embodiments, the liver injury is acute liver injury, specifically acute drug-induced liver injury caused by overdose of acetaminophen (APAP).

[0017] In some embodiments, the disease caused by liver damage is selected from liver fibrosis, cirrhosis, hepatitis, liver cancer, fatty liver, and liver failure.

[0018] Application of trefoil or its extract in the preparation of medicines or health products for preventing and / or treating abnormal transaminase.

[0019] The transaminase includes at least one of alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

[0020] Use of trefoil or its extract in preparing a medicine for preventing and / or treating hepatocyte necrosis.

[0021] Application of trefoil or its extract in preparing medicine or health care product for improving the antioxidant capacity of liver tissue.

[0022] In some embodiments, the drug is prepared from Herba Trifoliatae or an extract thereof and pharmaceutically acceptable excipients.

[0023] In some embodiments, the pharmaceutically acceptable excipients include at least one of a filler, a disintegrant, a diluent, a lubricant, a binder, a wetting agent, a flavoring agent, a suspending agent, a solvent, a sustained-release agent, an emulsifier, an absorption enhancer, a surfactant or a preservative.

[0024] In some embodiments, the dosage form of the drug is granules, tablets, capsules, powders, pills, suspensions or liquid preparations, or one of decoctions, tablets, capsules, granules, pills, oral liquids, powders and pastes.

[0025] In some embodiments, the drug further comprises other liver-protecting drugs, and the liver-protecting drugs are chemical drugs, traditional Chinese medicines or Chinese patent medicine preparations.

[0026] In some embodiments, the drug is a composition obtained by mixing the raw materials after crushing; or an extract obtained by mixing the raw materials, or an extract obtained by mixing the raw materials separately.

[0027] A method for preventing or treating liver damage comprises administering Herba Tripterygii or an extract thereof to a person, patient or animal in need thereof.

[0028] The beneficial effects of this application are:

[0029] The present application provides the use of three-leaf herb or its extract in the prevention and / or treatment of liver damage. The present application study shows that three-leaf herb or its extract can significantly and rapidly reduce transaminase, improve liver function indicators, have the ability to resist oxidative stress levels, protect the histological morphology of the liver, can comprehensively regulate, be gentle and safe, can prevent and / or treat liver damage, and thus have a protective effect on diseases caused by liver damage, and is expected to become a new therapeutic drug for liver damage diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Shown are the changes in serum alanine aminotransferase (ALT) in different groups of male and female mice.

[0031] Figure 2 Shown are the changes in serum aspartate aminotransferase (AST) in different groups of male and female mice.

[0032] Figure 3 Shown are the changes in serum lactate dehydrogenase (LDH) in different groups of male and female mice.

[0033] Figure 4 Shown are changes in liver appearance in different groups of male and female mice.

[0034] Figure 5 H&E sections of livers from male and female mice in different groups.

[0035] Figure 6 Shows the changes in SOD activity in different groups of male mice.

[0036] Figure 7 Shows the changes in GSH content in male mice of different groups.

[0037] Figure 8 Shows the changes in MDA content in male mice of different groups.

[0038] Fig. 9 It is a three-leaf herringbone grass. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0040] Three-leaf herbaceous grass, also known as chicken eye grass and herbaceous grass, is the dried whole herb of the leguminous plant Three-leaf herbaceous grass. It is mainly produced in Guangdong, Guangxi, Fujian and other places. It was first included as a food in the "Compendium of Materia Medica for Famine Relief". Three-leaf herbaceous grass is cool in nature and sweet in taste. It has the effects of cooling blood and detoxifying, clearing away heat and relieving exterior symptoms, dispersing blood stasis and eliminating accumulation. It can be used to treat sore throat, colds, sores, swelling and pain, dysentery and other symptoms.

[0041] The Latin name of the three-leaf herbaceous grass is: Kummerowia striata (Thunb.) Schindl, as shown in the picture Fig. 9 shown.

[0042] Liver damage mainly refers to damage to liver cells, abnormal liver structure and function. Liver damage can stimulate the body to produce too many highly reactive oxygen free radicals, induce oxidative stress, trigger an inflammatory response, lead to liver cell damage and apoptosis, and release too much transaminase into the blood, thereby affecting normal liver function. If not prevented and treated in time, it may induce a series of complications or promote the development of other diseases. The present application has found through a large number of experiments that three-leaf herringbone grass or its extract can be used to prevent and / or treat liver damage, thereby having a preventive and / or therapeutic effect on diseases caused by liver damage.

[0043] Drug-induced liver injury (DILI) refers to liver damage caused by drugs or their metabolites. It can be divided into acute DILI and chronic DILI according to the course and duration of the disease.

[0044] 1. Acute drug-induced liver injury

[0045] Acute drug-induced liver injury generally refers to liver damage that occurs within a short period of time (usually within a few days to a few weeks after drug use). It may be caused by the direct toxic effects of the drug or an individual's allergic reaction to the drug. The characteristics of acute drug-induced liver injury include:

[0046] Rapid onset: Symptoms and liver function abnormalities usually appear soon after the drug is used.

[0047] Symptoms: May include fatigue, nausea, vomiting, jaundice (yellowing of the skin and eyes), abdominal pain, etc.

[0048] Laboratory tests: Liver function tests (such as ALT, AST, ALP, total bilirubin) usually show abnormalities.

[0049] Treatment: The drug that caused the liver injury usually needs to be stopped, and supportive care, such as liver-protective drugs and nutritional support, may be needed.

[0050] 2. Chronic drug-induced liver injury

[0051] Chronic drug-induced liver injury is when drug-induced liver damage lasts for more than 6 months. It may be caused by long-term exposure to certain drugs, which gradually damages the liver. Characteristics of chronic drug-induced liver injury include:

[0052] Slow onset: Symptoms and liver function abnormalities may develop gradually and may not be noticed.

[0053] Symptoms: May include fatigue, jaundice, ascites, spider angiomas, liver palms and other symptoms of chronic liver disease.

[0054] Laboratory tests: Liver function tests may show persistent abnormalities.

[0055] Treatment: In addition to discontinuation of the drug causing the liver injury, long-term medical management may be required, including treatment of complications of cirrhosis.

[0056] Chronic drug-induced liver injury may develop into more serious liver disease, such as cirrhosis or liver failure. Therefore, regular monitoring of liver function is very important for patients who take drugs that may cause liver injury for a long time.

[0057] In the present application, the terms "alanine aminotransferase (ALT)" and "aspartate aminotransferase (AST)" are enzymes whose blood levels increase when the liver is damaged, and are enzymes used as liver function indicators using this property.

[0058] In the present application, the term "lactate dehydrogenase (LDH)" is a key enzyme in glycolysis, which is widely distributed throughout the body and can indirectly reflect the degree of cell damage.

[0059] In the present application, the terms "reduced glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD)" are important indicators of oxidative stress.

[0060] Specifically, the present application verifies the effect of clover or its extract on the levels of ALT, AST, and LDH in serum through an acute liver injury model experiment in mice.

[0061] Examination of biochemical indices in the liver showed that 24 hours after intraperitoneal injection of APAP, the levels of ALT, AST, and LDH in the serum of the model group mice were significantly increased; compared with the control group, the levels of ALT, AST, and LDH in the serum of the APAP model group mice were significantly increased, and the difference was statistically significant (P<0.0001); compared with the APAP model group, the levels of ALT, AST, and LDH in the serum of the mice in the herbaceous clover extract group were significantly decreased, and the difference was statistically significant (P<0.0001).

[0062] The present application further found through liver pathology and histological examination that the APAP model group showed significant enlargement of liver sinusoids with congestion, a large number of inflammatory cell infiltration and increased eosinophilia in multiple hepatocyte cytoplasms, showing eosinophilic changes, and bridging necrosis in the liver lobules. Compared with the APAP model group, the liver lobule structure of the herbaceous clover extract group was restored, the hepatocyte structure was clear, and it was densely arranged in cords. The degree of coagulative necrosis in the central venous area of ​​the liver tissue was significantly reduced, and the degree of hepatocyte nucleus fragmentation was significantly reduced.

[0063] Experiments have shown that three-leaf herb or its extract can reduce substantial damage such as inflammatory response and eosinophilia of liver cells, effectively reduce the levels of AST and AST, LDH in serum, and improve SOD, MDA, GSH in oxidative stress indicators. It has a good preventive and therapeutic effect on liver damage, especially acute chemical liver damage, drug-induced liver damage and alcoholic liver damage.

[0064] In the prior art, commonly used drugs for treatment or prevention, such as NAC (N-acetylcysteine), can only have a good therapeutic effect on drug-induced liver injury when used in early and high doses, and may also cause side effects such as bleeding, nausea and vomiting, diarrhea or constipation. As a small molecule compound, NAC has poor stability in the body, fast clearance, and no specific distribution, resulting in low bioavailability, which greatly limits its clinical treatment effect. However, the three-leaf herb or its extract of the present application has a significant and rapid decrease in transaminase, protects the histological morphology of the liver, can be comprehensively conditioned, and is gentle and safe.

[0065] Therefore, the present application provides the following uses of herba trefoil or its extract:

[0066] Use of trefoil or its extract in preparing medicine or health care product for preventing and / or treating liver damage.

[0067] In some embodiments, the liver injury is chemical liver injury, drug-induced liver injury, or alcoholic liver injury.

[0068] In some preferred embodiments, the liver injury is acute liver injury or chronic liver injury.

[0069] As mentioned above, if liver damage is not prevented and treated in time, it may induce a series of complications or promote the development of other diseases. Trifoliate herb or its extract can be used to prevent and / or treat liver damage, and then prevent and treat other related diseases caused by liver damage.

[0070] Therefore, the present application also provides the use of three-leaf herbaceous grass or its extract in the preparation of a medicine or health product for preventing and / or treating diseases caused by liver damage or diseases or conditions related to liver damage. The diseases or conditions related to liver damage refer to other diseases or conditions caused by liver damage, which may occur due to liver damage, or be alleviated or disappear after the liver damage is treated.

[0071] The specific types of diseases caused by liver damage are not limited in this application. As long as the disease is caused by liver damage, it is within the protection scope of this application, including but not limited to liver fibrosis, cirrhosis, hepatitis, liver cancer, fatty liver, liver failure, etc., for example, liver damage, fatty liver, hepatitis, cirrhosis, liver failure, etc. caused by factors such as alcohol.

[0072] The liver injury described in the present application is acute liver injury; preferably, it is acute drug-induced liver injury caused by overdose of acetaminophen (APAP).

[0073] There are many causes of liver damage, including drug poisoning, viral infection, excessive alcohol intake, immune response, contact with chemical toxic substances in the environment, radiation damage, etc. The three-leaf herbaceous grass or its extract of the present application has a therapeutic or preventive effect on diseases caused by liver damage. The cause of liver damage is not limited. As long as the liver cells are damaged, the liver structure and function are abnormal, the liver damage is within the scope of the present application.

[0074] In some preferred embodiments, the liver damage is liver damage caused by alcohol, virus, drug and chemical toxic substances.

[0075] The term "prevention and / or treatment" means that the disease or disorder described herein can be treated, prevented, alleviated and / or alleviated in a subject.

[0076] The term "disease and / or condition" refers to a physical condition of the subject, which is associated with the disease and / or condition described herein.

[0077] The medicine or health product can be used for patients or other animals that receive the medicine or health product of the present application to treat, prevent, alleviate and / or relieve the diseases or conditions described in the present application. In some preferred embodiments, the medicine or health product is used for mammals, including but not limited to humans, cattle, horses, sheep, pigs, goats, rabbits, cats, dogs, mice and any other mammals that have livers and can cause damage.

[0078] In one preferred embodiment, the medicine or health product is for human use.

[0079] The dosage of the drug or health product of the present application depends on many factors, such as the nature and severity of the disease to be prevented or treated, the gender, age, weight and individual response of the patient or animal, the route of administration and the number of times of administration, etc. The above dosage can be administered in a single dosage form or divided into several dosage forms, such as two, three or four dosage forms. The dosage level must be selected based on the specific route of administration, the severity of the condition to be treated, and the condition and previous medical history of the patient to be treated.

[0080] However, it should be recognized that the total daily dosage of the drug or health product of the present application must be determined by the attending physician within the scope of sound medical judgment. For any specific patient, the specific therapeutically effective dosage level must be determined based on a variety of factors, including the disorder being treated and the severity of the disorder; the specific composition used; the patient's age, weight, general health, sex and diet; administration time, route of administration and excretion rate; duration of treatment; drugs used in combination or concurrently; and similar factors known in the medical field. For example, it is common practice in the art to start the administration of a dose lower than the level required to obtain the desired therapeutic effect and gradually increase the dose until the desired effect is obtained.

[0081] In some preferred embodiments, the drug or health product is administered in a single dose form, i.e., a single dose form. In a further preferred embodiment, the body weight of a person is 60 kg, and the single dose administered is equivalent to 5g-200g of the herb of three-leaf herb, for example, it can be 5g, 8g, 10g, 15g, 20g, 30g, 40g, 50g, 60g, 70g, 80g, 90g, 100g, 110g, 120g, 130g, 140g, 150g, 160g, 170g, 180g, 190g. It should be understood by those skilled in the art that, generally speaking, the body weight of a person is 60 kg. If the body weight of the person is not 60 kg, it can be converted according to this standard.

[0082] The medicine or health product of the present application can be administered in a unit dosage form, and the administration route can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneum or rectum, etc. The dosage form is, for example, tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. It can be a common preparation, a sustained-release preparation, a controlled-release preparation and various microparticle delivery systems.

[0083] The Herba Trifoliata extract described in the present application may be a Herba Trifoliata water extract or a Herba Trifoliata alcohol extract, or may be a mixture of water-alcohol extracts.

[0084] As a preferred embodiment, the Herba Lycopodii Thunbergii extract is the Herba Lycopodii Thunbergii extract prepared by using dried Herba Lycopodii Thunbergii.

[0085] The extraction method of the water extract of Herba Trifoliatae can use any water extraction method in the art. In a preferred embodiment, the present application adopts the following method to prepare the water extract of Herba Trifoliatae:

[0086] (1) Grinding: Take three-leaf herringbone grass and grind it into coarse powder;

[0087] (2) Water heating reflux extraction: Take the crude powder and add water, reflux extraction, filter and add water again, repeat the above reflux extraction steps, and then combine the filtrate;

[0088] (3) Concentrating and drying the filtrate: The filtrate is concentrated and dried to obtain the water extract of Herba Trifoliatae.

[0089] The water can be any form of water in this field, such as purified water, deionized water, secondary water, etc.

[0090] The number of reflux extractions is not limited in this application and can be one, two, three, four or more times.

[0091] The concentration method may be any method known in the art, such as reduced pressure concentration.

[0092] The extraction method of the herbaceous clover extract can use any alcohol extraction method in the art. In a preferred embodiment, the present application adopts the following method to prepare the herbaceous clover alcohol extract:

[0093] (1) Grinding: Take three-leaf herringbone grass and grind it into coarse powder;

[0094] (2) Alcohol heating reflux extraction: Take the crude powder and add alcohol, reflux extraction, filter and add alcohol again, repeat the above reflux extraction steps, and then filter the solution;

[0095] (3) Concentrating and drying the filtrate: The filtrate is concentrated and dried to obtain the Herba Trifoliatae alcohol extract.

[0096] The alcohol may be any common alcohol in the art, such as methanol, propanol, butanol, etc.

[0097] The number of reflux extractions is not limited in this application and can be one, two, three, four or more times.

[0098] The concentration method may be any method known in the art, such as reduced pressure concentration.

[0099] In a preferred embodiment, the medicine or health product is prepared from trefoil or its extract and pharmaceutically / health product acceptable excipients. The pharmaceutically / health product acceptable excipients include at least one of fillers, disintegrants, diluents, lubricants, adhesives, wetting agents, flavoring agents, suspending agents, solvents, sustained release agents, emulsifiers, absorption promoters, surfactants or preservatives. For example: lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gel, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, hydroxybenzene, methyl formate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, but are not limited thereto.

[0100] In a preferred embodiment, the medicine or health product used in the present application for preventing and / or treating diseases caused by liver damage may further contain one or more other active substances. The other active substances refer to other substances that are active and produce certain functions other than trefoil or its extract. The active substances may be active substances for preventing or treating diseases caused by liver damage, or active substances for preventing or assisting in treating diseases caused by liver damage, or active substances that do not treat diseases caused by liver damage, but it may have a positive effect or function in other convenient ways. For example, the active substance may be one or more of Artemisia capillaris, Radix Rehmanniae, Cortex Moutan, Rhizoma Coptidis, Radix Rehmanniae, Radix Bupleuri, Herba Prunellae, Radix Achyranthis Bidentatae, Radix Scutellariae, etc. or their extracts, and the other active substances may be original medicinal materials or extracts thereof (e.g., water extracts, alcohol extracts, or extracts of alcohol-water mixtures).

[0101] The present application further provides a composition for preventing and / or treating diseases caused by liver damage, wherein the composition comprises Herba Trifoliatae or an extract thereof as the sole active ingredient.

[0102] Experimental Materials:

[0103] Animals: SPF male C57BL / 6 mice, weighing 20 (± 2) g, 6 weeks old, provided by the Animal Experiment Center of Guilin Medical College;

[0104] APAP reagent with reagent purity (AR) of 99% was purchased from MCE (MedChemExpress), batch number 114541.

[0105] Liver function test kits (AST, ALT, LDH) were purchased from Shenzhen Mindray Bio-Medical Electronics Co., Ltd.

[0106] The oxidative stress test kit MDA was purchased from Elabscience, and the GSH and SOD test kits were purchased from Nanjing Jiancheng Company.

[0107] The three-leaf herbaceous grass is the whole plant of the three-leaf herbaceous grass, including stems and leaves, and is a dry product obtained by drying after fresh picking.

[0108] The present invention is described below in conjunction with the examples. Unless otherwise specified, other reagents and materials used in the following examples are commercially available.

[0109] Example 1 Preparation of Herba Trifoliatae Extract

[0110] (1) Preliminary crushing: Take the whole plant of Herba Tripterygii and crush it into coarse medicinal powder.

[0111] (2) Pure water heating reflux extraction: Take the dried crude powder of the Chinese medicine, add 8 times the weight of pure water, reflux extract at 120°C for 1 hour, filter, add 8 times the weight of pure water again, reflux at 120°C for 1 hour, filter, and combine the two filtrates.

[0112] (3) Concentration and drying of the filtrate: The filtrate is concentrated under reduced pressure at 60°C and then spray-dried to obtain a Herba Tripterygii extract. 1 g of the Herba Tripterygii extract is equivalent to 50 g of Herba Tripterygii medicinal material.

[0113] Example 2 Study on the effect of Herba Trifolii extract in preventing acetaminophen-induced acute liver injury in mice

[0114] Acetaminophen (APAP), also known as paracetamol, is a widely used antipyretic and analgesic. Its chemical name is N-(4-hydroxyphenyl)acetamide, and its CAS number is 103-90-2. APAP is an over-the-counter drug commonly used to treat symptoms such as colds, fever, joint pain, neuralgia and migraine.

[0115] The purpose of this experimental design is preventive administration. Compared with conventional liver protection drugs (NAC), the inventors determined the administration time and frequency through preliminary experiments. Modeling was performed 1 hour after the last administration of the herbaceous plant extract, and the transaminase index was significantly reduced 24 hours after modeling.

[0116] The inventor believes that prevention is closer to the application of liver protection drugs in clinical practice than treatment. Many liver protection drugs on the market are administered preventively, and liver protection drugs are often prescribed to patients together with drugs that easily induce liver damage. Therefore, through this experimental design, we can determine the role of three-leaf herb in preventing and / or treating liver damage.

[0117] (1) Grouping and drug administration

[0118] aGroup

[0119] Twenty-four 6-week-old C57BL / 6J male mice weighing between 18 and 22 g were randomly divided into 4 groups, namely, blank group (NC), model group (Model, i.e., APAP), Ks H high-dose administration group (Ks H, i.e., APAP+Ks high-dose), with 7 mice in each group, and positive drug group (NAC, i.e., APAP+100 mg / kg NAC), with 6 mice in each group.

[0120] Fifteen 6-week-old C57 BL / 6J female mice weighing between 16 and 20 g were divided into 5 groups, namely, blank group (nc), model group (model, i.e., APAP), high-dose Ksop administration group (Ks h, i.e., APAP+Ks high dose), medium-dose Ksop administration group (Ksm, i.e., APAP+Ks medium dose), and low-dose Ksop administration group (Ks l, i.e., APAP+Ks low dose), with 3 mice in each group.

[0121] The corresponding doses of the three-leaf herbaceous grass extract group were gavage, specifically: a high dose of 240 mg / kg of three-leaf herbaceous grass extract, which is equivalent to 12000 mg / kg of the original medicinal material concentrated and purified; b medium dose of 120 mg / kg of three-leaf herbaceous grass extract, which is equivalent to 6000 mg / kg of the original medicinal material concentrated and purified; c low dose of 60 mg / kg of three-leaf herbaceous grass extract, which is equivalent to 3000 mg / kg of the original medicinal material concentrated and purified. Once a day, for 3 consecutive days.

[0122] b. Administration

[0123] The normal group (NC) and the model group (Model) were gavaged with distilled water of corresponding doses according to body weight, once a day for 3 consecutive days.

[0124] The positive drug group (NAC) was intragastrically administered with 100 mg / kg NAC according to body weight, once a day for 3 consecutive days.

[0125] The group receiving treatment with Herba Tripterygii was given oral administration at three doses: high, medium and low, once a day for 3 consecutive days.

[0126] Then the acute liver injury model was established.

[0127] (2) Establishment of APAP acute liver injury model

[0128] The next day, the mice were fasted for 16 hours at night after the end of the drug administration. After the fasting, the mice were gavaged for the last time according to the groups. The normal group (NC) and the model group (Model) were gavaged with distilled water, the positive drug (NAC) was gavaged with 100 mg / kg NAC, and the drug-treated group was gavaged with the corresponding doses (240 mg / kg, 120 mg / kg, 60 mg / kg) of herbaceous clover.

[0129] In the three-day continuous drug administration, the mice were fasted for 16 hours after the second day of drug administration, and the drug administration was carried out on the third day after the fasting. The fasting was to ensure that the metabolites in the body of each group of mice, especially glutathione, were at the same level before modeling, so as to reduce the influence of metabolites in the body on modeling.

[0130] One hour after the last administration, the blank control group was intraperitoneally injected with normal saline, and the other groups were intraperitoneally injected with 300 mg / kg APAP (dissolved in normal saline) to induce drug-induced acute liver injury in mice.

[0131] (3) Sample collection

[0132] 24 hours after intraperitoneal injection of APAP or saline, mice in each group were weighed, blood was collected from the eyeball, and the blood was centrifuged at 4°C, 3000rpm for 15 minutes after standing at room temperature for 1 hour. The supernatant, i.e. serum, was collected and stored at -80°C. Mice were killed by cervical dislocation, the liver was removed, washed with saline, the surface liquid was absorbed with filter paper, and the body weight was measured; the largest lobe of the liver tissue was divided into 3 parts, fixed in 4% paraformaldehyde solution for subsequent pathological analysis, and the remaining liver lobes were chopped and quickly frozen in liquid nitrogen, and then stored at -80°C.

[0133] (4) Determination of biochemical indicators

[0134] Alanine aminotransferase (ALT) was detected using an automatic biochemical analyzer purchased from Shenzhen Mindray Bio-Medical Electronics Co., Ltd., batch number 140124009.

[0135] Aspartate aminotransferase (AST) was purchased from Shenzhen Mindray Bio-Medical Electronics Co., Ltd., batch number 140224004.

[0136] Lactate dehydrogenase (LDH) was purchased from Shenzhen Mindray Bio-Medical Electronics Co., Ltd., batch number 142723011.

[0137] (5) Pathological section processing

[0138] The liver tissues of the largest right lobe (with the largest cross section, portal vein area and central vein area) of different mice were fixed in 4% paraformaldehyde, dehydrated, transparent, embedded and stored at room temperature. When needed, the slices (4um) were cut and placed in an oven at 70°C for 2h before staining. After dewaxing with xylene, staining with hematoxylin-eosin (H&E), gradient dehydration with different concentrations of ethanol, transparent treatment with xylene, and sealing with neutral resin, the slices were observed under an optical microscope.

[0139] (6) Detection of oxidative stress indicators

[0140] Weigh 1 g of liver tissue accurately, add 9 times the volume of physiological saline, prepare 10% liver tissue homogenate under ice bath conditions, centrifuge at 4°C and 2500r / min for 15 minutes, and collect the supernatant. Strictly follow the instructions of the assay kit to determine SOD activity and GSH and MDA content.

[0141] (7) Statistical processing methods

[0142] Excel tables were used to summarize the data, and GraphPad 8.0.2 statistical software was used for data analysis and plotting. Paired t-test was used for pairwise comparisons. P < 0.05 was considered statistically significant.

[0143] 2. Results

[0144] (1) Changes in serum ALT, AST, and LDH activities in male and female mice in each group

[0145] Results Figure 1 .exist Figure 1 The ALT activity in the serum of the model group was significantly higher than that of the blank control group, and the difference was statistically significant (P<0.0001), indicating that the model was successfully established.

[0146] In the prior art, there is no clear limit on the dosage of APAP in animal model mice. According to the "Clinical Practice Guidelines for Cancer Pain in Adults" (2013 edition), the maximum daily dosage of APAP recommended in humans should not exceed 4,000 mg, 650 mg, taken once every 4 hours, or 1 g, taken once every 6 hours, and long-term use should not exceed 3,000 mg / d. At present, journals generally recognize that the APAP dosage of 300 mg / kg is the regeneration dose. At this dosage, the mouse liver can show obvious liver damage and the damage is the most serious at 24 hours. After 24 hours, the liver can slowly recover on its own. Judging by the experimental results such as liver appearance, biochemical indicators, and pathological sections, if the model group is compared with the blank group, there is a significant change, which can prove that the drug-induced liver injury in mice is successfully constructed. However, at this time (24 hours after modeling), there is currently no recognized unified biochemical indicator. In the study, the inventor obtained that the biochemical indicators related to the model group were significantly increased by nearly 10 times compared with the blank group, and the hepatocyte necrosis area visible in the pathological section was also significantly increased, so the modeling was considered successful. The dose commonly used in journals is 600 mg / kg as a non-regenerative dose, but this dose can cause liver failure and mouse death, so the inventors did not choose this dose in this study.

[0147] References include: Kotulkar M, Paine-Cabrera D, Abernathy S, et al. Role of HNF4alpha-cMyc interaction in liver regeneration and recovery after acetaminophen-induced acute liver injury. Hepatology. 2023;78(4):1106-1117.

[0148] and Figure 1 The relevant data are shown in Table 1.

[0149] Table 1. Changes in serum ALT activity in each group of mice (U / L)

[0150]

[0151] Compared with the NC group, **** P<0.0001; compared with the Model group, #### P<0.0001

[0152]

[0153] Compared with the nc group, ** P<0.001; compared with the model group, ## P<0.001

[0154] Each value on the horizontal axis in the table represents a mouse, and the vertical axis represents different groups. The differences in serum indicators in the normal group (NC) are based on individual differences, and the normal group (NC) did not undergo APAP modeling.

[0155] Figure 1 The results in Table 1 show that in male mice, the ALT activity in the model group was significantly increased (P<0.0001). Compared with the model group, the high-dose group of Herba Trifoliatae significantly reduced the ALT activity (P<0.0001), and the value was close to that of the blank group. This conclusion was also verified in female mice, indicating that the active ingredients of Herba Trifoliatae can effectively prevent APAP-induced acute liver injury.

[0156] The results of changes in serum AST activity of mice in each group are shown in Figure 2 and Table 2.

[0157] Table 2. Changes in serum AST activity in each group of mice (U / L)

[0158]

[0159] Compared with the NC group,**** P<0.0001; compared with the Model group, #### P<0.0001

[0160]

[0161]

[0162] Compared with the nc group, * P<0.05; compared with the model group, # P<0.05

[0163] exist Figure 2 As shown in Table 2, the AST activity in the serum of the model group of male mice was significantly higher than that of the blank control group, and the difference was statistically significant (P<0.0001), indicating that the model was successfully established. Compared with the model group, the high-dose group of Herba Trifoliatae significantly reduced the activity of AST (P<0.0001), and this conclusion was also verified in female mice, indicating that the active ingredients of Herba Trifoliatae can effectively prevent APAP-induced acute liver injury.

[0164] The results of serum LDH activity changes in each group of mice are shown in Figure 3 and Table 3.

[0165] Table 3. Changes in serum LDH activity in each group of mice (U / L)

[0166]

[0167] Compared with the NC group, **** P<0.0001; compared with the Model group, #### P<0.0001

[0168]

[0169] Compared with the nc group, * P<0.05; compared with the model group, # P<0.05

[0170] exist Figure 3 As shown in Table 3, the serum LDH activity of the male mouse model group was significantly higher than that of the blank control group, and the difference was statistically significant (P<0.0001), indicating that the model was successfully established. Compared with the model group, the high-dose group of Herba Trifoliatae significantly reduced the activity of LDH (P<0.0001), and this conclusion was also verified in female mice, indicating that the active ingredients of Herba Trifoliatae can effectively prevent APAP-induced acute liver injury.

[0171] (2) Changes in liver morphology of male and female mice in each group

[0172] Changes in liver appearance of male and female mice in different groups were shown in Figure 4 .

[0173] exist Figure 4 In the experiment, compared with the blank group, the model group showed obvious liver enlargement, light red uneven color, spot-like hemorrhagic necrosis foci on the surface, and the dorsal membrane was tense with a large number of millet-like white spots. It showed an acute liver injury, indicating that drug-induced acute liver injury was fierce. The high-dose Ks administration group significantly improved this phenomenon, the liver color tended to the normal group, the swelling was reduced, and no obvious hemorrhagic necrosis foci were observed. The results showed that the active ingredients of the three-leaf herb extract can protect the appearance of the mouse liver.

[0174] (3) Changes in pathological sections of male and female mice in each group

[0175] H&E sections of livers of male and female mice from different groups are shown in Figure 5 .

[0176] exist Figure 5 In the blank group, hepatocytes were arranged radially with the central vein as the center, the hepatic cord structure was clear, and the hepatic sinusoids were not dilated. No obvious hepatocyte necrosis or degeneration was observed, and there was no inflammatory cell infiltration and fibrosis in the hepatic lobule and portal area. Compared with the blank group, the hepatic sinusoids in the model group were significantly enlarged and congested, with a large number of inflammatory cell infiltrations and multiple hepatocyte cytoplasmic eosinophilic changes. Bridging necrosis appeared in the hepatic lobules. The necrotic area was mainly concentrated around the central vein. Congestion bands were seen in some parts of the necrotic area. Chromatin condensation, cytoplasmic concentration, apoptotic bodies and vacuoles, and large bilirubin-like particles were deposited at the edge of the necrotic area. The central vein destroyed the endothelium of the cell nucleus, and the nucleus dissolved and condensed. The high-dose Ks administration group significantly reversed this change. The hepatic cords were arranged neatly, there was no congestion and enlargement in the hepatic sinusoids, no obvious necrotic area was found, and only a small amount of inflammatory cell infiltration and eosinophilic changes appeared. The results showed that the extract of Trifolium ternariae could improve the pathological damage of liver tissue in mice with acute liver injury induced by APAP.

[0177] (4) Changes of oxidative stress-related indicators in male mice in each group

[0178] The changes in SOD activity, GSH content and MDA content of male mice in different groups are shown in Figure 6 , Figure 7 and Figure 8 .

[0179] Depend on Figure 6 , 78 It can be seen that compared with the NC group, the SOD activity and GSH content in the liver of the Model group mice were significantly reduced (P<0.05), and MDA was significantly increased. The Ks high-dose group restored the SOD activity and significantly increased the GSH content and reduced the MDA content. This shows that the extract of Herba Trifoliatae can improve the antioxidant capacity of liver tissue and alleviate oxidative stress. The relevant specific data are shown in Table 4.

[0180] Table 4. Changes of oxidative stress-related indicators in male mice in each group

[0181]

[0182] Compared with the NC group, * P<0.05; compared with the Model group, # P<0.05

[0183]

[0184] Compared with the NC group, * P<0.05; compared with the Model group, ### P<0.0001

[0185]

[0186]

[0187] Compared with the NC group, ** P<0.001; compared with the Model group, ### P<0.0001

[0188] From the above results, it can be seen that trifoliate herb or its extract can obviously and rapidly reduce transaminase and lactate dehydrogenase, improve liver function indicators, protect liver appearance and tissue morphology, reduce oxidative stress, and can achieve multi-faceted conditioning. It is gentle and safe and can be used to prevent and / or treat liver damage, thereby preventing liver damage from further developing into more serious diseases. It is expected to become a new drug for the treatment of liver damage diseases.

[0189] Although the embodiments of the present invention are described above, the present invention is not limited to the above specific embodiments and application fields, and the above specific embodiments are only illustrative and instructive, but not restrictive. A person skilled in the art can make many forms under the guidance of this specification and without departing from the scope of protection of the claims of the present invention, all of which belong to the protection of the present invention.

Claims

1. Use of Kummerowia striata (Thunb.) Schindl or an extract thereof in the preparation of a medicine or health product for preventing and / or treating liver damage, diseases caused by liver damage, or diseases related to liver damage.

2. The use according to claim 1, characterized in that: The extract of Herba Trifoliatae is a water extract, an alcohol extract, or a water-alcohol extract of Herba Trifoliatae.

3. The use according to claim 1, characterized in that: The liver damage includes drug-induced liver damage, chemical liver damage, and alcoholic liver damage.

4. The use according to claim 1, characterized in that: The liver damage is acute liver damage or chronic liver damage.

5. The use according to claim 1 or 2, characterized in that: The medicine is prepared from the herbaceous clover or its extract as described in claim 1 or 2 and pharmaceutically acceptable excipients.

6. The use according to claim 5, characterized in that: The medicine also includes other liver protection drugs, which are chemical drugs, traditional Chinese medicines or Chinese patent medicine preparations.

7. Use of Herba Tripterygii or its extract in the preparation of medicines or health products for preventing and / or treating abnormal transaminase.

8. The use according to claim 7, characterized in that: The transaminase includes at least one of alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

9. Use of Herba Tripterygii or its extract in the preparation of a medicament for preventing and / or treating hepatocyte necrosis.

10. Use of Herba Tripterygii or its extract in the preparation of medicines or health products for improving the antioxidant capacity of liver tissue.

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