Application of tripterygium wilfordii quinones natural product in preparation of liver protection medicine

Abstract

The invention relates to application of tripterygium wilfordii quinones natural products in preparation of liver protection drugs. The natural product disclosed by the invention can effectively promote the expression of normal hepatocytes, so that the liver injury is relieved or treated.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to the use of quinone natural products of Tripterygium wilfordii Hook. f. in the preparation of drugs for liver protection. Background Art

[0002] Liver injury is the pathological result of various liver diseases and is also a major global health problem, causing 2 million deaths worldwide every year. Non-alcoholic fatty liver disease (NAFLD) and alcohol-related liver disease (ALD) are the most common chronic liver diseases in clinical diagnosis and have mild symptoms. However, in ALD, acute episodes triggered by increased alcohol intake can exacerbate the disease pathological process, presenting as an acute clinical syndrome called alcoholic hepatitis (AH). Both ALD and NAFLD / non-alcoholic steatohepatitis (NASH) can lead to fibrosis and cirrhosis, increasing the risk of hepatocellular carcinoma. In addition, drug-induced liver injury (DILI) is the main cause of acute liver failure (ALF) and is also one of the main indications for liver transplantation in Western societies. Given the widespread use of prescription and over-the-counter drugs, DILI has also become a global concern, and there is an urgent need to find novel and effective therapies for the treatment of DILI. Summary of the Invention

[0003] The purpose of the present invention is to provide a class of safe and effective drugs for promoting hepatocyte proliferation to treat liver injury and their applications.

[0004] In the first aspect of the present invention, there is provided the use of quinone natural products of Tripterygium wilfordii Hook. f. for preparing a preparation or composition, wherein the preparation or composition is used for the following uses selected from the group:

[0005] (a) Promoting the proliferation of hepatocytes;

[0006] (b) Inhibiting drug-induced liver injury;

[0007] (c) Maintaining the normal function of the liver;

[0008] Wherein, the quinone natural products of Tripterygium wilfordii Hook. f. are selected from the group: tripterygium quinone A or its pharmaceutically acceptable salt, tripterygium quinone B or its pharmaceutically acceptable salt, or a combination thereof.

[0009] In another preferred embodiment, the hepatocytes are normal hepatocytes.

[0010] In another preferred embodiment, the hepatocytes are derived from rodents (such as mice, rats), primate mammals (humans).

[0011] In another preferred embodiment, the proliferation rate of the hepatocytes is ≥1%, preferably ≥5%, more preferably ≥10%, more preferably ≥20%, more preferably ≥30%.

[0012] In another preferred embodiment, the composition is a pharmaceutical composition.

[0013] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: oral preparations, injections, patches, and transdermal agents.

[0014] In another preferred embodiment, the oral preparations are selected from the group consisting of: capsules, tablets, pills, powders, granules, emulsions, solutions, suspensions, syrups, and tinctures.

[0015] In another preferred embodiment, the administration route of the pharmaceutical composition is selected from the group consisting of: oral, intravenous, intramuscular, parenteral, transdermal, intraperitoneal, or a combination thereof.

[0016] In another preferred embodiment, the preparation is a preparation for laboratory use.

[0017] In a second aspect of the present invention, a method for promoting hepatocyte proliferation in vitro is provided, the method comprising:

[0018] (i) Culturing hepatocytes in the presence of tripterygium p-benzoquinone natural products, thereby promoting the proliferation of hepatocytes,

[0019] wherein the tripterygium p-benzoquinone natural products are selected from the group consisting of: tripterygium p-benzoquinone A or a pharmaceutically acceptable salt thereof, tripterygium p-benzoquinone B or a pharmaceutically acceptable salt thereof.

[0020] In another preferred embodiment, the hepatocytes are normal hepatocytes.

[0021] In another preferred embodiment, the hepatocytes are derived from rodents (such as mice, rats), primate mammals (humans).

[0022] In another preferred embodiment, the hepatocytes are selected from the group consisting of: mouse hepatic parenchymal cells AML12 (Alpha Mouse Liver 12), human normal hepatocytes THLE-2.

[0023] In another preferred embodiment, the concentration of the tripterygium p-benzoquinone natural products is 1 - 1000 μg / ml; preferably 5 - 500 μg / ml; more preferably 5 - 100 μg / ml.

[0024] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0025] In a third aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising: (A) an active ingredient; the active ingredient is selected from the group consisting of: tripterygium p-benzoquinone A or a pharmaceutically acceptable salt thereof, tripterygium p-benzoquinone B or a pharmaceutically acceptable salt thereof; and (B) a pharmaceutically acceptable carrier.

[0026] In another preferred example, the active ingredient is the only active ingredient in the pharmaceutical composition.

[0027] In a fourth aspect of the present invention, there is provided a combination of active ingredients, the combination comprising:

[0028] (a1) A first active ingredient selected from the group consisting of tripteronquinone A or a pharmaceutically acceptable salt thereof, and tripteronquinone B or a pharmaceutically acceptable salt thereof;

[0029] (a2) A second active ingredient, which is a therapeutic agent different from the first active ingredient and used for inhibiting liver injury.

[0030] In another preferred example, the second active ingredient includes: glycyrrhizic acid derivatives, silymarin derivatives, glutathione, N-acetylcysteine, bicyclol, tiopronin, polyene phosphatidylcholine, ursodeoxycholic acid derivatives, ademetionine, obeticholic acid, silymarin.

[0031] In another preferred example, the concentration ratio of the first active ingredient to the second active ingredient is from 1000:1 to 1:1000; preferably, from 100:1 to 1:100; more preferably, from 10:1 to 1:10.

[0032] In another preferred example, the mass ratio of the first active ingredient to the second active ingredient is from 1:500 to 500:1; preferably, from 100:1 to 1:100; more preferably, from 1:50 to 50:1.

[0033] In a fifth aspect of the present invention, there is provided a pharmaceutical composition, the pharmaceutical composition comprising:

[0034] (1) The combination of active ingredients according to the fourth aspect of the present invention; and

[0035] (2) A pharmaceutically acceptable carrier.

[0036] In a sixth aspect of the present invention, there is provided a method for treating liver injury, comprising: administering to a subject in need a therapeutically effective amount of a liver injury inhibitor selected from the group consisting of tripteronquinone A or a pharmaceutically acceptable salt thereof, and tripteronquinone B or a pharmaceutically acceptable salt thereof.

[0037] In another preferred example, the subjects include rodents (such as mice, rats), and primate mammals (humans).

[0038] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Detailed implementation mode

[0039] Through extensive and in - depth research and extensive screening of natural products, the inventors unexpectedly found that tripterygium quinone A and tripterygium quinone B can promote the proliferation of normal hepatocytes, indicating that these two natural products may repair damaged tissues and restore liver function. For example, in acute liver injury, accelerating cell proliferation helps to quickly recover. In chronic liver diseases, it may delay the progression of the disease, such as fibrosis or cirrhosis, because continuous damage leads to cell death, and proliferation may replenish the lost cells and reduce the formation of scar tissue. It can also help the liver repair, maintain function, delay the progression of the disease, and reduce the need for transplantation. Based on this, the present invention was completed.

[0040] Terms

[0041] Tripterygium quinone A and tripterygium quinone B are two natural organic compounds, which are quinone compounds extracted from Tripterygium wilfordii. It is reported that the active ingredients in Tripterygium wilfordii have anti - inflammatory and anti - tumor effects, etc., but there is certain hepatotoxicity.

[0042] Tripterygium quinone A (Triptoquinone A), CAS: 142950 - 86 - 5, its structure is shown as follows,

[0043]

[0044] Tripterygium quinone B (Triptoquinone B), CAS: 142937 - 50 - 6, its structure is shown as follows,

[0045]

[0046] Drug - induced liver injury

[0047] During the use of drugs, liver injury caused by the drug itself or / and its metabolites, or due to special physical constitution with hypersensitivity or reduced tolerance to the drug is called drug - induced liver injury, also known as drug - induced liver disease, including liver injury caused by chemical drugs, biological products, Chinese patent medicines, etc. managed as prescription or over - the - counter drugs, as well as products such as Chinese medicinal materials, natural drugs, health products, dietary supplements, etc., or their metabolites, and even their excipients, pollutants, impurities, etc. Clinical manifestations include acute or chronic liver diseases, including liver injury.

[0048] Drug composition

[0049] The present invention also provides a drug composition for preparing a drug for promoting the proliferation of hepatocytes and liver protection, and the active ingredient it contains is selected from tripterygium quinone A or its pharmaceutically acceptable salt, tripterygium quinone B or its pharmaceutically acceptable salt, or a combination thereof.

[0050] The present invention also provides a pharmaceutical composition, which comprises an active ingredient within a safe and effective amount range, and a pharmaceutically acceptable carrier.

[0051] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" herein means that the components in the composition can be blended with the active ingredient of the present invention and with each other without significantly reducing the efficacy of the active ingredient. Some examples of pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyhydric alcohols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0052] There is no particular limitation on the administration mode of the active ingredient or pharmaceutical composition of the present invention. Representative administration modes include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), etc.

[0053] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.

[0054] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers. For example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, etc. In addition to these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweetening agents, flavoring agents and fragrances.

[0055] In addition to the active ingredient, suspensions may contain suspending agents, such as ethoxylated isooctadecanol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum monostearate and agar or mixtures of these substances, etc.

[0056] Compositions for parenteral injection may contain physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstituting into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyhydric alcohols and their suitable mixtures.

[0057] The main advantages of the present invention include:

[0058] (1) The active ingredient of the present invention has an excellent effect of promoting the proliferation of normal hepatocytes.

[0059] (2) The drug of the present invention is simple and convenient to obtain, has a low price, and is suitable for large-scale production.

[0060] (3) Compared with synthetic drugs, natural drugs are more easily metabolized by the body, reducing the risk of toxic accumulation.

[0061] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0062] Instruments and materials:

[0063] Cell counting slides (Model: JSP-GM, Guangzhou Boda Boju Technology Co., Ltd.); cell counters (Model: JSY-FL-045N, Guangzhou Boda Boju Technology Co., Ltd.); cell culture incubators (Thermo Fisher Scientific, USA); inverted fluorescence microscopes (Model: TS100, Nikon Corporation); digital display constant temperature water bath (Xicheng Xinrui Instrument Factory, Jintan District); 96-well cell culture plates (Corning Life Sciences, USA); cell culture dishes (Corning Life Sciences, USA); DMEM medium (Thermo Fisher Scientific, USA); cell proliferation-toxicity detection kit Cell Counting Kit-8 (Langjieke Technology Co., Ltd.); fetal bovine serum FBS (Merck, Germany)

[0064] Example 1

[0065] By the CCK-8 method, more than a dozen natural products were screened, and their proliferation or inhibitory effects on normal hepatocytes and hepatoma cells were measured respectively. Among them, unexpectedly, it was found that two natural products, tripteronquinone A and tripteronquinone B, have an obvious effect of promoting the proliferation of normal hepatocytes.

[0066] Subsequently, the effects of tripteronquinone A and tripteronquinone B on the activities of normal mouse liver cells AML12 and THLE-2 cells, as well as liver cancer cells hepG2 and hep1-6, were measured. It was found that tripteronquinone A and tripteronquinone B had a significant promoting effect on the proliferation of normal liver cells, while the promoting effect on the proliferation of liver cancer cells hepG2 and hep1-6 was not obvious. After comparing with the OD values of normal cells, the proliferation rates of tripteronquinone A / B on different types of liver cells were determined.

[0067] 1.1 Test method

[0068] In this experiment, a CCK-8 kit was used to measure the promoting effect of tripteronquinone A / B on normal mouse liver cells AML12 and THLE-2 cells.

[0069] The CCK-8 kit is a rapid, highly sensitive, and non-radioactive colorimetric detection kit based on WST-8 and widely used in cell proliferation and cytotoxicity. After adding the CCK-8 solution for color development, the cell survival rate is calculated by measuring its absorbance to reflect the drug activity.

[0070] Calculation formula

[0071] Cell survival rate = [(As - Ab) / (Ac - Ab)] × 100%

[0072] Inhibition rate = [(Ac - As) / (Ac - Ab)] × 100%

[0073] As: Absorbance of the experimental well (containing cells, medium, CCK-8 solution, and drug solution);

[0074] Ac: Absorbance of the control well (containing cells, medium, CCK-8 solution, without drug);

[0075] Ab: Absorbance of the blank well (containing medium, CCK-8 solution, without cells, drug);

[0076] 1.1.1 Experimental procedure

[0077] Culture and passage the cells, count them and inoculate them in a 96-well plate (the cell concentration per well is about 10 5 / 100 μl). After the cells cover more than 50%, administer the drug. After 24 hours of culture, add 10% CCK-8 reagent (100 μl), incubate in the dark for 2 hours, measure the absorbance (450 nm) with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the cell survival rate and its IC 50 value

[0078] 1.1.2 Drug preparation

[0079] The concentration of the drug stock solution is 1 mg / ml, and the experimental well concentration range (50, 25, 12.5, 6.25 μg / ml) is prepared by serial dilution. Three replicate wells are set for each concentration.

[0080] 1.2 Experimental results

[0081] Table 1. Proliferation-promoting effects of tripterygium quinone A on normal hepatocytes at different drug administration concentrations

[0082]

[0083] Table 2. Proliferation-promoting effects of tripterygium quinone B on normal hepatocytes at different drug administration concentrations

[0084]

[0085] As shown in Tables 1 and 2, tripterygium quinone A and tripterygium quinone B at different concentrations have a promoting effect on the proliferation of hepatocytes.

[0086] Table 3. Survival rates of liver cancer cells at different tripterygium quinone A administration concentrations

[0087]

[0088] Table 4. Survival rates of liver cancer cells at different tripterygium quinone B administration concentrations

[0089]

[0090] All documents mentioned in the present invention are cited herein by reference as if each document was individually cited as reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. The use of Tripterygium wilfordii for quinone natural products, characterized in that: For preparing a preparation or composition, wherein the preparation or composition is used for a purpose selected from the group consisting of: (a) Promote the proliferation of hepatocytes; (b) inhibit drug-induced liver injury; (c) Maintain normal liver function; Wherein, the tripterygium wilfordii quinone natural product is selected from the following group: tripterygium wilfordii quinone A or a pharmaceutically acceptable salt thereof, tripterygium wilfordii quinone B or a pharmaceutically acceptable salt thereof, or a combination thereof.

2. The use according to claim 1, characterized in that The hepatocytes are normal hepatocytes.

3. The use according to claim 1, characterized in that The hepatocytes are derived from rodents (such as mice and rats) and primates (humans).

4. The use according to claim 1, characterized in that The proliferation rate of the hepatocytes is ≥1%, preferably ≥5%, more preferably ≥10%, more preferably ≥20%, and more preferably ≥30%.

5. The use according to claim 1, characterized in that A method for promoting hepatocyte proliferation in vitro, characterized in that the method comprises: (i) culturing hepatocytes in the presence of a natural product of tripterygium wilfordii quinones, thereby promoting the proliferation of the hepatocytes, Wherein, the tripterygium wilfordii quinone natural product is selected from the following group: tripterygium wilfordii quinone A or a pharmaceutically acceptable salt thereof, tripterygium wilfordii quinone B or a pharmaceutically acceptable salt thereof.

6. The use according to claim 5, characterized in that The hepatocytes are normal hepatocytes.

7. The use according to claim 5, characterized in that The concentration of the tripterygium wilfordii quinone natural product is 1-1000 μg / ml, preferably 5-500 μg / ml, and more preferably 5-100 μg / ml.

8. A pharmaceutical composition comprising: (A) an active ingredient; the active ingredient is selected from the following group: tripterygium wilfordii quinone A or a pharmaceutically acceptable salt thereof, tripterygium wilfordii quinone B or a pharmaceutically acceptable salt thereof; and (B) a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, characterized in that The active ingredient is the only active ingredient in the pharmaceutical composition.

10. An active ingredient combination, comprising: (a1) a first active ingredient, wherein the first active ingredient is selected from the group consisting of tripterygium wilfordii quinone A or a pharmaceutically acceptable salt thereof, tripterygium wilfordii quinone B or a pharmaceutically acceptable salt thereof; (a2) a second active ingredient, the second active ingredient being a therapeutic agent for inhibiting liver damage different from the first active ingredient.

Images