Application of hypochaulinin and alpha-amyrin in anti-hepatic fibrosis

Abstract

The invention discloses an application of hypochaulinin and alpha-amyrin in anti-hepatic fibrosis, discovers and verifies a new application of hypochaulinin and alpha-amyrin as a hepatic fibrosis treatment drug for the first time, and provides a new direction for research and development of the hepatic fibrosis treatment drug. The hypochaulinin and the alpha-amyrin are expected to become a safer and more effective medicine for treating the hepatic fibrosis, and have a wide application prospect in the field of hepatic fibrosis treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular, relates to the application of pyralidin and α-amyrin in preventing liver fibrosis. Background Art

[0002] Hepatic fibrosis (HF) is a process in which multiple factors (such as HBV, HCV, alcohol, etc.) act on the liver, causing an imbalance in the extracellular matrix (ECM). Its further development will lead to cirrhosis and liver cancer, seriously affecting the life and health of patients. Hepatic stellate cells (HSC) are the main source of liver ECM and the cellular basis for the formation of liver fibrosis. They play a central role in ECM metabolism and the production of various cytokines. When the liver is damaged, HSC is activated and secretes a variety of cytokines, which further act on HSC to activate, proliferate, and migrate; myofibroblast-like phenotype transformation occurs, thereby synthesizing and secreting a large amount of type I collagen, type III collagen, fibronectin and other ECM components, which accumulate in the liver. Scar tissue gradually replaces the liver parenchyma, and eventually forms liver fibrosis.

[0003] Hydnocarpin is a flavonoid compound that can be isolated from the Hydnocarpin tree species. As a natural product, hydnocarpin has significant biological activity and anti-cancer potential, especially in the treatment of triple-negative breast cancer, but the relationship between hydnocarpin and liver fibrosis has not been found yet.

[0004] α-Amyrin is a triterpenoid compound that can be isolated from a variety of plants. It has a variety of biological activities, such as anti-inflammatory activity, antispasmodic properties, relaxant effects and analgesic effects. However, no studies have reported the relationship between α-Amyrin and liver fibrosis. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the present invention aims to provide the use of pyralidin and α-amyrin in anti-liver fibrosis.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: The first aspect of the present invention provides the use of pyruvate in the preparation of a medicament for preventing and / or treating liver fibrosis.

[0007] Furthermore, the sub-hyperchloric acid can be directly extracted from natural products or prepared by chemical synthesis; the molecular formula of the sub-hyperchloric acid is C 25 H 20O9, molecular weight 464.421, chemical name 5,7-dihydroxy-2-[2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-2,3-dihydro-1.4-benzodioxin-6-yl]chromen-4-one.

[0008] Furthermore, the drug for preventing and / or treating liver fibrosis includes an inhibitor of liver fibrosis markers in hepatic stellate cells.

[0009] Preferably, the dosage form of the drug includes tablets, capsules, injections, granules, powders, solutions, ointments, suppositories, inhalants, gels, microspheres and / or aerosols.

[0010] The present invention has found through research that pyruvate can achieve the effect of anti-liver fibrosis by inhibiting the expression level of liver fibrosis marker mRNA in hepatic stellate cells. In a specific embodiment of the present invention, the liver fibrosis markers include α-SMA and Col-I.

[0011] α-SMA (α-smooth muscle actin) is a marker of hepatic stellate cell (HSC) activation. During liver injury, quiescent HSCs are activated and transformed into α-SMA-positive myofibroblasts, which are the major contributors of collagen-producing cells in fibrotic livers. Therefore, increased expression of α-SMA is closely associated with the degree of liver fibrosis.

[0012] Col-I (collagen type I) is one of the main components of the liver extracellular matrix (ECM). During liver fibrosis, the synthesis and deposition of Col-I increase significantly, leading to abnormal liver structure and function. Therefore, the content of Col-I is also one of the important indicators reflecting the degree of liver fibrosis. Its increased level is usually associated with the progression and severity of liver fibrosis.

[0013] In the present invention, "liver fibrosis" is defined as the excessive accumulation of connective tissue or scar tissue in the liver. The accumulation of connective tissue / scar tissue in liver fibrosis is excessive compared to the connective tissue level in a normal healthy liver. This fibrosis is often accompanied by necrosis and / or inflammation of liver tissue. In particular, hepatic stellate cells (HSCs) storing vitamin α in a normal liver are transformed into myofibroblasts by acute and chronic liver damage, rapidly proliferate and synthesize excessive connective tissue by the increase of synthesis and migration of extracellular matrix such as collagen, proteoglycans or hyaluronic acid, which leads to the stimulation of the progression of liver fibrosis.

[0014] In the present invention, "anti-liver fibrosis" refers to the prevention of liver fibrosis or the treatment of liver fibrosis. "Treatment" refers to clinical intervention that attempts to change the disease process of the individual or cell being treated, and can be performed during clinical pathology. The therapeutic effects of treatment include, but are not limited to, limiting, preventing disease recurrence, alleviating symptoms, eliminating any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, alleviating or alleviating the disease state, and exempting or improving prognosis.

[0015] In some embodiments, the drug can be prepared according to any preparation method known in the art. For this purpose, if necessary, the hypochlorite can be combined with one or more solid or liquid excipients to prepare a suitable administration form or dosage form for human use.

[0016] In some embodiments, the drug described in the first aspect of the present invention can be prepared into a pharmaceutical preparation of an injection or oral preparation, including but not limited to: oral liquid preparations, granules, injections, tablets, capsules, pills, powders, sustained-release preparations, suppositories, aerosols, nano preparations, tinctures, syrups, wine preparations, and dew preparations. Those skilled in the art can select a suitable dosage form known in the art according to actual needs (e.g., to improve the bioavailability of the drug, to improve the patient's medication compliance, etc.).

[0017] In some embodiments, the hyaluronic acid provided by the present invention, the pharmaceutical composition containing hyaluronic acid or the pharmaceutical preparation containing hyaluronic acid can be administered in the form of unit dosage, and the administration route includes but is not limited to: enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneum or rectum, etc. The dosage form includes but is not limited to tablets, pills, powders, granules, solutions, capsules, dripping pills, aerosols, suspensions, emulsions, 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.

[0018] In some embodiments, in order to prepare the unit dosage form containing pyruvate into tablets, various excipients known in the art can be widely used. Examples of excipients include, but are not limited to: diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, calcium carbonate, microcrystalline cellulose, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, polyvinyl pyrrolidone, etc.; disintegrants, such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, sodium dodecyl sulfate, etc.; disintegration inhibitors, such as sucrose, tristearate, cocoa butter, etc.; absorption promoters, such as quaternary ammonium salts, sodium dodecyl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, etc. The tablets can be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

[0019] In some embodiments, in order to prepare the unit dosage form containing pyruvate into a pill, various excipients known in the art can be widely used. Examples of excipients include, but are not limited to: diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, Gelucire, kaolin, talc, etc.; binders, such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants, such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.

[0020] In some embodiments, in order to prepare the unit dosage form containing pyruvate into suppositories, various excipients known in the art can be widely used. Examples of excipients include, but are not limited to, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, and the like.

[0021] In some embodiments, in order to prepare the unit dosage form containing hydoxycycline into capsules, the active ingredient (hydoxycycline) is mixed with the above-mentioned various excipients, and the resulting mixture is placed in a hard gelatin capsule or a soft capsule. The active ingredient (hydoxycycline) can also be made into microcapsules, suspended in an aqueous medium to form a suspension, or loaded into a hard capsule or prepared as an injection for use.

[0022] In some embodiments, in order to prepare the unit dosage form containing hyaluronic acid into an injectable preparation, such as a solution, emulsion, lyophilized powder injection and suspension, all diluents commonly used in the art can be used, including but not limited to: water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol can be added to the injectable preparation. In addition, conventional cosolvents, buffers, pH regulators, etc. can also be added.

[0023] In certain embodiments, colorants, preservatives, perfumes, flavorants, sweeteners or other materials may be added to the pharmaceutical preparations as necessary.

[0024] It should be noted that the present invention does not have any special restrictions on the specific dosage of the sub-hepatic fibrosis agent. Any dosage that can produce corresponding liver fibrosis treatment and / or prevention effects on subjects or other subjects (such as in vitro cells, in vitro tissues, in vitro organoids, etc.) is within the protection scope of the present invention.

[0025] The second aspect of the present invention provides the use of α-amyrin in the preparation of a medicament for preventing and / or treating liver fibrosis.

[0026] Furthermore, the α-amyrin can be directly extracted from natural products or prepared by chemical synthesis; the molecular formula of the α-amyrin is C 30 H 50 O, molecular weight is 426.72.

[0027] Furthermore, the drug for preventing and / or treating liver fibrosis includes an inhibitor of liver fibrosis markers in hepatic stellate cells.

[0028] Preferably, the dosage form of the drug includes tablets, capsules, injections, granules, powders, solutions, ointments, suppositories, inhalants, gels, microspheres and / or aerosols.

[0029] The present invention has no particular limitation on the specific dosage of the α-amyrin. Any dosage that can produce corresponding liver fibrosis treatment and / or prevention effects on subjects or other subjects (such as in vitro cells, in vitro tissues, in vitro organoids, etc.) is within the protection scope of the present invention.

[0030] In the present invention, "subject" is used interchangeably with "subject", "patient" and refers to animals, preferably mammals such as non-primates (e.g., cows, pigs, horses, cats, dogs, rats, etc.) and primates (e.g., monkeys and humans), and most preferably humans.

[0031] A third aspect of the present invention provides a pharmaceutical composition for preventing and / or treating liver fibrosis.

[0032] Furthermore, the pharmaceutical composition comprises an effective amount of pyralidin and / or an effective amount of α-amyrin.

[0033] Furthermore, the pharmaceutical composition further comprises a second therapeutic agent.

[0034] Preferably, the second therapeutic agent is other drugs used to treat liver fibrosis.

[0035] Preferably, the other drugs for treating liver fibrosis include colchicine, adenosine analogs, liver-protecting drugs, salvia miltiorrhiza, astragalus, and angelica.

[0036] Furthermore, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0037] In the present invention, the terms "therapeutically effective amount" and "effective amount" are used interchangeably to refer to an amount of pyruvate or α-amyrin of the present invention that is sufficient to cause prevention of the development or onset of liver fibrosis or one or more symptoms thereof, enhance or improve the effect of another treatment, and / or alleviate one or more symptoms of liver fibrosis. For subjects suffering from liver fibrosis, a preferred therapeutically effective amount is an amount that effectively reduces fibrosis and / or improves liver function.

[0038] The therapeutically effective amount can be administered to the patient in one or more doses, the dosage being sufficient to alleviate, relieve, stabilize, reverse or slow the progression of the disease, or otherwise alleviate the pathological consequences of the disease, or alleviate the symptoms of the disease. The alleviation or mitigation does not need to be lasting, but can be within a certain time frame (at least one hour, at least one day, or at least one week or longer). The effective amount is usually determined by the doctor according to the individual case, and is within the skill range of those skilled in the art. When determining the appropriate dosage for achieving an effective amount, several factors are usually considered. These factors include the patient's age, sex and weight, the condition being treated, the severity of the condition, and the route of administration, dosage form and regimen, and the desired result.

[0039] Treating liver fibrosis or anti-liver fibrosis refers to administering the pyruvate or α-amyrin described herein to treat subjects with liver fibrosis. One result of the treatment of liver fibrosis is to reduce the formation of connective tissue. Another result of the treatment of liver fibrosis is to reduce inflammation and the infiltration of immune cells. Another result of the treatment of liver fibrosis is to reduce liver tissue necrosis. Another result of the treatment of liver fibrosis is to improve liver function.

[0040] In the present invention, the second therapeutic agent is not particularly limited, and any drug that can be used to prevent and / or treat liver fibrosis, or to assist in the prevention and / or treatment of liver fibrosis, falls within the scope of the second therapeutic agent. Those skilled in the art can combine the hyaluronic acid or α-amyrin with other drugs for the treatment of liver fibrosis according to actual needs. Therefore, using hyaluronic acid or α-amyrin as the only active ingredient or combining hyaluronic acid or α-amyrin with the other drugs as active ingredients in the preparation of drugs for the treatment of liver fibrosis falls within the protection scope of the present invention.

[0041] In some embodiments, the pharmaceutically acceptable excipients are those recognized in the art and include, for example, pharmaceutically acceptable materials, compositions or excipients that participate in carrying or transporting any subject composition from one organ or part of the body to another organ or part of the body, such as liquid or solid fillers, diluents, solvents or encapsulating materials. Each excipient must be "acceptable" in the sense of being compatible with the other ingredients of the subject composition and not injurious to the patient.

[0042] In certain embodiments, the pharmaceutically acceptable excipient is non-pyrogenic. Some examples of materials that can be used as pharmaceutically acceptable excipients include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and Soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer; (21) other non-toxic compatible substances used in pharmaceutical preparations.

[0043] The dosage of the pharmaceutical composition containing pyralidin or α-amyrin of the present invention depends on many factors, such as the nature and severity of the disease to be prevented and / or treated (in the present invention, especially liver fibrosis), the sex, age, weight and individual response of the patient or animal, the specific compound used, the route of administration and the number of administrations, etc. The above dosage can be administered in a single dosage form or divided into several, such as two, three or four dosage forms.

[0044] Furthermore, a variety of prescriptions can be made based on factors such as the formulation method, administration method, patient's age, weight, sex, morbidity, diet, administration time, administration route, excretion rate and reaction sensitivity, and a skilled physician can usually easily determine the prescription and the dosage that is effective for the desired prevention and / or treatment.

[0045] A fourth aspect of the present invention provides an in vitro method for inhibiting hepatic stellate cell fibrosis for non-therapeutic purposes.

[0046] Furthermore, the method comprises the following step: treating hepatic stellate cells with an effective amount of pyralidin and / or an effective amount of α-amyrin.

[0047] The fifth aspect of the present invention provides a method for evaluating whether a drug to be screened has the effect of preventing and / or treating liver fibrosis.

[0048] Furthermore, the method includes using pyralidin or α-amyrin as a positive drug to evaluate the effect of the drug to be screened.

[0049] Compared with the prior art, the present invention has the following beneficial effects: The present invention discovers and confirms for the first time the new use of pyruvate and α-amyrin as drugs for the treatment of liver fibrosis. Pyruvate and α-amyrin can significantly inhibit the expression of hepatic stellate cell fibrosis markers and can be used in the preparation of drugs for the treatment of liver fibrosis. The present invention provides a new direction for the research and development of drugs for the treatment of liver fibrosis. Pyruvate and α-amyrin are expected to become safer and more effective drugs for the treatment of liver fibrosis, and have broad application prospects in the field of liver fibrosis treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The results of expression of genes activated by stimulation of LX-2 cells under TGF-β1 induction are shown; Figure 2 It shows the effect of different concentrations of daphne on the LDH level of LX-2 cells induced by TGF-β1 stimulation; Figure 3 The effect of α-amyrin at different concentrations on LDH levels in LX-2 cells induced by TGF-β1 stimulation is shown; Figure 4 The results show the effect of pyruvate on the expression levels of α-SMA and Col-Ⅰ mRNA in LX-2 cells induced by TGF-β1. Compared with the blank control group, # P <0.05,## P <0.01,### P <0.001; compared with 5ng / mL TGF-β1 group,* P<0.05,** P <0.01,*** P <0.001; Figure 5 The results show the effect of α-amyrin on the expression of α-SMA and Col-Ⅰ mRNA in LX-2 cells induced by TGF-β1. Compared with the blank control group, # P <0.05,## P <0.01,### P <0.001; compared with 5ng / mL TGF-β1 group,* P <0.05,** P <0.01,*** P <0.001. DETAILED DESCRIPTION

[0051] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0052] The drugs, reagents and raw materials used in the present invention are easily obtained by ordinary technicians in the field and can be obtained from commercial channels unless otherwise specified. The experimental methods in the present invention that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way.

[0053] Example Study on the Protective Effects of Asterin and α-amyrin on Hepatic Fibrosis Cell Model 1. Experimental Methods Human hepatic stellate cells (LX-2 cells) were activated by 5ng / mL and 10ng / mL TGF-β1 for 24h, and the expression levels of α-SMA and Col-Ⅰ in the cells were detected by PCR to screen the TGF-β1 concentration that can significantly stimulate the expression of activated genes for subsequent experiments. After TGF-β1 induced LX-2 cells to construct a liver fibrosis cell model, the optimal concentrations of α-amyrin (CAS No.: 638-95-9) and hydoxycycline (CAS No.: 51419-48-8) were added, and the LDH content in the cell supernatant was detected by ELISα. The expression levels of α-SMA and Col-Ⅰ mRNA were detected by RT-PCR to evaluate the anti-fibrotic effects of α-amyrin and hydoxycycline on LX-2 cells induced by TGF-β1.

[0054] 2. Experimental Results The results showed that compared with the blank control group, the expression levels of α-SMA and Col-Ⅰ in LX-2 cells stimulated by 5ng / mL TGF-β1 and 10ng / mL TGF-β1 were significantly increased ( Figure 1 ), indicating that the model was successfully established. Because the activation degree of LX-2 cells under the stimulation concentration of 5ng / mL TGF-β1 was higher than that under 10ng / mL, 5ng / mL TGF-β1 was used for model establishment in the future.

[0055] Compared with the 5ng / mL TGF-β1 group, there was no significant change in LDH content in the positive drug group and the sub-hormone group ( Figure 2 ). There was no significant change in LDH content in the α-amyrin group and the positive drug group. Compared with the 5ng / mL TGF-β1 group, 15μM and 40μM α-amyrin were added on the basis of modeling ( Figure 5 ), 0.4μM, 20μM hyoscyamine ( Figure 4 ) can significantly reduce the expression levels of α-SMA and Col-Ⅰ mRNA in LX-2 cells. In summary, the results show that α-amyrin and hyoscyamine show comparable or better anti-liver fibrosis potential than the positive control drug (TGF-β1 inhibitor). Therefore, these two monomers are considered to be small molecules with anti-liver fibrosis potential.

[0056] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of chloranthone in the preparation of drugs for preventing and / or treating liver fibrosis.

2. The use according to claim 1, characterized in that: The sub-hyperchloric acid can be directly extracted from natural products or prepared by chemical synthesis; the molecular formula of the sub-hyperchloric acid is C 25 H 20 O9, molecular weight 464.421, chemical name 5,7-dihydroxy-2-[2-(4-hydroxy-3-methoxyphenyl)-3- (hydroxymethyl)-2,3-dihydro-1.4-benzodioxin-6-yl]chromen-4-one.

3. The use according to claim 1, characterized in that: The drug for preventing and / or treating liver fibrosis includes an inhibitor of liver fibrosis markers in hepatic stellate cells; Preferably, the dosage form of the drug includes tablets, capsules, injections, granules, powders, solutions, ointments, suppositories, inhalants, gels, microspheres and / or aerosols.

4. Use of α-amyrin in the preparation of drugs for preventing and / or treating liver fibrosis.

5. The use according to claim 4, characterized in that: The α-amyrin can be directly extracted from natural products or prepared by chemical synthesis; the molecular formula of the α-amyrin is C 30 H 50 O, molecular weight is 426.

72.

6. The use according to claim 1, characterized in that: The drug for preventing and / or treating liver fibrosis includes an inhibitor of liver fibrosis markers in hepatic stellate cells; Preferably, the dosage form of the drug includes tablets, capsules, injections, granules, powders, solutions, ointments, suppositories, inhalants, gels, microspheres and / or aerosols.

7. A pharmaceutical composition for preventing and / or treating liver fibrosis, characterized in that: The pharmaceutical composition comprises an effective amount of pyralidin and / or an effective amount of α-amyrin.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition further comprises a second therapeutic agent; Preferably, the second therapeutic agent is other drugs used to treat liver fibrosis; Preferably, the other drugs for treating liver fibrosis include colchicine, adenosine analogs, liver-protective drugs, salvia miltiorrhiza, astragalus, and angelica; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

9. A method for inhibiting hepatic stellate cell fibrosis in vitro for non-therapeutic purposes, characterized in that: The method comprises the following steps: treating hepatic stellate cells with an effective amount of pyralidin and / or an effective amount of α-amyrin.

10. A method for evaluating whether a drug to be screened has the effect of preventing and / or treating liver fibrosis, characterized in that: The method comprises taking pyralidin or α-amyrin as a positive drug to evaluate the effect of the drug to be screened.

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