Composition containing miltirone A, miltirone and salvianolic acid H and application thereof

Abstract

The invention discloses a pharmaceutical composition containing miltirone A, miltirone and salvianolic acid H, wherein the molar concentration ratio of the miltirone A to the miltirone to the salvianolic acid H is (0.1 to 200): (0.1 to 200): (0.1 to 200). According to the pharmaceutical composition disclosed by the invention, liver inflammation and liver fibrosis are inhibited by improving liver functions, reducing damage of liver cells, relieving infiltration of liver inflammatory cells and collagen deposition.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a composition comprising danshenquinone A, danshenone and salvianolic acid H and uses thereof. Background Art

[0002] The liver is an important organ in the human digestive system that is responsible for the metabolism and transformation of a variety of substances. It is not only involved in the metabolism of sugars, lipids, and proteins, but also plays a key role in detoxification, bile secretion, and vitamin storage. Liver health is crucial for maintaining the homeostasis of the body's internal environment. However, because the liver is directly exposed to endogenous metabolites and exogenous toxic substances (such as drugs, alcohol, viruses, etc.), it is extremely susceptible to damage from a variety of acute or chronic factors. Although the liver has a strong self-repair ability, when the damaging factors persist, the tissue remodeling in the liver repair process may be out of control, leading to the occurrence of fibrosis.

[0003] Liver fibrosis is a pathological response of the liver to long-term damage. Its core characteristics are the abnormal deposition of extracellular matrix (ECM) and the formation of scar tissue in the liver. This process not only destroys the normal function of the liver, but also changes the structure of liver tissue, eventually leading to cirrhosis and even further development of hepatocellular carcinoma (HCC). This pathological process seriously threatens the life and health of patients, but there is currently a lack of effective treatment for liver fibrosis. Therefore, in-depth research on the pathogenesis of liver fibrosis and the development of targeted treatment strategies are one of the important directions of current medical research.

[0004] Previous studies have suggested that the sustained activation of hepatic stellate cells (HSCs) is a central component of liver fibrosis. In the normal liver, HSCs are primarily distributed in the perisinusoidal space, remain quiescent, and have a star-shaped morphology. Their primary function is to store vitamin A and its esters and to maintain liver matrix homeostasis. However, under the stimulation of liver injury, such as viral infection, alcohol toxicity, metabolic disorders, or cholestasis, HSCs are rapidly activated and transform into a myofibroblast-like phenotype. This transformation is accompanied by significant changes in cell morphology, function, and gene expression, including high expression of α-smooth muscle actin (α-SMA), excessive synthesis and secretion of extracellular matrix (ECM) components such as type I and type III collagen, and a significant increase in cell proliferation. Furthermore, activated HSCs secrete a variety of proinflammatory cytokines and chemokines, further recruiting immune cells and forming an inflammatory microenvironment, thereby exacerbating liver injury and fibrosis. Studies have shown that HSCs are the primary source of excessive extracellular matrix deposition during liver fibrosis. Their activation is not only a key event in the development of liver fibrosis but also a key driving force in the progression of fibrosis to cirrhosis and even liver cancer. Therefore, inhibiting HSC activation or reversing its activation state has become an important strategy and research hotspot for anti-liver fibrosis treatment.

[0005] Liver fibrosis falls under the categories of "side pain" and "accumulation" in Traditional Chinese Medicine. Clinically, it can be manifested by side pain, fullness and pain under the side, and dull complexion. "Blood stasis blocking the collaterals" is its basic pathogenesis, and promoting blood circulation and removing blood stasis is an important treatment for liver fibrosis. Long-term clinical practice, especially research in recent years, has fully demonstrated the significant advantages of traditional Chinese medicine in the treatment of liver fibrosis and cirrhosis, and its application prospects are extremely broad. Due to their diverse ingredients, traditional Chinese medicine compound prescriptions or traditional Chinese medicine monomer compositions usually have the characteristics of comprehensive effects of multiple pathways, multiple links, multiple levels, and multiple targets. This holistic regulatory mechanism is particularly suitable for coping with the complex pathological process of liver fibrosis, providing a unique and effective solution for the prevention and treatment of the disease.

[0006] The pharmaceutical composition comprising danshenxinquinone A, danshenxinone and salvianolic acid H as claimed in the present invention has not been found in the prior art. Summary of the Invention

[0007] Based on this, the present invention provides a pharmaceutical composition comprising danshenquinone A, danshenone and salvianolic acid H, wherein the molar concentration ratio of danshenquinone A, danshenone and salvianolic acid H is (0.1-200): (0.1-200): (0.1-200).

[0008] Furthermore, the molar concentration ratio of the danshenquinone A, the danshenone and the salvianolic acid H is (0.1-25): (0.1-23): (0.1-126).

[0009] Furthermore, the molar concentration ratio of the danshenquinone A, the danshenone and the salvianolic acid H is (0.15-10): (0.1-10): (0.11-40).

[0010] Furthermore, the molar concentration ratio of the danshenquinone A, the danshenone and the salvianolic acid H is (0.2-5): (0.1-5): (0.12-30).

[0011] Furthermore, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is about 2.6: about 2.3: about 25.045.

[0012] Furthermore, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is about 1.2: about 1: about 20.

[0013] Furthermore, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is about 1.2: about 1: about 5.2.

[0014] Furthermore, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is about 0.245: about 2.3: about 12.6.

[0015] Furthermore, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is about 2.6: about 4.486: about 12.6.

[0016] Furthermore, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is about 4: about 1: about 20.

[0017] Furthermore, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is about 2.6: about 2.3: about 0.154.

[0018] Furthermore, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is about 2.6: about 0.114: about 12.6.

[0019] Further, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is not about 4.955: about 2.3: about 12.6.

[0020] Furthermore, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is not about 1.2: about 3.6: about 20.

[0021] Further, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is not about 2.6: about 2.3: about 12.6.

[0022] Furthermore, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is not about 4: about 3.6: about 5.2.

[0023] Further, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is not about 4: about 3.6: about 20.

[0024] Furthermore, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is not about 4: about 1: about 5.2.

[0025] According to another aspect of the present invention, there is provided a pharmaceutical preparation comprising the above-mentioned pharmaceutical composition and one or more optional pharmaceutically acceptable excipients.

[0026] Furthermore, the auxiliary material is selected from one or more of the following: diluent, wetting agent, binder, disintegrant, inclusion agent, flavoring agent, sustained-release agent, glidant, lubricant, dispersant, plasticizer, opacifier and antioxidant.

[0027] Furthermore, the dosage form of the pharmaceutical preparation is powder, tablet, pill, capsule, film, lozenge, granule, liposome nanoparticle, injection or oral solution.

[0028] Furthermore, the pharmaceutical composition or the pharmaceutical preparation further comprises one or more drugs and / or extracts for preventing and / or treating liver damage.

[0029] Furthermore, the liver damage is hepatitis and / or liver fibrosis.

[0030] Furthermore, the drug is a liver-protecting drug and / or an anti-liver fibrosis drug.

[0031] Furthermore, the hepatoprotective drug is selected from one or more of the following: anti-inflammatory hepatoprotective drugs, liver cell membrane repairing hepatoprotective drugs, antioxidant hepatoprotective drugs, choleretic hepatoprotective drugs, enzyme lowering hepatoprotective drugs, energy metabolism promoting hepatoprotective drugs and liver cell regeneration promoting hepatoprotective drugs.

[0032] Furthermore, the anti-liver fibrosis drug is selected from one or more of the following: Fuzheng Huayu Tablets, Fuzheng Huayu Capsules, Fufang Biejia Ruangan Tablets, Fufang Biejia Ruangan Capsules and Anluo Huaxian Pills.

[0033] Furthermore, the anti-inflammatory liver-protecting drug is selected from one or more of the following: prednisone, methylprednisolone, diammonium glycyrrhizinate, magnesium isoglycyrrhizinate, compound monoammonium glycyrrhizinate and compound glycyrrhizin.

[0034] Furthermore, the liver cell membrane repairing liver protection drug is polyene phosphatidylcholine.

[0035] Furthermore, the antioxidant liver-protecting drug is selected from one or more of the following: vitamin E, resveratrol, caffeic acid, dihydromyricetin, silymarins, bicyclol, glutathione and soybean lecithin.

[0036] Furthermore, the choleretic hepatoprotective drug is ursodeoxycholic acid and / or adenosine succinate.

[0037] Furthermore, the enzyme-lowering liver-protecting drug is bifendate and / or pentaester capsule.

[0038] Furthermore, the liver-protecting drug that promotes energy metabolism is selected from one or more of the following: adenosine triphosphate, coenzyme A, inosine, and coenzyme Q10.

[0039] Furthermore, the liver-protecting drug that promotes liver cell regeneration is a naked plasmid injection of recombinant human hepatocyte growth factor.

[0040] Furthermore, the extract is selected from one or more of the following: salvia miltiorrhiza extract, white peony root extract, angelica root extract, ligusticum chuanxiong extract, capillaris wormwood extract, sophora flavescens extract, astragalus root extract, schisandra chinensis extract and milk thistle extract.

[0041] Furthermore, the drug or the extract is a monomer compound or a mixture thereof obtained from a natural product.

[0042] According to another aspect of the present invention, there is provided a method for preparing the above-mentioned pharmaceutical preparation, the method comprising the following steps:

[0043] (1) mixing the above composition with a pharmaceutically acceptable excipient, and heating until the pharmaceutically acceptable excipient is melted to obtain a mixture; wherein the mass ratio of the composition to the pharmaceutically acceptable excipient is about 1:(0.1-100); and

[0044] (2) mixing the mixture uniformly, cooling it, and pulverizing it to obtain the pharmaceutical preparation.

[0045] According to another aspect of the present invention, there is provided a use of the above-mentioned composition or pharmaceutical preparation in the preparation of a medicament for preventing and / or treating liver injury.

[0046] Furthermore, the liver damage includes hepatitis and / or liver fibrosis.

[0047] Furthermore, the hepatitis is liver inflammation with abnormal serum liver function.

[0048] Furthermore, the serum liver function test is selected from one or more of the following: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and glutamyl transpeptidase.

[0049] Furthermore, the serum liver function is further selected from one or more of the following: total bile acid, albumin, total bilirubin, direct bilirubin and indirect bilirubin.

[0050] Furthermore, the liver fibrosis is virus- and / or drug-induced liver fibrosis.

[0051] Furthermore, the drug-induced liver fibrosis is liver fibrosis induced by antitumor drugs, antituberculosis drugs, antipyretic analgesics, immunosuppressants, hypoglycemic drugs, lipid-lowering drugs, antibacterial drugs, antifungal drugs and / or antiviral drugs.

[0052] Furthermore, the liver fibrosis is liver fibrosis induced by carbon tetrachloride and / or dimethylnitrosamine.

[0053] Furthermore, the liver fibrosis is liver fibrosis induced by hepatitis B virus and / or hepatitis C virus.

[0054] Furthermore, the liver fibrosis is liver fibrosis associated with activation of hepatic stellate cells.

[0055] Furthermore, the hepatic stellate cell activation is hepatic stellate cell activation with increased expression of α-smooth muscle actin.

[0056] Furthermore, the composition or pharmaceutical preparation prevents and / or treats liver damage in one or more of the following ways: inhibiting hepatic stellate cell activation, improving abnormal serum biochemical indicators, reducing liver tissue hydroxyproline content, and improving liver tissue pathological abnormalities.

[0057] Furthermore, the hepatic stellate cells are LX-2 hepatic stellate cells.

[0058] Furthermore, the inhibition of hepatic stellate cell activation includes: reducing the level of α-SMA in LX-2, JS-1, and T6 hepatic stellate cells.

[0059] Furthermore, the abnormal serum biochemical indicators include increased serum alanine aminotransferase level, increased serum aspartate aminotransferase level and decreased serum albumin content.

[0060] Furthermore, the liver tissue pathological abnormalities include liver tissue inflammation and liver tissue collagen deposition.

[0061] Beneficial effects of the present invention:

[0062] The pharmaceutical composition of the present invention improves liver function, reduces liver cell damage, alleviates liver inflammatory cell infiltration and collagen deposition, and inhibits liver inflammation and liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0064] Figure 1 Schematic diagram of the toxic effects of danshenquinone A, danshenone and danshenolic acid H on LX-2 cells.

[0065] Figure 2 Schematic diagram of the effects of DMS-8 on serum liver function and Hyp content in liver tissue of rats with DMN-induced liver fibrosis.

[0066] Figure 3 Schematic diagram of the effects of DMS-8 on liver tissue inflammation and collagen deposition in rats with DMN-induced liver fibrosis. DETAILED DESCRIPTION

[0067] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work shall fall within the scope of protection of the present invention.

[0068] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those skilled in the art in the field of the present invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar or equivalent to those disclosed herein can be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0069] The present invention is intended to encompass all alternatives, modifications, and equivalents that may come within the scope of the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0070] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0071] In the present invention, the term "comprising" is synonymous with "including." As used herein, the terms "comprises," "including," "having," "containing," or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0072] As described in the background technology section, existing common drugs used to treat liver damage, such as liver fibrosis, are prone to hepatotoxicity, poor efficacy, and lack therapeutic specificity. To address these issues, the present invention provides a pharmaceutical composition comprising danshenxinquinone A, danshenxinone, and salvianolic acid H, wherein the molar concentration ratio of danshenxinquinone A, danshenxinone, and salvianolic acid H is (0.1-200):(0.1-200):(0.1-200).

[0073] The following are the chemical structural formulas of danshenxinquinone A, danshenxinone and salvianolic acid H:

[0074]

[0075] In the present invention, when the molar concentration ratio, mass ratio, or other value or parameter is expressed as a range, a preferred range, or a range limited by a series of upper preferred values and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when the range "0.1-200" is disclosed, the described range should be interpreted as including the range "0.1-200", "0.1-160", "0.1-120", "0.1-80", "0.1-40", "40-200", "40-160", "40-120", "40-80", "80-200", "80-160", "80-120", "120-200", "120-160", "160-200", etc. When a numerical range is described in this article, unless otherwise stated, the range is intended to include its end values and all integers and fractions within the range.

[0076] The pharmaceutical composition of the present invention comprising three traditional Chinese medicine monomers, namely, danshenquinone A, danshenxinone and salvianolic acid H, can significantly reduce serum transaminase levels in mice with liver fibrosis model, and alleviate liver tissue inflammation and collagen deposition.

[0077] In a preferred embodiment, the molar concentration ratio of the danshenquinone A, the danshenone and the salvianolic acid H is (0.1-25): (0.1-23): (0.1-126).

[0078] In a preferred embodiment, the molar concentration ratio of the danshenquinone A, the danshenone and the salvianolic acid H is (0.15-10): (0.1-10): (0.11-40).

[0079] In a preferred embodiment, the molar concentration ratio of the danshenquinone A, the danshenone and the salvianolic acid H is (0.2-5): (0.1-5): (0.12-30).

[0080] In a preferred embodiment, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is about 2.6: about 2.3: about 25.045.

[0081] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2.6" includes ±5% of 2.6, or from 2.47 to 2.73; "about 2.3" includes ±5% of 2.3, or from 2.185 to 2.415; "about 25.045" includes ±5% of 25.045, or from 23.79275 to 26.29725.

[0082] In a preferred embodiment, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is about 1.2: about 1: about 20.

[0083] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 1.2" includes ±5% of 1.2, or from 1.14 to 1.26; "about 1" includes ±5% of 1, or from 0.95 to 1.05; "about 20" includes ±5% of 20, or from 19 to 21.

[0084] In a preferred embodiment, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is about 1.2: about 1: about 5.2.

[0085] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.2" includes ±5% of 1.2, or from 1.14 to 1.26; "about 1" includes ±5% of 1, or from 0.95 to 1.05; "about 5.2" includes ±5% of 5.2, or from 4.94 to 5.46.

[0086] In a preferred embodiment, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is about 0.245: about 2.3: about 12.6.

[0087] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.245" includes ±5% of 0.245, or from 0.23275 to 0.25725; "about 2.3" includes ±5% of 2.3, or from 2.185 to 2.415; "about 12.6" includes ±5% of 12.6, or from 11.97 to 13.23.

[0088] In a preferred embodiment, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is about 2.6: about 4.486: about 12.6.

[0089] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2.6" includes ±5% of 2.6, or from 2.47 to 2.73; "about 4.486" includes ±5% of 4.486, or from 4.2617 to 4.7103; "about 12.6" includes ±5% of 12.6, or from 11.97 to 13.23.

[0090] In a preferred embodiment, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is about 4: about 1: about 20.

[0091] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2; "about 1" includes ±5% of 1, or from 0.95 to 1.05; "about 20" includes ±5% of 20, or from 19 to 21.

[0092] In a preferred embodiment, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is about 2.6: about 2.3: about 0.154.

[0093] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2.6" includes ±5% of 2.6, or from 2.47 to 2.73; "about 2.3" includes ±5% of 2.3, or from 2.185 to 2.415; "about 0.154" includes ±5% of 0.154, or from 0.1463 to 0.1617.

[0094] In a preferred embodiment, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is about 2.6: about 0.114: about 12.6.

[0095] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2.6" includes ±5% of 2.6, or from 2.47 to 2.73; "about 0.114" includes ±5% of 0.114, or from 0.1083 to 0.1197; "about 12.6" includes ±5% of 12.6, or from 11.97 to 13.23.

[0096] In a preferred embodiment, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is not about 4.955: about 2.3: about 12.6.

[0097] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4.955" includes ±5% of 4.955, or from 4.70725 to 5.20275; "about 2.3" includes ±5% of 2.3, or from 2.185 to 2.415; "about 12.6" includes ±5% of 12.6, or from 11.97 to 13.23.

[0098] In a preferred embodiment, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is not about 1.2: about 3.6: about 20.

[0099] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.2" includes ±5% of 1.2, or from 1.14 to 1.26; "about 3.6" includes ±5% of 3.6, or from 3.42 to 3.78; "about 20" includes ±5% of 20, or from 19 to 21.

[0100] In a preferred embodiment, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is not about 2.6: about 2.3: about 12.6.

[0101] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2.6" includes ±5% of 2.6, or from 2.47 to 2.73; "about 2.3" includes ±5% of 2.3, or from 2.185 to 2.415; "about 12.6" includes ±5% of 12.6, or from 11.97 to 13.23.

[0102] In a preferred embodiment, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is not about 4: about 3.6: about 5.2.

[0103] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2; "about 3.6" includes ±5% of 3.6, or from 3.42 to 3.78; "about 5.2" includes ±5% of 5.2, or from 4.94 to 5.46.

[0104] In a preferred embodiment, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is not about 4: about 3.6: about 20.

[0105] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2; "about 3.6" includes ±5% of 3.6, or from 3.42 to 3.78; "about 20" includes ±5% of 20, or from 19 to 21.

[0106] In a preferred embodiment, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is not about 4: about 1: about 5.2.

[0107] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2; "about 1" includes ±5% of 1, or from 0.95 to 1.05; "about 5.2" includes ±5% of 5.2, or from 4.94 to 5.46.

[0108] According to another aspect of the present invention, there is provided a pharmaceutical preparation comprising the above-mentioned pharmaceutical composition and one or more optional pharmaceutically acceptable excipients.

[0109] In a preferred embodiment, the excipient is selected from one or more of the following: diluents, wetting agents, binders, disintegrants, inclusion agents, flavoring agents, sustained-release agents, glidants, lubricants, dispersants, plasticizers, opacifiers and antioxidants.

[0110] In a preferred embodiment, the pharmaceutical preparation is in the form of powder, tablet, pill, capsule, film, lozenge, granule, liposome nanoparticle, injection or oral solution.

[0111] In a preferred embodiment, the pharmaceutical composition or the pharmaceutical preparation further comprises one or more drugs and / or extracts for preventing and / or treating liver damage.

[0112] In a preferred embodiment, the liver injury is hepatitis and / or liver fibrosis.

[0113] In a preferred embodiment, the drug is a hepatoprotective drug and / or an anti-hepatic fibrosis drug.

[0114] In a preferred embodiment, the hepatoprotective drug is selected from one or more of the following: anti-inflammatory hepatoprotective drugs, liver cell membrane repairing hepatoprotective drugs, antioxidant hepatoprotective drugs, choleretic hepatoprotective drugs, enzyme lowering hepatoprotective drugs, energy metabolism promoting hepatoprotective drugs and liver cell regeneration promoting hepatoprotective drugs.

[0115] In a preferred embodiment, the anti-hepatic fibrosis drug is selected from one or more of the following: Fuzheng Huayu Tablets, Fuzheng Huayu Capsules, Fufang Biejia Ruangan Tablets, Fufang Biejia Ruangan Capsules and Anluo Huaxian Pills.

[0116] In a preferred embodiment, the anti-inflammatory hepatoprotective drug is selected from one or more of the following: prednisone, methylprednisolone, diammonium glycyrrhizinate, magnesium isoglycyrrhizinate, compound monoammonium glycyrrhizinate and compound glycyrrhizin.

[0117] In a preferred embodiment, the liver cell membrane repairing hepatoprotective drug is polyene phosphatidylcholine.

[0118] In a preferred embodiment, the antioxidant hepatoprotective drug is selected from one or more of the following: vitamin E, resveratrol, caffeic acid, dihydromyricetin, silymarins, bicyclol, glutathione and soybean lecithin.

[0119] In a preferred embodiment, the choleretic hepatoprotective drug is ursodeoxycholic acid and / or adenosine succinate.

[0120] In a preferred embodiment, the enzyme-lowering liver-protecting drug is bifendate and / or pentazocet capsule.

[0121] In a preferred embodiment, the liver-protecting drug that promotes energy metabolism is selected from one or more of the following: adenosine triphosphate, coenzyme A, inosine, and coenzyme Q10.

[0122] In a preferred embodiment, the liver-protecting drug that promotes liver cell regeneration is a naked plasmid injection of recombinant human hepatocyte growth factor.

[0123] In a preferred embodiment, the extract is selected from one or more of the following: salvia miltiorrhiza extract, white peony root extract, angelica root extract, ligusticum chuanxiong extract, capillaris wormwood extract, sophora flavescens extract, astragalus root extract, schisandra chinensis extract and milk thistle extract.

[0124] In a preferred embodiment, the drug or the extract is a monomeric compound or a mixture thereof obtained from a natural product.

[0125] According to another aspect of the present invention, there is provided a method for preparing the above-mentioned pharmaceutical preparation, the method comprising the following steps:

[0126] (1) mixing the above composition with a pharmaceutically acceptable excipient, and heating until the pharmaceutically acceptable excipient is melted to obtain a mixture; wherein the mass ratio of the composition to the pharmaceutically acceptable excipient is about 1:(0.1-100); and

[0127] (2) mixing the mixture uniformly, cooling it, and pulverizing it to obtain the pharmaceutical preparation.

[0128] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0129] According to another aspect of the present invention, there is provided a use of the above-mentioned composition or pharmaceutical preparation in the preparation of a medicament for preventing and / or treating liver injury.

[0130] In a preferred embodiment, the liver damage includes hepatitis and / or liver fibrosis.

[0131] In a preferred embodiment, the hepatitis is liver inflammation with abnormal serum liver function.

[0132] In a preferred embodiment, the serum liver function test is selected from one or more of the following: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and glutamyl transpeptidase.

[0133] In a preferred embodiment, the serum liver function is further selected from one or more of the following: total bile acid, albumin, total bilirubin, direct bilirubin and indirect bilirubin.

[0134] In a preferred embodiment, the liver fibrosis is virus- and / or drug-induced liver fibrosis.

[0135] In a preferred embodiment, the drug-induced liver fibrosis is liver fibrosis induced by antitumor drugs, antituberculosis drugs, antipyretic analgesics, immunosuppressants, hypoglycemic drugs, lipid-lowering drugs, antibacterial drugs, antifungal drugs and / or antiviral drugs.

[0136] In a preferred embodiment, the liver fibrosis is liver fibrosis induced by carbon tetrachloride and / or dimethylnitrosamine.

[0137] In a preferred embodiment, the liver fibrosis is liver fibrosis induced by hepatitis B virus and / or hepatitis C virus.

[0138] In a preferred embodiment, the liver fibrosis is liver fibrosis associated with hepatic stellate cell activation.

[0139] In a preferred embodiment, the hepatic stellate cell activation is the activation of hepatic stellate cells with increased expression of α-smooth muscle actin.

[0140] In a preferred embodiment, the composition or the pharmaceutical preparation prevents and / or treats liver damage in one or more of the following ways: inhibiting hepatic stellate cell activation, improving abnormal serum biochemical indicators, reducing liver tissue hydroxyproline content, and improving liver tissue pathological abnormalities.

[0141] In a preferred embodiment, the hepatic stellate cells are LX-2 hepatic stellate cells.

[0142] In a preferred embodiment, the inhibition of hepatic stellate cell activation comprises: reducing the level of α-SMA in LX-2, JS-1, and T6 hepatic stellate cells.

[0143] In a preferred embodiment, the abnormal serum biochemical indicators include elevated serum alanine aminotransferase levels, elevated serum aspartate aminotransferase levels, and decreased serum albumin levels.

[0144] In a preferred embodiment, the liver tissue pathological abnormality includes liver tissue inflammation and liver tissue collagen deposition.

[0145] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or conditions recommended by the manufacturer.

[0146] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0147] The above-mentioned features of the present invention or the features described in the embodiments may be combined in any combination. All features disclosed in this patent specification may be used in any combination, and each feature disclosed in the specification may be replaced by any alternative feature that can provide the same, equal, or similar purpose. Therefore, unless otherwise specified, the features disclosed are only general examples of equal or similar features.

[0148] Example

[0149] Experimental Materials

[0150] The three monomer compositions of danshenxinquinone A, danshenxinone, and salvianolic acid H used in the following examples were all provided by MedChemExpress. Their purity, as measured by HPLC-DAD peak area normalization, was greater than 99%. Each monomer was dissolved in dimethyl sulfoxide (DMSO), mixed, and diluted to the desired concentration using DMEM medium. The final DMSO concentration of the composition was less than or equal to 0.1%.

[0151] Example 1

[0152] Cytotoxic effects of danshenquinone A, danshenone, and salvianolic acid H on LX-2 human HSC cell line

[0153] LX-2 cells were seeded in 96-well plates, 5000 cells / well. After the cells adhered, different concentrations of danshenquinone A (1.5625-25 μM), danshenxinone (1.5-24 μM) and danshenolic acid H (7.75-124 μM) were added, and 3 replicates were set up for each concentration. A normal control group was set up, and 0.01% DMSO was added. After the cells were continuously incubated with the drugs for 24 hours, the incubation was terminated. The cell viability was detected by the CCK8 kit. The results showed that 1.5625-25 μM danshenquinone A, 1.5-24 μM danshenxinone and 7.75-124 μM danshenolic acid H had no obvious toxicity to LX-2 cells (results as shown in Figure 2). Figure 1 shown).

[0154] Example 2

[0155] Effects of salvianolic acid H and danshenquinone A on the survival rate of LX-2 cells activated by TGF-β1

[0156] LX-2 cells were seeded in 96-well plates at 3,000 cells / well. After cell attachment, they were stimulated with 2.5 ng / ml TGF-β1 for 16 hours to induce an HSC activation model. Simultaneously, they were treated with safe concentrations of danshenquinone A (0.245-4.955 μM), danshenxinone (0.114-4.486 μM), and salvianolic acid H (0.154-25.045 μM) for 24 hours. A normal control group (N) and a model control group (M) were set up, each receiving 0.01% DMSO. A response surface-central composite design (RS-CCD) with three factors and five levels (-1.682, -1, 0, 1, 1.682) was performed using Design-expert 13.0 software. After 24 hours of co-incubation with each drug group, the cells were terminated and cell viability was measured using a CCK8 assay. The RS-CCD three-factor five-level design codes are shown in Table 1 ; the specific experimental combinations of the RS-CCD three-factor five-level design and the effects of each group of conditions on the survival rate of LX-2 cells induced by TGF-β1 are shown in Table 2 .

[0157] The results showed that different molar ratios of danshenxinkun A (D), miltirone (M), and salvianolic acid H (S) in the DMS-2, DMS-4, DMS-5, DMS-8, DMS-9, DMS-12, DMS-13, DMS-14, DMS-15, DMS-17, DMS-19, and DMS-20 combinations all inhibited the survival rate of LX-2 cells activated by TGF-β1 to varying degrees. Among them, DMS-8 (danshenxinkun A:miltirone:salvianolic acid H = 1.2 μM:1 μM:5.2 μM) had the most significant inhibitory effect on the survival rate of LX-2 cells activated by TGF-β1 (P < 0.001).

[0158] Table 1 RS-CCD three-factor five-level codes

[0159]

[0160] Table 2 RS-CCD three-factor five-level experimental combinations and results

[0161]

[0162]

[0163] Note: Compared with group N, * P<0.05; compared with group M, #P<0.05, ## P<0.001.

[0164] Example 3

[0165] Effects of DMS-8 on dimethylnitrosamine (DMN)-induced hepatic fibrosis in rats

[0166] Given that the aforementioned experiments revealed that a combination of 1.2 μM danshenquinone A, 1 μM danshenxinone, and 5.2 μM salvianolic acid H effectively inhibited TGF-β1-induced HSC activation in vitro, the molar mass ratio of danshenquinone A:danshenxinone:salvianolic acid H (1.2 μM:1 μM:5.2 μM) was converted to a mass ratio of 1:0.794:7.874 based on the molecular weights of danshenquinone A, danshenxinone, and salvianolic acid H (danshenquinone A, 296.32; danshenxinone, 282.38; and salvianolic acid H, 538.46). Furthermore, animal experiments were conducted to investigate the anti-hepatic fibrosis effects of DMS-8 in vivo.

[0167] Forty-eight male SPF Wistar rats, weighing 200 g ± 10 g, were randomly divided into a normal control group (n = 10), a model control group (n = 14), a low-dose DMS-8 group (n = 12), and a high-dose DMS-8 group (n = 12). The model control, low-dose DMS-8, and high-dose DMS-8 groups were intraperitoneally injected with dimethylnitrosamine (DMN) at a dose of 10 μg / kg for 3 consecutive days per week for 5 weeks to induce liver fibrosis. The initial injection dose was 2 / 3 of the normal dose. Starting from the fourth week of model establishment, the low-dose DMS-8 group was given 2.586 mg / kg of danshenquinone A, 2.053 mg / kg of danshenxinone, and 20.361 mg / kg of salvianolic acid H by gavage; the high-dose DMS-8 group was given 5.171 mg / kg of danshenquinone A, 4.107 mg / kg of danshenxinone, and 40.722 mg / kg of salvianolic acid H by gavage, seven days a week for two consecutive weeks. The normal control group and the model control group were given an equal volume of double-distilled water by gavage. Hepatic inflammation and collagen deposition were observed by HE staining and collagen staining. Serum liver function tests were performed using a kit, and liver hydroxyproline (HYP) content was measured by the Jamall method.

[0168] The results showed that compared with the normal control group, the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and liver tissue HYP content in the model control group were significantly increased (P < 0.05), and the serum albumin (Alb) content was significantly decreased (P < 0.05); liver tissue inflammation and collagen deposition were aggravated. The serum ALT, AST levels and liver tissue hydroxyproline content in the low-dose and high-dose DMS-8 groups were significantly decreased (P < 0.05), the serum Alb level was significantly increased (P < 0.05), and liver tissue inflammation and collagen deposition were significantly alleviated. Compared with the low-dose DMS-8 group, the serum ALT, AST and liver tissue Hyp content in the high-dose DMS-8 group were significantly decreased (P < 0.05), and liver tissue inflammation and collagen deposition were significantly alleviated. Results Figure 2 and Figure 3 shown.

[0169] Finally, when the above-mentioned active components of the present invention are used, they can also be used together with pharmaceutically acceptable carriers, excipients or diluents, or together with other natural medicines or chemicals to prepare pharmaceutical preparations. The dosage form of the pharmaceutical preparations can be capsules, granules, tablets, injections, liposome nanoparticles, sustained-release agents, controlled-release agents, dispersible tablets or intravenous preparations, etc., wherein the excipients can include one or a combination of two or more of solvents, disintegrants, flavoring agents, preservatives, colorants and adhesives, and the various carriers, excipients and diluents are conventional excipients in the art.

[0170] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only intended to help understand the method of the present invention and its core concept. At the same time, changes or modifications made by those skilled in the art based on the concept of the present invention, the specific implementation methods of the present invention, and the scope of application are all within the scope of protection of the present invention. In summary, the contents of this specification should not be understood as limiting the present invention.

Claims

1. A pharmaceutical composition comprising danshenquinone A, danshenone and salvianolic acid H, characterized in that: The molar concentration ratio of the danshenquinone A, the danshenxinone and the salvianolic acid H is (0.1-200): (0.1-200): (0.1-200).

2. The pharmaceutical composition according to claim 1, characterized in that The molar concentration ratio of the danshenquinone A, the danshenxinone and the salvianolic acid H is (0.1-25): (0.1-23): (0.1-126); Preferably, the molar concentration ratio of the danshenquinone A, the danshenone and the salvianolic acid H is (0.15-10): (0.1-10): (0.11-40); More preferably, the molar concentration ratio of the danshenquinone A, the danshenone and the salvianolic acid H is (0.2-5): (0.1-5): (0.12-30).

3. The pharmaceutical composition according to claim 1, characterized in that The molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is about 2.6: about 2.3: about 25.045; Preferably, the molar concentration ratio of the danshenquinone A, the danshenone and the salvianolic acid H is about 1.2: about 1: about 20; Preferably, the molar concentration ratio of the danshenquinone A, the danshenone and the salvianolic acid H is about 1.2: about 1: about 5.2; Preferably, the molar concentration ratio of the danshenquinone A, the danshenone and the salvianolic acid H is about 0.245: about 2.3: about 12.6; Preferably, the molar concentration ratio of the danshenquinone A, the danshenxinone and the salvianolic acid H is about 2.6: about 4.486: about 12.6; Preferably, the molar concentration ratio of the danshenquinone A, the danshenone and the salvianolic acid H is about 4: about 1: about 20; Preferably, the molar concentration ratio of the danshenquinone A, the danshenone and the salvianolic acid H is about 2.6: about 2.3: about 0.154; Preferably, the molar concentration ratio of the danshenquinone A, the danshenone and the salvianolic acid H is about 2.6: about 0.114: about 12.6; More preferably, the molar concentration ratio of the danshenquinone A, the danshenone and the salvianolic acid H is not about 4.955: about 2.3: about 12.6; More preferably, the molar concentration ratio of the danshenquinone A, the danshenone and the salvianolic acid H is not about 1.2: about 3.6: about 20; More preferably, the molar concentration ratio of the danshenquinone A, the danshenone and the salvianolic acid H is not about 2.6: about 2.3: about 12.6; More preferably, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is not about 4: about 3.6: about 5.2; More preferably, the molar concentration ratio between the danshenquinone A, the danshenone and the salvianolic acid H is not about 4: about 3.6: about 20; More preferably, the molar concentration ratio of the danshenquinone A, the danshenone, and the salvianolic acid H is not about 4: about 1: about 5.

2.

4. A pharmaceutical preparation, characterized in that A pharmaceutical composition comprising any one of claims 1 to 3, and one or more optional pharmaceutically acceptable excipients; Preferably, the excipient is selected from one or more of the following: diluent, wetting agent, binder, disintegrant, inclusion agent, flavoring agent, sustained-release agent, glidant, lubricant, dispersant, plasticizer, opacifier and antioxidant.

5. The pharmaceutical preparation according to claim 4, characterized in that The dosage form of the pharmaceutical preparation is powder, tablet, pill, capsule, film, lozenge, granule, liposome nanoparticle, injection or oral solution.

6. The pharmaceutical composition according to any one of claims 1 to 3 or the pharmaceutical preparation according to claim 4 or 5, characterized in that The pharmaceutical composition or the pharmaceutical preparation further comprises one or more drugs and / or extracts for preventing and / or treating liver damage; Preferably, the liver injury is hepatitis and / or liver fibrosis; Preferably, the drug is a hepatoprotective drug and / or an anti-hepatic fibrosis drug; More preferably, the hepatoprotective drug is selected from one or more of the following: anti-inflammatory hepatoprotective drugs, hepatoprotective drugs that repair liver cell membranes, antioxidant hepatoprotective drugs, choleretic hepatoprotective drugs, enzyme-lowering hepatoprotective drugs, energy metabolism-promoting hepatoprotective drugs, and hepatoprotective drugs that promote liver cell regeneration; More preferably, the anti-hepatic fibrosis drug is selected from one or more of the following: Fuzheng Huayu Tablets, Fuzheng Huayu Capsules, Fufang Biejia Ruangan Tablets, Fufang Biejia Ruangan Capsules and Anluo Huaxian Pills; Preferably, the anti-inflammatory hepatoprotective drug is selected from one or more of the following: prednisone, methylprednisolone, diammonium glycyrrhizinate, magnesium isoglycyrrhizinate, compound monoammonium glycyrrhizinate and compound glycyrrhizin; Preferably, the liver cell membrane repairing hepatoprotective drug is polyene phosphatidylcholine; Preferably, the antioxidant hepatoprotective drug is selected from one or more of the following: vitamin E, resveratrol, caffeic acid, dihydromyricetin, silymarin, bicyclol, glutathione and soybean lecithin; Preferably, the choleretic hepatoprotective drug is ursodeoxycholic acid and / or adenosine succinate; Preferably, the enzyme-lowering hepatoprotective drug is bifendate and / or pentazocet capsule; Preferably, the energy metabolism-promoting hepatoprotective drug is selected from one or more of the following: adenosine triphosphate, coenzyme A, inosine, and coenzyme Q10; Preferably, the liver-protecting drug that promotes liver cell regeneration is a naked plasmid injection of recombinant human hepatocyte growth factor; Still more preferably, the extract is selected from one or more of the following: Danshen extract, Baishao extract, Angelica extract, Chuanxiong extract, Artemisia capillaris extract, Sophora flavescens extract, Astragalus extract, Schisandra chinensis extract and Milk thistle extract; Particularly preferably, the drug or the extract is a monomeric compound or a mixture thereof obtained from a natural product.

7. A method for preparing the pharmaceutical preparation according to any one of claims 4 to 6, characterized in that: The preparation method comprises the following steps: (1) mixing the composition of any one of claims 1 to 3 with a pharmaceutically acceptable excipient, and heating until the pharmaceutically acceptable excipient is melted to obtain a mixture; wherein the mass ratio of the composition to the pharmaceutically acceptable excipient is about 1:(0.1-100); and (2) mixing the mixture uniformly, cooling it, and crushing it to obtain the pharmaceutical preparation.

8. Use of the composition according to any one of claims 1 to 3 or the pharmaceutical preparation according to any one of claims 4 to 6 in the preparation of a medicament for preventing and / or treating liver damage.

9. The use according to claim 8, characterized in that The liver damage includes hepatitis and / or liver fibrosis; Preferably, the hepatitis is liver inflammation with abnormal serum liver function; More preferably, the serum liver function test is selected from one or more of the following: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and glutamyl transpeptidase; More preferably, the serum liver function is further selected from one or more of the following: total bile acid, albumin, total bilirubin, direct bilirubin and indirect bilirubin; Furthermore, preferably, the liver fibrosis is virus- and / or drug-induced liver fibrosis; Still more preferably, the drug-induced liver fibrosis is liver fibrosis induced by antitumor drugs, antituberculosis drugs, antipyretic analgesics, immunosuppressants, hypoglycemic drugs, lipid-lowering drugs, antibacterial drugs, antifungal drugs and / or antiviral drugs; More preferably, the liver fibrosis is liver fibrosis induced by carbon tetrachloride and / or dimethylnitrosamine; Still more preferably, the liver fibrosis is liver fibrosis induced by hepatitis B virus and / or hepatitis C virus; Still more preferably, the liver fibrosis is liver fibrosis associated with hepatic stellate cell activation; Particularly preferably, the hepatic stellate cell activation is hepatic stellate cell activation with increased expression of α-smooth muscle actin.

10. The use according to claim 8, characterized in that The composition or pharmaceutical preparation prevents and / or treats liver damage in one or more of the following ways: inhibiting hepatic stellate cell activation, improving abnormal serum biochemical indicators, reducing liver tissue hydroxyproline content, and improving liver tissue pathological abnormalities; Preferably, the hepatic stellate cells are LX-2 hepatic stellate cells; Preferably, the inhibiting of hepatic stellate cell activation comprises: reducing the level of α-SMA in LX-2, JS-1, and T6 hepatic stellate cells; Preferably, the abnormal serum biochemical indicators include elevated serum alanine aminotransferase levels, elevated serum aspartate aminotransferase levels, and decreased serum albumin levels; Preferably, the liver tissue pathological abnormalities include liver tissue inflammation and liver tissue collagen deposition.

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