A pharmaceutical composition for preventing and / or treating liver disease

Abstract

The present application provides a kind of pharmaceutical composition, the composition includes when medicine glycoside, when medicine alcohol glycoside and gentiopicroside, the mass ratio of the medicine glycoside, when medicine alcohol glycoside and gentiopicroside is 1-6:1-6:1.The present application carries out efficacy screening to effective component in Tibetan plantain medicinal plant, and different effective components are combined, and the result shows that when the combination of when medicine alcohol glycoside, medicine glycoside and gentiopicroside is applied, it has obvious effect on treating and / or preventing liver disease.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a pharmaceutical composition for the prevention and / or treatment of liver diseases, and particularly to a pharmaceutical composition containing active ingredients such as gentianin, gentianin and gentiopicrin from Artemisia capillaris medicinal plants. Background Technology

[0002] Tibetan Artemisia capillaris is a class of medicinal plants that are annual, low-growing herbaceous medicinal plants belonging to the Gentianaceae family. In the history of Tibetan medicine, it is one of the eight precious Tibetan medicines. There are more than ten kinds of original plants that can be used as medicine, mainly Gentianaceae plants, including Swertia japonica, Swertia semperflorens, Swertia macrophylla, Swertia semperflorens, Swertia japonica var. florida, and Swertia semperflorens var. florida.

[0003] Artemisia capillaris, a type of medicinal plant, is cool in nature and bitter in taste. It has the effects of clearing the liver and gallbladder, and reducing fever. It is mainly used to treat acute and chronic hepatitis, chronic cholecystitis, etc., with significant efficacy. Its main chemical components are... Ketones, flavonoids, iridoids, triterpenoids, etc. The non-patent literature "High-performance liquid chromatography determination of major medicinal components in 15 species of Swertia" (Northwest Botanical Journal, 2004, 24(7): 1298-1302) analyzed the major components of 15 species of Swertia from the Qinghai-Tibet Plateau and the Yunnan-Guizhou Plateau, finding that gentiopicrin was the most abundant among bitter glycosides, followed by swertiapicrin, and gentiopicrin was the least abundant. Among ketone glycosides, mangiferin is the most abundant, followed by dextrin.

[0004] The application of extracts or monomeric compounds from Artemisia capillaris, a type of medicinal plant, in the treatment of liver diseases has been reported in the prior art, for example:

[0005] CN107875191A discloses a composition for reducing the toxicity of drugs that cause liver damage, comprising a group of effective parts of Swertia japonica, wherein the group of effective parts of Swertia japonica includes swertiamarin, gentiopicrin, swertiamarin, mangiferin, and isorhizoma swertiamarin, and the mass ratio of the five components is (0.04-0.71):(20-40):(1-15):(1.6-26):(0.01-0.16).

[0006] CN107875163A discloses another effective component of Artemisia capillaris for treating acute and chronic hepatitis. This effective component mainly contains four active ingredients: gentiopicrin, swertiamarin, mangiferin, and swertiamarin, with a total content of 50-95%. The mass ratio of the four active ingredients is (40-90):(1.0-10):(0.5-5):(0.5-5).

[0007] CN102138966A discloses an extract of Artemisia capillaris, containing 10-50% iridoids and 10-50%... Ketones and 5-20% triterpenoids, wherein the iridoids include swertiamarin, gentiopicrin, etc.; Ketones include mangiferin, dansperidin, etc. The extract of Artemisia capillaris has a significant protective and improving effect on acute liver injury and obstructive jaundice caused by chemical substances.

[0008] CN104650024A disclosed The uses of ketone monomer compounds in the treatment of liver fibrosis and cirrhosis were further disclosed, including six specific compounds. Ketone monomeric compounds, including 1,7-hydroxy-3,4-methoxy Ketone, 1,5-dihydroxy-3-methoxy Ketones, dangyao alcohol glycosides, etc.

[0009] However, there remains an urgent need to obtain compositions with more clearly defined components and superior therapeutic effects from the numerous active ingredients and extracts of Artemisia capillaris. Summary of the Invention

[0010] This invention screens the efficacy of active ingredients in Artemisia capillaris and combines different active ingredients. Surprisingly, it was found that when a combination of gentianin, gentianin and gentiopicrin is applied, it has a significant effect on the treatment and / or prevention of liver disease.

[0011] A first aspect of the present invention provides a pharmaceutical composition comprising gentianin, gentianolin and gentiopicrin.

[0012] Wherein, the mass ratio of gentianoside, gentianool glycoside and gentiopicrin is 1-6:1-6:1; preferably, in the above three-component composition, based on the mass of gentiopicrin, the mass ratio of gentianoside to gentiopicrin is specifically 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, and the mass ratio of gentianool glycoside to gentiopicrin is specifically 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6.

[0013] The gentianin, gentianolin, or gentianin described in this invention can be chemically synthesized, biosynthesized, or extracted from plants. Preferably, the gentianin, gentianolin, or gentianin of this invention are extracts obtained from plants, such as medicinal plants of the *Swertia* genus (including *Swertia serratifolia*, *Swertia szechuanensis*, *Swertia macrophylla*, etc.) and *Swertia hygroscopica*. The plants can also be non-*Swertia* medicinal plants; for example, the gentianin can be extracted from *Lonicera japonica* and *Swertia serratifolia*; the gentianolin can be extracted from *Swertia serratifolia* and *Gentiana*; and the gentianin can be extracted from *Gentiana*, *Gentiana scabra*, *Gentiana macrophylla*, etc. Preferably, the gentianin, gentianolin, or gentianin is an extract obtained from *Swertia serratifolia* or *Swertia macrophylla*.

[0014] The gentianin, gentianol glycoside, or gentianin described in this invention can be prepared using conventional plant extraction methods in the prior art, such as solvent extraction, ultrasonic extraction, supercritical fluid extraction, membrane extraction and separation technology, etc.; preferably, solvent extraction can be used, and the extraction solvent can be: low molecular weight alcohol (such as methanol, ethanol, butanol, or propanol), acetate (such as methyl acetate or ethyl acetate), ketone (such as acetone), ether (such as diethyl ether), low boiling point aliphatic hydrocarbons, aromatic hydrocarbons, or chlorinated hydrocarbons, etc.; the extraction process can be heating reflux, soaking extraction, etc., and can be combined with one or more of other extraction and separation methods such as extraction, distillation, crystallization, chromatographic separation, etc. to further purify and separate the extract. Exemplary extraction methods are described, for example, in CN101830892A, CN103275147A, "Separation and purification of iridoid glycosides and gentianin from Swertia japonica in western Sichuan using macroporous resin". Research on the processing of ketone components (9th National Symposium on Traditional Chinese Medicine and Natural Products), etc.

[0015] The gentianin, gentianolin, or gentiopicrin described in this invention can be extracted from different plants, or extracted separately or simultaneously from the same plant. In an embodiment of this invention, the pharmaceutical composition is prepared by mixing the separately extracted gentianin, gentianolin, or gentiopicrin.

[0016] The compositions of the present invention may also contain pharmaceutically acceptable excipients. Therefore, the compositions can be formulated into specific dosage forms and administered via any suitable route, such as oral, rectal, nasal, pulmonary, local (including oral and sublingual), transdermal, intracerebrospinal, intraperitoneal, vaginal, and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous, and intradermal) routes, preferably oral.

[0017] The oral administration composition includes solid dosage forms, such as capsules, tablets, pills, lozenges, powders, and granules.

[0018] The oral administration composition also includes liquid dosage forms, such as solutions, emulsions, suspensions, syrups, and elixirs.

[0019] Other suitable dosage forms include suppositories, sprays, ointments, creams, gels, inhalers, skin patches, implants, etc.

[0020] To prepare the above-described dosage form composition, methods known in the art may be used, wherein the pharmaceutically acceptable excipients may be any pharmaceutically acceptable carrier, diluent, excipient or other additive commonly used in the art.

[0021] Examples of carriers include lactose, kaolin, sucrose, cyclodextrin, talc, agar, pectin, gum arabic, stearic acid, lower alkyl ethers of cellulose, corn starch, potato starch, magnesium stearate, and gelatin.

[0022] Any other excipients or additives commonly used for coloring, flavoring, preservation, etc., may be used, provided they are compatible with the active ingredient or the ingredient already used.

[0023] The amount of active compound per unit dose may vary depending on the nature of the active compound and the intended dosing regimen. Generally, it ranges from 0.1 mg to 5000 mg per unit dose.

[0024] The compositions of the present invention may also contain other active ingredients, wherein the other active ingredients are selected from those containing iridoids, terpenes, etc. Ketones and triterpenoids. Preferably, the other active ingredients are selected from swertiamarin, swertiamarin, oleanolic acid, neomangiferin, isomangiferin, luteolin, ursolic acid, isovitexin, mangiferin, sennain, and 3-oxodesylbisphenol A. Ketone, 7-O-[α-L-rhamnopyranose-(1→2)-β-D-xylanose]-1,8-dihydroxy-3-methoxy Ketone, 7-O-β-D-xylanose-1,8-dihydroxy-3-methoxy Ketone, 3-O-β-D-glucopyranose-1,8-dihydroxy-5-methoxy One or more of ketones or isohypericin, more preferably, the other active ingredients are selected from one or more of neomangiferin, luteolin or oleanolic acid.

[0025] Other active ingredients in the compositions of the present invention may be chemically synthesized, biosynthesized, or extracted from plants. Preferably, the other active ingredients of the present invention are extracts obtained from plants, such as medicinal plants of the *Artemisia* genus, for example, *Swertia* genus (including *Swertia serratifolia*, *Swertia szechuanensis*, *Swertia macrophylla*, etc.) and *Swertia hygroscopica* buds. The other active ingredients of the present invention and gentianin may be extracted from different plants, or they may be extracted separately or simultaneously from the same plant.

[0026] In the compositions of the present invention, the mass ratio of other active ingredients to gentiopicrin is 0.1-20:1, for example, 0.1:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1.

[0027] In an embodiment of the present invention, the composition comprises duracil, duracil alcohol, gentiopicrin and pharmaceutically acceptable excipients, wherein the mass ratio of duracil, duracil alcohol and gentiopicrin is 1-6:1-6:1.

[0028] In an embodiment of the present invention, the composition comprises datura glycoside, datura alcohol glycoside, gentiopicrin and pharmaceutically acceptable excipients, wherein the mass ratio of datura glycoside, datura alcohol glycoside and gentiopicrin is 1-6:1-6:1.

[0029] In embodiments of the present invention, the composition comprises swertiamarin, swertiamarin alcohol, gentiopicrin, other active ingredients, and pharmaceutically acceptable excipients. The mass ratio of swertiamarin, swertiamarin alcohol, other active ingredients, and gentiopicrin is 1-6:1-6:0.1-20:1. The other active ingredients are selected from one or more of swertiamarin, swertiamarin, oleanolic acid, neomangiferin, isomangiferin, luteolin, ursolic acid, isovitexin, mangiferin, or isorhamnetin.

[0030] Another aspect of the present invention provides the use of a composition of gentiopicroside, gentiopicroside and gentiopicroside in the preparation of a medicament for the treatment and / or prevention of liver disease.

[0031] Another aspect of the present invention provides the use of the combined use of gentianoside, gentianool glycoside, and gentiopicrin in the preparation of medicaments for the treatment and / or prevention of liver diseases. The gentianoside, gentianool glycoside, and gentiopicrin described in this invention can be administered simultaneously, sequentially, or at intervals.

[0032] The liver diseases described in this invention are selected from liver injury, hepatitis, cirrhosis, ascites, alcoholic liver disease, fatty liver disease, and liver fibrosis. The liver injury is selected from acute liver necrosis and liver failure; the hepatitis is selected from jaundice hepatitis, viral hepatitis (e.g., hepatitis B, hepatitis A, and hepatitis C), and alcoholic hepatitis; the cirrhosis is selected from viral hepatitis cirrhosis, alcoholic cirrhosis, metabolic cirrhosis, cholestatic cirrhosis, hepatic venous reflux obstruction cirrhosis, autoimmune cirrhosis, toxic and drug-induced cirrhosis, malnutrition-related cirrhosis, and cryptogenic cirrhosis; the fatty liver disease is selected from alcoholic fatty liver disease and non-alcoholic fatty liver disease; the liver fibrosis is selected from liver fibrosis caused by cirrhosis, alcohol-induced liver fibrosis, liver fibrosis caused by non-alcoholic fatty liver disease, liver fibrosis caused by bile duct injury, liver fibrosis caused by primary biliary cirrhosis, and liver fibrosis caused by autoimmune hepatitis.

[0033] This invention conducts pharmacodynamic studies on the active ingredients in extracts of Artemisia capillaris, a type of medicinal plant, and obtains compositions with more clearly defined components and excellent therapeutic effects by studying the proportions of different active ingredients.

[0034] The term "treatment" as used in this invention includes the suppression, delay, mitigation, reduction, limitation, relief or extinction of a disease, disorder, symptom or condition, its occurrence and / or process, and / or its symptoms.

[0035] The term "prevention" as used in this invention includes reducing the risk of having, being infected with, or experiencing a disease, disorder, symptom, or condition, its occurrence and / or progression, and / or its symptoms.

[0036] The term "comprising" as used in this invention means "open" or "inclusive" in such a way that it includes the listed elements, and also allows for the inclusion of additional, unmentioned elements. Attached Figure Description

[0037] Figure 1 The results show the hepatocyte survival rate induced by different concentrations of CCl4 in Example 1.

[0038] Figure 2 This is a dose-effect curve of the monomer compound solution and the mixture of the composition in Example 3.

[0039] Figure 3 The image shows the Fa-CI curve of the mixture in Example 3. Detailed Implementation

[0040] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Example 1: Comparative Results of the Protective Effects of 10 Components from Artemisia capillaris on CCl4-Induced Hepatocellular Damage

[0042] 1. Determination of the optimal concentration of CCl4 for inducing hepatocyte injury

[0043] Log-grown WRL68 human hepatocytes were harvested, digested, and cultured in RPMI-1640 medium to adjust the cell density to 5 × 10⁻⁶ cells / year. 4 Cells / mL, hepatocyte viability greater than 90%. Add 100 μL of cell suspension to each well of a 96-well plate and incubate at 37°C with 5% CO2 for 24 h until all cells adhere. Prepare a 5M CCl4 stock solution by dissolving 4.82 mL of CCl4 stock solution in 5.18 mL of DMSO. Dilute this stock solution with RPMI-1640 medium containing 10% fetal bovine serum to prepare solutions containing 16.7, 23.1, 28.6, 33.3, and 37.5 mM CCl4. Add these solutions to 96-well plates, using CCl4-free RPMI-1640 medium as a blank control. Perform five parallel runs for each concentration. After 6 h of incubation, assess hepatocyte viability using the MTT assay. Figure 1 As shown, the MTT assay results after 6 hours showed that when the CCl4 concentration was ≥23.1 mM, the hepatocyte survival rate decreased significantly (P<0.05); the mortality rate reached 42% at 37.5 mM. Therefore, the CCl4 modeling concentration selected for subsequent experiments was 28.6 mM.

[0044] 2.10 Experiments on the Protective Effects of Components Against CCl4-Induced Hepatocyte Damage

[0045] Ten common components from the medicinal plant Artemisia capillaris were selected: swertiamarin, swertiamarin, oleanolic acid, isomangiin, swertiamarin, ursolic acid, gentiopicrin, isovitexin, mangiferin, and isorhizoma serotonin. Standards were purchased for the following experiments.

[0046] Log-phase WRL68 human hepatocytes were harvested and cultured in 1640 medium containing 10% fetal bovine serum, with the cell density adjusted to 5 × 10⁶ cells / year. 4Cells were seeded at a density of 100 μL / mL in 96-well plates and incubated in a CO2 incubator for 12–24 h. Then, 50 μL of 1% FBS-1640 culture medium with different components was added, and the cells were incubated for another 24 h. The culture medium was then discarded, and 150 μL of culture medium containing 28.6 mM CCl4 and different components was added. After incubation for another 6 h, the cell supernatant was collected. Five series concentrations were set for each component (see Table 1), and each concentration was replicated in five wells.

[0047] Table 1-10 Experimental Concentrations of Components on CCl4-Induced Hepatocellular Injury

[0048] Ingredient name Series concentrations (μM) Isorhizonin 5、15、50、100、200 mangiferin 25、50、100、200、400 Isomangin 0.3、1.5、8、40、200 Isovitilione 2、6、18、50、150 When drug glycosides 10、25、50、100、200 Swertia triterpenoids 10、30、100、200、400 Gentianoside 5、10、20、40、100 When drug alcohol glycoside 10、25、50、100、200 Ursolic acid 10、20、40、80、150 Oleanolic acid 5、12.5、25、50、100

[0049] The absorbance (OD) values ​​of LDH were read using an ELISA reader, and the LDH content was calculated. The inhibition rate of the component was calculated using CCl4 content as the standard. The fitted curve and IC50 were then analyzed. 50 value.

[0050] The LDH value (U / L) is calculated using the following formula:

[0051] (Measured OD - Control OD) / (Standard OD - Blank OD) × Standard concentration (0.2 μmol / mL) × 1000;

[0052] The inhibition rate is calculated using the following formula:

[0053] (LDH value in Model group - LDH value in treatment group) / LDH value in Model group x 100%;

[0054] The Model group refers to the CCl4-induced liver injury model group (the culture medium contains only CCl4 inducer, without any other drugs).

[0055] IC with 10 components 50 The results are shown in Table 2, and the order of activity is as follows: oleanolic acid > dangyao glycoside > dangyao glycoside > mangiferin > gentianin > isomangiferin > isohypericin > ursolic acid > swertiamarin > isovitexin.

[0056] Table 2-10 Experimental Results of the Effects of 10 Components on CCl4-Induced Hepatocellular Injury

[0057] Ingredient name IC50 (μM) Oleanolic acid 3.16 When drug alcohol glycoside 7.7 When drug glycosides 10.3 mangiferin 26.9 Gentianoside 69.8 Isomangin 136 Isorhizonin 181 Ursolic acid 187.2 Swertia triterpenoids 247 Isovitilione 380

[0058] Example 2: Comparative Results of the Protective Effects of Different Combinations of Active Ingredients from Artemisia capillaris on CCl4-Induced Hepatocellular Damage

[0059] Based on the results of Example 1, five highly active ingredients were selected and arranged in different combinations to compare the effects of different combinations of active ingredients in Artemisia capillaris on CCl4-induced hepatocyte damage, and the optimal combination scheme was screened out.

[0060] According to the formulation in Table 3, different combination solutions were prepared using standards of each component. Each combination solution had the same concentration (15 μM), and cell activity comparison experiments were conducted according to the method described in section 2 of Example 1. Cells were added to 50 μL of 1% FBS-1640 culture medium containing different combination solutions, with five wells in parallel. After culturing for 24 h, the culture medium was discarded, and 150 μL of culture medium containing 28.6 mM CCl4 and different drugs was added. After incubation for another 6 h, the cell supernatant was collected, and the absorbance (OD value) of each group was measured using a microplate reader. The LDH content was calculated.

[0061] Table 3 - Experimental results of different combinations of active ingredients from Artemisia capillaris-like medicinal plants on CCl4-induced hepatocyte damage.

[0062]

[0063]

[0064] The results showed that the combination of dangyao glycoside, dangyao glycoside and gentiopicrin had significantly better activity than the combination of three components in other Tibetan Artemisia capillaris medicinal plants.

[0065] Example 3: Synergistic verification of the protective effect of the combination of dangyao glycoside, dangyao glycoside, and gentiopicrin against CCl4-induced hepatocyte damage.

[0066] Based on the results of Example 2, the combination of dangyao alcohol glycoside, dangyao glycoside and gentiopicrin was selected as the optimal active composition, and the synergistic effect among dangyao alcohol glycoside, dangyao glycoside and gentiopicrin was further investigated.

[0067] Following the method described in section 2 of Example 1, logarithmic growth phase WRL68 human hepatocytes were harvested and cultured in 1640 medium containing 10% fetal bovine serum to adjust the cell density to 5 × 10⁶ cells / mL. 4 Cells were seeded at a density of 100 μL / mL in 96-well plates and incubated in a CO2 incubator for 12–24 h. Then, 1% FBS-1640 medium containing different drugs was added according to the formulations in Table 4. After incubation for another 24 h, the medium was discarded, and 150 μL of medium containing 28.6 mM CCl4 and the different drugs was added. After incubation for another 6 h, the cell supernatant was collected, and the results were replicated in 5 parallel wells. The LDH absorbance (OD value) was read using a microplate reader, and the LDH content was calculated. The drug inhibition rate was calculated using the CCl4 content as the standard.

[0068] Table 4 - Relative changes in LDH values ​​of monomeric compound solutions and composition mixtures

[0069]

[0070]

[0071] Based on the relative changes in LDH and drug concentrations obtained above, the CI (Combination Index) was calculated using Compusyn software (a CI value below 1 indicates a synergistic effect between drugs, while a CI value above 1 indicates an antagonistic effect). The results are shown in Table 5. Figure 2-3 The results show that the combination described in this invention falls within the synergistic effect region.

[0072] Table 5 - CI Co-index of the Composition Mixture

[0073]

[0074] Example 4: Capsule Composition Formulation

[0075] Weigh the ingredients according to the following prescription:

[0076] 6g of dangyao alcohol glycoside

[0077] When the drug glycoside 6g

[0078] Gentianoside 1g

[0079] Starch: 50g

[0080] Mix the thymol glycoside, gentiopicroside and gentiopicroside, add starch, mix well, and make 100 capsules.

[0081] Example 5: Tablet Composition Formulation

[0082] Weigh the ingredients according to the following prescription:

[0083] 4g of dangyao alcohol glycoside

[0084] When the drug glycoside 4g

[0085] Gentianoside 1g

[0086] Microcrystalline cellulose: 30g

[0087] Mannitol: 10g

[0088] 30g of croscarmellose sodium cellulose

[0089] Magnesium stearate: 1g

[0090] Mix gentianoside, gentianoside, and gentiopicroside, add microcrystalline cellulose, mannitol, and croscarmellose sodium, mix well, form a soft mass, pass through a 20-80 mesh sieve, and granulate. Dry the wet granules, measure and control the moisture content, granulate again, and collect the dry granules through a 20-80 mesh sieve. Add magnesium stearate to the dry granules from the previous step, mix well, and compress to obtain 100 tablets.

[0091] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0092] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A pharmaceutical composition consisting of quercitrin, quercetagetin and gentiopicroside, wherein the mass ratio of quercitrin, quercetagetin and gentiopicroside is 2-4:2-4:

1.

2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

3. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition is a capsule, a tablet, a pill, a lozenge, a powder, a granule, a solution, an emulsion, a suspension, a syrup, an elixir, a suppository, a spray, an ointment, an inhalant, a skin patch or an implant.

4. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition is a cream or a gel.

5. Use of the pharmaceutical composition according to any one of claims 1-4 in the preparation of a medicament for the treatment and / or prevention of liver diseases, wherein the liver diseases are liver injuries.

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