Medicine containing anthocyanin and schizophyllum commune beta-glucan and having anti-inflammatory effect as well as preparation method and application of medicine

Abstract

The invention relates to the field of medicines, in particular to an anti-inflammatory medicine containing anthocyanin and schizophyllum commune beta-glucan as well as a preparation method and application of the anti-inflammatory medicine. The concentration of the schizophyllum commune beta-glucan solution is 4-120 [mu] g / mL, the concentration of the anthocyanin solution is 0.16-4 [mu] g / mL, and the schizophyllum commune beta-glucan solution has no toxic influence on BJ cells and can be applied to drugs; furthermore, the Schizophyllum commune beta-glucan solution and the anthocyanin solution are compounded according to the mass ratio of (20: 1)-(40: 1), so that the inhibition effect on an inflammatory factor IL-1beta of the BJ cells can be remarkably improved; the obvious synergistic interaction effect is realized, so that the use dosage of a single drug can be obviously reduced. According to the technical scheme, it is found for the first time that the anthocyanin and schizophyllum commune beta-glucan are compatible and combined to have the synergistic anti-inflammatory effect, and a new candidate composition source is provided for improving inflammation.

Description

Technical Field

[0001] The present application relates to the field of medicine, and in particular, to a medicine comprising anthocyanins and Schizophyllum β-glucan having anti-inflammatory effects, and a preparation method and application thereof. Background Art

[0002] Inflammation is a defensive response of the body to stimulation, manifested as redness, swelling, heat, pain and dysfunction. Inflammation can be infectious inflammation caused by infection or non-infectious inflammation not caused by infection.

[0003] Inflammation in the body is very common, and anti-inflammatory drugs are a class of drugs that can be effective against inflammation.

[0004] However, for the anti-inflammatory drugs currently on the market, when facing severe inflammation, the dosage of the anti-inflammatory active ingredients needs to be increased. Summary of the invention

[0005] The purpose of the embodiments of the present application is to provide a drug containing anthocyanins and Schizophyllum β-glucan with anti-inflammatory effect, and a preparation method and application thereof.

[0006] In a first aspect, the present application provides a drug comprising anthocyanins and Schizophyllum β-glucan having an anti-inflammatory effect, comprising:

[0007] Schizophyllum β-glucan solution and anthocyanin solution;

[0008] The concentration of the Schizophyllum β-glucan solution is 4 μg / mL to 120 μg / mL;

[0009] The concentration of anthocyanin solution is 0.16 μg / mL to 4 μg / mL;

[0010] The mass ratio of the Schizophyllum β-glucan solution to the anthocyanin solution is 20:1 to 40:1.

[0011] In the above technical scheme, the concentration of the Schizophyllum β-glucan solution is 4μg / mL~120μg / mL, and the concentration of the anthocyanin solution is 0.16μg / mL~4μg / mL, which has no toxic effect on BJ cells and can be used as medicine; further, compounding the Schizophyllum β-glucan solution and the anthocyanin solution in a mass ratio of 20:1~40:1 can significantly improve the inhibitory effect on the inflammatory factor IL-1β of BJ cells; achieve a significant synergistic effect, thereby significantly reducing the dosage of a single drug.

[0012] Furthermore, the technical solution of the present application discovered for the first time that the combination of anthocyanins and Schizophyllum β-glucan has a synergistic anti-inflammatory effect, providing a new source of candidate compositions for improving inflammation.

[0013] Furthermore, the technical solution of the present application discovered for the first time that anthocyanins and Schizophyllum β-glucan, when in the above-mentioned ratio range, can play a significant synergistic role in inhibiting the expression of the inflammatory factor IL-1β, thereby reducing the dosage and toxicity of a single active ingredient and improving the pharmacological efficacy of SPG.

[0014] In other embodiments of the present application, the concentration of the above-mentioned Schizophyllum β-glucan solution is 4.8 μg / mL to 96 μg / mL;

[0015] The concentration of anthocyanin solution is 0.4 μg / mL to 4 μg / mL;

[0016] The mass ratio of the Schizophyllum β-glucan solution to the anthocyanin solution is 20:1 to 40:1.

[0017] In other embodiments of the present application, the concentration of the Schizophyllum β-glucan solution is 12 μg / mL to 72 μg / mL;

[0018] The concentration of anthocyanin solution is 0.8 μg / mL to 3.2 μg / mL;

[0019] The mass ratio of the Schizophyllum β-glucan solution to the anthocyanin solution is 20:1 to 40:1.

[0020] In other embodiments of the present application, the preparation of the Schizophyllum β-glucan solution comprises:

[0021] The Schizophyllum seed liquid is inoculated into the culture medium for culturing to obtain a fermentation product;

[0022] The culture medium includes: a promoting substance;

[0023] Based on the total volume of the culture medium, the promoting substances include:

[0024] 10mg / L ~ 20mg / L of vitamin B1 and 1mg / L ~ 10mg / L of folic acid.

[0025] In other embodiments of the present application, the culture medium includes: a basic culture substance;

[0026] Calculated by mass percentage, the basic culture material includes: 1% to 5% glucose, 0.1% to 2% tryptone, 0.1% to 1% yeast extract powder, 0.01% to 0.1% magnesium sulfate, 0.01% to 0.2% potassium dihydrogen phosphate; the balance is water.

[0027] In other embodiments of the present application, preparing anthocyanin solution comprises:

[0028] Dissolve cyanidin-3-O-glucoside in a solvent.

[0029] In other embodiments of the present application, the solvent includes: dimethyl sulfoxide and water.

[0030] In a second aspect, the present application provides a method for preparing a drug containing anthocyanins and Schizophyllum β-glucan having an anti-inflammatory effect, comprising:

[0031] The Schizophyllum β-glucan solution and the anthocyanin solution were mixed.

[0032] In a third aspect, the present application provides a composition comprising anthocyanins and Schizophyllum β-glucan having an anti-inflammatory effect, comprising:

[0033] Schizophyllum β-glucan solution and anthocyanin solution;

[0034] The concentration of the Schizophyllum β-glucan solution is 4 μg / mL to 120 μg / mL;

[0035] The concentration of anthocyanin solution is 0.16 μg / mL to 4 μg / mL;

[0036] The mass ratio of the Schizophyllum β-glucan solution to the anthocyanin solution is 20:1 to 40:1.

[0037] In a fourth aspect, the present application provides a composition for use in medicine.

[0038] In a fifth aspect, the present application provides an application of a composition in cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0040] Figure 1 The dose-effect curve of SPG, C3G and the combination of the two in inhibiting the expression of inflammatory factor IL-1β in BJ cells;

[0041] Figure 2 The graph is the inhibition rate-combination index (Fa-CI) curve of the combination of SPG and C3G at a mass concentration ratio of 30:1. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments.

[0043] Therefore, the following detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0044] The present application provides a drug comprising anthocyanins and Schizophyllum β-glucan having an anti-inflammatory effect, including:

[0045] Schizophyllum β-glucan solution and anthocyanin solution;

[0046] The concentration of the Schizophyllum β-glucan solution is 4 μg / mL to 120 μg / mL;

[0047] The concentration of anthocyanin solution is 0.16 μg / mL to 4 μg / mL;

[0048] The mass ratio of the Schizophyllum β-glucan solution to the anthocyanin solution is 20:1 to 40:1.

[0049] The present invention firstly finds that the combination of Schizophyllum β-glucan solution and anthocyanin solution has a synergistic anti-inflammatory effect, and provides a new candidate composition source for improving inflammation. It firstly finds that after the Schizophyllum β-glucan solution and anthocyanin solution are matched in a certain proportion and within a relatively low concentration range, they can play a significant synergistic role in inhibiting the expression of the inflammatory factor IL-1β, reducing the dosage and toxicity of a single active ingredient, and improving the pharmacological efficacy of Schizophyllum β-glucan.

[0050] In the above technical scheme, the concentration of the Schizophyllum β-glucan solution is 4μg / mL~120μg / mL, and the concentration of the anthocyanin solution is 0.16μg / mL~4μg / mL, which has no toxic effect on BJ cells and can be used as medicine; further, compounding the Schizophyllum β-glucan solution and the anthocyanin solution in a mass ratio of 20:1~40:1 can significantly improve the inhibitory effect on the inflammatory factor IL-1β of BJ cells; achieve a significant synergistic effect, thereby significantly reducing the dosage of a single drug.

[0051] Furthermore, the median effect principle and Chou-Talalay combined index method were used to determine the synergistic inhibitory effect of the combination of Schizophyllum β-glucan solution and anthocyanin solution on the expression of inflammatory factor IL-1β mRNA, and the combination index (CI) was calculated. When CI < 1, the combined effect of the combination is a synergistic effect; when CI = 1, the combined effect of the combination is an additive effect; when CI > 1, the combined effect of the combination is an antagonistic effect; in the above technical scheme of the present application, within the above range, the CI value of the combination of Schizophyllum β-glucan solution and anthocyanin solution is lower than 1, which has a significant synergistic effect on the expression of inflammatory factor IL-1β in BJ cells and can significantly reduce the dosage of a single drug.

[0052] Further illustratively, in some embodiments of the present application, the drug containing anthocyanins and Schizophyllum β-glucan having anti-inflammatory effects includes:

[0053] Schizophyllum β-glucan solution and anthocyanin solution;

[0054] The concentration of the Schizophyllum β-glucan solution is 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL or a range between any two of the foregoing values.

[0055] The concentration of the anthocyanin solution is 0.16 μg / mL, 0.18 μg / mL, 0.2 μg / mL, 0.22 μg / mL, 0.24 μg / mL, 0.4 μg / mL, 0.8 μg / mL, 1 μg / mL, 1.4 μg / mL, 2 μg / mL, 2.4 μg / mL, 3 μg / mL, 3.4 μg / mL, 4 μg / mL, 4.4 μg / mL, 4 μg / mL or a range between any two of the foregoing values.

[0056] The mass ratio of the Schizophyllum β-glucan solution to the anthocyanin solution is 20:1, 22:1, 25:1, 30:1, 35:1, 38:1, 40:1 or a range between any two of the aforementioned values.

[0057] Further optionally, in some embodiments of the present application, the above-mentioned drug containing anthocyanins and Schizophyllum β-glucan having anti-inflammatory effect includes:

[0058] The concentration of the Schizophyllum β-glucan solution is 4.8 μg / mL to 96 μg / mL;

[0059] The concentration of anthocyanin solution is 0.4 μg / mL to 4 μg / mL;

[0060] The mass ratio of the Schizophyllum β-glucan solution to the anthocyanin solution is 20:1 to 40:1.

[0061] Further optionally, in some embodiments of the present application, the above-mentioned drug containing anthocyanins and Schizophyllum β-glucan having anti-inflammatory effect includes:

[0062] The concentration of the Schizophyllum β-glucan solution is 12 μg / mL to 72 μg / mL;

[0063] The concentration of anthocyanin solution is 0.8 μg / mL to 3.2 μg / mL;

[0064] The mass ratio of the Schizophyllum β-glucan solution to the anthocyanin solution is 20:1 to 40:1.

[0065] Further, in some embodiments of the present application, preparing a Schizophyllum β-glucan solution comprises:

[0066] The Schizophyllum seed liquid is inoculated into the culture medium for culturing to obtain a fermentation product;

[0067] The culture medium includes: a promoting substance;

[0068] Based on the total volume of the culture medium, the promoting substances include:

[0069] 10mg / L ~ 20mg / L of vitamin B1 and 1mg / L ~ 10mg / L of folic acid.

[0070] In the above technical solution, by adding vitamin B1 and folic acid to the culture medium, the growth of Schizophyllum species can be promoted; and more Schizophyllum β-glucan can be obtained.

[0071] Illustratively, in some embodiments of the present application, preparing a Schizophyllum β-glucan solution comprises:

[0072] The Schizophyllum seed liquid is inoculated into the culture medium for culturing to obtain a fermentation product;

[0073] The culture medium includes: a promoting substance;

[0074] Based on the total volume of the culture medium, the promoting substances include:

[0075] Vitamin B1 at a content of 10mg / L, 11mg / L, 12mg / L, 13mg / L, 14mg / L, 15mg / L, 16mg / L, 17mg / L, 18mg / L, 19mg / L, 20mg / L or in a range between any two of the aforementioned values, and folic acid at a content of 1mg / L, 2mg / L, 3mg / L, 4mg / L, 5mg / L, 6mg / L, 7mg / L, 8mg / L, 9mg / L, 10mg / L or in a range between any two of the aforementioned values.

[0076] Further, in some embodiments of the present application, the culture medium includes: a basic culture substance;

[0077] Calculated by mass percentage, the basic culture material includes: 1% to 5% glucose, 0.1% to 2% tryptone, 0.1% to 1% yeast extract powder, 0.01% to 0.1% magnesium sulfate, 0.01% to 0.2% potassium dihydrogen phosphate; the balance is water.

[0078] The above basic culture medium is conducive to the growth of Schizophyllum species and is conducive to obtaining Schizophyllum β-glucan.

[0079] Further optionally, illustratively, in some embodiments of the present application, the culture medium includes: a basic culture substance;

[0080] In terms of mass percentage, the basic culture material includes: 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5% or a range between any two of the aforementioned values ​​of glucose, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, 1%, 1.2%, 1.5%, 1.6%, 1.8%, 2% or a range between any two of the aforementioned values ​​of tryptone, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% or any two of the aforementioned values ​​of tryptone. Yeast extract powder in an amount ranging between the above numerical values, magnesium sulfate in an amount ranging between any two of the above numerical values, potassium dihydrogen phosphate in an amount ranging between 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1% or any two of the above numerical values; and the balance water.

[0081] Further illustratively, in some embodiments of the present application, preparing a Schizophyllum β-glucan solution comprises:

[0082] The schizophyllum (strain from Guangdong Microbial Culture Collection Center, numbered GDMCC 5.43) was transferred to the slant culture medium for activation, and cultured at 28°C for 5-7 days until the mycelium covered the slant; then the slant strain was transferred to the liquid culture medium, expanded and cultured for 2 days to obtain the fermentation seed liquid; then the fermentation medium was prepared: the fermentation medium included the following components: 2% glucose, 1% tryptone, 0.5% yeast extract powder, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, 15mg / L vitamin B1 and 5mg / L folic acid, pH natural (about 6.0); 10% schizophyllum strains were inoculated into the above culture medium, and fermented at 28°C, 160r / min for 50 hours. The fermentation product was centrifuged to remove the bacteria to obtain the fermentation product. Subsequently, 2 times the volume of anhydrous ethanol of the fermentation product was added for alcohol precipitation, filtered, the precipitated crude polysaccharide was collected, and washed with anhydrous ethanol for multiple times, and vacuum freeze-dried to obtain SPG. Weigh 100 mg of solid SPG, add 100 mL of ultrapure water to dissolve it completely, prepare a 1 mg / mL SPG solution, and store it at 4°C for use in combination.

[0083] Further, in some embodiments of the present application, preparing anthocyanin solution comprises:

[0084] Dissolve cyanidin-3-O-glucoside in a solvent.

[0085] Cyanidin-3-O-glucoside has excellent anthocyanin active ingredients and can obtain high-quality anthocyanin active ingredients.

[0086] Further optionally, in some embodiments of the present application, the above-mentioned cyanidin-3-O-glucoside chloride (C3G) can be purchased from MCE Company.

[0087] Further optionally, in some embodiments of the present application, the purity of the above-mentioned cyanidin-3-O-glucoside is >99%.

[0088] Illustratively, in some embodiments of the present application, the purity of the above-mentioned cyanidin-3-O-glucoside is 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or a content in the range between any two of the aforementioned values.

[0089] Furthermore, in some embodiments of the present application, the solvent includes: dimethyl sulfoxide and water.

[0090] For example, in some embodiments of the present application, the preparation of anthocyanin solution includes: accurately weighing 20 mg of C3G powder, dissolving it to 1 mg / mL with DMSO to obtain a C3G stock solution, and storing it at -20°C. Taking 1 mL, diluting it to 50 μg / mL with ultrapure water to obtain a C3G working solution.

[0091] In some embodiments of the present application, the above-mentioned drug containing anthocyanins and Schizophyllum β-glucan with anti-inflammatory effect includes an acceptable drug carrier. For example, the acceptable drug or cosmetic carrier may include: edible / external gel, emulsion gel, etc.

[0092] In some embodiments of the present application, the dosage form of the above-mentioned drug containing anthocyanins and Schizophyllum β-glucan with anti-inflammatory effect can be an oral solution or a lyophilized powder; the dosage form of the drug or cosmetic for external application can be a patch, paste or ointment for external application.

[0093] Some embodiments of the present application provide a method for preparing a drug containing anthocyanins and Schizophyllum β-glucan having an anti-inflammatory effect, comprising:

[0094] The Schizophyllum β-glucan solution and the anthocyanin solution were mixed.

[0095] The above technical scheme can compound Schizophyllum β-glucan and anthocyanin by mixing the Schizophyllum β-glucan solution and the anthocyanin solution, and is used to prepare a drug containing anthocyanin and Schizophyllum β-glucan and having an anti-inflammatory effect.

[0096] Further, illustratively, in some embodiments of the present application, the Schizophyllum β-glucan solution and the anthocyanin solution can be compounded according to different mass concentrations. illustratively, the mass concentration ratios of SPG to C3G are 20:1, 30:1 and 40:1.

[0097] Some embodiments of the present application provide a composition comprising anthocyanins and Schizophyllum β-glucan having an anti-inflammatory effect, comprising:

[0098] Schizophyllum β-glucan solution and anthocyanin solution;

[0099] The concentration of the Schizophyllum β-glucan solution is 4 μg / mL to 120 μg / mL;

[0100] The concentration of anthocyanin solution is 0.16 μg / mL to 4 μg / mL;

[0101] The mass ratio of the Schizophyllum β-glucan solution to the anthocyanin solution is 20:1 to 40:1.

[0102] In the above technical scheme, the schizophyllum β-glucan solution and the anthocyanin solution are compounded in the above concentration range and according to the above mass ratio, and the synergistic inhibitory effect on BJ cell inflammation can be analyzed: the synergistic inhibitory effect of the C3G+SPG combination on the expression of inflammatory factor IL-1βmRNA is determined by the median effect principle and the Chou-Talalay combination index method, and the combination index (CI) is calculated. When CI<1, the combination effect of the combination is a synergistic effect; when CI=1, the combination effect of the combination is an additive effect; when CI>1, the combination effect of the combination is an antagonistic effect.

[0103] Further optionally, exemplarily, in some embodiments of the present application, the composition comprising anthocyanins and Schizophyllum β-glucan having anti-inflammatory effect comprises:

[0104] Schizophyllum β-glucan solution and anthocyanin solution;

[0105] The concentration of the Schizophyllum β-glucan solution is 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL or a range between any two of the foregoing values.

[0106] The concentration of the anthocyanin solution is 0.16 μg / mL, 0.18 μg / mL, 0.2 μg / mL, 0.22 μg / mL, 0.24 μg / mL, 0.4 μg / mL, 0.8 μg / mL, 1 μg / mL, 1.4 μg / mL, 2 μg / mL, 2.4 μg / mL, 3 μg / mL, 3.4 μg / mL, 4 μg / mL, 4.4 μg / mL, 4 μg / mL or a range between any two of the foregoing values.

[0107] The mass ratio of the Schizophyllum β-glucan solution to the anthocyanin solution is 20:1, 22:1, 25:1, 30:1, 35:1, 38:1, 40:1 or a range between any two of the aforementioned values.

[0108] Some embodiments of the present application provide an application of a composition in medicine and cosmetics.

[0109] In the above technical solution, it was found for the first time that the combination of C3G and SPG has a synergistic anti-inflammatory effect, providing a new source of candidate compositions for improving inflammation.

[0110] It was discovered for the first time that C.3G and SPG, when combined in a certain proportion and within a relatively low concentration range, can exert a significant synergistic inhibitory effect on the expression of the inflammatory factor IL-1β, thereby reducing the dosage and toxicity of a single active ingredient and improving the pharmacological efficacy of SPG.

[0111] The features and performance of the present application are further described in detail below in conjunction with the embodiments:

[0112] Example 1

[0113] Provided is a composition comprising anthocyanins and Schizophyllum β-glucan having anti-inflammatory effects, which is prepared according to the following steps:

[0114] (1) Preparation of Schizophyllum β-glucan solution:

[0115] Step S1, activation of Schizophyllum strains: transferring the Schizophyllum strains to a slant culture medium for activation, and culturing at 28° C. and 160 r / min for 5-7 days until the mycelium covers the slant; then transferring the slant strains to a liquid culture medium, and expanding the culture for 2 days to obtain a fermentation seed liquid;

[0116] Step S2, preparing fermentation medium:

[0117] Fermentation medium includes: basic culture substances and promoting substances.

[0118] The basic culture material includes the following components in percentage by mass: 2% glucose, 1% tryptone, 0.5% yeast extract powder, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate; the balance is water;

[0119] Calculated based on the total volume of the fermentation medium, 15 mg / L of vitamin B1 and 5 mg / L of folic acid were added, and the pH was natural (about 6.0);

[0120] Step S3, fermentation of Schizophyllan β-glucan (SPG): based on the total mass of the fermentation medium obtained in step S2, 10% of the fermentation seed liquid obtained in step S1 is added to the fermentation medium obtained in the above step S2, and fermentation is carried out at 28° C. and 160 r / min for 50 hours to obtain a fermentation mixture.

[0121] Step S4, SPG purification: The fermentation mixture obtained in step S3 is centrifuged to remove the bacterial cells to obtain a fermentation product. Then, anhydrous ethanol twice the volume of the fermentation product is added for alcohol precipitation, filtered, the precipitated polysaccharide is collected, and washed with anhydrous ethanol for multiple times, and vacuum freeze-dried to obtain SPG.

[0122] Step S5, preparing SPG solution: weigh 100 mg of the dried SPG obtained in step S4, add ultrapure water to completely dissolve it, and prepare an SPG working solution with a concentration of 80 μg / mL.

[0123] (2) Preparation of anthocyanin solution:

[0124] Cyanidin-3-O-glucoside chloride (C3G, purchased from MCE, purity>99%) was used to prepare anthocyanin solution.

[0125] Accurately weigh 20 mg of C3G powder and dissolve it in DMSO to 1 mg / mL to obtain C3G stock solution, which is stored at -20°C. Take 1 mL and dilute it to 4 μg / mL in ultrapure water to obtain anthocyanin solution.

[0126] (3) Preparing a composition comprising anthocyanins and Schizophyllum β-glucan having anti-inflammatory effects:

[0127] The Schizophyllum β-glucan solution prepared in the above step (1) and the anthocyanin solution prepared in the above step (2) are compounded in a mass ratio of 20:1.

[0128] Example 2

[0129] Provided is a composition comprising anthocyanins and Schizophyllum β-glucan having anti-inflammatory effects, which is prepared according to the following steps:

[0130] (1) Preparation of Schizophyllum β-glucan solution:

[0131] Step S1, activation of Schizophyllum strains: transferring the Schizophyllum strains to a slant culture medium for activation, and culturing at 28° C. and 160 r / min for 5-7 days until the mycelium covers the slant; then transferring the slant strains to a liquid culture medium, and expanding the culture for 2 days to obtain a fermentation seed liquid;

[0132] Step S2, preparing fermentation medium:

[0133] Fermentation medium includes: basic culture substances and promoting substances.

[0134] The basic culture material includes the following components in percentage by mass: 2% glucose, 1% tryptone, 0.5% yeast extract powder, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate; the balance is water;

[0135] Calculated based on the total volume of the fermentation medium, 15 mg / L of vitamin B1 and 5 mg / L of folic acid were added, and the pH was natural (about 6.0);

[0136] Step S3, fermentation of Schizophyllan β-glucan (SPG): based on the total mass of the fermentation medium obtained in step S2, 10% of the fermentation seed liquid obtained in step S1 is added to the fermentation medium obtained in the above step S2, and fermentation is carried out at 28° C. and 160 r / min for 50 hours to obtain a fermentation mixture.

[0137] Step S4, SPG purification: The fermentation mixture obtained in step S3 is centrifuged to remove the bacterial cells to obtain a fermentation product. Then, anhydrous ethanol twice the volume of the fermentation product is added for alcohol precipitation, filtered, the precipitated polysaccharide is collected, and washed with anhydrous ethanol for multiple times, and vacuum freeze-dried to obtain SPG.

[0138] Step S5, preparing SPG solution: weigh 100 mg of the dried SPG obtained in step S4, add ultrapure water to completely dissolve it, and prepare an SPG working solution with a concentration of 120 μg / mL.

[0139] (2) Preparation of anthocyanin solution:

[0140] Cyanidin-3-O-glucoside chloride (C3G, purchased from MCE, purity>99%) was used to prepare anthocyanin solution.

[0141] Accurately weigh 20 mg of C3G powder and dissolve it in DMSO to 1 mg / mL to obtain C3G stock solution, which is stored at -20°C. Take 1 mL and dilute it to 4 μg / mL in ultrapure water to obtain anthocyanin solution.

[0142] (3) Preparing a composition comprising anthocyanins and Schizophyllum β-glucan having anti-inflammatory effects:

[0143] The Schizophyllum β-glucan solution prepared in the above step (1) and the anthocyanin solution prepared in the above step (2) are compounded in a mass ratio of 30:1.

[0144] Example 3

[0145] Provided is a composition comprising anthocyanins and Schizophyllum β-glucan having anti-inflammatory effects, which is prepared according to the following steps:

[0146] (1) Preparation of Schizophyllum β-glucan solution:

[0147] Step S1, activation of Schizophyllum strains: transferring the Schizophyllum strains to a slant culture medium for activation, and culturing at 28° C. and 160 r / min for 5-7 days until the mycelium covers the slant; then transferring the slant strains to a liquid culture medium, and expanding the culture for 2 days to obtain a fermentation seed liquid;

[0148] Step S2, preparing fermentation medium:

[0149] Fermentation medium includes: basic culture substances and promoting substances.

[0150] The basic culture material includes the following components in percentage by mass: 2% glucose, 1% tryptone, 0.5% yeast extract powder, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate; the balance is water;

[0151] Calculated based on the total volume of the fermentation medium, 15 mg / L of vitamin B1 and 5 mg / L of folic acid were added, and the pH was natural (about 6.0);

[0152] Step S3, fermentation of Schizophyllan β-glucan (SPG): based on the total mass of the fermentation medium obtained in step S2, 10% of the fermentation seed liquid obtained in step S1 is added to the fermentation medium obtained in the above step S2, and fermentation is carried out at 28° C. and 160 r / min for 50 hours to obtain a fermentation mixture.

[0153] Step S4, SPG purification: The fermentation mixture obtained in step S3 is centrifuged to remove the bacterial cells to obtain a fermentation product. Then, anhydrous ethanol twice the volume of the fermentation product is added for alcohol precipitation, filtered, the precipitated polysaccharide is collected, and washed with anhydrous ethanol for multiple times, and vacuum freeze-dried to obtain SPG.

[0154] Step S5, preparing SPG solution: weigh 100 mg of the dried SPG obtained in step S4, add ultrapure water to completely dissolve it, and prepare an SPG working solution with a concentration of 120 μg / mL.

[0155] (2) Preparation of anthocyanin solution:

[0156] Cyanidin-3-O-glucoside chloride (C3G, purchased from MCE, purity>99%) was used to prepare anthocyanin solution.

[0157] Accurately weigh 20 mg of C3G powder and dissolve it in DMSO to 1 mg / mL to prepare C3G stock solution, which is stored at -20°C. Take 1 mL and dilute it to 3 μg / mL in ultrapure water to prepare anthocyanin solution.

[0158] (3) Preparing a composition comprising anthocyanins and Schizophyllum β-glucan having anti-inflammatory effects:

[0159] The Schizophyllum β-glucan solution prepared in the above step (1) and the anthocyanin solution prepared in the above step (2) are compounded in a mass ratio of 40:1.

[0160] Experimental Example 1 Cytotoxicity Assay (CCK-8 Method)

[0161] Place BJ cells in DMEM high-glucose medium containing 10% fetal bovine serum and 1% double antibody, and culture at 37°C and 5% carbon dioxide. When the cells are in good growth condition, inoculate RAW264.7 cells in a 96-well plate at a density of 1×104 cells per well, add 150 μL of culture medium to each well, and incubate at 37°C for 24 hours. Use the SPG working solution and C3G working solution prepared in Example 1; and add the SPG working solution at the final concentration set in Table 1 and the C3G working solution at the final concentration set in Table 2, and co-culture with BJ cells, and set 5 parallel replicates for each concentration.

[0162] According to the dosing requirements in Tables 1 and 2, the culture medium was replaced, and after continuing to culture for 24 h, 15 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C in the dark for 0.5 h. The OD value was detected at 450 nm to calculate the cell viability.

[0163] Cell viability % = (OD of each experimental group - OD of solvent control group / normal group - OD of solvent control group) × 100%

[0164] Table 1 Effect of various concentrations of Example 1 on BJ cell survival rate

[0165] SPG concentration (μg / mL) BJ cell survival rate (%) Solvent control group 0 100.0 Experimental Group 4 101.3 Experimental Group 12 110.4 Experimental Group 24 107.2 Experimental Group 48 99.7 Experimental Group 72 99.7 Experimental Group 96 99.1 Experimental Group 120 99.8

[0166] As shown in Table 1, SPG at a concentration of 4 μg / mL to 120 μg / mL has no toxic effect on BJ cells and can be used in combination with C3G within this concentration range.

[0167] Table 2 Effect of various concentrations of Example 2 on BJ cell survival rate

[0168] C3G concentration (μg / mL) BJ cell survival rate (%) Control group 0 100.0 Experimental Group 0.16 101.5 Experimental Group 0.4 102.4 Experimental Group 0.8 100.3 Experimental Group 1.6 104.7 Experimental Group 2.4 101.9 Experimental Group 3.2 99.9 Experimental Group 4 99.1

[0169] As shown in Table 2, C3G at a concentration of 0.16 μg / mL to 4 μg / mL has no toxic effect on BJ cells and can be used in combination with SPG within this concentration range.

[0170] Experimental Example 2 Detection of inflammatory factor IL-1β content (RT-qPCR method)

[0171] BJ cells with good growth status were taken and inoculated into 6-well plates at a density of 1×106 cells per well. 2 mL of culture medium was added to each well and incubated at 37°C for 24 h.

[0172] The experiment was divided into a normal group, a model group, an SPG experimental group, a C3G experimental group, and SPG+C3G (Examples 1 to 3).

[0173] Normal group: cells, no lipopolysaccharide (LPS), no SPG or C3G;

[0174] Model group: with cells and LPS (10 μg / mL), but without SPG or C3G;

[0175] SPG experimental group: according to the drug addition requirements, the culture medium was replaced, and LPS with a final concentration of 10 μg / mL was added after 2 h of drug action, and the cells were collected after 2-4 hours of continuous culture;

[0176] C3G experimental group: according to the drug addition requirements, the culture medium was replaced, and LPS with a final concentration of 10 μg / mL was added after 2 h of drug action, and the cells were collected after 2 to 4 hours of continuous culture;

[0177] SPG+C3G experimental group: according to the drug addition requirements, the culture medium was replaced, and LPS with a final concentration of 10 μg / mL was added after the drug action for 2 h. The cells were collected after the culture action continued for 2 to 4 h.

[0178] A nucleic acid extraction kit was used to extract total RNA from cells in each experimental group and reverse transcribed into cDNA. The sequence and related information of the human inflammatory factor IL-1β were searched, and primers were synthesized by the company. RT-PCR was performed according to the steps of the kit to detect the mRNA expression level of IL-1β in each group, and the relative expression level of the inflammatory factor IL-1β was calculated using the 2-△△Ct method.

[0179] The test results are shown in the table below and the attached figure of the instruction manual.

[0180] Table 3 Effects of different concentrations of SPG, CG3 and their combination on the relative expression of IL-1β

[0181]

[0182] It can be seen from the test results in the above table that the relative expression level of the inflammatory factor IL-1β in the BJ cells of each embodiment is lower than that of the model group, which shows that the scheme of the embodiment of the present application can effectively improve the anti-inflammatory effect.

[0183] Furthermore, by comparing the various embodiments, it can be seen that when the ratio of the SPG+CG3 composition is 30:1, the relative expression level of the inflammatory factor IL-1β of BJ cells is 1.1, which is significantly lower than 3.2 in the model group and is the lowest among the different ratios, indicating that when the ratio of the SPG+CG3 composition is 30:1, the inhibitory effect on the inflammatory factor IL-1β of BJ cells is the best.

[0184] Instructions attached Figure 1The dose-effect curve of SPG, C3G and the combination of the two in inhibiting the expression of inflammatory factor IL-1β in BJ cells; Note: the drug mass concentration of the combination is plotted based on the mass concentration of SPG in the combination.

[0185] Depend on Figure 1 It can be seen that the inhibition rate of the combination of SPG and C3G on the expression of the inflammatory factor IL-1β is dose-dependent, and compared with the use of SPG and C3G separately, when the same IL-1β inhibition rate is achieved, the concentration of the single drug in the combination of SPG and C3G is significantly reduced, indicating that there is a synergistic effect.

[0186] Experimental Example 3 Analysis of the synergistic inhibitory effect of SPG, C3G and their combination on the inflammatory factor IL-1β

[0187] The median effect principle and Chou-Talalay combined index method were used to determine the synergistic inhibitory effect of SPG, C3G and their combination on the expression of inflammatory factor IL-1β mRNA. The dosage used under the specific inhibition rate of a single or two drugs was calculated according to the median effect equation Fa / Fu=(D / Dm)m. The median effect equation was converted into a linear equation log(Fa / Fu)=mlogD-mlogDm, where D represents the drug dose, Fa represents the decimal inhibition rate, Fu=1-Fa, Dm is the median effect concentration, representing the drug concentration at 50% effect, and m is the slope of the linear equation line.

[0188] The combination index (CI) of chlorinated anthocyanidins and crude extracts of blueberry anthocyanins was calculated according to the Chou-Talalay equation (CI = (D1) / (Dx1)+(D2) / (Dx2)). D1 and D2 were the concentrations of the combination of SPG and C3G at the drug effect X (inhibition rate of inflammatory factor IL-1β mRNA expression), and Dx1 and Dx2 were the concentrations of SPG and C3G when the above drug effect X was achieved. The synergistic inhibitory effect of the two on the expression of inflammatory factor IL-1β mRNA was evaluated. When CI < 1, the combined effect of the two drugs is a synergistic effect; when CI = 1, the combined effect of the two drugs is an additive effect; when CI > 1, the combined effect of the two drugs is an antagonistic effect.

[0189] The test results are shown in the instruction manual. Figure 2 and Table 4.

[0190] Figure 2 The graph is the inhibition rate-combination index (Fa-CI) curve of the combination of SPG and C3G at a mass concentration ratio of 30:1.

[0191] Table 4 When the IL-1β inhibition rate Fa = 0.5, the drug dosage table of SPG, C3G and the combination of the two (30:1)

[0192] Drug / combination CI index (Fa=0.5) Drug dosage (μg / mL) SPG / 92.5 C3G / 3.7 SPG+CG3(30:1) 0.72 50.5+1.7

[0193] Depend on Figure 2 As shown in Table 4, when the mass concentration ratio is 30:1 and within a certain concentration range, the CI value of the combination of SPG and C3G is lower than 1, which has a significant synergistic effect on the expression of the inflammatory factor IL-1β in BJ cells and can significantly reduce the dosage of a single drug.

[0194] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A drug comprising anthocyanins and Schizophyllum β-glucan having anti-inflammatory effects, characterized in that: include: Schizophyllum β-glucan solution and anthocyanin solution; The concentration of the Schizophyllum β-glucan solution is 4 μg / mL to 120 μg / mL; The concentration of the anthocyanin solution is 0.16 μg / mL to 4 μg / mL; The mass ratio of the Schizophyllum β-glucan solution to the anthocyanin solution is 20:1 to 40:

1.

2. The drug comprising anthocyanins and Schizophyllum β-glucan having anti-inflammatory effect according to claim 1, characterized in that: The concentration of the Schizophyllum β-glucan solution is 4.8 μg / mL to 96 μg / mL; The concentration of the anthocyanin solution is 0.4 μg / mL to 4 μg / mL; The mass ratio of the Schizophyllum β-glucan solution to the anthocyanin solution is 20:1 to 40:

1.

3. The drug comprising anthocyanins and Schizophyllum β-glucan having anti-inflammatory effect according to claim 1, characterized in that: The concentration of the Schizophyllum β-glucan solution is 12 μg / mL to 72 μg / mL; The concentration of the anthocyanin solution is 0.8 μg / mL to 3.2 μg / mL; The mass ratio of the Schizophyllum β-glucan solution to the anthocyanin solution is 20:1 to 40:

1.

4. The drug comprising anthocyanins and Schizophyllum β-glucan having anti-inflammatory effect according to any one of claims 1 to 3, characterized in that: The preparation of the Schizophyllum β-glucan solution comprises: The Schizophyllum seed liquid is inoculated into the culture medium for culturing to obtain a fermentation product; The culture medium comprises: a promoting substance; Based on the total volume of the culture medium, the promoting substance comprises: 10mg / L ~ 20mg / L of vitamin B1 and 1mg / L ~ 10mg / L of folic acid.

5. The drug comprising anthocyanins and Schizophyllum β-glucan having anti-inflammatory effect according to claim 4, characterized in that: The culture medium comprises: basic culture material; Calculated by mass percentage, the basic culture material includes: 1% to 5% glucose, 0.1% to 2% tryptone, 0.1% to 1% yeast extract powder, 0.01% to 0.1% magnesium sulfate, 0.01% to 0.2% potassium dihydrogen phosphate; and the balance is water.

6. The drug comprising anthocyanins and Schizophyllum β-glucan having anti-inflammatory effect according to any one of claims 1 to 3, characterized in that: The preparation of the anthocyanin solution comprises: Dissolve cyanidin-3-O-glucoside in a solvent.

7. The drug comprising anthocyanins and Schizophyllum β-glucan having anti-inflammatory effect according to claim 6, characterized in that: The solvent includes dimethyl sulfoxide and water.

8. The method for preparing the anti-inflammatory drug comprising anthocyanins and Schizophyllum β-glucan according to any one of claims 1 to 7, characterized in that: include: The Schizophyllum β-glucan solution and the anthocyanin solution are mixed.

9. A composition comprising anthocyanins and Schizophyllum β-glucan having an anti-inflammatory effect, characterized in that: include: Schizophyllum β-glucan solution and anthocyanin solution; The concentration of the Schizophyllum β-glucan solution is 4 μg / mL to 120 μg / mL; The concentration of the anthocyanin solution is 0.16 μg / mL to 4 μg / mL; The mass ratio of the Schizophyllum β-glucan solution to the anthocyanin solution is 20:1 to 40:

1.

10. Use of the composition according to claim 9 in medicine or cosmetics.

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