A drug containing anthocyanin and Schizophyllum commune β-glucan with anti-inflammatory effects, its preparation method and application

Abstract

This application relates to the field of drugs. Specifically, it relates to a drug containing anthocyanin and Schizophyllum commune β-glucan with anti-inflammatory effects, its preparation method, and applications. The concentration of the Schizophyllum commune β-glucan solution is 4 μg / mL to 120 μg / mL, and the concentration of the anthocyanin solution is 0.16 μg / mL to 4 μg / mL. It has no toxic effect on BJ cells and can be applied to drugs. Further, when the Schizophyllum commune β-glucan solution and the anthocyanin solution are compounded according to a mass ratio of 20:1 to 40:1, the inhibitory effect on the inflammatory factor IL-1β of BJ cells can be significantly improved; a significant synergistic effect is achieved, thereby significantly reducing the single drug dosage. The technical solution of this application first discovers that the combination of anthocyanin and Schizophyllum commune β-glucan has a synergistic anti-inflammatory effect, providing a new candidate composition source for improving inflammation.

Description

Technical Field

[0001] The present application relates to the field of drugs, and in particular, to a drug containing anthocyanin and Schizophyllum commune β-glucan with anti-inflammatory effects, its preparation method, and its application. Background Art

[0002] Inflammation is a defensive response of the body to stimuli, 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 of the body is very common, and anti-inflammatory drugs are a class of drugs that can have an effect on inflammation.

[0004] However, for the current anti-inflammatory drugs on the market, when facing severe inflammation, the dose of the anti-inflammatory active ingredient needs to be increased. Summary of the Invention

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

[0006] In a first aspect, the present application provides a drug containing anthocyanin and Schizophyllum commune β-glucan with anti-inflammatory effects, including:

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

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

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

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

[0011] In the above technical solution, the concentration of the Schizophyllum commune β-glucan solution is 4 μg / mL to 120 μg / mL, and the concentration of the anthocyanin solution is 0.16 μg / mL to 4 μg / mL, which has no toxic effect on BJ cells and can be applied to drugs; further, when the Schizophyllum commune β-glucan solution and the anthocyanin solution are compounded according to a mass ratio of 20:1 to 40:1, the inhibitory effect on the inflammatory factor IL-1β of BJ cells can be significantly improved; a significant synergistic effect is achieved, thereby significantly reducing the single-drug usage dose.

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

[0013] Furthermore, for the first time, it is found in the technical solution of this application that when anthocyanins and Schizophyllum commune β-glucan are within the above-mentioned ratio range, they can significantly synergistically inhibit the expression of inflammatory factor IL-1β, reduce the dosage and toxicity of a single active ingredient, and improve the pharmacological efficacy of SPG.

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

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

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

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

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

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

[0020] In other embodiments of this application, preparing the Schizophyllum commune β-glucan solution includes:

[0021] Inoculating the Schizophyllum commune seed liquid into a culture medium for cultivation 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 substance includes:

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

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

[0026] Based on the mass percentage, the basic culture substance 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 this application, preparing the anthocyanin solution includes:

[0028] Dissolving 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 preparation method of a drug containing anthocyanins and Schizophyllum commune β-glucan with anti-inflammatory effects, including:

[0031] Mix the Schizophyllum commune β-glucan solution and the anthocyanin solution.

[0032] In a third aspect, the present application provides a composition containing anthocyanins and Schizophyllum commune β-glucan with anti-inflammatory effects, including:

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

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

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

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

[0037] In a fourth aspect, the present application provides an application of the composition in drugs.

[0038] In a fifth aspect, the present application provides an application of the composition in cosmetics. Description of the Drawings

[0039] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

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

[0041] Figure 2 It is the inhibition rate-combination index (Fa-CI) curve diagram of the combination of SPG and C3G at a mass concentration ratio of 30:1. Detailed Embodiments

[0042] To make the objectives, 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 some, but not all, of the embodiments of the present application.

[0043] Accordingly, the following detailed description of the embodiments of the present application is not intended to limit the scope of the claimed present application, but merely represents selected embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative efforts shall fall within the scope of protection of the present application.

[0044] An embodiment of the present application provides a drug containing anthocyanin and Schizophyllum commune β-glucan with an anti-inflammatory effect, including:

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

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

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

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

[0049] The present invention first discovers that the combination of Schizophyllum commune β-glucan solution and anthocyanin solution has a synergistic anti-inflammatory effect, providing a new candidate composition source for improving inflammation. It is first discovered that after the Schizophyllum commune β-glucan solution and anthocyanin solution are combined in a certain ratio and within a relatively low concentration range, they can significantly play a role in synergistically 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 commune β-glucan.

[0050] In the above technical solution, the concentration of the Schizophyllum commune β-glucan solution is 4 μg / mL to 120 μg / mL, and the concentration of the anthocyanin solution is 0.16 μg / mL to 4 μg / mL, which has no toxic effect on BJ cells and can be applied to drugs; further, when the Schizophyllum commune β-glucan solution and the anthocyanin solution are compounded according to a mass ratio of 20:1 to 40:1, the inhibitory effect on the inflammatory factor IL-1β of BJ cells can be significantly improved; a significant synergistic effect is achieved, thereby significantly reducing the single drug dosage.

[0051] Furthermore, the medium effect principle and the Chou-Talalay combined index method were used to measure the synergistic inhibitory effect of the combination of Schizophyllum commune 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 composition is a synergistic effect; when CI = 1, the combined effect of the composition is an additive effect; when CI > 1, the combined effect of the composition is an antagonistic effect. In the above technical solution of the present application, within the above range, the CI value of the combination of Schizophyllum commune β-glucan solution and anthocyanin solution is lower than 1, and it has an obvious synergistic effect on the expression of inflammatory factor IL-1β in BJ cells, and can significantly reduce the single drug dosage.

[0052] Further exemplarily, in some embodiments of the present application, drugs containing anthocyanin and Schizophyllum commune β-glucan with anti-inflammatory effects include:

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

[0054] The concentration of Schizophyllum commune 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 the range between any two of the foregoing values.

[0055] The concentration of 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 the range between any two of the foregoing values.

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

[0057] Further optionally, in some embodiments of the present application, the above drugs containing anthocyanin and Schizophyllum commune β-glucan with anti-inflammatory effects include:

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

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

[0060] The mass ratio of the Schizophyllum commune 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 anthocyanin and Schizophyllum commune β-glucan with anti-inflammatory effects includes:

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

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

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

[0065] Furthermore, in some embodiments of the present application, preparing the Schizophyllum commune β-glucan solution includes:

[0066] Inoculating the Schizophyllum commune seed liquid into a culture medium for cultivation to obtain a fermentation product;

[0067] The culture medium includes: promoting substances;

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

[0069] 10 mg / L to 20 mg / L of vitamin B1 and 1 mg / L to 10 mg / 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 commune can be promoted; more Schizophyllum commune β-glucan can be obtained.

[0071] Exemplarily, in some embodiments of the present application, preparing the Schizophyllum commune β-glucan solution includes:

[0072] Inoculating the Schizophyllum commune seed liquid into a culture medium for cultivation to obtain a fermentation product;

[0073] The culture medium includes: promoting substances;

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

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

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

[0077] By mass percentage, the basal culture substance includes: 1% - 5% glucose, 0.1% - 2% tryptone, 0.1% - 1% yeast extract powder, 0.01% - 0.1% magnesium sulfate, 0.01% - 0.2% potassium dihydrogen phosphate; the balance is water.

[0078] The above-mentioned basal culture medium is beneficial to the growth of Schizophyllum commune strains; it is beneficial to obtain Schizophyllum commune β-glucan.

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

[0080] By mass percentage, the basal culture substance includes: glucose with a content of 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5% or in the range between any two of the foregoing values, tryptone with a content of 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 in the range between any two of the foregoing values, yeast extract powder with a content of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% or in the range between any two of the foregoing values, magnesium sulfate with a content of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1% or in the range between any two of the foregoing values, potassium dihydrogen phosphate with a content of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.08%, 0.1%, 0.11%, 0.12%, 0.15%, 0.16%, 0.17%, 0.18%, 0.2% or in the range between any two of the foregoing values; the balance is water.

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

[0082] The Schizophyllum commune (the strain is from the Guangdong Provincial Culture Collection Center of Microorganisms, numbered GDMCC 5.43) was transferred to a slant medium for activation and cultured at 28 °C for 5 - 7 days until the slant was covered with mycelia; then the slant culture was transferred to a liquid medium and cultured for 2 days to obtain a fermentation seed liquid; then a 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, 15 mg / L of vitamin B1 and 5 mg / L of folic acid, with the pH being natural (about 6.0); 10% of the Schizophyllum commune strain was inoculated into the above medium and fermented at 28 °C and 160 r / min for 50 hours. The fermentation product was centrifuged to remove the thalli, obtaining the fermentation product. Then, anhydrous ethanol with a volume twice that of the fermentation product was added for alcohol precipitation, filtered, and the precipitated crude polysaccharide was collected and washed repeatedly with anhydrous ethanol, and then vacuum freeze-dried to obtain SPG. 100 mg of solid SPG was weighed and added to 100 mL of ultrapure water to completely dissolve it, preparing an SPG working solution with a concentration of 1 mg / mL, which was stored at 4 °C for compatibility use.

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

[0084] Dissolving 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 (Cyanidin-3-O-glucoside chloride, C3G) can be selected to 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 > 99%.

[0088] Exemplarily, 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 the content within the range between any two of the aforementioned values.

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

[0090] Exemplarily, in some embodiments of the present application, the preparation of the anthocyanin solution includes: accurately weighing 20 mg of C3G powder, dissolving it in DMSO to 1 mg / mL to obtain a C3G stock solution, and storing it at -20°C. Take 1 mL and dilute 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 anthocyanin and Schizophyllum commune β-glucan with anti-inflammatory effects includes an acceptable pharmaceutical carrier. Exemplarily, acceptable pharmaceutical or cosmetic carriers may include: edible / topical gels, emulsion gels, etc.

[0092] In some embodiments of the present application, the dosage form of the above-mentioned drug containing anthocyanin and Schizophyllum commune β-glucan with anti-inflammatory effects can be oral liquid, freeze-dried powder; the dosage form of the externally applied drug or cosmetic can be patches, pastes or ointments for external application, etc.

[0093] Some embodiments of the present application provide a preparation method of a drug containing anthocyanin and Schizophyllum commune β-glucan with anti-inflammatory effects, including:

[0094] Mix the Schizophyllum commune β-glucan solution and the anthocyanin solution.

[0095] Through the above technical solution, by mixing the Schizophyllum commune β-glucan solution and the anthocyanin solution, the Schizophyllum commune β-glucan and anthocyanin can be compounded for preparing a drug containing anthocyanin and Schizophyllum commune β-glucan with anti-inflammatory effects.

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

[0097] Some embodiments of the present application provide a composition containing anthocyanin and Schizophyllum commune β-glucan with anti-inflammatory effects, including:

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

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

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

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

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

[0103] Further optionally, by way of example, in some embodiments of the present application, a composition comprising anthocyanin and Schizophyllan β-glucan having anti-inflammatory effects includes:

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

[0105] The concentration of the Schizophyllan 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 the 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 the range between any two of the foregoing values.

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

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

[0109] In the above technical solution, it is first discovered that the C3G and SPG combination has a synergistic anti-inflammatory effect, providing a new candidate composition source for improving inflammation.

[0110] It was first discovered that after C.3G and SPG were combined in a certain proportion and within a relatively low concentration range, they could significantly synergistically inhibit the expression of the inflammatory factor IL-1β, reducing the dosage and toxicity of a single active ingredient and improving the pharmacological efficacy of SPG.

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

[0112] Example 1

[0113] Provide a composition containing anthocyanin and Schizophyllum commune β-glucan with anti-inflammatory effects, which is prepared according to the following steps:

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

[0115] Step S1: Activation of Schizophyllum commune strain: Transfer the Schizophyllum commune strain to a slant medium for activation, and culture it at 28°C and 160 r / min for 5 - 7 days until the mycelium covers the slant; then transfer the slant strain to a liquid medium and expand the culture for 2 days to obtain a fermentation seed liquid;

[0116] Step S2: Preparation of fermentation medium:

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

[0118] Calculated by mass percentage, the basic culture substances include the following components: 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, add 15 mg / L of vitamin B1 and 5 mg / L of folic acid, and the pH is natural (about 6.0);

[0120] Step S3: Fermentation of Schizophyllum commune β-glucan (SPG): Based on the total mass of the fermentation medium obtained in Step S2, inoculate 10% of the fermentation seed liquid obtained in Step S1 into the fermentation medium obtained in Step S2 above, and carry out fermentation at 28°C and 160 r / min for 50 hours to obtain a fermentation mixture.

[0121] Step S4: Purification of SPG: Centrifuge the fermentation mixture obtained in Step S3 to remove the thalli and obtain a fermentation product. Then add anhydrous ethanol with a volume twice that of the fermentation product for alcohol precipitation, filter, collect the precipitated polysaccharide, and wash it multiple times with anhydrous ethanol. After vacuum freeze-drying, it is SPG.

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

[0123] (2) Preparation of anthocyanin solution:

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

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

[0126] (3) Preparation of a composition containing anthocyanin and Schizophyllum commune β-glucan with anti-inflammatory effects:

[0127] Mix the Schizophyllum commune β-glucan solution prepared in the previous step (1) and the anthocyanin solution prepared in the previous step (2) according to a mass ratio of 20:1.

[0128] Example 2

[0129] Provide a composition containing anthocyanin and Schizophyllum commune β-glucan with anti-inflammatory effects, which is prepared according to the following steps:

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

[0131] Step S1: Activation of Schizophyllum commune strain: Transfer Schizophyllum commune to a slant medium for activation, and culture it at 28°C and 160 r / min for 5 - 7 days until the slant is covered with mycelia; then transfer the slant strain to a liquid medium for subculture for 2 days to obtain a fermentation seed liquid;

[0132] Step S2: Preparation of fermentation medium:

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

[0134] Calculated by mass percentage, the basic culture substances include the following components: 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, add 15 mg / L of vitamin B1 and 5 mg / L of folic acid, and the pH is 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 was inoculated into the fermentation medium obtained in step S2 above, and fermentation was carried out at 28 °C and 160 r / min for 50 hours to obtain a fermentation mixture.

[0137] Step S4. Purification of SPG: The fermentation mixture obtained in step S3 was centrifuged to remove the thalli, and a fermentation product was obtained. Subsequently, anhydrous ethanol with a volume twice that of the fermentation product was added for alcohol precipitation, filtration, the precipitated polysaccharide was collected, and it was washed repeatedly with anhydrous ethanol and then vacuum freeze-dried to obtain SPG.

[0138] Step S5. Preparation of SPG solution: Weigh 100 mg of the dried SPG prepared 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] An anthocyanin solution was prepared using cyanidin-3-O-glucoside chloride (C3G, purchased from MCE Company, purity > 99%).

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

[0142] (3) Preparation of a composition containing anthocyanin and Schizophyllan β-glucan with anti-inflammatory effects:

[0143] The Schizophyllan β-glucan solution prepared in the previous step (1) and the anthocyanin solution prepared in the previous step (2) were compounded according to a mass ratio of 30:1.

[0144] Example 3

[0145] A composition containing anthocyanin and Schizophyllan β-glucan with anti-inflammatory effects was provided and prepared according to the following steps:

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

[0147] Step S1. Activation of Schizophyllum commune strain: The Schizophyllum commune strain was transferred to a slant medium for activation and cultured at 28 °C and 160 r / min for 5 - 7 days until the slant was covered with mycelia; subsequently, the slant strain was transferred to a liquid medium for subculture for 2 days to obtain a fermentation seed liquid;

[0148] Step S2: Prepare the fermentation medium:

[0149] The fermentation medium includes: a basic culture substance and a promoting substance.

[0150] By mass percentage, the basic culture substance includes the following components: 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, add 15 mg / L of vitamin B1 and 5 mg / L of folic acid, and the pH is 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, inoculate 10% of the fermentation seed liquid obtained in Step S1 into the fermentation medium obtained in the above Step S2, and carry out fermentation at 28 °C and 160 r / min for 50 hours to obtain a fermentation mixture.

[0153] Step S4: Purification of SPG: Centrifuge the fermentation mixture obtained in Step S3 to remove the thalli to obtain a fermentation product. Subsequently, add anhydrous ethanol with a volume twice that of the fermentation product for alcohol precipitation, filter, collect the precipitated polysaccharide, and wash it multiple times with anhydrous ethanol. After vacuum freeze - drying, it is SPG.

[0154] Step S5: Prepare the SPG solution: Weigh 100 mg of the dried SPG prepared 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 the anthocyanin solution:

[0156] Use cyanidin - 3 - O - glucoside chloride (C3G, purchased from MCE Company, purity > 99%) to prepare the anthocyanin solution.

[0157] Accurately weigh 20 mg of C3G powder, dissolve it in DMSO to 1 mg / mL to obtain a C3G stock solution, and store it at - 20 °C. Take 1 mL and dilute it with ultrapure water to 3 μg / mL to obtain the anthocyanin solution.

[0158] (3) Prepare a composition containing anthocyanin and Schizophyllan β - glucan with anti - inflammatory effects:

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

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

[0161] BJ cells were placed in DMEM high-glucose medium containing 10% fetal bovine serum and 1% double antibody and cultured in an environment of 37 °C and 5% carbon dioxide. When the cell growth state was good, RAW264.7 cells were inoculated into a 96-well plate at a density of 1×104 cells per well, and 150 μL of medium was added to each well and incubated at 37 °C for 24 h. The SPG working solution and C3G working solution prepared in Example 1 were used; and the SPG working solution added according to the final concentration set in Table 1 and the C3G working solution added according to the final concentration set in Table 2 were co-cultured with BJ cells, and 5 parallel replicates were set for each concentration.

[0162] According to the drug addition requirements in Table 1 and Table 2, the medium was changed, and after continuous culture for 24 h, 15 μL of CCK-8 reagent was added to each well, incubated at 37 °C in the dark for 0.5 h, and the OD value was detected at 450 nm, and the cell viability was calculated.

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

[0164] Table 1 Effects of various concentrations in Example 1 on the survival rate of BJ cells

[0165]

[0166] It can be seen from Table 1 that SPG at a concentration of 4 μg / mL to 120 μg / mL has no toxic effect on BJ cells and can be formulated with C3G within this concentration range.

[0167] Table 2 Effects of various concentrations in Example 2 on the survival rate of BJ cells

[0168]

[0169] It can be seen from Table 2 that C3G at a concentration of 0.16 μg / mL to 4 μg / mL has no toxic effect on BJ cells and can be formulated with SPG within this concentration range.

[0170] Experimental Example 2 Detection of Inflammatory Factor IL-1β Content (RT-qPCR Method)

[0171] Take BJ cells in good growth state and inoculate BJ cells into a 6-well plate at a density of 1×106 cells per well. Add 2 mL of culture medium to each well and incubate at 37 °C for 24 h.

[0172] The experiments were divided into a normal group, a model group, an SPG experimental group, a C3G experimental group, and an SPG + C3G (Examples 1 - 3).

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

[0174] Model group: There are cells, there is LPS (10 μg / mL), no SPG or C3G;

[0175] SPG experimental group: According to the drug addition requirements, change the culture medium. After the drug acts for 2 h, add LPS with a final concentration of 10 μg / mL and continue to culture for 24 h, then collect the cells;

[0176] C3G experimental group: According to the drug addition requirements, change the culture medium. After the drug acts for 2 h, add LPS with a final concentration of 10 μg / mL and continue to culture for 24 h, then collect the cells;

[0177] SPG + C3G experimental group: According to the drug addition requirements, change the culture medium. After the drug acts for 2 h, add LPS with a final concentration of 10 μg / mL and continue to culture for 24 h, then collect the cells.

[0178] Use a nucleic acid extraction kit to extract the total RNA of the cells in each experimental group; and reverse transcribe it into cDNA; search for the human inflammatory factor IL-1β sequence and related information, synthesize primers by the company, and perform RT-PCR to detect the mRNA expression level of IL-1β in each group according to the kit steps, and calculate the relative expression level of the inflammatory factor IL-1β by the 2-△△Ct method.

[0179] The test results are shown in the following table and the accompanying drawings of the specification.

[0180] Table 3 Effects of different concentrations of SPG, CG3, and their combination on the relative expression level 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 example is lower than that of the model group. Thus, it can be shown that the solutions of the embodiments of the present application can effectively improve the anti-inflammatory effect.

[0183] Furthermore, by comparing different 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 in BJ cells is 1.1, which is significantly lower than 3.2 in the model group and is the lowest among different ratios. This indicates that when the ratio of the SPG+CG3 composition is 30:1, the inhibitory effect on the inflammatory factor IL-1β in BJ cells is the best.

[0184] Instruction manual attachment Figure 1 is the dose-effect relationship curve of SPG, C3G and their combination inhibiting the expression of inflammatory factor IL-1β in BJ cells; Note: The drug mass concentration of the composition is plotted with the mass concentration of SPG in the combination.

[0185] It can be Figure 1 seen that the inhibitory rate of the combination of SPG and C3G on the expression of inflammatory factor IL-1β shows a dose dependence. And compared with the separate use of SPG and C3G respectively, when reaching the same IL-1β inhibitory rate, the concentration of the single drug in the SPG and C3G combination is significantly reduced, indicating its synergistic effect.

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

[0187] The median-effect principle and the Chou-Talalay combination 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. According to the median-effect equation Fa / Fu=(D / Dm)m, the dosage of a single or two drugs at a specific inhibition rate was calculated. The median-effect equation was transformed into a linear equation log(Fa / Fu)=mlogD-mlogDm, where D represents the drug dosage, Fa represents the fractional inhibition rate, Fu = 1 - Fa, Dm is the median concentration, representing the drug concentration at 50% effect, and m is the slope of the straight line of the linear equation.

[0188] According to the Chou-Talalay equation (CI=(D1) / (Dx1)+(D2) / (Dx2)), the combination index (CI) of Schizophyllan β-glucan and cyanidin-3-O-glucoside was calculated. D1 and D2 are the respective concentrations of the SPG and C3G combination at the X drug effect (inhibitory rate of inflammatory factor IL-1β mRNA expression), and Dx1 and Dx2 are the concentrations of SPG and C3G when used alone to achieve the above X drug effect. 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 attachment Figure 2 and Table 4.

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

[0191] Table 4 Drug dosage table of SPG, C3G and their composition (30:1) when the inhibition rate of IL - 1β, Fa = 0.5

[0192]

[0193] From Figure 2 and Table 4, it can be seen that when the mass concentration ratio is 30:1 and within a certain concentration range, the CI value of the composition of SPG and C3G is less than 1, and it has an obvious synergistic effect on the expression of the inflammatory factor IL - 1β in BJ cells, and can significantly reduce the single - drug dosage.

[0194] The above - mentioned are only the preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A drug comprising cyanidin-3-O-glucoside and Schizophyllum β-glucan having an anti-inflammatory effect, characterized in that: include: Schizophyllum β-glucan solution and cyanidin-3-O-glucoside solution; The concentration of the Schizophyllum β-glucan solution is 80 μg / mL to 120 μg / mL; The concentration of the cyanidin-3-O-glucoside solution is 3 μg / mL to 4 μg / mL; The mass ratio of the Schizophyllum β-glucan solution to the cyanidin-3-O-glucoside solution is 30:1; the drug has an inhibitory effect on the inflammatory factor IL-1β of BJ cells.

2. The drug comprising cyanidin-3-O-glucoside and Schizophyllum β-glucan and having anti-inflammatory effect according to claim 1, 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.

3. The drug comprising cyanidin-3-O-glucoside and Schizophyllum β-glucan and having anti-inflammatory effect according to claim 2, characterized in that: The culture medium comprises: basic culture material; In terms of 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.

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

5. The drug comprising cyanidin-3-O-glucoside and Schizophyllum β-glucan and having anti-inflammatory effect according to claim 4, characterized in that: The solvent includes dimethyl sulfoxide and water.

6. A method for preparing a drug having anti-inflammatory effect comprising cyanidin-3-O-glucoside and Schizophyllum β-glucan according to any one of claims 1 to 5, characterized in that: include: The Schizophyllum β-glucan solution and the cyanidin-3-O-glucoside solution are mixed.

7. A composition comprising cyanidin-3-O-glucoside and Schizophyllum β-glucan having an anti-inflammatory effect, characterized in that: include: Schizophyllum β-glucan solution and cyanidin-3-O-glucoside solution; The concentration of the Schizophyllum β-glucan solution is 80 μg / mL to 120 μg / mL; The concentration of the cyanidin-3-O-glucoside solution is 3 μg / mL to 4 μg / mL; The mass ratio of the Schizophyllum β-glucan solution to the cyanidin-3-O-glucoside solution is 30:1.

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