阅读说明：本技术 一种具有抗炎效果的溶菌酶制剂 (Lysozyme preparation with anti-inflammatory effect ) 是由 陈生红 于 2021-07-30 设计创作，主要内容包括：本发明涉及一种具有抗炎效果的溶菌酶制剂,其中活性成分溶菌酶和白毛藤提取物的重量比为1：(1-5),所述白毛藤提取物为白毛藤生物碱、白毛藤多糖或其混合物。本发明含溶菌酶的制剂制备方法简单,配伍合理,多种活性成分在发挥各自功效的同时,又能产生协同增效作用,具有显著的抗炎效果。本发明的溶菌酶和白毛藤提取物来源天然,无毒副作用。(The invention relates to a lysozyme preparation with anti-inflammatory effect, wherein the weight ratio of active ingredients of lysozyme to solanum lyratum extract is 1: (1-5), the solanum lyratum extract is solanum lyratum alkaloid, solanum lyratum polysaccharide or a mixture thereof. The preparation method of the lysozyme-containing preparation is simple, the compatibility is reasonable, the various active ingredients can generate the synergistic interaction while playing respective effects, and the lysozyme-containing preparation has a remarkable anti-inflammatory effect. The lysozyme and the solanum lyratum extracts have natural sources and no toxic or side effect.)

1. A lysozyme preparation with anti-inflammatory effect is characterized in that the weight ratio of active ingredients of lysozyme to solanum lyratum extract is 1: (1-5).

2. The lysozyme formulation according to claim 1, wherein the Solanum lyratum extract is Solanum lyratum alkaloid, Solanum lyratum polysaccharide or a mixture thereof.

3. The lysozyme formulation according to claim 2, wherein the Solanum lyratum extract is a mixture of Solanum lyratum alkaloids and Solanum lyratum polysaccharides.

4. The lysozyme formulation according to claim 3, wherein the solanum lyratum alkaloid is prepared by a method comprising: taking the dry powder of the solanum lyratum thunb, adding ethanol for reflux extraction, decompressing and concentrating, adding acid to adjust the pH value to 3, fully stirring and dissolving, standing, and filtering; extracting the filtrate with n-hexane, adding alkali into the extracted acidic solution to adjust the pH to 10, and extracting with dichloromethane; then extracting the alkaline mother liquor with ethyl acetate, decompressing, concentrating and recovering the solvent to obtain the solanum lyratum alkaloid.

5. The lysozyme formulation according to claim 3, wherein the solanum lyratum polysaccharide is prepared by a method comprising: mixing herba Solani Lyrati dry powder with water, extracting at high temperature, vacuum concentrating the supernatant, precipitating with ethanol, dissolving the precipitate in appropriate amount of water, deproteinizing by Sevag method, precipitating with ethanol, and vacuum drying to obtain herba Solani Lyrati polysaccharide.

6. A lysozyme formulation according to any one of claims 1 to 5, wherein the weight ratio of lysozyme, solanum lyratum alkaloid and solanum lyratum polysaccharide is 1: (1-2): (1-2).

7. The lysozyme formulation according to claim 6, wherein the weight ratio of lysozyme to Mucuna birdwoodiana alkaloid to Mucuna birdwoodiana polysaccharide is 1:1: 2.

8. the lysozyme formulation of claim 7, further comprising a pharmaceutically acceptable excipient.

9. The lysozyme formulation of claim 8, wherein the lysozyme formulation is in the form of a tablet, a capsule, or an injection.

10. Use of a lysozyme formulation according to any one of claims 1 to 9 for the preparation of an anti-inflammatory medicament.

Technical Field

The invention relates to the field of medicines, and particularly relates to a lysozyme preparation.

Background

Inflammatory responses are a common pathological phenomenon associated with a variety of disease states, and when tissues or cells with vascularity respond to damaging stimuli, they have the effects of killing pathogens, limiting infection, repairing damage, etc. by a defensive response that occurs through the release of inflammatory mediators. The inflammatory response usually needs to be accurately regulated and controlled, and the disturbance of the response can cause tissue damage to the body and can endanger life in severe cases.

Lysozyme is present in biological secretions such as egg white, saliva, etc. and is capable of catalyzing the hydrolysis of the beta-1, 4 glycosidic bond between bacterial cell wall peptidoglycan N-acetylglucosamine and N-acetylmuramic acid. When bound to negatively charged viral proteins, lysozyme is capable of forming double salts with DNA, RNA, apoproteins, which double salts can inactivate viruses.

The lysozyme has obvious antibacterial action on gram-positive bacteria such as aerobic spore forming bacteria, bacillus subtilis, lichen bacillus and the like, has good biocompatibility to human bodies, has no immunogenicity, and is widely applied to clinical application with antibacterial, antiviral, hemostatic, immunity enhancing, tissue recovery accelerating and the like functions. In particular, lysozyme has been allowed to be applied to cheese and fermented wine as a food preservative by the international committee on food law and the national health department, and is now widely applied to the field of food processing.

Solanum lyratum also called solanum lyratum, solanum lyratum and solanum schoenoprasum are whole plants of solanaceae solanum lyratum, are perennial sprawl herb plants, are widely distributed in Zhejiang, Jiangsu, Jiangxi, Anhui, Hunan and other provinces, are mainly harvested in summer, and have quite abundant resources. The Chinese medicinal herb of Solanum lyratum Thunb has been used as traditional Chinese medicine for over 2000 years, and according to records of Shennong herbal medicine, Solanum lyratum Thunb has sweet and cold taste. It is mainly used for treating cold and heat, diabetes, invigorating spleen and replenishing qi, reducing weight and prolonging life after long-term taking. The solanum lyratum thunb is taken as a medicine by whole herbs and roots, has the functions of clearing heat and promoting diuresis, detoxifying and reducing swelling, resisting cancer and the like, is mainly used for treating cold and fever, icteric hepatitis, cholecystitis, gonorrhea, rheumatic arthritis, cholelithiasis, malaria, uterine erosion, leucorrhea, furunculosis, nephritic edema and other symptoms, is also clinically used for treating various cancers, and particularly has certain curative effects on cervical cancer, lung cancer, esophageal cancer, liver cancer, gastric cancer, vocal cord cancer and the like. The main chemical components of the solanum lyratum are beta-hydroxy steroid alkaloid, and the secondary chemical components are polysaccharide, caffeic acid, vanillic acid and the like.

At present, the application of lysozyme and solanum lyratum extract in combination for anti-inflammatory drugs has not been reported.

Disclosure of Invention

The invention aims to solve the technical problem of providing a medicinal preparation which has the advantages of effective anti-inflammation, no toxic or side effect and natural active ingredient source.

The technical scheme adopted by the invention for solving the problems is to provide a lysozyme preparation with an anti-inflammatory effect, wherein the weight ratio of active ingredients of lysozyme to solanum lyratum thunb extract is 1: (1-5).

Preferably, the solanum lyratum thunb extract is solanum lyratum thunb alkaloid, solanum lyratum thunb polysaccharide or a mixture thereof.

More preferably, the solanum lyratum extract is a mixture of solanum lyratum alkaloids and solanum lyratum polysaccharides.

Preferably, the preparation method of the solanum lyratum alkaloid comprises the following steps: taking the dry powder of the solanum lyratum thunb, adding ethanol for reflux extraction, decompressing and concentrating, adding acid to adjust the pH value to 3, fully stirring and dissolving, standing, and filtering; extracting the filtrate with n-hexane, adding alkali into the extracted acidic solution to adjust the pH to 10, and extracting with dichloromethane; then extracting the alkaline mother liquor with ethyl acetate, decompressing, concentrating and recovering the solvent to obtain the solanum lyratum alkaloid.

More preferably, the preparation method of the solanum lyratum alkaloid comprises the following steps: taking the solanum lyratum dry powder, adding 90% ethanol with 2 times of weight, performing reflux extraction at 50 ℃ for 6h, performing reduced pressure concentration, adding dilute hydrochloric acid to adjust the pH to 3, fully stirring for dissolution, standing for 12h, and filtering; extracting the filtrate with equal amount of n-hexane for 2 times, adjusting pH of the extracted acidic solution to 10 with concentrated ammonia water, and extracting with dichloromethane for 3 times; extracting the alkaline mother liquor with ethyl acetate for 3 times, and concentrating under reduced pressure to recover solvent to obtain herba Solani Lyrati alkaloid.

Preferably, the preparation method of the solanum lyratum thunb polysaccharide comprises the following steps: mixing herba Solani Lyrati dry powder with water, extracting at high temperature, vacuum concentrating the supernatant, precipitating with ethanol, dissolving the precipitate in appropriate amount of water, deproteinizing by Sevag method, precipitating with ethanol, and vacuum drying to obtain herba Solani Lyrati polysaccharide.

More preferably, the preparation method of the solanum lyratum polysaccharide comprises the following steps: taking the solanum lyratum thunb dry powder, adding 10 times of water by weight, mixing uniformly, leaching for 4 hours at 70 ℃, taking the supernatant, concentrating in vacuum, precipitating by using 95% ethanol with 6 times of the volume of the concentrated solution, dissolving the precipitate in proper amount of water again, deproteinizing by using a Sevag method, adding proper amount of 95% ethanol for precipitation, and drying in vacuum to obtain solanum lyratum thunb polysaccharide.

Preferably, in the lysozyme preparation, the weight ratio of lysozyme to solanum lyratum alkaloid to solanum lyratum polysaccharide is 1: (1-2): (1-2).

More preferably, in the lysozyme preparation, the weight ratio of lysozyme to solanum lyratum alkaloid to solanum lyratum polysaccharide is 1:1: 2.

preferably, the lysozyme is extracted from egg white.

Preferably, the lysozyme preparation further comprises pharmaceutically acceptable auxiliary materials.

Preferably, the lysozyme preparation is in the form of tablets, capsules or injections.

The invention also provides an application of the lysozyme preparation in preparing anti-inflammatory drugs.

The invention has the positive and beneficial effects that: surprisingly, through repeated experiments, the invention unexpectedly discovers that the combination of lysozyme, solanum lyratum alkaloid and solanum lyratum polysaccharide has synergistic effect on treating inflammation. The preparation method of the lysozyme-containing preparation is simple, the compatibility is reasonable, the various active ingredients can generate the synergistic interaction while playing respective effects, and the lysozyme-containing preparation has a remarkable anti-inflammatory effect. In addition, the lysozyme and the solanum lyratum extracts are natural in source and have no toxic or side effect.

Detailed Description

The present invention will be further described with reference to the following examples, but the embodiments of the present invention are not limited thereto. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. The lysozyme used in examples and experimental examples was lysozyme extracted from egg white.

Example 1 preparation of Solanum lyratum alkaloid extract

Taking 100g of solanum lyratum dry powder, adding 2 times of 90% ethanol by weight, performing reflux extraction at 50 ℃ for 6h, performing reduced pressure concentration, adding dilute hydrochloric acid to adjust the pH to 3, fully stirring for dissolution, standing for 12h, and filtering; extracting the filtrate with equal amount of n-hexane for 2 times, adjusting pH of the extracted acidic solution to 10 with concentrated ammonia water, and extracting with dichloromethane for 3 times; extracting the alkaline mother liquor with ethyl acetate for 3 times, and concentrating under reduced pressure to recover solvent to obtain herba Solani Lyrati alkaloid.

Example 2 preparation of Mucuna birdwoodiana polysaccharide extract

Taking 100g of solanum lyratum dry powder, adding 10 times of water by weight, mixing uniformly, leaching for 4 hours at 70 ℃, taking supernatant fluid, concentrating in vacuum, precipitating with 95% ethanol with 6 times of the volume of concentrated solution, dissolving precipitate in proper amount of water again, deproteinizing by a Sevag method, adding proper amount of 95% ethanol for precipitation, and drying in vacuum to obtain solanum lyratum polysaccharide.

Example 3 capsules containing Lysozyme and Solanum lyratum alkaloid

0.4g of lysozyme, 1.2g of solanum lyratum alkaloid prepared in example 1, 1g of microcrystalline cellulose, 0.3g of sodium carboxymethyl starch and 0.1g of magnesium stearate are fully mixed, dried and prepared into granules, and the granules are filled into gelatin capsules to obtain 10 capsules with the unit weight of 300 mg.

Example 4 capsules containing Lysozyme and Mucuna birdwoodiana polysaccharide

0.4g of lysozyme, 1.2g of solanum lyratum polysaccharide prepared in example 2, 1g of microcrystalline cellulose, 0.3g of sodium carboxymethyl starch and 0.1g of magnesium stearate are fully mixed, dried and prepared into granules, and the granules are filled into gelatin capsules to obtain 10 capsules with the unit weight of 300 mg.

Example 5 capsules containing Lysozyme, Solanum lyratum alkaloid and Solanum lyratum polysaccharide (1:1.5:1.5)

0.4g of lysozyme, 0.6g of solanum lyratum alkaloid prepared in example 1, 0.6g of solanum lyratum polysaccharide prepared in example 2, 1g of microcrystalline cellulose, 0.3g of sodium carboxymethyl starch and 0.1g of magnesium stearate are fully mixed, dried and prepared into granules, and the granules are filled into gelatin capsules to obtain 10 capsules with the unit weight of 300 mg.

Example 6 capsules containing Lysozyme, Solanum lyratum alkaloid and Solanum lyratum polysaccharide (1:2:1)

0.4g of lysozyme, 0.8g of solanum lyratum alkaloid prepared in example 1, 0.4g of solanum lyratum polysaccharide prepared in example 2, 1g of microcrystalline cellulose, 0.3g of sodium carboxymethyl starch and 0.1g of magnesium stearate are fully mixed, dried and prepared into granules, and the granules are filled into gelatin capsules to obtain 10 capsules with the unit weight of 300 mg.

Example 7 capsules containing Lysozyme, Solanum lyratum alkaloid and Solanum lyratum polysaccharide (1:1:2)

0.4g of lysozyme, 0.4g of solanum lyratum alkaloid prepared in example 1, 0.8g of solanum lyratum polysaccharide prepared in example 2, 1g of microcrystalline cellulose, 0.3g of sodium carboxymethyl starch and 0.1g of magnesium stearate are fully mixed, dried and prepared into granules, and the granules are filled into gelatin capsules to obtain 10 capsules with the unit weight of 300 mg.

Test example 1 test of anti-inflammatory Effect of lysozyme-containing preparation of the present invention

1. Establishment of cell inflammation model

Grouping experiments: DMEM cell culture solutions with LPS concentrations of 0.25mg/L, 0.5mg/L, 1mg/L, 2mg/L and 4mg/L were prepared. To a 24-well plate, 1mL (1X 10 per well) of well-mixed cell suspension was added⁵And each cell) is cultured for 24 hours, then the culture medium is removed, 1mL of sample solution is added according to the above groups, and the cells are cultured for 24 hours, and each group is provided with 3 multiple holes. And (3) determining the NO content of the cell supernatant by adopting a Griess kit, and selecting the LPS concentration with the NO release amount having a significant difference with that of a control group for subsequent experiments.

2. Determination of NO content

To a 24-well plate, 1mL (1X 10 per well) of well-mixed cell suspension was added⁵Individual cells), culturing for 24h, discarding the culture medium, and performing a grouping experiment, wherein each group is provided with 3 multiple holes. The experimental groups were as follows: normal control group (without drug and LPS), LPS model group (1mg/L LPS), test group 1(1mg/L LPS +1mg/mL lysozyme solution), test group 2(1mg/L LPS +1mg/mL solanum lyratum alkaloid solution prepared in example 1), test group 3(1mg/LLPS +1mg/mL solanum lyratum polysaccharide solution prepared in example 2), test group 4(1mg/L LPS +0.25mg/mL lysozyme solution +0.75mg/mL solanum lyratum alkaloid solution prepared in example 1), test group 5(1mg/LLPS +0.25mg/mL lysozyme solution +0.75mg/mL solanum lyratum polysaccharide solution prepared in example 2), test group 6(1mg/L LPS +0.25mg/mL lysozyme solution +0.375mg/mL solanum lyratum alkaloid solution prepared in example 1 +0.375mg/mL solanum polysaccharide solution prepared in example 2) Liquid), test group 7(1mg/L LPS +0.25mg/mL lysozyme solution +0.5mg/mL solanum lyratum alkaloid solution prepared in example 1 +0.25mg/mL solanum lyratum polysaccharide solution prepared in example 2), test group 8(1mg/L LPS +0.25mg/mL lysozyme solution +0.25mg/mL solanum lyratum alkaloid solution prepared in example 1 +0.5mg/mL solanum lyratum polysaccharide solution prepared in example 2). Adding 1ml DMEM cell culture solution containing 1mg/L LPS (i.e. LPS addition amount is 1mg) into each group except normal control group, culturing for 24 hr, discarding culture solution, and grouping according to the above stepsAdding 1ml of DMEM cell culture solution containing lysozyme and/or solanum lyratum extract into each group of samples (namely lysozyme, solanum lyratum polysaccharide and solanum lyratum alkaloid are all dissolved in the DMEM cell culture solution, the total adding amount of the lysozyme and/or the solanum lyratum extract in each test group is 1mg), culturing for 24h, collecting cell supernatant, and determining the content of NO released by different samples on RAW264.7 cells by adopting a Griess kit, wherein the specific test results are shown in the following table 1.

TABLE 1 Effect of lysozyme formulations of the present invention on NO content released from RAW264.7 cells

Test group	NO content (μmol/L)
		Normal control group	6.2
LPS model group	35.5
		Test group 1 (Lysozyme)	19.4
Test group 2 (Solanum lyratum alkaloid)	24.3
		Test group 3 (Solanum lyratum polysaccharide)	26.9
Test group 4 (Lysozyme: Solanum lyratum alkaloid ═ 1:3)	16.4
		Test group 5 (lysozyme: solanum lyratum polysaccharide ═ 1:3)	15.3
Test group 6 (Lysozyme: Solanum lyratum alkaloid: Solanum lyratum polysaccharide 1:1.5:1.5)	14.9
		Test group 7 (Lysozyme: Solanum lyratum alkaloid: Solanum lyratum polysaccharide: 1:2:1)	15.7
Test group 8 (lysozyme: solanum lyratum alkaloid: solanum lyratum polysaccharide 1:1:2)	9.8

Inflammation is an immune response. When the organism is stimulated by toxic substances, inflammatory substances can be found in inflammatory reaction, a series of immune reactions can be made, and the damage of organism tissues and cells can be repaired, so that the organism is prevented from being damaged, and the function of protecting the organism is achieved. NO is a free radical species that can be produced by macrophages or other immune-activated cells. A model of cellular inflammation can be established by measuring the amount of NO released. The induction of inflammation by LPS in RAW264.7 cells is a commonly used model of cellular inflammation. As shown in Table 1, after LPS acts on RAW264.7 cells for 24 hours through the detection of a Griess kit, compared with a normal control group, the NO release amount of the LPS is very significantly different, which indicates that the modeling is successful.

The lysozyme and solanum lyratum extract test groups with different prescriptions can obviously reduce the NO release amount of RAW264.7 cells, and show that the lysozyme and solanum lyratum extract test groups have obvious improvement effect on RAW264.7 cell inflammation. Particularly, under the condition that the total amount of active ingredients is kept unchanged, compared with the single use of the lysozyme, the solanum lyratum alkaloid or the solanum lyratum polysaccharide, the NO release amount of RAW264.7 cells is reduced to different degrees by using the composition containing the lysozyme and the solanum lyratum alkaloid and/or the solanum lyratum polysaccharide, and the combination use of the lysozyme and the solanum lyratum extract is proved to generate a synergistic effect, so that the use amount of anti-inflammatory ingredients is reduced.