CN113521262B - Lysozyme preparation with anti-inflammatory effect - Google Patents
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Abstract
The invention relates to a lysozyme preparation with anti-inflammatory effect, wherein the weight ratio of active ingredients lysozyme to solanum lyratum thunb extract is 1: (1-5), wherein the white vine extract is white vine alkaloid, white vine polysaccharide or a mixture thereof. The lysozyme-containing preparation disclosed by the invention is simple in preparation method, reasonable in compatibility, and capable of generating a synergistic effect while playing respective effects of various active ingredients, and has a remarkable anti-inflammatory effect. The lysozyme and the solanum lyratum thunb extract of the invention are natural in source and have no toxic or side effect.
Description
Technical Field
The invention relates to the field of medicines, in particular to a lysozyme preparation.
Background
Inflammatory response is a common pathological phenomenon associated with a variety of disease states, and when a tissue or cell with vascularity responds to damaging stimuli, it has the effects of killing pathogens, limiting infection, repairing damage, etc. by releasing the defensive response that occurs in the inflammatory mediators. The inflammatory reaction usually needs to be accurately regulated, and the reaction disorder can cause tissue injury to harm the organism, and can endanger life when serious.
Lysozyme exists in biological secretions such as egg white, saliva and the like, and can catalyze the hydrolysis of beta-1, 4 glycosidic bonds between bacterial cell wall peptidoglycan N-acetylglucosamine and N-acetylmuramic acid. When bound to negatively charged viral proteins, lysozyme is able to form double salts with DNA, RNA, apoproteins, which can deactivate the virus.
The lysozyme has obvious antibacterial effect on gram-positive bacteria such as aerobic spore forming bacteria, bacillus subtilis, bacillus licheniformis and the like, has good biocompatibility on human bodies and no immunogenicity, and is widely applied to the clinic for resisting bacteria, resisting viruses, stopping bleeding, enhancing immunity, accelerating tissue recovery and the like. In particular, lysozyme has been allowed by the international food code committee and the health sector in China to be applied as a food preservative to cheese and fermented wines, and has been widely used in the field of food processing.
The solanum lyratum, which is also called solanum lyratum, is named as the solanum lyratum, is named as Mao Fengteng, calabash and talent, is the whole herb of solanaceae plant solanum lyratum, is a perennial tendril herb, is widely distributed in Zhejiang, jiangsu, jiangxi, anhui, hunan and other provinces, is mainly collected in summer, and has quite abundant resources. The dry whole herb of the solanum lyratum thunb has more than 2000 years history as a traditional Chinese medicine, and Bai Yingwei is sweet and cold according to the description of Shennong herbal meridian. Mainly cold and heat, quench thirst, tonify middle-jiao and qi, and lighten and delay life after long-term administration. The glabrous greenbrier rhizome is used as a medicine with whole herb and root, has the functions of clearing heat and promoting diuresis, detoxifying and detumescence, resisting cancer and the like, is mainly used for treating symptoms such as common cold and fever, icteric hepatitis, cholecystitis, gonorrhea, rheumatic arthritis, cholelithiasis, malaria, uterine erosion, leucorrhea, furuncle, nephritic edema and the like, and is also clinically used for treating various cancers, in particular to cervical cancer, lung cancer, esophagus cancer, liver cancer, gastric cancer, vocal cord cancer and the like. The main chemical components of the solanum lyratum are beta-hydroxysteroid alkaloid, and then polysaccharide, caffeic acid, vanillic acid and the like.
At present, the application of combination of lysozyme and solanum lyratum thunb extract in anti-inflammatory medicaments has not been reported yet.
Disclosure of Invention
The invention aims to solve the technical problem of providing a pharmaceutical preparation which can effectively resist inflammation, has no toxic or side effect and has natural active ingredient sources.
The invention solves the problems by adopting the technical scheme that a lysozyme preparation with anti-inflammatory effect is provided, wherein the weight ratio of active ingredients lysozyme to solanum lyratum thunb extract is 1: (1-5).
Preferably, the white vine extract is white vine alkaloid, white vine polysaccharide or a mixture thereof.
More preferably, the hirsute sinensis extract is a mixture of hirsute sinensis alkaloids and hirsute sinensis polysaccharides.
Preferably, the preparation method of the solanum lyratum alkaloid comprises the following steps: collecting dry powder of herba Solani Lyrati, extracting with ethanol under reflux, concentrating under reduced pressure, adding acid to adjust pH to 3, stirring thoroughly to dissolve, 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, concentrating under reduced pressure, and recovering solvent to obtain the solanum lyratum alkaloid.
More preferably, the preparation method of the solanum lyratum alkaloid comprises the following steps: reflux-extracting herba Solani Lyrati dry powder with 2 times of 90% ethanol at 50deg.C for 6 hr, concentrating under reduced pressure, adding dilute hydrochloric acid to adjust pH to 3, stirring thoroughly, dissolving, standing for 12 hr, and filtering; extracting the filtrate with n-hexane for 2 times, adding ammonia water to adjust pH to 10, and extracting with dichloromethane for 3 times; then extracting the alkaline mother liquor with ethyl acetate for 3 times, concentrating under reduced pressure, and recovering solvent to obtain the solanum lyratum alkaloid.
Preferably, the preparation method of the solanum lyratum thunb polysaccharide comprises the following steps: collecting dry powder of herba Solani Lyrati, mixing with water, extracting at high temperature, vacuum concentrating supernatant, precipitating with ethanol, dissolving precipitate in 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 thunb polysaccharide comprises the following steps: mixing the dried powder of herba Solani Lyrati with 10 times of water, leaching at 70deg.C for 4 hr, collecting supernatant, vacuum concentrating, precipitating with 95% ethanol of 6 times of the volume of the concentrate, dissolving the precipitate in appropriate amount of water, deproteinizing by Sevag method, precipitating with 95% ethanol, and vacuum drying to obtain herba Solani Lyrati polysaccharide.
Preferably, in the lysozyme preparation, the weight ratio of lysozyme to white vine alkaloid to white vine polysaccharide is 1: (1-2): (1-2).
More preferably, in the lysozyme preparation, the weight ratio of lysozyme to white vine alkaloid to white vine 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 a tablet, a capsule or an injection.
The invention also provides application of the lysozyme preparation in preparation of anti-inflammatory drugs.
The invention has the positive beneficial effects that: surprisingly, through repeated experiments, the invention surprisingly finds that the combination of lysozyme, the solanum lyratum alkaloid and the solanum lyratum polysaccharide has a synergistic effect on treating inflammation. The lysozyme-containing preparation disclosed by the invention is simple in preparation method, reasonable in compatibility, and capable of generating a synergistic effect while playing respective effects of various active ingredients, and has a remarkable anti-inflammatory effect. In addition, lysozyme and the glabrous greenbrier rhizome extract are natural in source, and have no toxic or side effect.
Detailed Description
The present invention will be further described with reference to examples, but the embodiments of the present invention are not limited thereto. The experimental methods used in the following examples are conventional methods unless otherwise specified. The lysozyme used in examples and test examples was lysozyme extracted from egg white.
Example 1 preparation of an alkaloid extract from Solanum lyratum
Taking 100g of glaucescent fissistigma root dry powder, adding 2 times of 90% ethanol by weight, extracting at 50 ℃ under reflux for 6 hours, concentrating under reduced pressure, adding dilute hydrochloric acid to adjust the pH to 3, standing for 12 hours after fully stirring and dissolving, and filtering; extracting the filtrate with n-hexane for 2 times, adding ammonia water to adjust pH to 10, and extracting with dichloromethane for 3 times; then extracting the alkaline mother liquor with ethyl acetate for 3 times, concentrating under reduced pressure, and recovering solvent to obtain the solanum lyratum alkaloid.
Example 2 preparation of Solanum lyratum polysaccharide extract
Mixing 100g of dried white vine powder with 10 times of water, leaching at 70 ℃ for 4 hours, collecting supernatant, concentrating in vacuum, precipitating with 95% ethanol with 6 times of the volume of the concentrate, dissolving the precipitate in a proper amount of water, deproteinizing by a Sevag method, precipitating with a proper amount of 95% ethanol, and drying in vacuum to obtain white vine polysaccharide.
Example 3 Capsule containing lysozyme and Solanum lyratum alkaloid
The preparation method comprises the steps of fully mixing 0.4g of lysozyme, 1.2g of the glaucescent fissistigma root alkaloid prepared in the embodiment 1, 1g of microcrystalline cellulose, 0.3g of sodium carboxymethyl starch and 0.1g of magnesium stearate, granulating by a dry method, and filling into gelatin capsules to obtain 10 capsules with the unit weight of 300 mg.
Example 4 Capsule containing lysozyme and Solanum lyratum Thunb polysaccharide
The preparation method comprises the steps of fully mixing 0.4g of lysozyme, 1.2g of the glaucescent fissistigma root polysaccharide prepared in the embodiment 2, 1g of microcrystalline cellulose, 0.3g of sodium carboxymethyl starch and 0.1g of magnesium stearate, granulating by a dry method, and filling into gelatin capsules to obtain 10 capsules with the unit weight of 300 mg.
Example 5 Capsule containing lysozyme, solanum lyratum alkaloid and Solanum lyratum polysaccharide (1:1.5:1.5)
The preparation method comprises the steps of fully mixing 0.4g of lysozyme, 0.6g of the glabrous greenbrier rhizome alkaloid prepared in the example 1, 0.6g of the glabrous greenbrier rhizome polysaccharide prepared in the example 2, 1g of microcrystalline cellulose, 0.3g of sodium carboxymethyl starch and 0.1g of magnesium stearate, granulating by a dry method, and filling into gelatin capsules to obtain 10 capsules with the unit weight of 300 mg.
Example 6 Capsule containing lysozyme, solanum lyratum alkaloid and Solanum lyratum polysaccharide (1:2:1)
The preparation method comprises the steps of fully mixing 0.4g of lysozyme, 0.8g of the glabrous greenbrier rhizome alkaloid prepared in the example 1, 0.4g of the glabrous greenbrier rhizome polysaccharide prepared in the example 2, 1g of microcrystalline cellulose, 0.3g of sodium carboxymethyl starch and 0.1g of magnesium stearate, granulating by a dry method, and filling into gelatin capsules to obtain 10 capsules with the unit weight of 300 mg.
Example 7 Capsule containing lysozyme, solanum lyratum alkaloid and Solanum lyratum polysaccharide (1:1:2)
The preparation method comprises the steps of fully mixing 0.4g of lysozyme, 0.4g of the glabrous greenbrier rhizome alkaloid prepared in the example 1, 0.8g of the glabrous greenbrier rhizome polysaccharide prepared in the example 2, 1g of microcrystalline cellulose, 0.3g of sodium carboxymethyl starch and 0.1g of magnesium stearate, granulating by a dry method, and filling into gelatin capsules to obtain 10 capsules with the unit weight of 300 mg.
Test example 1 anti-inflammatory Effect test of lysozyme-containing preparation of the present invention
1. Cell inflammation model establishment
Experimental grouping: DMEM cell culture solutions were prepared at LPS concentrations of 0.25mg/L, 0.5mg/L, 1mg/L, 2mg/L and 4mg/L, respectively. 1mL of well-mixed cell suspension (1X 10 per well) was added to a 24-well plate 5 Cells), the medium was removed after 24h of incubation, 1mL of sample solution was added in the above groups, and incubated for 24h, each group was provided with 3 duplicate wells. The NO content of the cell supernatant was determined using the Griess kit and the LPS concentration with significant difference in NO release from the control was selected for subsequent experiments.
2. NO content determination
1mL of well-mixed cell suspension (1X 10 per well) was added to a 24-well plate 5 Individual cells), culturing for 24 hours, discarding the culture medium, and performing grouping experiments, wherein 3 compound holes are arranged in each group. The experimental groupings were as follows: normal control group (without drug and LPS), LPS model group (1 mg/L LPS), test group 1 (1 mg/L LPS+1mg/mL lysozyme solution), test group 2 (1 mg/L LPS+1mg/mL Solanum lyratum thunb biological alkali solution prepared in example 1), test group 3 (1 mg/LLPS+1mg/mL Solanum lyratum thunb polysaccharide solution prepared in example 2), test group 4 (1 mg/L LPS+0.25mg/mL Solanum lyratum thunb biological alkali solution prepared in example 1+0.75 mg/mL), test group 5 (1 mg/LLPS+0.25mg/mL Solanum lyratum thunb polysaccharide solution prepared in example 2+0.75 mg/mL Solanum lyratum thunb polysaccharide solution prepared in example 2), test group 6 (1 mg/L LPS+0.25mg/mL Solanum lyratum thunb polysaccharide solution prepared in example 1+0.25 mg/mL Solanum lyratum thunb polysaccharide solution prepared in example 1), test group 7 (1 mg/L LPS+0.25mg/mL Solanum lyratum embodiment 1+0.25 mg/mL Solanum lyratum lycex polysaccharide solution prepared in example 2), test group 7 (1 mg/L LPS+0.25mg/mL Solanum lyratum polysaccharide solution prepared in example 1+0.25 mg/mL Solanum lyratum polysaccharide solution prepared in example 2). Except for the normal control group, 1ml of DMEM cell culture solution containing 1mg/L LPS (namely, the addition amount of LPS is 1 mg) is firstly added into each group, the culture solution is removed after 24 hours of culture, and then the culture solution is added according to the groupsA total of 1ml of the DMEM cell culture solution containing lysozyme and/or Solanum lyratum extract (i.e. lysozyme, solanum lyratum polysaccharide and Solanum lyratum alkaloid are dissolved in the DMEM cell culture solution, and the total addition amount of the active ingredient lysozyme and/or Solanum lyratum extract in each test group is 1 mg) was added, and the samples were cultured for 24 hours, and cell supernatants were collected, and the NO release amounts of the different samples on RAW264.7 cells were measured by using a Griess kit, and specific test results are shown in Table 1 below.
TABLE 1 Effect of the lysozyme formulation of the present invention on NO content released by RAW264.7 cells
| Test group | NO content (mu mol/L) |
| Normal control group | 6.2 |
| LPS model group | 35.5 |
| Test group 1 (lysozyme) | 19.4 |
| Test group 2 (white Mucuna alkaloid) | 24.3 |
| Test group 3 (white Mucuna polysaccharide) | 26.9 |
| Test group 4 (lysozyme: white Mao vine alkaloid=1:3) | 16.4 |
| Test group 5 (lysozyme: white Mao Teng polysaccharide=1:3) | 15.3 |
| Test group 6 (lysozyme: white Mucuna alkaloid: white Mucuna polysaccharide=1:1.5:1.5) | 14.9 |
| Test group 7 (lysozyme: white glaucescent fissistigma root alkaloid: white glaucescent fissistigma root polysaccharide=1:2:1) | 15.7 |
| Test group 8 (lysozyme: white glaucescent fissistigma root alkaloid: white glaucescent fissistigma root 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 injury of organism tissues and cells can be repaired, so that the organism is prevented from being injured, and the effect of protecting the organism is achieved. NO is a radical-like substance that can be produced by macrophages or other immunocompetent cells. A cellular inflammation model can be established by measuring the amount of NO released. Induction of inflammation by RAW264.7 cells with LPS is a common model of cellular inflammation. As shown in Table 1, after LPS acted on RAW264.7 cells for 24 hours by Griess kit, the NO release amounts were all significantly different from that of the normal control group, indicating that the modeling was successful.
The lysozyme and solanum lyratum extract test groups with different prescriptions can obviously reduce the NO release amount of RAW264.7 cells, which shows 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 composition singly using lysozyme, white-hair-vine alkaloid or white-hair-vine polysaccharide, the composition containing lysozyme and white-hair-vine alkaloid and/or white-hair-vine polysaccharide has the advantages that the NO release amount of RAW264.7 cells is reduced to different degrees, and the combined use of lysozyme and white-hair-vine extract has a synergistic effect, so that the use amount of anti-inflammatory ingredients is reduced.
Claims (4)
1. The lysozyme preparation with anti-inflammatory effect is characterized in that the weight ratio of active ingredients lysozyme, glabrous greenbrier rhizome alkaloid and glabrous greenbrier rhizome polysaccharide is 1:1:2;
the preparation method of the solanum lyratum alkaloid comprises the following steps: collecting dry powder of herba Solani Lyrati, extracting with ethanol under reflux, concentrating under reduced pressure, adding acid to adjust pH to 3, stirring thoroughly to dissolve, 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; extracting the alkaline mother liquor with ethyl acetate, concentrating under reduced pressure, and recovering solvent to obtain radix Solani Lyrati alkaloid;
the preparation method of the glaucescent fissistigma root polysaccharide comprises the following steps: collecting dry powder of herba Solani Lyrati, mixing with water, extracting at high temperature, vacuum concentrating supernatant, precipitating with ethanol, dissolving precipitate in water, deproteinizing by Sevag method, precipitating with ethanol, and vacuum drying to obtain herba Solani Lyrati polysaccharide.
2. The lysozyme formulation of claim 1 further comprising a pharmaceutically acceptable excipient.
3. The lysozyme formulation according to claim 2, characterized in that the formulation of the lysozyme formulation is a tablet, capsule or injection.
4. Use of a lysozyme formulation according to any one of claims 1 to 3 for the manufacture of an anti-inflammatory medicament.
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