[0134] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.
[0135] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The term "and/or" as used herein includes any and all combinations of one or more of the related listed items.
[0136] 1. Component screening
[0137] Anti-allergic components WK2 (first anti-allergy composition), Comthing Tetra (second anti-allergy composition), Bisabolol (bisabolol), Defensil (fallen bell extract), deep sea two-section mustard oil, Formulated into emulsion and evaluated for efficacy.
[0138] Table 1
[0139]
[0140]
[0141]
[0142] 2. Formula ratio
[0143] In order to better evaluate the efficacy of anti-allergic and soothing components, the anti-allergic components are used to prepare anti-allergic emulsions. The formula and process are as follows:
[0144]
[0145]
[0146] 3. Formulation technology
[0147] The comparative groups 1-10 and Examples 1-3 were added to the formulations respectively to prepare anti-allergic soothing emulsions for efficacy evaluation.
[0148] The preparation process of anti-allergic soothing emulsion is as follows:
[0149] 1) Weigh the corresponding percentages of glycerin, propylene glycol, butylene glycol, betaine, allantoin, chitooligosaccharides, complex amino acids, oligomeric sodium hyaluronate, and sodium pyrrolidone carboxylate according to the formula ratio;
[0150] 2) Weigh Carbo U20 and Arginine according to the ratio;
[0151] 3) Add the deionized water of the corresponding proportion, raise the temperature to 80-85°C, stir at 250-300 rpm, and stir for 30 min.
[0152] 4) Add caprylic acid and capric acid triglyceride, Dow corning, temperature control at 80-85℃, homogenize at 6500-7500rpm for 3min.
[0153] 5) Decrease the temperature by 55-60°C, add ceramide liposomes and extreme repair peptides, and stir for 180-250rpm for 30 minutes.
[0154] 6) Cooling down to 45 degrees Celsius, control sample components (or example components), p-hydroxyacetophenone and phenoxyethanol, stir at 80-100 rpm, stir for 10 minutes, and cool to room temperature.
[0155] 4. Efficacy evaluation
[0156] 4.1 LPS induces NO release from RAW264.7 cells
[0157] 4.1.1 Experimental principle and method
[0158] LPS (Lipopolysaccharide):
[0159] The thicker (8-10nm) layer of lipopolysaccharide is located in the outermost layer of the cell wall of gram-negative bacteria. It is composed of lipid A, core polysaccharide and O-specific side chain. Lipopolysaccharides are endotoxins and important group-specific antigens (O antigens). Lipopolysaccharide is composed of three parts. Lipid A (Lipid A) is a glycolipid that constitutes endotoxin activity and is covalently linked to the heteropolysaccharide chain. It has two parts: one is the core polysaccharide, which is constant in the relevant strain; the other O-specific chain (O-specific chain) is highly variable. The lipopolysaccharide of Escherichia coli is a commonly used B-cell mitogen, namely polyclonal activator, in laboratory immunology. This experiment uses LPS to induce allergic reactions in RAW264.7 cells and release NO (nitric oxide).
[0160] Griess assay–NO content test method:
[0161] NO is easily oxidized to NO in the body or aqueous solution 2 Under acidic conditions, NO and diazonium salt sulfonamides undergo a diazonium reaction to form diazonium compounds, which further react with naphthyl vinyl diamine, and the product concentration generated by this reaction has a linear relationship with the NO concentration. There is a maximum absorption peak at 540nm.
[0162] Experimental materials and methods:
[0163] Material: DMEM, FBS, LPS, MTT, Griess reagent
[0164] experimental method:
[0165] RAW 264.7 cell culture
[0166] Pipette RAW264.7 cells into DMEM medium to disperse the cells, use Hemacytometer to count the cells, and then use DMEM to dilute the cells to a concentration of 5×104 cells/ml.
[0167] The diluted cell solution was seeded into 96wells, each well was 100ul, that is, 5×103 cells/well.
[0168] Incubate for 24 hours in a 37°C, 5% CO2 incubator.
[0169] Preparation of test sample and blank sample: The test sample is diluted with DMEM medium, and the concentration after dilution is: 1% (the concentration passed the previous MTT test, and the result is non-toxic)
[0170] Sample composition LPS 10ppm LPS 10ppm+1%sample
[0171] After the cells are cultured for 24 hours, observe whether the cells are fully adherent and grow. If the cells are fully adherent, remove the primordium and wash with DPBS.
[0172] After removing the DPBS, add the samples prepared previously. The solubility of the sample is selected as a safe concentration of 1% (20ul/well) based on the results of the toxicity test.
[0173] After the sample was added, it was placed in a 37°C, 5% CO2 incubator for 20 hours.
[0174] After 20 hours, transfer 50ul of medium to a new 96Well, add the same volume of 1% Griessreagent, mix well, and react for 10 minutes in the dark.
[0175] Use ELISAreader to test the absorbance value of the sample at 540nm.
[0176] NO inhibition:
[0177] NO Inhibition%=((〖OD〗_Control-〖OD〗_Sample)/〖OD〗_Control)*100%
[0178] Among them: 〖OD〗_Control-Absorbance value of blank sample
[0179] 〖OD〗_Sample-The absorbance value of the sample
[0180] 4.1.2 Evaluation of component efficacy
[0181] According to Table 1, the control sample or the example sample was added to the anti-allergy soothing emulsion, and the macrophage nitric oxide inhibited inflammation model was used to evaluate the efficacy (as shown in Table 2 and image 3 Shown).
[0182] Table 2 Results of inhibiting NO release amount of anti-allergy soothing composition
[0183]
[0184]
[0185] 4.2 Inhibition of histamine release in P815 mouse macrophages
[0186] 4.2.1 Experimental principle:
[0187] 1) HPLC detection method for histamine content
[0188] Histamine has neither a fluorescent chromophore group nor an ultraviolet chromophore group, but dansyl chloride can react with the active hydrogen on the primary or secondary amine group of histamine, and the HCl with one part removed will generate fluorescence and ultraviolet light. The reaction formula is as follows:
[0189]
[0190] 2) Experimental materials and methods:
[0191] Material: DMEM, FBS, MTT, Dansyl chloride, NaHCO3, ammonia, acetonitrile, ammonium acetate
[0192] experimental method:
[0193] P815 cell culture:
[0194] (1) Use a pipette to suck DMEM medium to blow the P815 cells to disperse the cells, use Hemacytometer to count the cells, and then use DMEM to dilute the cells to a concentration of 5×10 4 cells/ml.
[0195] (2) The released cell solution is respectively inoculated into 6well petri dish, each hole is 1ml, which is 0.5×10 7 cells/well.
[0196] (3) Incubate for 24 hours in an incubator at 37°C and 5% CO2.
[0197] (4) Preparation of test sample and blank sample: The test sample is diluted with DMEM medium, and the concentrations after dilution are: 2% (the concentration passed the previous MTT test, and the result is non-toxic).
[0198] Name
[0199] (5) After 24 hours, place the 6well petri dish under a 15W ultraviolet lamp and irradiate it for 20 seconds to induce an allergic reaction in P815 cells and release histamine.
[0200] (6) Since P815 cells grow in a semi-adherent manner, without changing the medium, add 1ml of DMEM medium to the blank sample, and add 1ml of medium containing 2% of the sample to the test sample to make the total medium The concentration of the sample reaches 1%.
[0201] (7) After the sample is added, it is placed in an incubator at 37°C and 5% CO2 for 24 hours.
[0202] (8) After 24 hours, remove 200ul of the culture medium for histamine derivatization reaction.
[0203] 3) HPLC analysis of histamine
[0204] (1) Sample derivation
[0205] Take 100ul culture medium into a 2ml tube, add 200ul saturated NaHCO3 solution and 400ul 10mg/ml dansyl chloride solution in sequence, mix well with cap and vortex, and react for 60 minutes in the dark at room temperature. After 60 minutes of reaction, 100 ul of 20% ammonia-acetonitrile solution was added, and the reaction was stopped for 30 minutes. Add 100ul of acetonitrile, vortex and mix well, and pass through a 0.45um filter membrane for HPLC analysis.
[0206] (2) Standard working solution derivation
[0207] Take appropriate amount of histamine standard stock solution to prepare 1, 5, 10, 50, and 100 ppm standard working solution, take 100ul into a 2ml tube, install the above method for derivatization reaction. After filtration through 0.45um membrane, HPLC analysis was performed.
[0208] (3) Chromatographic conditions
[0209] Column: Agilent C18 reversed phase chromatographic column, 250mm×4.6mm, 5um
[0210] Mobile phase: acetonitrile: 0.1mol/L ammonium acetate=80:20(v:v)
[0211] Flow rate: 1ml/min
[0212] Column temperature: 35℃
[0213] Injection volume: 20ul
[0214] UV detection wavelength: 254nm
[0215] 4) Experimental results and analysis
[0216] (1) Standard curve
[0217] Under the chromatographic conditions established in this experiment, with the histamine chromatographic peak area (Y) as the ordinate and the corresponding concentration (X) as the abscissa, draw a standard curve (such as figure 1 Shown).
[0218] Peak areas of histamine standard solutions of different concentrations:
[0219] Concentration (ppm)
[0220] Within the concentration range of 1-100ppm for histamine standard working solution, the peak area has a good linear relationship with the concentration. The linear regression equation is y=267.8x+180.58, R 2 = 0.9999. The standard solution chromatogram of histamine is as follows figure 2 Shown.
[0221] 4.2.2 Efficacy evaluation results (as shown in Table 3 and Figure 4 Shown)
[0222] Table 3 Histamine release inhibitory results of the anti-allergic soothing composition combination
[0223]
[0224]
[0225] Conclusion: The anti-allergy component WK2 screened by the anti-allergy and soothing composition of the present invention has the best addition concentration of 0.5%, Comthingtetra 0.5%; ultimate anti-allergy peptide 0.05%, Bisabolol best addition concentration 0.5%, Defensil best addition The concentration of 0.5%, the best concentration of mustard seed oil in the deep sea is 0.5%. For emulsion formulation development, through macrophage nitric oxide inflammatory release model evaluation, the inhibition rate of nitric oxide release can reach 46.07%, and the inhibition rate of histamine is as high as 61.59%.
[0226] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, all possible combinations of the various technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, All should be considered as the scope of this specification.
[0227] The above-mentioned embodiments only express several implementation modes of the present invention, and the description is relatively specific and detailed, but it should not be understood as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.
