| Objective:1.Hyaluronic acid in the skin of Rana temporaria chensinensis were extracted with double enzymes,exploring its optimum extraction conditions by response surface analysis.2.The hyaluronic acid crude product was purified by purification,deproteinization and decolorization to obtain a higher purity hyaluronic acid product.3.The product was prepared into hyaluronic acid toning lotion to explore its moisturizing property as a moisturizing ingredient cosmetic.Methods:1.Rana chensinensis David skin as the research object,with the hyaluronic acid(HA)rate as a measure indicator.Three extraction parameters including enzyme temperature,enzyme hydrolysis time(time of double enzyme was equal)and enzyme amount(dosage ratio of double enzyme was 1:1)of double enzyme(alkaline proteinase and trypsin enzyme)hydrolysis method as the respective variables and hyaluronic acid rate as response value were explored by using Box-Benhnken central composite design and response surface analysis theory based on single factor experiment,so as to study the interaction of the respective variables and their influence on the HA extraction rate and set up the simulated quadratic polynomial regression equation of prediction model.2.First,the extracted hyaluronic acid crude product was purified by quaternary ammonium salt precipitation,secondly deproteinized by Sevag method(chloroform and n-butanol were in a 5:1 ratio in the separating funnel,shaking 20 minutes,taking the organic phase layer,and extracted three times as described above method.Combined the organic phase layer and concentrated to the appropriate volume,Finally,Decolorized with hydrogen peroxide and dried to get hyaluronic acid boutique.3.Analyzed and determined for HA.The appearance of HA was observed and the physical properties were measured.Molecular weight of HA was determined by the Gel chromatography method.The absorbance was measured at 595 nm by Coomassie brilliant blue method and the protein content was determined.4.Hyaluronic acid boutique was solvent with water,then added preservatives and thickeners into the solution to obtain HA toning lotion.Conducting toxicology experiments taking Kunming mice(23±2g)as experimental subjects.The hairs of the back of mice were removed by small scissors and scalpels.The different concentrations of hyaluronic acid toning lotion were applied to the mice.The back of the dermis explored the moisturizing properties of the lotion by means of short-term tests(6 hours)and long-term trials(15 days).Results:1.Comparing the two methods,the HA yield of the optimum extraction process by response surface analysis is higher.The optimum extraction condition was enzyme amount 55000 U/g under the optimal temperature 50℃ for a duration of 14 h.Under these conditions,the average extraction yield of HA was up to 27.68 mg/g in close agreement with the predicted value 27.46 mg/g,the relative error of 0.85%.2.The purity of hyaluronic acid obtained after purification was 92.0%,Glucuronic acid accounted for 42.2%and protein content<10.0%,it was consistent with food and cosmetic grade.3.It conformmed the requirements by the external quality standards such as appearance,traits,pH,and water solubility.The relative molecular mass of HA was 8.5×105 and 9.2×105 respectively by Gel Chromatography.The molecular weight was in the range of 40～100×104,belong to low molecular category,the total number of bacteria test results were 60 cfu/mL,which met the standard.4.Results of toxicity showed that it was non-toxic side effects on mice,Through the acute toxicity test of the skin and oral administration of mice,we didn’t found poisoning and death mice,indicating that the hyaluronic acid product obtained from the Rana temporaria chensinensis skin had no toxic-side effects on mice.The skin moisture content(%)of experimental group 1(5mL/Kg),2(10mL/Kg)and blank group were 71.66±1.68,72.61 ± 1.65 and 69.54± 1.30 respectively,positive control group was 72.32± 2.06.The skin moisture content of mice was significantly higher than the blank control group,no significant difference compared with the positive control.Indicating that the hyaluronic acid product obtained from the Rana temporaria chensinensis skin had a moisturizing property.Hyaluronic acid toning lotion made up a prescription according to the following weight composition:hyaluronic acid powder 0.5-1,A 0.1,PEG-150 distearate 0.1,water 98.8-99.3.Hyaluronic acid homemade(0.5%,1%)and hyaluronic acid purchasing(0.5%)were not significantly different from the blank group by short-term(6h)experiment,which shows hyaluronic acid toning lotion had no Moisturizing effect in the short term(P<0.05).Hyaluronic acid homemade(0.5%,1%)and hyaluronic acid purchasing(0.5%)were a few significantly different from the blank group by long-term(15d)experiment,which showed that HA toning lotion had a significant moisturizing effect.The concentration was the higher,significant moisturizing effect was the more.In addition,the moisturizing effect of homemade HA lotion was basically the same with the purchase of HA lotion under the same concentration,indicating homemade hyaluronic acid toning lotion had moisturizing effect.Conclusion:1.The yield of hyaluronic acid was 27.68 mg/g,and the conditions were accurate and feasible by the method of response surface analysis.2.Hyaluronic acid quality,purity of 92.0%,glucuronic acid accounted for 42.2%.The relative molecular mass of HA was 8.5 × 105 and 9.2 × 105,and the molecular weight was in the range of 40-100 × 104,the total number of bacteria test results met the relative standard3.Oral administration of the skin and toxicity experiments showed that hyaluronic acid was non-toxic to mice,a mouse skin experiment(gravimetric method)showed that the hyaluronic acid extracted from the Rana temporaria chensinensis skin had a moisturizing effect.4.We made hyaluronic acid toning lotion whose formular was consist of hyaluronic acid powder 0.5-1,A 0.1,PEG-150 distearate 0.1,water 98.8-99.3.it showed hyaluronic acid toning lotion had moisturizing effect by the mouse skin smear experiment. |
PDF Full Text Request