Extraction And Separation Whitening Active Ingredient From Paeonia Lactiflora Flowers And Its Preparation In Whitening Products

Objective:Paeonia lactiflora flowers is the dried flower of Paeonia lactiflora Pall,which has the effects of nourishing blood and softening the liver,regulating blood and menstruation,and moisturizing the skin.In this paper,Paeonia lactiflora flowers was taken as the research object,and modern traditional Chinese medicine extraction and separation technology combined with activity tracing method was used to find its whitening effect active ingredients,qualitative analysis of the active ingredients,on the basis of which three Paeonia lactiflora flowers whitening products are designed and prepared,and optimize its prescription and preparation process to expand the application range of Paeonia lactiflora flowers in skin care products.Methods:（1）Optimization of Paeonia lactiflora flowers extraction process:the comprehensive score of tyrosinase activity inhibition rate and 1,1-diphenyl-2-trinitrophenylhydrazine（DPPH）free radical scavenging rate was used as indicators,the effects of ethanol volume fraction,extraction temperature,material-liquid ratio and extraction time on the comprehensive score were investigated,and the best extraction process of Paeonia lactiflora flowers was selected by single-factor experiment and orthogonal experiment.（2）Separation of Paeonia lactiflora flowers whitening active substances:three different polar solvents of petroleum ether,ethyl acetate and n-butanol were used to extract Paeonia lactiflora flowers extract,and the DPPH radical scavenging rate and tyrosinase activity inhibition of each extract was compared,select extracts with the highest scavenging rate and inhibition for silica gel column chromatographic separation,according to the method of activity tracking of DPPH free radical scavenging and tyrosinase activity inhibition rate obtained the most active components and used Ultra-high performance liquid chromatography-quadrupole-orbitrap mass spectrometry（UPLC-QE-Orbitrap-MS）performed compositional qualitative analysis of this component.（3）Preparation of Paeonia lactiflora flowers whitening products:using Paeonia lactiflora flowers extract as functional raw materials,three whitening products were designed and prepared:Paeonia lactiflora flowers whitening gel,Paeonia lactiflora flowers whitening cream and Paeonia lactiflora flowers lotion.The optimal prescription and preparation process of three Paeonia lactiflora flowers whitening products were determined by single factor experiments and orthogonal experiments,and the sensory,efficacy,stability and safety of three whitening products of Paeonia lactiflora flowers were evaluated.Results:（1）The optimal extraction process of Paeonia lactiflora flowers was 70%by volume fraction of ethanol,extraction temperature of 80°C,material-liquid ratio of 1:20g/ml,and extraction time of 2 h by combining tyrosinase activity inhibition rate and DPPH radical scavenging rate.（2）The DPPH free radical scavenging rates of each extract was:Vitamin C>ethyl acetate extract>crude extract>n-butanol extract>petroleum ether extract>aqueous phase,and the tyrosinase activity inhibition rate of each extract was:Vitamin C>ethyl acetate extract>crude extract>aqueous phase>petroleum ether extract>n-butanol extract,ethyl acetate extract had the highest activity,and the DPPH radical scavenging IC₅₀ was9.16μg/ml,tyrosinase activity inhibition rate IC₅₀ was 0.58 mg/ml,ethyl acetate extract was determined to be the primary fraction Points.Ethyl acetate extract silica gel column chromatography separated 7 components,and the DPPH radical scavenging rate of each component was:E component>F component>C component>G component>D component>B component>A component,the inhibition rate of tyrosinase activity of each component was:E component>F component>G component>C component>B component>D component>A component,The E component had the highest activity,and the DPPH radical scavenging rate was 80.97%,the tyrosinase activity inhibition rate was61.48%,and the E component was determined to be the next separation fraction.Four components were obtained by chromatography of the E component silica gel column,and the DPPH radical scavenging rates of each component was:K component>J component>I component>H component,and the inhibition rate of tyrosinase activity of each component was:K component>J component>I component>H component,and K component had the highest activity,and the DPPH radical scavenging rate was 82.84%,and the tyrosinase activity inhibition rate was 70.43%.The chemical composition of component K was qualitatively analyzed using UPLC-QE-Orbitrap-MS,and the possible structures of eight of the components were determined:methyl gallate,2,2’-methylenebis-（4-Methyl-6-tert-butylphenol）,2,5-di-tert-butylhydroquinone,azelaic acid,dibutyl phthalate,monooctyl phthalate,cyclopenta（iso）leucine,cyclohepta（iso）leucine.（3）The best prescription of Paeonia lactiflora flowers whitening gel was Paeonia lactiflora flowers extract 1%,carbomer 0.8%,glycerol 15%,propylene glycol 20%,triethanolamine 1.7%,ethylparaben 0.1%,methylparaben 0.1%,its p H value is6.763±0.080,and the moisturizing rate is 76.94%and 94.60%for 48 h in the environment of relative humidity of 31%and 81%,the average UV absorption rates of UVA region and UVB region were 51.33%and 96.86%,respectively,the DPPH radical scavenging rate IC₅₀=1.140 mg/ml,and the tyrosinase activity inhibition rate IC₅₀=182.694 mg/ml.The best prescription for Paeonia lactiflora flowers Whitening Cream is olive oil 1%,white petrolatum 2%,liquid paraffin 2%,cetyl alcohol 4%,silicone oil 1%,glyceryl monostearate 1.5%,glycerol 10%,propylene glycol 7.5%,triethanolamine 0.5%,carbomer 0.4%,Tween 80 5%,Paeonia lactiflora flowers flower extract 0.8%,methylparaben 0.1%,ethylparaben 0.1%,rose fragrance 0.1%,its p H value is6.295±0.091,the moisturization rate of 48 h in the environment of relative humidity of31%and 81%is 68.06%and 87.45%,the average UV absorption rate of UVA region and UVB region is 54.20%and 93.62%,respectively,and the DPPH radical scavenging rate IC₅₀=1.490 mg/ml.The optimal treatment of Paeonia lactiflora flowers lotion is ethanol20%,ethylparaben 0.1%,methylparaben 0.1%,rose fragrance 0.1%,glycerol 5%,propylene glycol 5%,PEG400 1%,Tween 20 2%,polyvinylpiratone 3%,sorbitol 2%,Paeonia lactiflora flowers extract 0.3%,EDTA-2Na 0.02%,triethanolamine 0.1%,its p H value was 6.113±0.020,the moisturization rate of 48 h was 68.33%and 88.33%in the environment of relative humidity of 31%and 81%,the average UV absorption rate of UVA region and UVB region was 23.66%and 75.03%,respectively,the DPPH radical scavenging rate IC₅₀=3.672 mg/ml,and the tyrosinase activity inhibition rate IC₅₀=613.124 mg/ml.The three whitening products of Paeonia lactiflora flowers have good appearance and sensory effects,no stratification deterioration was found in centrifugation experiment at room temperature,cold resistance experiment,heat resistance experiment,cold and heat cycle experiment and room temperature retention experiment,and the stability meets the requirements of cosmetics,in acute oral toxicity test,eye irritation test,skin irritation test and human patch test,no dead skin lesions,allergies and other phenomena,can be used safely.Conclusion:The extraction process of Paeonia lactiflora flowers selected in this paper is stable and feasible,and the whitening activity of Paeonia lactiflora flowers extract may be different from methyl gallate,2,2’-methylenebis-（4-methyl-6-tert-butylphenol）,2,5-di-tert-butylhydroquinone,azelaic acid,dibutyl phthalate,monooctyl phthalate,cyclopenta（iso）leucine,cycloheta（iso）leucine and other active ingredients are related,Paeonia lactiflora flowers whitening products have good whitening,moisturizing and antioxidant properties,and have broad research and development prospects in the cosmetics market.