Extraction Skin-whitening Agent From Radix Puerariae And Its Application In Cosmatic

This paper focused on studying the skin-whitening agent from radix puerariae and its inhibitory capability against tyrosinase. The active agents were extracted from radix puerariae and the processing conditions were optimized with the orthogonal design test. After the process of separation and purifying, the skin-whitening agent was obtained, and the inhibitory effect of puerarin on tyrosinase was studied, using enzymological kinetic method.The water and ethanol extracts of radix puerariae, Lonicera japonica Thunb., Artemisia anomala S. Moore and Dispyrosl kaki Lf. were obtained by refluxing with both water and 75%(v/v)ethanol, concentration and drying, respectively. The inhibitory capability of water and 75%(v/v)ethanol extracts against tyrosinase were investigated. The results showed that extract of radix puerariae has the stronger inhibition than others.The optimum reaction conditions of skin-whitening agent's inhibition on tyrosinase determined by the single factor experiment method were as follows: the pH value of Phosphate Buffer Solution(PBS) was 6.85, the volume of L-Tyrosine was 1.0mL, the volume of tyrosinase was 0.4mL,time in water bath before tyrosinase added was 10 minutes and reacting time was 5 minutes.The processing conditions of ethanol extract of radix puerariae were optimized with the orthogonal design test. The optimum extracting condition was obtained as the concentration of ethanol was 75%; time for refluxing was 4h, the ratio of material and solvent was l:8 and repeat the process for 2 times. The amount of flavonoid from extract of radix puerariae was investigated, results showed that the largest amount of flavonoid was13.74%.The ethanol extract was further separated with solvents of different polar such as petroleum ether, trichloromethane, ethyl acetate and n-butylalcohol and their inhibitory capability against tyrosinase were compared. It has been found that the inhibitory capability of ethyl acetate-extracted fraction against tyrosinase was the strongest. The extract of ethyl acetate was purified by silica gel column chromatography and tracked by TLC. 10 eluants were obtained and the inhibitory capability of each eluant against tyrosinase was investigated. The results showed that the inhibition of B was 65.7% and the inhibition of D was 79.2%. B and D was further purified by silica gel column chromatography and tracked by TLC. It has been found that the inhibition of B3 obtained from B was 92.3%, and the inhibition of D4 obtained from D was 98.5%.To identify the skin-whitening agent from radix puerariae, HPLC, UV and LCMS were used. It has been confirmed that daidzein and puerarin were the two main skin-whitening agents obtained from radix puerariae.The mechanism of puerarin's inhibition on tyrosinase was studied, using enzymological kinetic method. The results showed that puerarin was a noncompetitive inhibitor proved by its Lineweaver-Burk plots.Puerarin was applied in cosmetic. The results confirmed that puerarin has skin-lightening effect after 3 months of trial by 15 people.The antimicrobial effect of radix puerariae extract and puerarin were studied. Results showed that radix puerariae extract has better antimicrobial effect than puerarin.