| According to the tyrosinase inhibition rate and clearance ratio of DPPH free radicals as the comprehensive score, this paper focused on study active agents extracted and purified from Danggui Buxue decoction and its safety. In addition, the inhibition type and mechanism of tyrosinase were further discussed, that provided ideas and theoretical basis for the explorations and promotion of whitening agent from Chinese herbal compound.The results showed as follows:1) Comprehensive score was used as the target to explore the best ratio of Danggui Buxue decoction for whitening efficacy by the uniform design test. The result showed that optimum proportion of Astragali Radix and Angelicae Sinensis Radix was 1:7. Its inhibition rate of tyrosinase achieved 89.65% and scavenging ratio of DPPH free radicals achieved 90.96%.2) Solid-liquid ratio, extraction solvents and extraction temperature was explored by single factor experiment with the comprehensive score as index. And three factors and three levels response surface analysis test was set up to establish regression model and polynomial fitting, which was draw process prediction model and validated. The result showed that the optimum condition of extraction technology were: the ratio of material and solvent was1:12 g/m L, the concentration of ethanol was 65%, the temperature was92 ℃. Respectively, that was exhibiting a good agreement with their predicted values.3) The active agents were purified from Danggui Buxue decoction and the processing conditions were optimized with macroporous resin. The optimum purification process were: the concentration was 0.4 g/m L, the p H value was 3, the appropriatestripping solvent was 70% ethanol, the ethanol p H value was 9, flow velocity was 2m L/min, the amount of elution was 200 m L. Bovine Corneal Opacity and Permeability Test and RBC Hemolytic Test was used to evaluate the safety of whitening essence. The results demonstrated that IVIS values was calculated to be less than 0 in BCOP test, the results was mild and no eye irritation. In the hemolytic test, the half hemolysis rate of whitening essence was 0.0308 g/m L, which could be considered without irritation.4) The active agents purified from the Danggui Buxue decoction has hardly suppression on monophenolase, but it has a reversible inhibition on diphenolase which were elucidated by means of enzyme kinetics. Its IC50 was 311.24 mg/m L. The mechanism was defined as a reversible mixed-type by Lineweaver-Burk plot. And the KI and KIS value was 0.0048 mg/m L and 0.0062 mg/m L.5) Based on the fundamental formulation, emulsifier, thickener and the content of active agent were explored their effect on skin feeling and produce stability. Then the Danggui Buxue decoction has been made and tested by human skin patch test and whitening effect evaluation. The results demonstrated that, the best emulsifier system is Olivem?1000 and enzymatically decomposed lecithin compounded, and the best thickener system was 1% Sepiplus 400 and 0.1% xanthan gum and 0.3% VP copolymer compounded. The active agents adding range was on 0.5~1.5%, its best effective was added 1.5%, it could make the skin melanin value lower about 37 after the use of 90 days. |
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