Study Of Persimmon Leaf's Effective Locus Influencing Melanin Metabolism

ObjectivePersimmon Leaf, the unique herb which the famous doctors of Beijing TCM Hospital used to cure moth-patch is chosen to study, it is extracted in the reflux and percolation method, separated by solvent extraction and polyamide column chromatography. The main components in each extractive are analyzed, and effects of the extractives to melanin metabolism are observed to explore the active site in Persimmon Leaf to cure moth-patch.MethodsPersimmon Leaf was extracted with water and different density ethanol in the reflux and percolation method; The extraction process of total flavones in Persimmon Leaf was optimized by orthogonal test; The acquired extractives were separated by solvent extraction and polyamide column chromatography; Methods of thin-layer chromatography and HPLC were used to analyze the main components in each extractive; MTT and L-dopa oxidation methods were employed to study the effects to proliferation and tyrosinase activity of B-16 melanoma cells cultured in vitro, after they were treated with different concentrations of Persimmon Leaf extractives.Results(1) The more effective way to extract total flavones in Persimmon Leaf was extraction with 25 times amount of 80% alcohol under reflux for 3 times, 1.5h, 1h and 1h.(2) Thin-layer chromatography showed that Persimmon Leaf extractives contained triterpenoid - Oleanolic Acid, flavonoids - Rutin, organic acid - ProtocatechuicAcid and tannin - Galic Acid.(3) HPLC results showed that flavones-Rutin, Quercetin and Kaempferol in 70% ethanol extractive were equal to 95% ethanol extractive, and both higher than water extractive when Persimmon Leaf was extrated with reflux method; Flavones in 70% ethanol percolate were higher than 70% ethanol reflux extractive; Above 80 percent of Rutin, Quercetin and approximately 50 percent of Kaempferol were enriched in the EtOAC part when the 70% ethanol percolate was separated by solvent extraction; Above 60 percent of the flavonones were enriched in the 95% ethanol eluate when the 70% ethanol percolate was purified by polyamide column .(4) Both aqueous and ethanol extractives of Persimmon Leaf had inhibitory effects to melanoma proliferation, and dose-effect relationship was submit. The 70 % ethanol percolate had the minimum IC50 which was correspondent to the positive control.(5) The inhibitory effect of lower concentration's Persimmon Leaf extractives was stronger than higher concentration's on tyrosinase activity in the range investigated, the best concentration scope was 12.5-50μg/mL. Total tendency of extractive's inhibitory rate in the same series was that: middle polarity parts>polarity parts>nonpolarity parts, For Example: F>I>H, F,F-B>F-C,F-E>F-A. The inhibitory effect was raised after the CHCl3 and EtOAC parts combined, but it was still weaker than the total 70% ethanol percolate. The inhibitory effect of 80% ethanol extractive of Persimmon Leaf was stronger than each part acquired by polyamide column chromatography. All of the above results showed that the effect of Persimmon Leaf extractive to tyrosinase activity in melanoma cells degraded after further separation.ConclusionsPersimmon Leaf inhibits melanin metablisom through synergistic effects of multicomponent, and polarity parts slightly surpass nonpolarity.