Regulation Of Yihei Formula And Cuhei Formula On The Melanin Synthesis And Tyrosinase Activity In Human Melanoma A375Cells

Objective: To screen herbal medicines for regulating melanin synthesis from theChinese medicines commonly used in chloasma and vitiligo treatment to composeinhibiting or promoting melanin synthesis prescriptions, and further to study theeffects of these prescriptions on melanin synthesis and its mechanism and TraditionalChinese Medicine pathogenesis to provide efficacious prescriptions to skin pigmentobstacles.Methods:270kinds of Chinese medicines common used in treating Chloasma andVitiligo was selected on China National Knowledge Infrastructure (CNKI) in recent30years. Every herbal extract was prepared by soaking herb in95%ethanol at Roomtemperature for one week. After human melanoma A375cells were treated with everyherbal ethanol extract at100μg/ml concentration for48hours, the cell growth activitywas measured by MTT and the cell melanin content was assayed by NaoH spallationto screen herbs with high inhibition or promoting ratio of melanin synthesis. Herbalcomposition was optimized by orthogonal test to form a inhibiting or promotingmelanin synthesis formulae, named Yihei formula and Cuhei formula. Effects of Yiheiformula and Cuhei formula on melanin synthesis were determined in dose-dependenceand time-dependence. Effects of these two formulae on tyrosinase activity weredetermined by oxyphenylalanine oxidation method in dose-dependence andtime-dependence. The morphology of human melanoma cells was observed andpicture-taken under a invertmicroscope.Results:1. herbal screening and prescriptions developing of regulating melaninsynthesis in human melanoma A375cells. After human melanoma A375cells weretreated with every herbal ethanol extract at100μg/ml concentration for48hours, theherbs with obvious cell melanin synthesis and without obvious cell growth wereselected by single-factor analysis of variance(One-Way ANOVA) of melanin contentand cell growth OD values difference between control group an treat group, in which Semen Litchi、Rhizoma Cyperi、Polyporus Umbellatus、Semen Coicis、Fructus Lyciiand Plastrum Testudinis were obtained with inhibiting melanin synthesis, RadixAucklandiae、Fructus Toosendan、Morinda Officinalis How、Eucommia Ulmoides、Chantui and Radix Angelicae Dahuricae were obtained with inhibiting melaninsynthesis. The herbal composition above two herbal groups was optimized byorthogonal test to form a inhibiting or promoting melanin synthesis formulae, namedYihei formula and Cuhei formula.2. Effects of Yihei formula and Cuhei formula on melanin synthesis in humanmalignant melanoma A375cells. When the concentration of Yihei formula andCuhei formula was between6.25and100ug/ml, the inhibition or promotion ratioto melanin synthesis showed an increasing with the concentration increment ofYihei formula and Cuhei formula，the effects reached a peak value when the drugconcentration to100μg/ml. while the drug concentration was between200and800ug/ml, the inhibition or promotion ratio to melanin synthesis showeddeclining with the increasing concentration of Yihei formula and Cuhei formula,when to800ug/ml, the melanin synthesis ratio decreased to minimum peakvalue.3. Effects of Yihei formula and Cuhei formula on tyrosinase activity in humanmalignant melanoma A375cells. When the concentration of Yihei formula andCCuhei formula was between6.25and100ug/ml, the inhibition or promotion ratio totyrosinase activity showed an increasing with the concentration increment of Yiheiformula and Cuhei formula， the effects reached a peak value when the drugconcentration to100μg/ml. while the drug concentration was between200and800ug/ml, the inhibition or promotion ratio to tyrosinase activity showed decliningwith the increasing concentration of Yihei formula and Cuhei formula, when to800ug/ml, the tyrosinase activity ratio decreased to minimum peak value.4. Normal human melanoma A375cells was fusiform shape under invert microscope.Compared with the control cells, the volume and dendrites of the cells decreased andintracellular melanin granule was reduced when treated with Yihei formula at100ug/ml. while the volume and dendrites of the cells and intracellular melaningranule increased, showed third grade or multilevel fusiform shape when treated withCuhei formula at100ug/ml.Conclusions:1.This study provides efficacious prescriptions to Yihei formula, Cuhei formula to treat Skin pigment obstacles.2. The effects of Yihei formula and Cuhei formula on melanin synthesis formulaeshow dose-and time-dependence.3. The effects of Yihei formula and Cuhei formula on melanin synthesis formulaeshow positive relationship.4.The literature analysis and experimental results indicate: there are the pathogenesisof liver depression、kidney deficiency、wind and dampness pathogen invading in skinpigment obstacles.