| Study Background and PurposePigment dysbolism skin diseases(such as vitiligo, chloasma)have a high incidence, Seriously affecting the patient's quality of life. with the improvement of living standards, People pay more attention to this kind of disease prevention, in recent years, research of the relationship between Traditional Chinese medicine and pigment metabolism has become a hot topic, the influence of many herbs on tyrosinase activity and melanin metabolization already had been preliminary revealed with the research methods and technologies increasingly maturing. However,most of the studies are confined to observe Single traditional Chinese medicine or compound Chinese medicine rarely to design multilayer systematic research from herbal medicine color-effect phenomenon theory now. The effects and intervention mechanism on color-effect herbal medicine and melanin metabolization research are still lacking. Traditional Chinese medicine accumulated "with white to black, with black to white" clinical experience in the long period of clinical practice. Professor yang created herbal medicine color-effect phenomenon theory treating pigment dermatosis getting enlightenment in analogism and formed characteristic theory and treatment methods in the long-term clinical exploration and theory research considering a certain correlation between herb color and pigment dermatosis curative effect, the correlation of color and curative effect was called"herbal medicine color-effect phenomenon"for the first time and color-effect herbs treating pigment disease was called "color-effect herbal medicine", Preliminary study herb medicinal materials color and pigment dermatosis curative effect and have achieved some results.In addition, Puncturervine Caltrop Fruit(PCF)can cure both hyperpigmentation and hypopigmentation disease, as a commonly used herb curing Pigment dysbolism skin diseases. Recently there have been many experimental research reports about PCF on tyrosinase activity. but the reports were very different, even opposite.our past preliminary research results showed the bi-directional regulating effect of PCF on tyrosinase activity and melanocytes proliferation. but the mechanism was not very clear. so the two aspects of researches on color-effect herbs medicine regulating melanoma cells pigment metabolism and bi-directional regulating effect of PCF on melanin metabolization is extremely necessary which has a good prospect and practical significance to explore Chinese medicine, TCM theory development, pigment disease theory innovation and clinical practice guidance.Based on this, this study screen out of every ten black and white kinds of high frequencyly used and effective in treating pigment herbs which chinese traditional treatment experience and modern research have confirmed, by means of consulting a great quantity of literature and clinical data combining with clinical practice experience, taking current mature research technology in tyrosinase, cell proliferation and melanin synthesis as observing indexes, research color-effect herbal medicine on the influence of melanoma cells in vitro tyrosinase activity and the bi-directional regulating effect of Puncturervine Caltrop Fruit(PCF) on Pigment metabolism adopting Chinese medicine serological methods, exploring the correlation of pigment metabolism and color-effect herbal medicine, combing the theory of traditional chinese medicine treating pigment disease to guide clinical treatment availably provide scientific basis for clinical dialectical medication and color-effect phenomenon theory and a new thinking for the study of traditional Chinese medicine curing pigment disease.Methods and contents1. This study screen out of high frequencyly used and effective in treating pigment herbs which traditional chinese treatment experience and modern research have confirmed, by means of consulting a great quantity of literature and clinical data combining with clinical practice experience, According to the herb color into black (dark) and white (light) groups, each of two groups choose 10 herbs as a contrast preparating each herb water extraction and diluting into excluding tire bovine serum medium, make the drug end concentration for 20g/ml,10μg/ml,5μg/ml.2. Prepare medicated serums from wistar mice after they have been feeded with PCF in different dosages consulting medical experimental zoology and herb serological methods, dilute it with excluding tire bovine serum culture medium, make the drug end concentration for 20μg/ml.3. Application of cell culture techniques breeding melanoma cells in vitro.the influence of black and white groups of different concentrations herb water extraction and medicated serums from wistar mice after they have been feeded with PCF in different dosages to tyrosinase activity is measured by enzymological method of dopa reaction; MTT is used to study the proliferation of cells with medicated serums from wistar mice which were feeded with PCF in different dosages, the synthesis of melanin with medicated serums from wistar mice which were feeded with PCF in different dosages is measured by NaOH-assay; each drug and concentration set three repeated holes, repeat 3 times tests, and establish the control group.4. Application of statistical software SPSS 13.0, each experiment was repeated 3 times, displayed by x±s, adoption of the single factor Variance analysis Dunnett test and LSD-t test statistical analysis, the experimental results of the analysis, areα=0.05, P<0.05, when the contractor for the difference was significant.Results1. Cells tyrosinase activity determination results:In black group herbs, Millettia Dielsiana, Salvia Miltiorrhiza, China Dodder, Malaytea Scurfpea Fruit at low, medium and high concentration (5,10,20μg/ml), Radix Polygoni Multiflori, Safflower, Fructus Ligustri Lucidi at medium, high concentration (10,20μg/ml), Chinese Foxglove, Herba Leonuri at high concentration(20μg/ml), Herba Ecliptae at low concentration (5μg/ml) can promote significantly tyrosinase activity (P<0.05 or P<0.01, with significant difference). In white group herbs, Bletilla,Dioscoreae at high concentration(20μg/ml), Tuckahoe,White Paeony at medium and high concentration (10,20μg/ml), Terrestris,Dahurian Angelicaroot,Angelica at low and medium concentration (5,10μg/ml) can inhibit significantly tyrosinase activity (P<0.05, with significant difference). But Dahurian Angelicaroot,Terrestris,Angelica at high concentration (20μg/ml), Peachseed at medium and high concentration (10,20μg/ml), Tuckahoe,White Paeony at low concentration (5μg/ml) can promote tyrosinase activity. of White Atractylodes,Coix Seed compared with control group (P>0.05), there are no significant differences.2. PCF medicated serums at low and medium concentration(10g,30g)depressed cell proliferation and melanin synthesis(P<0.01), while high concentration(60g) were found activate tendency to cell proliferation,melanin synthesis (P<0.01).The full text of the conclusions 1. All of the black (depth) herbs have more apparently stimulative effect, with drug concentrations increasing trends to increase on cells of tyrosinase activity. Seven of every ten white (shallow) herbs on cells of tyrosinase activity, melanoma cell proliferation and melanin synthesis have apparently inhibiting effect, in a way explain that black (depth) herbs on cells of tyrosinase activity, melanoma cell proliferation and melanin synthesis totally have more apparently stimulative effect with white (shallow) herbs apparently inhibiting effect.2. Terrestris,Dahurian Angelicaroot,Tuckahoe,White Paeony,Chinese Foxglove revealed two-way regulating functions on tyrosinase activity. Terrestris,Dahurian Angelicaroot at low concentration inhibiting and high concentration promoting, but Tuckahoe,White Paeony,Chinese Foxglove at low concentration promoting and high concentration inhibiting.3. Peachseed showed the promoting effect on cells of tyrosinase activity, even though it is white. It revealed the opposite effect with Color-effect correlation.5. In white (shallow) herbs, Atractylodes macrocephala Koidz and Semen Coicis on cells of tyrosinase activity did not have the role of promoting or inhibiting.6. The effect of PCF medicated serums on melanoma cell proliferation and melanin synthesis was associated with mice administration dosage. It presented a two-way regulation that low and medium dosage (10g,30g) can decrease tyrosinase activity while high dosage (60g) can increase tyrosinase activity. When use PCF to cure different pigiment disease, in order to improve the effect of cure, we must use the dialectic method with different dosages, such as use low and medium dosage to cure disorders of hyperpigmentation, while use high dosage to cure disorders of hypopigmentation.
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