| Study Background and PurposePigment dysbolism skin diseases(such as vitiligo, chloasma) are clinical common dermatosis. but the mechanism and laws of science have not been thoroughly expounded, causing a lack of relatively ideal treatment means and methods. 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 which our preliminary results and past scholars' research showed the relationship of medicinal materials color and efficacy. So the research of herbal medicine color-effect phenomenon theory, especially in modern scientific research technique, color-effect herbal medicine regulation and intervention of modern molecular mechanism is necessary to research. the further study has a good prospect and practical significance to explore Chinese medicine, TCM theory development, TCM development research, 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 human A375 melanoma cells in vitro tyrosinase, cell proliferation and melanin synthesis, 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 20μg/ml,10μg/ml, 5μg/ml.2. Application of cell culture techniques breeding human A375 melanoma cells in vitro.the influence of black and white groups of different concentrations herb water extraction to tyrosinase activity is measured by enzymological method of dopa reaction; MTT is used to study the proliferation of cells, the synthesis of melanin is measured by NaOH-assay; each drug and concentration set three repeated holes, repeat 3 times tests, and establish the control group:pure medium group, Chinese traditional medicine group (medium+Chinese medicine) and control group (medium+cells).3. Application of statistical software SPSS 13.0, adoption of the single factor Variance analysis Dunnett test statistical analysis, the experimental results of the analysis, displayed byx±s, are a=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, 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), Tuckare,White Paeony at low concentration (5μg/ml) can promote tyrosinase activity. White Atractylodes,Coix Seed compared with control group, there are no significant differences (P>0.05).2. Melanoma cell proliferation determination results:Compared with the control group, black group herbs in addition to Herba Leonuri, the rest nine herbs of them showed the promoting effect on melanoma cell proliferation with drug concentrations increasing trends to increase (P≤0.05). white group herbs in addition to Peachseed,Coix Seed,Dioscoreae, the rest seven herbs showed the inhibiting effect on melanoma cell proliferation with drug concentrations increasing trends to increase (P<0.05). In addition, research results showed that Terrestris,Dahurian Angelicaroot,Tuckahoe,White Paeony,Chinese Foxglove, the five drugs have two-way regulating functions. Although Peachseed is white, it showed the promoting effect. Herba Leonur,Coix Seed,Dioscoreae, compared with control group, there are no significant differences (P>0.05).3. Melanoma Cells melanin content determination results:Compared with control group, black group herbs in addition to Herba Leonuri, the rest showed promoting effect on melanin synthesis with drug concentrations increasing trends to increase (P<0.05). White group herbs in addition to Peachseed,Coix Seed, the rest eight herbs showed obviously the inhibiting effect on melanin synthesis (P≤0.05). In addition, research results showed that Terrestris,Dahurian Angelicaroo,Tuckahoe,White Paeony,Chinese Foxglove, the five drugs have two-way regulating functions. Although Peachseed is white, it showed the promoting effect. Herba Leonuri,Coix Seed compared with control group, there are no significant differences (P>0.05).The full text of the conclusions1. Nine of every ten black (depth) herbs on cells of tyrosinase activity, melanoma cell proliferation and melanin synthesis totally have stimulative effect, 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, melanoma cell proliferation and melanin synthesis. 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, melanoma cell proliferation and melanin synthesis, even though it is white. It revealed the opposite effect with Color-effect correlation. 4. Leonurus heterophyllus Sweet,Atractylodes macrocephala Koidz,Semen Coicis,Paeonia lactiflora Pall on cells of tyrosinase activity, melanoma cell proliferation and melanin synthesis did not have the role of promoting or inhibiting, or only on one aspect. |
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