Effects Of Single Herbs Decoloring And Medicated Serum With Compound Chinese Drugs On Melanin Metabolization In Human A375Melanoma Cells In Vitro

Study Background and PurposeSkin pigment metabolism disorders are related closely to various body factors and environmental factors.Pigment dysbolism skin diseases(such as vitiligo.chloasma) are easy diagnosis and difficult to be treated. in recent years, along with the wide application of molecular biology technology, the research in pigment metabolism rapidly is developing. domestic and overseas scholars actively explore pigment metabolism of the mechanism and influencing factors, expounds the regulation mechanism of traditional Chinese medicine on pigment metabolism, finding a safe, effective medicine ingredient or prescription for pigmentation metabolic diseases. but the mechanism and laws of science have not been thoroughly expounded, causing a lack of relatively ideal treatment means and methods. 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. 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.In addition,Vitiligo is easy to diagnosis and difficult to cure as a typical diseases in clinical, Traditional Chinese medicine has distinct characteristics in the treatment, emphasizing syndrome type points with syndrome differentiation. but Syndromes classification and herbs of Vitiligo are too various.moreover, in clinical practice many vitiligo patients appear " No symptoms can discriminate" or "litte symptoms can discriminate", besides the skin spots, no other special symptoms. So my teacher put forward a "dispeling the wind, promoting blood circulation, filling kidney" as the basic principle, combining the staging treatment.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, single herb Chinese medicine after the active carbon decoloring and medicated serum with compound Chinese drugs with four different effects using serological methods on the influence of human A375melanoma cells in vitro melanin metabolization. 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 five herbs as a contrast preparating each herb water extraction,take the traditional Chinese medicine water extract10ml,with purple spectrophotometric method determinating absorbency valuevia Orthogonal Design Screening decoloring technology of every traditional Chinese medicine in order to best decoloring conditions, taking clear liquid in decoloring and filtering in reserve and diluting into excluding tire bovine serum medium, make the drug end concentration for10μg/ml.2. With reference to medical experiments zoology and traditional Chinese medicine serological methods, preparation of medicated serum with compound Chinese drugs with four different effects, diluting into excluding tire bovine serum medium, make the medicated serum end concentration for five percent,ten percent, twenty percent, thirty percent.3. Application of cell culture techniques breeding human A375melanoma cells in vitro.the influence of black and white groups of before and after decoloring herb water extraction and compound Chinese drugs with four different effects in different concentrations 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, repeat3times tests, and establish the control group:pure medium group, Chinese traditional medicine group (medium+Chinese medicine) and control group (medium+cells).4. Application of statistical software SPSS13.0, adoption of the single factor Variance analysis Dunnett test statistical analysis and factorial design material of variance analysis, the experimental results of the analysis, displayed by x±s, are a=0.05, P≤0.05, when the contractor for the difference was significant.Results1. the results of single flavour Chinese medicine adjust melanin metabolization①herbs Without decoloring, Radix Polygoni Multiflori,Malaytea Scurfpea Fruit, Salvia Miltiorrhiza,Millettia Dielsiana, Herba Ecliptae, Peachseed can promote significantly tyrosinase activity (P<0.05,P<0.01with significant difference). Tuckahoe、Dahurian Angelicaroot、Bletilla can inhibit significantly tyrosinase activity (P<0.05, P<0.01with significant difference) compared with control group; and the herbs after decoloring, Herba Ecliptae，Radix Polygoni Multiflori can’t promote tyrosinase activity (P>0.05),activation function of Salvia Miltiorrhiza,Millettia Dielsiana,Malaytea Scurfpea Fruit reduce, there are significant differences with decoloring group(P<0.05, P<0.01with significant difference). Peachseed、Tuckahoe、 Dahurian Angelicaroot、Bletilla、White Atractylodes are no significant differences with decoloring group (P>0.05)。②compared with control group, herbs Without decoloring, Radix Polygoni Multiflori,Malaytea Scurfpea Fruit, Salvia Miltiorrhiza, Millettia Dielsiana,Peachseed can promote significantly melanoma cell proliferation (P≤0.05,P≤0.01with significant difference),Dahurian Angelicaroot、Bletilla、White Atractylodes inhibit melanoma cell proliferation(P<0.01, with significant difference), and the herbs after decoloring, Salvia Miltiorrhiza, Millettia Dielsiana can’t promote melanoma cell proliferation (P>0.05), activation function of Bletilla、White Atractylodes Malaytea Scurfpea Fruit reduce, there are significant differences with decoloring group(P<0.05, P<0.01with significant difference). Peachseed、Herba Ecliptae,、Dahurian Angelicaroot、 White Atractylodes there are no significant differences with decoloring group (P>0.05)。③herbs Without decoloring,Malaytea Scurfpea Fruit.Salvia Miltiorrhiza, Millettia Dielsiana, Peachseed can promote significantly melanocytes synthesis (P<0.01,with significant difference),White Atractylodes、Bletilla can inhibit significantly melanocytes synthesis(P<0.05, with significant difference) compared with control group; and the herbs after decoloring, Millettia Dielsiana,Peachseed can’t promote melanocytes synthesis (P>0.05),activation function of Salvia Miltiorrhiza, Malaytea Scurfpea Fruit reduce, there are significant differences with decoloring group(P<0.05, P<0.01with significant difference). Herba Ecliptae, Tuckahoe can promote significantly melanocytes synthesis (P<0.01,with significant difference), Dahurian Angelicaroot after decoloring inhibit significantly melanocytes synthesis (P<0.01,with significant difference)with before decoloring.Radix Polygoni Multiflori,Bletilla、White Atractylodes there are no significant differences with decoloring group (P>0.05)。2、the results of medicated serum with compound Chinese drugs adjust melanin metabolization①Between the comparison of medicated serum with compound Chinese drugs in the same concentration, the Prescription Ⅰ,Ⅱ,Ⅲ medicated serum in five percent concentration couldn’t promote tyrosinase activity,there was no statistical significance compared with blank control group (p>0.05). the promotional action on tyrosinase activity of prescription Ⅵ was more obvious than other prescription,(P=0.014<0.05, with significant difference), there was no statistical significance between Prescription Ⅵ and PrescriptionⅢ (P=0.233>0.05); there was no statistical significance between prescriptions in ten percent concentration (P=0.124>0.05) Prescription Ⅰ,Ⅲ,Ⅵ in high concentration increased the activity of tyrosinase significantly(P<0.05, P<0.01with significant difference). The promotional action on tyrosinase activity of prescription VI was more obvious than other prescription, the difference has statistics significance (P<0.001,with significant difference).Between the comparison of concentration in the same medicated serum with compound Chinese drugs, The promotional action on tyrosinase activity of medicated serum was more obvious along with the increase of concentration, the comparison in Prescription Ⅰ, there was no statistical significance between twenty percent concentration and thirty percent medicated serum concentration (P=0.203>0.05) there was statistical significance compared with five percent and ten percent medicated serum concentration; the comparison of concentration in Prescription Ⅱ; the comparison in Prescription Ⅱ, the promotional action on tyrosinase activity of five percent, twenty percent, thirty percent was more obvious than ten percent medicated serum concentration(P<0.05,P<0.01with significant difference), there was no statistical significance among the three concentrations, there was statistical significance in each concentration in Prescription Ⅲ,Ⅵ(P<0.01, with significant difference),The promotional action on tyrosinase activity of medicated serum was more obvious along with the increase of concentration.②There was no statistical significance between prescriptions in five and ten percent concentration with blank control group (P>0.05), there was no statistical significance among the prescriptions in promoting melanoma cell proliferation. Prescription Ⅰ,Ⅱ,Ⅲ,Ⅵ in twenty concentration can increased the melanoma cell proliferation significantly,the promotional action on melanoma cell proliferation of prescription Ⅵ was more obvious than other prescription, the difference has statistics significance (P<0.001) prescription Ⅰ,Ⅲ,Ⅵin thirty concentration increased the melanoma cell proliferation significantly(P<0.05,P<0.01with significant difference). PrescriptionⅡ has statistical significance compared with prescription Ⅵ (P<0.01, with significant difference).Between the comparison of concentration in the same medicated serum with compound Chinese drugs, the promotional action on melanoma cell proliferation of medicated serum was more obvious along with the increase of concentration, the comparison in Prescription Ⅰ, there was no statistical significance in promoting melanoma cell proliferation.between twenty percent concentration and thirty percent medicated serum concentration (P=0.831>0.05), there was statistical significance compared with five percent and ten percent medicated serum concentration; the comparison of concentration in Prescription Ⅱ; the comparison in Prescription Ⅱ, the promotional action on melanoma cell proliferation of ten percent, twenty percent, thirty percent was more obvious than five percent medicated serum concentration(P<0.05, P<0.01with significant difference), the promotional action on melanoma cell proliferation of twenty percent was more obvious than other concentrations(P<0.01, with significant difference);there was statistical significance in each concentration of Prescription Ⅲ,Ⅵ(P<0.01, with significant difference),the promotional action on melanoma cell proliferation of medicated serum was more obvious with other concentrations. ③There was statistical significance in promoting melanocytes synthesis compared with blank control group in Prescriptions (P<0.001,with significant difference). The promotional action on melanocytes synthesis of prescription VI was more obvious than other prescriptions. there are no significant differences of Prescription Ⅰ, Ⅱ,Ⅲ (P>0.05).the promotional action on melanocytes synthesis of medicated serum was more obvious along with the increase of concentration, the comparison in each Prescription there was statistical significance in promoting melanocytes synthesis. there was no statistical significance between twenty percent and thirty percent medicated serum concentration (P=0.831>0.05), there was statistical significance compared with five percent and ten percent medicated serum concentration.The full text of the conclusions1. after decoloring, the most Black (depth) herbs and White (shallow) herbs lost or reduce the function on melanin metabolization, Black (depth) herbs was more obvious than White (shallow) herbs. there are certain correlation between herbs color pigment metabolism.2. The prescription Ⅵ with effect of dispel wind and eliminate dampness, nourishing the blood and promoting blood circulation and tonifying liver and kidney, the promotional action on tyrosinase activity was more obvious compared with prescription of Ⅰ, Ⅱ,Ⅲ in20%concentration.