| 1.Background Poliosis is a common hair pathological lesion,affecting the patient's appearance and mental health.Genetic factors,mental factors and immune disorders will lead to the occurrence of the disease,but the mechanism is not very clear.Recorded in the seventeenth volumes of traditional Chinese medicine prescription preparation,Shengfa tablets consist of fleece-flower root,fructus ligustri lucidi,rehmannia glutinosa,ink dry lotus,mulberries,yam,cortex moutan,rhizoma alismatis,poria cocos,dwarf lilyturf,dateplum persimmon and black beans.Nourishing liver and kidney and tonifying blood,Shengfa tablets have the effect of ufa and rising hair.The prescription is used in poliosis,alopecia areata,all bald,and seborrheic alopecia caused by liver and kidney deficiency,blood deficiency.Shengfa tablets have entered the market with good reputation and curative effect for the treatment of poliosis since1880 s.The original quality standard only has reducing sugar identification,lack of specificity,and the treatment mechanism research is blank.Therefore,in order to expand market,ensure its safety,effectiveness and quality controlling,this research carried out to improve the quality standard of Shengfa tablet,the TLC method was used for the identification of four medicinal materials and two content determination detected by HPLC method.On the basis of ensuring the quality stability,choosing B16 melanoma cells as the model,promoting melanin synthesis effect and the mechanism of the prescription were discussed,which provide reliable experimental data support for clinical application.2.Purpose Having improved Shengfa tablets quality standard and researched the role of melanin synthesis and mechanism for the first time,which ensure the safety,effectiveness and quality control,to provide reference for the marketing and clinical application.3.Methods3.1 The improvement of Shengfa tablets quality standard.(1)Established TLC method to identify the fleece-flower root,ink dry lotus,dwarf lilyturf,and radix rehmanniae through the selections of preparation method of sample,developing agent and absorption spectroscopic conditions.(2)Establish HPLC method for content determination of Stilbene glycosides and Specnuezhenide through the selection of preparation method of sample and chromatographic condition,which were tested by methodology validation.3.2 The effect of Shengfa tablets on melanin synthesis.(1)Different concentrations of Shengfa tablets water extraction liquid acted on B16 melanoma cells for 24 h,48 h,respectively.The cell proliferation was determined by MTT method,to screen the appropriate concentration.(2)The activation of tyrosinase was detected by dopa oxidation method,melanin content was detected by sodium hydroxide pyrolysis method after acted for 48 h.The results showed that promoting melanin synthesis effect of Shengfa tablet is obvious.3.3 The influence of Shengfa tablets on MITF and TYR protein expression in B16 melanoma cells.To research the effect deeper,different concentrations of Shengfa tablets water extraction liquid acted on B16 melanoma cells for 24 h,MITF and TYR protein expression were detected by Western blot. 3.4 The study on melanin synthesis mechanism of Shengfa tablets.(1)To research further,different concentrations of Shengfa tablets water extraction liquid acted on B16 melanoma cells for 30 min,p-CREB protein expression were detected by Western blot.(2)The PKA signaling pathway inhibitor H89·2HCl pretreated cells for 1 h to block PKA pathway,then treated the cells with drug,to detect the tyrosinase activity and content of melanin synthesis.(3)The PKA signaling pathway inhibitor H89·2HCl pretreated cells for 1 h to block PKA pathway,then treated the cells with drug,to detect MITF,TYR and p-CREB protein expression with Western blot technique.The results further illustrated Shengfa tablet's promoting melanin synthesis function is related to PKA signal pathway.4.Results4.1 The improvement of Shengfa tablets quality standard.(1)Based on the preparation of test sample,selection of chromatographic conditions,strong specificity TLC methods were established to identify the traditional Chinese medicine fleece-flower root,the ink dry lotus,radix ophiopogonis,radix rehmanniae.Results showed that the samples and reference substance or reference crude drug had the same spots in the corresponding position,the spots were clear and negative control had no interference.This established TLC identification method has advantages of easy operation,stable result,etc.(2)The linear range of Specnuezhenide was 8.16~122.40 mg/m L(R2=0.9998),and the average recovery was 100.77% with RSD of 1.12%(n=6).The linear range of Stilbene glycosides was 2.43~46.80 mg/m L(R2=0.9994),and the average recovery was 100.4% with RSD of 0.94%(n=6).The method is simple and accurate for the quality control of Shengfa Tablets.4.2 The effect of Shengfa tablets on melanin synthesis.(1)The MTT assays data showed that 0.25~1 mg/m L Shengfa tablet water extract had no obvious effect on the proliferation of B16 cells,2~4 mg/m L inhibits cells proliferation significantly,P<0.05.Based on that,choose 0.25,0.5,1 mg/m L as the following concentration.(2)Compared with control group,the activity of tyrosinase and the synthesis of melanin were obviously promoted in low concentration group,P<0.05.The results were significant in medium and high concentration group,P<0.01.4.3 The influence of Shengfa tablets on MITF and TYR protein expression in B16 melanoma cells.The results showed that,compared with control group,MITF and TYR protein expression increased in 0.25 mg/m L,P<0.05,and significantly raised in 0.5 and 1mg/m L,P<0.01.It proved that Shengfa tablet can increase the expression of protein related melanin synthesis.4.4 The study on melanin synthesis mechanism of Shengfa tablets.(1)Gray value data showed that Shengfa tablets water extract could upregulate concentration-dependently expression of p-CREB protein compared with the control group in B16 cells(P<0.05,P<0.01).(2)H89·2HCl pretreatment group can significantly reduced the tyrosinase activity,melanin synthesis,P<0.01.(3)The Western blot assays showed that MITF,TYR and p-CREB protein expression in H89·2HCl pretreatment group were significantly less than dosing group,P<0.01.5.Conclusions5.1 Established Shengfa tablets TLC identification methods and HPLC quantitative methods for the first time.It provides the basis for the quality control of the products,and ensures the stability.5.2 The water extract of Shengfa tablets can activate tyrosinase activity in B16 melanoma cells and promote melanogenesis.5.3 The effect of Shengfa tablets on melanogenesis is through the up-regulation of the expression of MITF and TYR protein.5.4 The study results showed that the promoting melanin synthesis mechanism of Shengfa tablets is through up-regulation the phosphorylation protein p-CREB in PKA pathway which can further increase the MITF protein expression,thereby activates tyrosinase gene family, promots the synthesis of melanin ultimately.6.Innovation points6.1 Improved Shengfa tablet quality standard for the first time.6.2 Studied the promoting melanin synthesis effect and mechanism of Shengfa tablet for the first time.6.3 Based on the PKA signal pathway to study melanin synthesis mechanism of Shengfa tablet,the regulation process may be PKA-p-CREB-MITF-TYR-melanin. |
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