Effect Of Ethanol Extracts From Traditional Chinese Medicines On The MRNA Level Of Guinea Pig's Tyrosinase

Background and Objective. Vitiligo is a skin disorder of depigmentation , a therapeutic effect on which may be obtained by promotion of melanogenesis. Tyrosinase is the key enzyme in melanin biosynthesis. In this study , we selected seven traditional Chinese drugs including Herba Ecliptae, reportedly effective in activating tyrosinase in vitro, to be investigated and used brownish guinea pigs as a experimental model. In situ hybridization(ISH) and reverse transcripitional-polymerase chain reaction(RT-PCR) were performed to explore the effects of traditional Chinese drugs on the level of tyrosinase mRNA. Dopa-oxygenase staining and Schmorl staining were used to observe the impact of these drugs on melanogenesis and proliferation of melanocytes. The aim is to elucidate the mechanism of melanogenesis of these Chinese traditional medicines.Materials and methods: Traditional Chinese medicines(TCM): Herba Ecliptae .Spica Prunellae.Caulis Spatholobi,Frwcfus Tribuli terrestris, Herba Menthae, Radix Glehniae.Fructus Psoraleae. These TCM were made into ethanol extracts. Brownish guinea pigs were used as a model. The sequence of guinea pigs mRNA ISH probe:l-acctgacttt agcaaaacat accatcagtg, 2-gccaaatgaa gaatggatca actcccatgt, 3-tctggatgca ttactatgtg tcaagagata. The sequence of RT-PCR primers: mice tyrosinase: 5'-TTCAAAGGGG TGGATGACCG-3',5'-GACACATAGT AATGCATCC-3'(319bp). Guinea pigs skin were made into frozen section after TCM applied topically for four weeks. Experiments were performed as follow:1 mRNA in situ hybridization: According to Genebank guinea pig's tyrosinase cDNA sequence ,the ISH probes were designed and labeled by DIG. The results of ISH were described as the number of positive cell per 100 basal layer cells and the grey scale of the hybridization signals. 2. RT-PCR: Total RNA was isolated by using TRIZOL agent. RT-PCR wrer performed according to the RNA PCR Kit(AMV)Ver.2.1 manufacturer's instruction. RT-PCRproducts were analysed on an ethidium bromide-stained gel(1.5%).3. Schmorl stain and dopa-oxygenase stain were performed .to observe the number of positive cell per 100 basal layer cells.Results :In mRNA ISH, the results suggest that these seven drugs, especially Spica Prunellae, Herba Ecliptae, Fructus Psoraleae can significantly increase the mRNA level of tyrosinase(P<0.01). Simular results were derived from the RT-PCR experiment. In schmorl stain and dopa-oxygenase stain, the number of cells containing melanin granules and dopa positive cells per 100 basal layer cells were significantly increased in TCM treated groups(P <0.01,<0.05). As for some TCM, these three effects are not absolutely parallel.Conclusion. The results of our study suggest that traditional Chinese medicines including Herba Ecliptae can increase the mRNA level of tyrosinase ,promote the proliferation of melanocytes and enhance the melanogenesis.