The Effects Of Andrographolide On Melanin Synthesis And Its Mechanism

The ozone hole forms and is enlarged, which results from the environmental pollution, leading to more ultraviolet radiation reaching the ground and human beings accepted it forcefully. Ultraviolet radiation can accelerate melanin synthesis in melanocyte inducing hyperpigmentation disorders, such as sunburn, resulting in negative effects on psychology. But the normal whiting agents, such as kojic acid and arbutin, have their own disadvantages. As a result, it is feasible to search for new whiting agents which can suppress melanin synthesis and have enormous potential applied to cosmetic.In this study, the murine B16F10 melanoma cells were used as cell model to screen whiting agents suppressing melanin synthesis. Then tyrosinase (TYR) activity, the transcriptional and protein level of TYR family and microphthalmia-associated transcription factor (MITF) and protein content of signal molecular involved in melanin synthesis was measured, we found that andrographolide inhibited melanin content resulting from inhibiting the phosphorylation of glycogen synthase kinase 3P(GSK3β) depending on Akt, accerlating the degradation of β-catenin, inhibiting the translocation of β-catenin to the nucleus, decreasing transcriptional and protein expression of MITF and TYR family, decreasing TYR activity and inhibiting melanin synthesis. The effects of andrographolide on melanin synthesis in human epidermis melanocyte (HEM) and brown guinea pigs induced by UVB irradiation was tested.All content is made up of four parts.The first part is "screening of melanin inhibitors".B16F10 cells were maintained in phenol red-free DMEM with materials using kojic acid as a positive material. When melanin released from B16F10 cells, the supernatant was measured at OD490nm to screen a probable whiting agent. Then a potential whiting agent named andrographolide was sought out.The second part is "the effects of andrographolide on melanin synthesis in vitro".The effects of andrographolide on melanin content in B16F10 and HEM cells were measured after the viability of andrographolide on B16F10 and HEM cells was tested using MTS. Data indicated that andrographolide decreased melanin content in B16F10 cells and HEM cells concentration in a dose dependent manner without marked viability on these two kinds of cells. The effects of andrographolide on TYR activity from B16F10 and HEM cells and tyrosinase from mushroom were measured and data showed that andrographolide could decrease TYR activity in cells resulting from lowering TYR content. Then transcriptional and protein content of TYR family and MITF was measured, which showed that andrographolide decreased transcriptional and protein expression of TYR family by inhibiting the transcription of MITF.The third part is "the effects of andrographolide on melanin synthesis in vivo".The hair in guinea pigs’back was shaved off using infant clipper and electric shaver to expose four same areas in size (2cm*2cm). Then guinea pigs were exposed to UVB (2J/cm2) and daubed with cream twice per day. When the color of four areas seemed different, the guinea pigs were taken photos and their skin tissues were made into skin sections. Darta showed 5%andrographolide reduced hyperpigmentation, melanin content, TYR content and TYR positive cell number in guinea pigs induced by UVB irradiation as 5% kojic acid did. As a result, andrographolide suppresses TYR content, the differentiation of melanocyte and melanin synthesis leading to an inhibiton of hyperpigmentation in brown guinea pigs induced by UVB irradiation.The fourth part is "the mechanism of andrographolide suppressing melanin synthesis".After treated with andrographolide in B16F10 cells, some signal molecular involved in melanin synthesis was tested. We found that total β-catenin content, p-GSK3β and p-Akt decreased after treated with andrographolide. Data in experiments disposed by MG132 or cycloheximide (CHX) demonstrated that andrographolide could accelerate the degradation of β-catenin. Data also showed that GSK3β inhibitor and Akt activator could reserve the decline of β-catenin content induced by andrographolide.Consequently, andrographolide can decrease the phosphorylation of GSK3β depending on Akt-dependent, increasing the degradation speed of β-catenin, inhibiting the translocation of β-catenin into the nucleus resulting in a decline to MITF.