Inhibitory Effect Of Cashew Nut Shell Oil Extract On Melanin Production At Animal Level And Mechanism Of Inhibiting Melanin Transfer

Moderate amounts of melanin can protect skin cells from ultraviolet radiation,but too much melanin can also lead to excessive skin pigmentation disorder,resulting in chloasma,senile moles,postoperative inflammatory pigmentation and many other skin problems.Skin pigmentation depends not only on the amount of melanosomes produced,but also on the amount of melanosomes transferred to adjacent keratinocytes.Inhibition of melanin production and transfer is of great significance to improve skin pigmentation.In this study,cashew nut shell oil extract(CNCE)was used to verify whether the drug could inhibit the production of melanin.The body surface of zebrafish was recorded by stereoscope at 24 h and 48 h after the addition of the drug;After the fish were lysed,the melanin in the fish body was dissolved with NaOH,and its content was directly determined.The remaining batch of zebrafish was extracted for protein,and the expression of proteins TYR,TRP1,TRP2 and α-MSH that related to melanin production in zebrafish was determined by Western blot.The results showed that with the increase of the concentration of CNCE(110,120,140,180 μg/mL),the melanin content of zebrafish decreased significantly,but not in a dose-dependent manner.Western blot results showed that the expression of DCT,TYR and α-MSH protein in zebrafish were down-regulated.These results suggested that CNCE could inhibit the production of melanin in zebrafish.B16F10 was used as the donor cells of melanin and HaCAT cells were used as the receptor cells.The B16F10-HaC AT co-culture system was used as the model of melanin transfer in vitro and the related studies were carried out.According to the previous research results in our laboratory,CADI,which has a better inhibitory activity on tyrosinase,was selected to further verify the mechanism of melanin transfer at the cellular level,and Arbutin was used as the positive control to observe the comparison of the inhibition effect of the two drugs on melanin transfer.We used flow cytometry to quantitatively detect the number of transferred melanin,and detected the changes in the content of calcium ions in HaCAT cells after the treatment of two drugs.It was found that the melanin transfer volume and intracellular calcium content in HaCAT cells were significantly reduced after treatment with CADI,which was better than that of arbutin control group.Then observed the effects of different drugs on three pathways of melanin transfer.In the phagocytosis related pathway,the expression of PAR-2 protein was detected in this experiment.Fluorescence microspheres were co-cultured with HaCAT,and the change of phagocytosis was observed by confocal microscopy.NMDAR protein was detected in the transfer-related pathway of filopodia,and the changes of filopodia in melanocytes and the contact points of the two types of cells in co-culture were observed by scanning electron microscopy.In the dendritic phagocytosis pathway,Racl and Cdc42 related to dendritic generation were detected by Western blot and qPCR at the protein and mRNA levels,respectively,and dendritic of cells were qualitatively observed by scanning electron microscopy and immunofluorescence staining.The results showed that CADI(20μM)down-regulated the expression of PAR-2,NMDAR,Racl,and Cdc42,and inhibited the production of dendrites and filopodia in melanocytes,which was better than that of Arbutin at the same concentration(20 μM)or even high concentration(110μM).In conclusion,the active ingredients from cashew nut shell oil can significantly inhibit the formation and transfer of melanin,and its essence extract CADI effectively inhibited melanin transfer.As a pure natural extract,cashew nut shell oil extracts have broad application prospects in improving skin pigmentation.