Enzymatic Synthesis And Biological Activity Of Acylated Arbutin

Arbutin,namely p-hydroxyphenyl-?-D-glucopyranoside,is a well-known tyrosinase inhibitor and has been widely used as a cosmetic whitening agent.It also plays an important role in scavenging free radicals,diuresis,antibacterial,antiphlogosis,antibechic and as an anticancer drug.However,the application of arbutin has been strongly limited by its poor cell membrane penetration.In order to overcome the shortcoming,many reports have focused on the regioselective acylation of arbutin.Enzymatic synthesis is more efficient and highly selective compared to the chemical approach.It's generally believed that acylation of arbutin can enhance both of its solubility and its stability in various media.Its biological activity can also be adjusted by the acylation.The regioselective acylation of arbutin catalyzed by lipase from Candida sp.(CSL)was successfully conducted in non-aqueous media.The effects of enzyme origin,acyl donor,organic solvent,water activity,temperature and enzyme dosage were investigated.Under the optimum conditions,the highest enzyme activity(3.71±0.13 ?mol/h/mg)could be obtained and the regioselectivity of the CSL-catalyzed acylation was highly specific at the C-6? position in the glucose moiety of arbutin(>99%).The maximum conversion could reach 91.42±2.43% after 24 h.The process still required long time to obtain satisfied enzyme activity.Microwave irradiation is a rapid developing technology in green chemistry and widely used in the enzyme catalytic reaction.In the current work,microwave was applied to improve the enzyme activity.The effects of various parameters on the enzyme activity has been investigated.The experimental results showed that microwave radiation can significantly improve the enzyme activity.Under the optimum conditions(microwave power,400 W;microwave temperature,60? and aw of 0.41),the enzyme activity can reached 15.68±0.15 ?mol/h/mg,which increased 4 times compared to traditional reaction.More than 90% of the conversion could be obtained when the reation was carried out for about 2 h.Antioxidant properties of arbutin and 6?-acetylated arbutin were evaluated on the basis of measuring scavenging activity for DPPH radicals,ABTS radicals cations,super oxygen free radicals,iron ion chelating ability and reducing total antioxidant capacity.The results showed that arbutin and 6?-acetylated arbutin have shown stronger free radical scavenging activity compared to Vc and BHT.However,6?-acetylated arbutin was weaker antioxidant than arbutin in vitro.The objectives of this study were to determine the effects of arbutin and acetylated arbutin on antioxidant in vivo using Caenorhabditis elegans as a model.Our findings indicated that,in C.elegans,6?-acetylated arbutin stronger slow aging,extend lifespan and enhance resistance to stress than arbutin.The mean lifespan of animals treated with 5.4 mM 6?-acetylated arbutin increased significantly in a dose-dependent manner compared with the arbutin(p<0.01).In addition,animals pre-treated with 6?-acetylated arbutin were more resistant to stresses such as heat,juglone and UV radiation,suggesting that cellular defense and immune system functions were also improved.Furthermore,semi-quantitative RT-PCR was used to analyze the genes expression of associated with aging.The results suggested that acetylated arbutin significantly extended the lifespan of C.elegans through up-regulating some genes' expression such as daf-16,skn-1,hsp-16.2,hsf-1 and hsp-70 which would provide important reference for anti-aging research of 6?-acetylated arbutin.In the present study,6?-acetylated arbutin was prepared in order to improve the biological effects of arbutin,and we investigated the effects of 6?-acetylated arbutin on melanin synthesis,tyrosinase activity and pro-apoptotic effects on B16 murine melanoma cells.Data revealed that 6?-acetylated arbutin significantly inhibited the biosynthesis of melanin and tyrosinase activity compared with parent arbutin in B16 murine melanoma cells.In addtion,the 6?-acetylated arbutin strongly inhibited the proliferation of B16 murine melanoma cells in a time-and dose-dependent manner.Both of them promoted cell apoptosis,caused G1 phase arrest in cell cycle,and induced mitochondria disruption in B16 murine melanoma cells.Furthermore,the reduced expressions of B-cell lymphoma-extra large(Bcl-xL)and B-cell lymphoma 2(Bcl-2)were observed in arbutin and 6?-acetylated arbutin-treated cells.Moreover,a detailed mechanistic analysis revealed that the cell apoptosis was mainly caused by the activation of the mitochondria-mediated pathway.Besides the anti-proliferative effects mediated,the wound healing assay and transwell migration assay was performed to check whether the 6?-acetylated arbutin could inhibit the cell migration.Compared with the control,obvious inhibition in cell migration was observed,indicating the inhibition of cell migration could be induced by both the arbutin and its acetylated derivative.All data suggested that the 6?-acetylated arbutin showed pro-apoptotic activities in B16 murine melanoma cells related to mitochondrial pathway.In sum,the 6?-acetylated arbutin could be used as a potential candidate not only for skin whitening,but also for melanoma treatment.