猕猴桃皮多酚提取及其对B16细胞黑色素的影响

Extraction of the polyphenols from kiwifruit peel and its effect on melanin in B16 cells

植物多酚类物质对皮肤增白和美容有着重要的作用,是美容护肤保健品中的主要成分,猕猴桃皮中多酚类物质含量丰富,可成为美容保健品的原料。为了探究猕猴桃皮中多酚类物质的提取及其美白作用,该研究采用深共熔溶剂(Deep-Eutectic Solvents,DESs)提取猕猴桃皮多酚,通过单因素试验和响应面试验确定优化提取工艺,用高效液相色谱分析猕猴桃皮多酚的成分,并研究猕猴桃皮多酚对酪氨酸酶的影响及对B16细胞黑色素的作用机制。结果表明:深共熔溶剂是提取猕猴桃皮多酚的较佳溶剂,提取猕猴桃皮多酚优化工艺为DES-1含水率18%、料液比1:29g/m L、提取时间22 min、提取温度69℃,猕猴桃皮多酚得率55.53 mg/g。猕猴桃皮多酚中的主要成分有(+)-儿茶素、绿原酸、咖啡酸和表儿茶素,并且猕猴桃皮多酚对体外酪氨酸酶活性有抑制作用。此外,当猕猴桃皮多酚浓度为100μg/m L时对B16细胞内黑色素生成有明显的抑制作用,能使细胞内黑色素相对含量降为75.08%,细胞内酪氨酸酶活性降至69.45%,且优于阳性对照组曲酸(P<0.05)。结果表明,猕猴桃皮多酚可作为一种天然美白剂应用于化妆品及其他保健品中。

Polyphenols are the main ingredients to whiten and beautify the human skin in beauty and health care products.Kiwifruit peel rich in polyphenols can be used as raw materials.This study aims to explore the extraction and whitening effect of polyphenols from the kiwifruit peel using Deep-Eutectic Solvents(DESs).A systematic investigation was made on the effects of DESs types,DES water contents,material-to-liquid ratio,extraction time,and temperature on the yield of total kiwifruit peel polyphenols.A Response Surface Method(RSM) experiment was combined to determine the best extraction.High-Performance Liquid Chromatography(HPLC) was selected to identify the composition of kiwifruit peel polyphenols.The inhibitory effect of kiwifruit peel polyphenols on the mushroom tyrosinase activity in vitro was evaluated,where the Kojic acid was taken as the positive control,while the L-tyrosine was used as the substrate.Besides,a model of melanin expression induced by α-melanocyte-stimulating hormone(α-MSH) was constructed to explore the action mechanism of kiwifruit peel polyphenols on the melanin in B16 cells.The results showed that the highest yield of kiwifruit peel polyphenols was achieved when the DESs solvent was assisted by ultrasonic extraction,where the molar ratio of choline chloride-ethylene glycol was 1:2.The best process parameters were as follows.The water content of DES-1 was 18%,the material-to-liquid ratio was 1:29 mg/L,the extraction time was 22 min,and the extraction temperature was 69 ℃.As such,the yield of kiwifruit peel polyphenols was(55.53±1.27) mg/g under the optimal conditions.10 kinds of polyphenol compounds were detected in the kiwifruit peel,including the gallic acid,protocatechin,(+)-catechin,chlorogenic acid,caffeic acid,epicatechin,paracoumaric acid,ferulic acid,rutin,and phloridin.The main components of kiwifruit peel polyphenols were(+)-catechin,chlorogenic acid,caffeic acid,and epicatechin,whose contents were 32.50,46.40,26.90,and 68.70 mg/L,respectively.The kiwifruit peel polyphenols presented an excellent inhibitory effect on the mushroom tyrosinase.The inhibition rate also gradually increased,as the mass concentration increased.When the mass concentration was 0.8 mg/mL,the inhibition rate reached 67.19%,which was lower than that of kojic acid at the same mass concentration(P<0.05).Compared with the blank group,the melanin content in B16cells increased significantly after α-MSH induced,indicating that the α-MSH induced melanin expression model was successfully constructed.The amount of melanin production was set as 100% in the model group.The relative content of melanin in the B16 cells decreased gradually in a dose-dependent manner,as the concentration increased within the range of kiwifruit peel polyphenols.The relative content of melanin decreased to 75.08% in the treated B16 cells with 100 μg/m L kiwifruit peel polyphenols.Both kiwifruit peel polyphenols and positive control group exhibited an inhibitory effect on the tyrosinase activity in the B16 cells,compared with the model group.The kiwifruit peel polyphenols greatly inhibited the intracellular tyrosinase activity in a dose-dependent manner within the mass concentration range in the experiment.In the kiwifruit peel polyphenols,the relative activity of tyrosinase decreased to 69.45% in the treated B16 cells with 100 μg/m L.These findings can provide the potential application of kiwifruit peel polyphenols as a natural whitening agent in cosmetic and health care products.