基于细胞水平评价白及多糖对黑色素生成的抑制作用

The inhibitory effect of Bletilla striata polysaccharide on melanin production based on cellular level

目的研究白及多糖对小鼠黑色素瘤细胞B16-F10中黑色素生成的抑制作用及其机制。方法采用CCK-8法检测白及多糖对B16-F10细胞活性的影响;采用NaOH裂解法测定白及多糖对B16-F10细胞内黑色素含量的影响;采用多巴氧化法和蛋白免疫印迹法(Western blot)分别检测B16-F10细胞中酪氨酸酶(TYR)活性和酪氨酸酶相关蛋白1(TRP-1)、酪氨酸酶相关蛋白2(TRP-2)的表达情况,并利用分光光度仪检测白及多糖的抗氧化能力。结果白及多糖在0.1~1.0 mg/mL浓度内对B16-F10细胞活性无影响。用0.5和1.0 mg/mL白及多糖处理B16-F10细胞,可降低B16-F10细胞内黑色素含量。1.0 mg/mL白及多糖可显著抑制α-MSH诱导的B16-F10细胞中TYR活性;0.5和1.0 mg/mL白及多糖可显著抑制α-MSH诱导的B16-F10细胞中TRP-1、TRP-2的蛋白表达。白及多糖在0.1~1.0 mg/mL浓度内,DPPH平均清除率为18.85%、27.10%、47.92%、60.65%,抗超氧阴离子平均活力为20.94、22.41、30.68、38.86 U/gprot,羟基自由基平均抑制能力为7.39、8.07、18.84、34.37 U/mgprot。结论白及多糖对B16-F10细胞中黑色素的生成具有良好的抑制作用,其机制可能与降低细胞内黑色素含量、抑制TYR活性和TRP-1、TRP-2的蛋白表达以及抗氧化作用有关。

Objective This research aimed to investigate the melanin production inhibition and its mechanism of Bletilla striata polysaccharide on melanoma cells(B16-F10).Methods CCK-8 was used to detect the effect of Bletilla striata polysaccharide on the activity of B16-F10 cells.NaOH cracking method was used to determine the effect of Bletilla striata polysaccharide on melanin content in B16-F10 cells.Dopa oxidation method and Western blot were used to determine the effects of Bletilla striata polysaccharide onα-MSH induced the activity of tyrosinase(TYR)and the expression levels of tyrosinase related protein 1(TRP-1)and tyrosinase related protein 2(TRP-2)in B16-F10 cells,respectively.The antioxidant properties of Bletilla striata polysaccharide were detected by spectrophotometry.Results The results showed that Bletilla striata polysaccharide did not affected the ac tivity of B16-F10 cells with the concentration range of 0.1~1.0 mg/mL.B16-F10 cells treated with 0.5 and 1.0 mg/mL Bletilla striata polysaccharide for 24 hours could reduce the content of intracellular melanin.1.0 mg/mL Bletilla striata polysaccharide could significantly inhibit the activity of TYR thatα-MSH induced in B16-F10 cells.And the 0.5 and 1.0 mg/mL Bletilla striata polysaccharide could significantly inhibit the expression levels of TRP-1,TRP-2 thatα-MSH induced in B16-F10 cells.The average clearance rate of DPPH are 18.85%,27.10%,47.92%,60.65%,the average activity of anti superoxide anion are 20.94,22.41,30.68,38.86 U/gprot,the average inhibition capacity of hydroxyl radical are 7.39,8.07,18.84,34.37 U/mgprot with the concentration range of 0.1~1.0 mg/mL.Conclusion Bletilla striata polysaccharide had a good inhibitory effect on melanin production,and its mechanism may be related to the content reduction of intracellular melanin,the inhibition of TYR activity,the decrease of the TRP-1 and TRP-2 expression levels and antioxidation capacity.