“四白”中药复方醇提液的安全性及护肤作用研究

Research of Biosafety and Skin-Care Efficacy on Alcoholic Extract of Chinese Herbal Compound“Four Whites”

目的研究白僵蚕、白蔹、白术及白芍(“四白”)中药复方醇提液的安全性及护肤(美白、抗氧化、抗炎)功效。方法采用红细胞溶血实验及MTT法检测细胞存活率,评估“四白”中药复方醇提液的眼刺激性及细胞安全性;通过羟自由基、1,1-二苯基-2-三硝基苯肼(DPPH)自由基清除实验、铁离子(Fe^(3+))还原实验和抗过氧化氢(H2O2)氧化损伤实验评估“四白”中药复方醇提液的抗氧化能力;通过酪氨酸酶活性抑制实验评估“四白”中药复方醇提液的美白功效;通过透明质酸酶活性抑制实验和采用ELISA法检测脂多糖(LPS)诱导RAW264.7细胞一氧化氮(NO)水平,评价“四白”中药复方醇提液的抗炎功效。结果(1)与0.1%十二烷基硫酸钠(SDS)组比较,30 g·L^(-1)“四白”中药复方醇提液组的红细胞溶血率显著降低(P<0.01)。与空白组比较,“四白”中药复方醇提液(5、10、15、20、25、30 g·L^(-1))不同浓度组的HaCaT、B16及RAW264.7细胞活力均显著降低(P<0.05,P<0.01);烟酰胺对B16细胞的IC50值为(26.3±1.3)g·L^(-1),低于“四白”中药复方醇提液对B16细胞的IC50值[(33.8±2.7)g·L^(-1)]。(2)与空白组比较,“四白”中药复方醇提液(2、4、8、16、32 g·L^(-1))的羟自由基清除率显著升高(P<0.01);“四白”中药复方醇提液(0.25~4 g·L^(-1))的DPPH自由基清除率显著升高(P<0.01);“四白”中药复方醇提液(5、10、15、20、25、30 g·L^(-1))对Fe^(3+)的还原力显著增强(P<0.01);与空白组比较,模型组HaCaT细胞活力显著降低(P<0.01),而“四白”中药复方醇提液(5、10、15、20、25、30 g·L^(-1))预保护后,HaCaT细胞活力有上升趋势,但差异无统计学意义(P>0.05)。(3)随着“四白”中药复方醇提液浓度的升高,酪氨酸酶活性抑制率逐渐升高,在30 g·L^(-1)浓度时,活性抑制率达到92.1%;与空白组比较,“四白”中药复方醇提液(15、20、25、30 g·L^(-1))对B16细胞的酪氨酸酶活性抑制率显著升高(P<0.05,P<0.01)。(4)随着“四白”中药复方醇提液浓度的升高,透明质酸酶活性抑制率逐渐升高;与空白组比较,LPS模型组RAW264.7细胞的NO含量显著增加(P<0.01),而与模型组比较,“四白”中药复方醇提液(5、10、15、20、25 g·L^(-1))不同浓度组RAW264.7细胞的NO含量显著降低(P<0.01)。结论“四白”中药复方醇提液具有较低的刺激性和细胞毒性,且有一定的抗氧化、美白及抗炎功效。

Objective To study the biosafety and skincare(whitening,anti-oxidatidant,anti-inflammatory)effects of the alcoholic extracts of the Chinese herbal compound Bombyx Batryticatus,Ampelopsis Radix,Atractylodis Macrocephalae Rhizoma and Paeoniae Radix Alba(namely,the"four whites")were investigated.MethodsThe cell viability and cell safety of alcoholic extract of"four whites"were assessed by red blood cell haemolysis assay and MTT assay;the antioxidant effect of alcoholic extract of"four whites"was assessed by hydroxyl radical,1,1-diphenyl-2-trinitrophenyl hydrazine(DPPH)radical scavenging assay,iron ion(Fe^(3+))reduction assay and antihydrogen peroxide(H_(2)O_(2))oxidative damage assay.The whitening efficacy of alcoholic extract of"four whites"was evaluated by tyrosinase activity inhibition assay;the anti-inflammatory efficacy of alcoholic extract of"four whites"was evaluated by hyaluronidase activity inhibition assay and ELISA assay to detect the NO level of LPS-induced RAW264.7 cells,and the anti-inflammatory efficacy of the alcoholic extract of"four whites"was evaluated.Results(1)Compared with the 0.1%SDS group,the haemolysis rate of erythrocytes in the 30 g·L^(-1)alcoholic extract of"four whites"group was significantly decreased(P<0.01).Compared with the blank group,the viability of HaCaT,B16 and RAW264.7 cells was significantly decreased(P<0.05,P<0.01)in the groups with different concentrations of alcoholic extract of"four whites"(5,10,15,20,25 and 30 g·L^(-1));the IC_(50)value of nicotinamide on B16 cells was(26.3±1.3)g·L^(-1),which was lower than that of alcoholic extract of"four whites"on B16 cells(33.8±2.7)g·L^(-1).(2)Compared with the blank group,the hydroxyl radical scavenging rate of alcoholic extract of"four whites"(2,4,8,16 and 32 g·L^(-1))was significantly increased(P<0.01);the DPPH radical scavenging rate of alcoholic extract of"four whites"(0.25-4 g·L^(-1))was significantly increased(P<0.01);the scavenging rate of DPPH radicals was significantly increased in the alcoholic extract of"four whites"(5,10,15,20,25 and 30 g·L^(-1));the reducing power of alcoholic extract of"four whites"against Fe^(3+)was significantly enhanced(P<0.01);compared with the blank group,the viability of HaCaT cells in the model group was significantly decreased(P<0.01),while HaCaT cell viability tended to increase after pre-protection with alcoholic extract of"four whites"(5,10,15,20,25 and 30 g·L^(-1)),but the difference was not statistically significant(P<0.05).(3)The inhibition rate of tyrosinase activity gradually increased with the increase of the concentration of the alcoholic extract of"four whites".At the concentration of 30 g·L^(-1),the activity inhibition rate reached 92.1%.Compared with the blank group,the inhibition rate of tyrosinase activity in B16 cells was significantly increased by the alcoholic extract of"four whites"(15,20,25,30 g·L^(-1))(P<0.05,P<0.01).(4)With the increase of the concentration of alcoholic extract of"four whites",the inhibition rate of hyaluronidase activity gradually increased;compared with the blank group,the NO content of RAW264.7 cells in the LPS model group was significantly increased,while compared with the model group,the NO content of RAW264.7 cells significantly decreased in different concentration groups of alcoholic extract of"four whites"(5,10,15,20,25 g·L^(-1)).Conclusion alcoholic extract of"four whites"has low irritation and cytotoxicity,and has certain antioxidant,whitening and anti-inflammatory effects.