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目的基于非动物测试理念,研究植物抗氧化剂复配后的皮肤抗老化功效。方法体外培养HaCaT角质细胞,紫外线照射诱发氧化损伤模型,分别加入白藜芦醇、原花青素和二者复配物,用MTT比色法、荧光素标记法和ELISA法检测细胞活性、活性氧簇自由基(ROS)、超氧化物岐化酶(SOD)、细胞周期及细胞凋亡的变化。对含两种植物原料的眼霜配方进行鸡胚尿囊膜血管试验(CAMVA)和牛角膜混浊渗透试验(BCOP)的组合测试评价其眼刺激性。选择平均年龄40岁的女性志愿者25名,眼部连续使用该眼霜60 d,于使用产品的第0、30和60 d分别检测皮肤的水份、水份流失、弹性和肤色等皮肤老化相关指标。结果白藜芦醇-原花青素复配的植物抗氧化剂可明显缓解紫外线造成的HaCaT细胞氧化损伤,SOD水平明显升高(P<0.001),ROS荧光强度、细胞凋亡率及S期细胞比例均明显降低(P<0.001),细胞修复作用强于单独作用。经过替代方法组合评价其安全性后进行临床志愿者测试,证明皮肤保湿、弹性和皮肤颜色等指标得到改善。结论白藜芦醇和原花青素两种植物抗氧化剂复配使用可有效减少皮肤氧化应激和明显改善皮肤性能。
Objective Based on the concept of non-animal testing, the anti-aging effect of the skin after the compounding of plant antioxidants was studied. Methods HaCaT keratinocytes were cultured in vitro and oxidative damage was induced by UV irradiation. Resveratrol, proanthocyanidins and their complexes were added respectively. Cell viability was measured by MTT assay, fluorescein labeling and ELISA. (ROS), superoxide dismutase (SOD), cell cycle and apoptosis. Eye irritation with two plant material formulations was tested for eye irritancy by a combined test of chick embryo allantoic vascular (CAMVA) and bovine corneal opacification (BCOP) tests. Twenty-five female volunteers aged 40 years were selected. The eyes were continuously treated with the eye cream for 60 days. The skin was tested for moisture, water loss, elasticity and skin color on days 0, 30 and 60, respectively index. Results Resveratrol-proanthocyanidin combined with plant antioxidants could significantly reduce the oxidative damage of HaCaT cells induced by ultraviolet light and the level of SOD was significantly increased (P <0.001). The ROS fluorescence intensity, apoptosis rate and the proportion of S phase cells were significantly increased Decreased (P <0.001), cell repair stronger than alone. After an alternative method to assess the safety of the combination of clinical volunteers tested to prove skin moisture, elasticity and skin color and other indicators have been improved. Conclusions Resveratrol and proanthocyanidin can effectively reduce skin oxidative stress and improve skin performance when combined with antioxidants.