南瓜中游离叶黄素的制备及抗氧化活性

Manufacture Process and Antioxidant Activity of Free Lutein from Cucurbita moschata Duch.

摘要:

为了探究南瓜提取物中叶黄素酯的皂化工艺条件和其体外抗氧化能力，在单因素试验基础上，以皂化液浓度、皂化温度和皂化时间为响应因子，采用Box-Behnken中心组合实验原理进行三因素三水平设计，优化南瓜叶黄素的皂化工艺；利用高效液相色谱（HPLC）和质谱（MS）分析检测皂化产物；经硅胶柱色谱分离得到纯化的南瓜叶黄素；采用分光光度法，通过比较DPPH·和·OH的清除能力评价南瓜中游离叶黄素的体外抗氧化活性。结果表明，南瓜提取物中叶黄素酯的最佳皂化工艺条件为皂化液质量浓度33 g/dL，温度33 ℃，时间5.5 h。在最优工艺条件下，南瓜提取物中游离叶黄素得率为0.4175%，与预测值相近，说明该优化工艺稳定可行。高效液相色谱（HPLC）和质谱（MS）分析检测表明皂化产物为叶黄素。硅胶柱色谱纯化后，游离叶黄素的纯度可提高到91.39%。南瓜中游离叶黄素清除DPPH·和·OH的半抑制质量浓度IC₅₀分别为0.021 4 mg/mL和0.038 3 mg/mL，抗氧化活性较高，可作为一种良好的天然抗氧化剂。

Abstract:

The saponification conditions of the extract from Cucurbita moschata Duch. was optimized and its antioxidant activity in vitro was evaluated. On the basis of single factor experiment, the saponification solution concentration, saponification temperature and saponification time were optimized as the response factors. The Box-Behnken Center combination experiment principle was carried out by the three-factor and three-level design to optimize the saponification process of Cucurbita moschata Duch. lutein. After saponification, high performance liquid chromatography (HPLC) and mass spectrometry (MS) analysis were used to detect the purified Cucurbita moschata Duch. lutein, which was separated by silica gel column chromatography. The free lutein in Cucurbita moschata Duch. was evaluated by spectrophotometry comparing the scavenging ability of DPPH· and ·OH in vitro antioxidant activity. The optimum saponification condition for lutein ester in Cucurbita moschata Duch. extract was set as 33% saponification at 33 ℃ for 5.5 h. Under the optimal conditions, the yield of free lutein in Cucurbita moschata Duch. extract was 0.4175%, which was similar to the predicted value, revealing that the optimization process was stable and feasible. HPLC and MS analysis showed that the saponified product was lutein. After purification by silica gel column chromatography, the purity of free lutein significantly increased to 91.39%. The half-inhibitory concentration IC₅₀ of DPPH· and ·OH in free lutein in Cucurbita moschata Duch. is 0.021 4 mg/mL and 0.038 3 mg/mL, respectively. It has high antioxidant activity and can be used as a good natural antioxidant.