熟地黄多糖的提取及其体外抗氧化活性研究

Extraction and Antioxidative Activity Analysis in Vitro of Shudihuang(Rehmanniae Radix Preparata)Polysaccharides

目的研究熟地黄多糖的最佳提取方法,评估熟地黄多糖的体外抗氧化活性,为进一步的体内活性研究提供依据。方法采用水提醇沉法、微波提取法和超声波提取法提取熟地黄多糖,筛选出得率最高的方法。通过Sevag法除去粗多糖中的蛋白成分,透析法除去小分子,活性炭除色素后,得到精制多糖,苯酚-硫酸比色法测定多糖含量。通过4种不同的抗氧化体系以及细胞水平的实验,检测了多糖的体外抗氧化活性,包括羟自由基、超氧阴离子自由基、1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基的清除效果、总抗氧化能力(Total antioxidant capacity,T-AOC)、对细胞内超氧化物歧化酶(SOD)和谷胱甘肽(GSH-px)活力以及丙二醛(MDA)水平的影响。结果精制的熟地黄多糖(Rehmanniae Radix Preparata polysaccharides,RGP)清除羟自由基EC_(50)值为1.51 mg/mL,清除超氧阴离子自由基EC_(50)值为3.04 mg/mL,清除DPPH自由基的EC_(50)值为0.26 mg/mL,其总抗氧化活性为18.85±0.80 U/mg。与阳性对照2,6-二叔丁基对甲酚(2,6-Di-tert-butyl-4-methylphenol,BHT)相比,熟地黄多糖具有较好的DPPH自由基清除作用、一定的羟自由基和超氧阴离子自由基的清除能力,并且与多糖浓度呈现出剂量效应。而且熟地黄多糖能够有效提高H2O2所导致的SOD和GSH-px活力降低的情况,以及降低H_(2)O_(2)诱导的MDA水平升高的情况。熟地黄多糖还具有很好的总抗氧化能力。结论传统中药熟地黄的多糖成分具有良好的体外抗氧化活性,具有开发为天然抗氧化剂和食品保健品的潜在价值。

Objective This study investigated the optimal extraction method of polysaccharides from Shudihuang(Rehmanniae Radix Preparata),and evaluated the anti-oxidative activity of polysaccharides in vitro,providing the basis for further study of its activities in vivo.Methods Water extraction and alcohol precipitation,microwave extraction and ultrasonic extraction were used to extract Shudihuang(Rehmanniae Radix Preparata)polysaccharides(RGP).The protein component in the crude polysaccharide was removed by sevag method.The small molecule was removed by dialysis method,and the pigment was removed by activated carbon.Then the refined polysaccharide was obtained.The contents of the refined polysaccharides were determined by phenol-sulfuric acid colorimetry.The antioxidant activities of RGP in vitro were measured by four different antioxidative systems and cell-based assays,including hydroxyl radicalscavenging effect,superoxide anion radicalscavenging effect,DPPH radicalscavenging effect,the total antioxidant capacity(T-AOC),and effects on activities of SOD and levels of GSH-px and MDA.Result The results showed that the EC;values of the refined polysaccharides were 1.51 mg/mL for scavenging hydroxyl radicals,3.04 mg/mL for scavenging superoxide anion radicals,0.26 mg/mL for scavenging DPPH radicals.Furthermore,T-AOC of RGP was(18.85±0.80)U/mg.Compared with the positive control BHT,RGP showed better scavenging capacity of DPPH free radical,certain scavenging ability of hydroxyl free radical and superoxide anion free radical.Furthermore,it has a dose effect with the concentration of RGP.RGP also had a good T-AOC capacity.Besides,RGP could significantly improve the abnormal decrease of SOD activity and GSH level and the abnormal increase of MDA level induced by H;O;in HeLa cells.Conclusion The polysaccharides of Shudihuang(Rehmanniae Radix Preparata)have good antioxidant activity in vitro and potential value for developing natural antioxidant,food and health products.