樟叶木脂素的提取纯化及其抗氧化性、抗癌活性

Extraction and Purification of Lignans from Cinnamomum camphora Leaves and Its Antioxidant Activity and Anticancer Activity

为了优化樟叶木脂素超声波技术提取条件,探究其抗氧化性、抗肝癌活性,本文通过单因素实验考察各个因素对木脂素提取量的影响,利用响应面试验优化提取工艺并建立相应的数学模型。粗品经过浓缩、萃取、柱色谱等步骤进行分离纯化;紫外色谱、红外光谱、质谱、核磁共振等技术进行结构鉴定。利用高效液相谱(High Efficiency Liquid Chromatography,HPLC)外标法进行纯度鉴定;同时利用1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)法,羟基自由基清除法研究其抗氧化性;MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)法研究其对肝癌细胞(HepG2)和人脐静脉内皮细胞(HUVEC)的毒性作用。结果表明:樟叶木脂素的最优超声提取条件为浸提时间78 min,超声时间3 min,料液比1:22 g/mL,乙醇浓度60%,在此条件下樟树叶中木脂素提取量为45.31 mg/g;经过纯化和结构鉴定最终得到芝麻素和松脂素-β-D-吡喃葡萄糖苷,其纯度分别为94.84%和90.14%;抗氧化实验结果表明芝麻素,松脂素-β-D-吡喃葡萄糖苷具有较好的清除DPPH自由基、羟基自由基效果,其中松脂素-β-D-吡喃葡萄糖苷清除DPPH自由基的IC_(50)为0.31 mg/mL,清除羟基自由基的IC_(50)为0.71 mg/mL;芝麻素清除DPPH自由基的IC_(50)为0.55 mg/mL,清除羟基自由基的IC_(50)为0.94 mg/mL。细胞毒性试验发现芝麻素、松脂素-β-D-吡喃葡萄糖苷均对HepG2有抑制作用,且芝麻素对肝癌细胞(HepG2)、人脐静脉内皮细胞(HUVEC)的作用大于松脂素-β-D-吡喃葡萄糖苷。综上可知响应面法可以用于樟叶木脂素的提取分离,且该提取物具有抗氧化和抗肝癌的功效。

The aim of this study was to evaluate the antioxidant and anticancer activities of lignans from Cinnamomum camphora leaves.Response surface methodology(RSM)was used to optimize ultrasonic wave assisted extraction of lignans based on single factor tests and establish the corresponding mathematical model.The lignans were separated and purified by the steps of concentration,extraction,chromatography-spectrum and so on.The structure was identified by ultraviolet chromatography,infrared spectroscopy,mass spectrometry and nuclear magnetia resonance.The purity of the compounds was determined using HPLC technique and they were used for further experiments.The antioxidant activity of lignans was studied in vitro by scavenging activity against DPPH·and·OH.The effects of lignans on HepG2 liver cancer cells and human umbilical vein endothelial cell(HUVEC)were compared by MTS assay.The results were as follows:Liquid-to-solid ratio 1 cells and human umbilical vein endothelial cell(HUVEC)were compared by MTS assay.The results were as follows:Liquid-to-solid ratio 1:22 g/mL,ultrasonic time 3 min,ethanol concentration 60%and extraction time 78 min,the yield of lignans was 45.31 mg/g.The purity of sesamin and pinoresinol-β-D-glucaside was 94.84%,90.14%respectively.And the lignans had strong scavenging activity against DPPH·and·OH.Among them,the half inhibitory concentration(IC_(50))of pinoresinol-β-D-glucaside for scavenging DPPH free radical and hydroxyl free radical was 0.31 mg/mL and 0.71 mg/mL,respectively;the IC_(50) of sesamin for scavenging DPPH free radical and hydroxyl radical was 0.55 mg/mL and 0.94 mg/mL,respectively.Anticancer activity experiments showed that sesamin had stronger inhibition against HepG2 and HUVEC than pinoresinol-β-D-glucaside.The extraction process by RSM was reliable and the optimal conditions were achieved to get sesamin and pinoresinol-β-D-glucaside,and they had antioxidant and anticancer activities.