龙眼多糖制备工艺及其抗氧化活性研究
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摘要
龙眼多糖(Longan polysaccharides, LP)是龙眼果肉中重要的活性物质之一,具有良好的抗氧化、抑制肿瘤和免疫调节等活性,近年来受到广泛关注。本文首先对7个广东省主栽龙眼品种的多糖含量和组成进行分析,筛选出高多糖含量品种;在此基础上优化和完善了龙眼多糖的高效提取工艺和制备工艺,并利用体外实验模型评价其抗氧化活性。主要结论如下:
7个广东省主栽的龙眼品种中,储良鲜果肉中粗多糖含量为6.37±0.16mg(10.0g鲜重),显著高于其它品种。龙眼多糖属于杂多糖,单糖组成包括果糖、甘露糖、鼠李糖、葡萄糖、半乳糖、木糖和阿拉伯糖,其中葡萄糖、半乳糖、木糖和阿拉伯糖在7个龙眼品种中均存在且含量较高。储良龙眼粗多糖的单糖组成为果糖、鼠李糖、葡萄糖、半乳糖、木糖和阿拉伯糖,对应含量百分比为3.88%:5.27%:74.96%:6.06%:2.81%:6.99%。
采用响应面法(Response Surface Methodology,RSM)对酶法提取和微波前处理-超声波提取龙眼多糖的工艺进行优化。酶法提取最佳工艺参数为:酶添加量1.2%、液料比6.0(mL/g)、酶解温度45.0℃、酶解时间187.0min;微波前处理-超声波提取最佳工艺参数为:微波功率640W,前处理时间60s,超声波功率为590W、液料比9.5(mL/g)、超声波时间8.5min。在此条件下,酶法提取龙眼多糖(Enzyme extraction longan polysaccharide,EELP)和微波前处理-超声波提取龙眼多糖(Microwave and ultrasonic extraction longan polysaccharide, MUELP)得率分别为12.23±0.15mg/g和11.03±0.38mg/g龙眼(干重)。
对龙眼多糖制备工艺中滤渣清洗、除蛋白和乙醇沉淀工艺进行优化。最终确定滤渣清洗次数2次;三氯乙酸除蛋白浓度2%,除蛋白时间2h;EELP的醇沉浓度65%、醇沉时间12h,MUELP的醇沉浓度75%、醇沉时间18h。此工艺流程下,最终EELP和MUELP得率分别为9.17±0.32mg/g和8.11±0.28mg/g龙眼(干重),其中多糖含量分别为71.26±2.41%和70.53±3.53%。评价了龙眼多糖的急性毒性和抗氧化活性。小鼠急性毒理实验表明龙眼多糖属于实际无毒。
抗氧化研究结果表明,两种龙眼多糖均具有良好的清除羟自由基、DPPH自由基和ABTS自由基功效,MUELP的IC50值分别为8.204±0.239、0.667±0.053和0.481±0.038mg/mL,EELP的IC50值分别为9.601±0.223、1.710±0.068和0.604±0.031mg/mL。龙眼多糖对超氧阴离子自由基清除较弱,多糖浓度50mg/mL时MUELP和EELP的自由基清除率分别为36.10%和18.94%;还原力测定方面,多糖浓度为25mg/mL时的,MUELP和EELP在700nm下吸光值分别为1.119±0.041和0.641±0.024。因此,MUELP比EELP具有更好的抗氧化活性。
Longan polysaccharides (LP) is one of the most important bioactive compounds in longan pulp. In recent years, it has been drawing close attention because of its bioactive function in immunoregulation, anti-tumor and antioxidant activities. In this research, the longan polysaccharides of 7 Guangdong main cultivars were studied and the high polysaccharide cultivar was chosen; the extraction and preparation of longan polysaccharide were optimized; the antioxidant activities of longan polysaccharide were evaluated. Main conclusions were summarized as follows:
Chuliang longan contained 6.37±0.16 mg polysaccharide per 10.0 gram pulp weight, which was significantly higher than that of the other 6 longan cultivars (p<0.05). Longan polysaccharide was heteropolysaccharides which were composed of monosaccharides including fructose, rhamnose, mannose, glucose, galactose, xylose and arabonose. Glucose, galactose, xylose and arabonose were the main monosaccharide components of longan polysaccharide in the 7 cultivars. The monosaccharide components of Chuliang longan polysaccharide were fructose, rhamnose, glucose, galactose, xylose and arabonose, and the ratio of percentage composition were 3.88%:5.27%:74.96%:6.06%:2.81%:6.99%. After comprehensive consideration, Chuliang longan was selected as the high polysaccharide content cultivar and was studied in the following research.
Single factor analysis and response surface methodology (RSM) were used to optimize the enzyme and microwave-ultrasonic extraction. The analysis result showed that, using enzymatic extraction method, the optimal conditions to obtain the highest yield were enzyme concentration 1.2%, water to raw material 6.0(mL/g), extraction temperature 45.0℃and extraction time 187.0min. Using microwave-ultrasonic extraction method, the optimal conditions to obtain the highest yield were microwave power 640W, microwave time 60s, ultrasonic power 590W, ratio of water to material 9.5(mL/g), ultrasonic time 8.5min. The actual yields of EELP and MUELP were 12.23±0.15mg/g and11.03±0.38mg/g, respectively.
The preparations of EELP and MUELP were optimized, the fruit residue was determined to be washed twice; the trichloroacetic acid (TCA) processing to remove protein were determined to be with the concentration of 2% and the time of 2h; the alcohol precipitation processing were determined to be with the concentration of 65% and the time of 12h in the EELP and the concentration of 75% and the time of 18h in the MUELP. Under these preparations, the final yields of EELP and MUELP were 9.17±0.32mg/g and 8.11±0.28mg/g, and the polysaccharides contents were 71.26±2.41% and 70.53±3.53%, respectively.
The acute toxicity and antioxidant activities of longan polysaccharide were evaluated. The result of acute toxicological study in mice showed that the longan polysaccharide is of actual non-toxic. The result of antioxidant activities showed that longan polysaccharides had good free radical scavenging abilities in removing hydroxyl radicals, 1,1-dipheny1-2-picrylhydrazyl(DPPH) radicals, 2,2’-azinobis-3-ethyl-benzothiazoline-6-sulphonic acid (ABTS) radicals. The IC50 value of MUELP were 8.204±0.239, 0.667±0.053 and 0.481±0.038mg/mL respectively; and the IC50 value of EELP were 9.601±0.223, 1.710±0.068 and 0.604±0.031mg/mL respectively. With the polysaccharides concentration of 50mg/mL, the superoxide anion radicals IC50 value of MUELP and EELP were 36.10% and 18.94% respectively. With the polysaccharides concentration of 25mg/mL, the OD value at 700nm of MUELP and EELP were 1.119±0.041 and 0.641±0.024, respectively. Therefore, MUELP had antioxidant activities better than EELP.
7个广东省主栽的龙眼品种中,储良鲜果肉中粗多糖含量为6.37±0.16mg(10.0g鲜重),显著高于其它品种。龙眼多糖属于杂多糖,单糖组成包括果糖、甘露糖、鼠李糖、葡萄糖、半乳糖、木糖和阿拉伯糖,其中葡萄糖、半乳糖、木糖和阿拉伯糖在7个龙眼品种中均存在且含量较高。储良龙眼粗多糖的单糖组成为果糖、鼠李糖、葡萄糖、半乳糖、木糖和阿拉伯糖,对应含量百分比为3.88%:5.27%:74.96%:6.06%:2.81%:6.99%。
采用响应面法(Response Surface Methodology,RSM)对酶法提取和微波前处理-超声波提取龙眼多糖的工艺进行优化。酶法提取最佳工艺参数为:酶添加量1.2%、液料比6.0(mL/g)、酶解温度45.0℃、酶解时间187.0min;微波前处理-超声波提取最佳工艺参数为:微波功率640W,前处理时间60s,超声波功率为590W、液料比9.5(mL/g)、超声波时间8.5min。在此条件下,酶法提取龙眼多糖(Enzyme extraction longan polysaccharide,EELP)和微波前处理-超声波提取龙眼多糖(Microwave and ultrasonic extraction longan polysaccharide, MUELP)得率分别为12.23±0.15mg/g和11.03±0.38mg/g龙眼(干重)。
对龙眼多糖制备工艺中滤渣清洗、除蛋白和乙醇沉淀工艺进行优化。最终确定滤渣清洗次数2次;三氯乙酸除蛋白浓度2%,除蛋白时间2h;EELP的醇沉浓度65%、醇沉时间12h,MUELP的醇沉浓度75%、醇沉时间18h。此工艺流程下,最终EELP和MUELP得率分别为9.17±0.32mg/g和8.11±0.28mg/g龙眼(干重),其中多糖含量分别为71.26±2.41%和70.53±3.53%。评价了龙眼多糖的急性毒性和抗氧化活性。小鼠急性毒理实验表明龙眼多糖属于实际无毒。
抗氧化研究结果表明,两种龙眼多糖均具有良好的清除羟自由基、DPPH自由基和ABTS自由基功效,MUELP的IC50值分别为8.204±0.239、0.667±0.053和0.481±0.038mg/mL,EELP的IC50值分别为9.601±0.223、1.710±0.068和0.604±0.031mg/mL。龙眼多糖对超氧阴离子自由基清除较弱,多糖浓度50mg/mL时MUELP和EELP的自由基清除率分别为36.10%和18.94%;还原力测定方面,多糖浓度为25mg/mL时的,MUELP和EELP在700nm下吸光值分别为1.119±0.041和0.641±0.024。因此,MUELP比EELP具有更好的抗氧化活性。
Longan polysaccharides (LP) is one of the most important bioactive compounds in longan pulp. In recent years, it has been drawing close attention because of its bioactive function in immunoregulation, anti-tumor and antioxidant activities. In this research, the longan polysaccharides of 7 Guangdong main cultivars were studied and the high polysaccharide cultivar was chosen; the extraction and preparation of longan polysaccharide were optimized; the antioxidant activities of longan polysaccharide were evaluated. Main conclusions were summarized as follows:
Chuliang longan contained 6.37±0.16 mg polysaccharide per 10.0 gram pulp weight, which was significantly higher than that of the other 6 longan cultivars (p<0.05). Longan polysaccharide was heteropolysaccharides which were composed of monosaccharides including fructose, rhamnose, mannose, glucose, galactose, xylose and arabonose. Glucose, galactose, xylose and arabonose were the main monosaccharide components of longan polysaccharide in the 7 cultivars. The monosaccharide components of Chuliang longan polysaccharide were fructose, rhamnose, glucose, galactose, xylose and arabonose, and the ratio of percentage composition were 3.88%:5.27%:74.96%:6.06%:2.81%:6.99%. After comprehensive consideration, Chuliang longan was selected as the high polysaccharide content cultivar and was studied in the following research.
Single factor analysis and response surface methodology (RSM) were used to optimize the enzyme and microwave-ultrasonic extraction. The analysis result showed that, using enzymatic extraction method, the optimal conditions to obtain the highest yield were enzyme concentration 1.2%, water to raw material 6.0(mL/g), extraction temperature 45.0℃and extraction time 187.0min. Using microwave-ultrasonic extraction method, the optimal conditions to obtain the highest yield were microwave power 640W, microwave time 60s, ultrasonic power 590W, ratio of water to material 9.5(mL/g), ultrasonic time 8.5min. The actual yields of EELP and MUELP were 12.23±0.15mg/g and11.03±0.38mg/g, respectively.
The preparations of EELP and MUELP were optimized, the fruit residue was determined to be washed twice; the trichloroacetic acid (TCA) processing to remove protein were determined to be with the concentration of 2% and the time of 2h; the alcohol precipitation processing were determined to be with the concentration of 65% and the time of 12h in the EELP and the concentration of 75% and the time of 18h in the MUELP. Under these preparations, the final yields of EELP and MUELP were 9.17±0.32mg/g and 8.11±0.28mg/g, and the polysaccharides contents were 71.26±2.41% and 70.53±3.53%, respectively.
The acute toxicity and antioxidant activities of longan polysaccharide were evaluated. The result of acute toxicological study in mice showed that the longan polysaccharide is of actual non-toxic. The result of antioxidant activities showed that longan polysaccharides had good free radical scavenging abilities in removing hydroxyl radicals, 1,1-dipheny1-2-picrylhydrazyl(DPPH) radicals, 2,2’-azinobis-3-ethyl-benzothiazoline-6-sulphonic acid (ABTS) radicals. The IC50 value of MUELP were 8.204±0.239, 0.667±0.053 and 0.481±0.038mg/mL respectively; and the IC50 value of EELP were 9.601±0.223, 1.710±0.068 and 0.604±0.031mg/mL respectively. With the polysaccharides concentration of 50mg/mL, the superoxide anion radicals IC50 value of MUELP and EELP were 36.10% and 18.94% respectively. With the polysaccharides concentration of 25mg/mL, the OD value at 700nm of MUELP and EELP were 1.119±0.041 and 0.641±0.024, respectively. Therefore, MUELP had antioxidant activities better than EELP.
引文
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