飞天蜈蚣七中多糖和黄酮类成分提取工艺及生物活性研究
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摘要
本课题以陕西产飞天蜈蚣七为研究对象,首先对飞天蜈蚣七多糖的提取、纯化工艺及结构分析进行了系统研究；其次对飞天蜈蚣七脱脂液中的总黄酮分离纯化工艺进行了研究；同时对飞天蜈蚣七多糖和总黄酮的体外抗氧化性、抑菌活性进行了初步探讨。主要研究成果如下：
(1)飞天蜈蚣七多糖提取工艺研究
分别对飞天蜈蚣七多糖的热水浸提法、超声辅助法两种提取工艺进行了研究,并优化了提取工艺参数。热水浸提法优化工艺参数：液料比10(mL/g),提取温度95℃,提取时间1.9h,提取次数2次,在上述工艺条件下,多糖提取率3.16%；超声辅助法优化工艺参数：超声时间32min,温度84℃,超声功率900W,液料比20(mL/g),提取次数2次;在上述工艺条件下,多糖提取率3.99%；试验结果表明超声辅助法有明显缩短提取时间、增加提取率的作用。
(2)飞天蜈蚣七多糖脱蛋白工艺研究
通过对4种多糖脱蛋白方法的比较试验表明,木瓜蛋白酶-Sevage法是最为理想的飞天蜈蚣七多糖脱蛋白方法。通过优化试验得到工艺条件为：酶用量2.7%,酶解温度60℃,酶解时间2h,pH=7,结合3次Sevage法操作,在此工艺条件下,多糖蛋白脱除率92.68%,多糖保留率86.91%。
(3)飞天蜈蚣七多糖脱色工艺研究
通过对活性炭法、双氧水法、反胶束溶液法、大孔树脂法四种脱色方法对比研究,得到了一条大孔树脂吸附法脱除飞天蜈蚣七多糖色素的工艺路线,并求得了飞天蜈蚣七多糖色素的吸附动力学、热力学等相关参数。通过静态、动态吸附和解吸试验表明：LSA-700B树脂是脱除飞天蜈蚣七多糖色素的理想树脂。试验结果表明该法最佳工艺条件为：上柱流速2BV/h,上样量2.OBV,上样浓度4mg/mL,温度为30℃,在此工艺条件下,多糖色素脱除率90.11%,多糖保留率90.75%。
(4)飞天蜈蚣七多糖结构分析
通过分级纯化试验,分别得到了水提纯化多糖(APS-1-1)和超声纯化多糖(UAPS-1-1)。结构解析研究结果表明：APS-1-1和UAPS-1-1均为均一多糖,结构十分相似。其中APS-1-1的分子量为1.28×106gmol-1,单糖组成为鼠李糖、阿拉伯糖及半乳糖,单糖的摩尔比为1：3.92：6.11,部分结构是(1→4)Gal的主链；UAPS-1-1的分子量为4.28×105Dal,单糖组成为鼠李糖、阿拉伯糖及半乳糖,单糖的摩尔比为1：1.19：1.25,部分结构是(1→4)Gal的主链。
(5)飞天蜈蚣七总黄酮纯化工艺
采用大孔树脂吸附法对飞天蜈蚣七脱脂液中的总黄酮的分离纯化进行了研究,得到了一条大孔树脂吸附法分离纯化飞天蜈蚣七总黄酮的工艺路线和吸附动力学及热力学的相关参数。通过静态、动态吸附和解吸试验表明：LX-38树脂是分离纯化总黄酮的理想树脂,纯化的优化条件为：上样液浓度为8.0mg/mL,流速为2BV/h, pH为5.5;以80%的乙醇为洗脱剂,用5BV的乙醇洗脱；纯化后纯度为30.87%。
(6)飞天蜈蚣七多糖和总黄酮的体外活性试验
试验结果表明：飞天蜈蚣七多糖和总黄酮均具有较好的体外抗氧化活性,其中多糖对DPPH、超氧阴离子(O2-)的清除效果较好；总黄酮对DPPH、超氧阴离子(O2-)及羟基自由基(·OH)的清除效果较好。
飞天蜈蚣七总黄酮在体外对肺炎链球菌、大肠埃希菌、铜绿假单胞菌以及金黄色葡萄球菌4种菌株具有较强的抑制活性；而飞天蜈蚣七多糖对上述四种菌株均无抑菌活性。
本课题的研究结果为飞天蜈蚣七多糖、总黄酮开发为药品和功能食品提供了一定的技术支撑和理论依据。
The thesis is aimed to develop valuable materials from the herbal resource of Province of Shaanxi, the Aralia chinensis. This thesis focused on the systematic study of the processes of the polysaccharide, extraction, purification and decolorization. Furthermore the sepeartion and purification of the flavonoids from the degreasing liquid were studied; Finally, the biologic activitites of the obtained polysaccharide and flavonoids were tested. The detailed achievements are as follows:
(1) The extraction of polysaccharide
Aralia chinensis polysaccharide was obtained by using hot water extraction and ultrasonic wave extraction. On the basis of mono-factor tests, the method of response surface analysis(RSA) was carried out to determine the optimal condition for the process of the extraction. The optimal process condition using hot water extraction was, the weight ratio of liquid and solid (10/1, water/dry weight), the extraction temperature of 95℃, the extraction time of 1.9 h and the extraction times of 2 times. The extraction rate of polysaccharide was 3.16% on the above mentioned condition. For the ultrasonic wave extraction, the optimal condition was:the extraction time of 32min, the extraction temperature of 84℃, the ultrasonic extraction power of 900 W, the weight ratio of liquid and solid (20/1, water/dry weight) and the extraction times of 2 times.Under the optimized condition, the extraction rate of polysaccharide was 3.99%. The comparison result shows that the ultrasonic method was more time-saving and the higher extraction rate of polysaccharide could be obtained.
(2) The separation process of protein
Through comparing the four methods of protein removal, papain-Sevage method was choosed as the optimal method. The optimal condition was the substrate concentration 2.7% based on papain, the separation temperature of 60℃, the hydrolysis time of 2 h, the pH value of 7.0 and the repeat times of 3 times. The amount of protein removal was 92.68% while the content of polysaccharide remained 86.91% after the deproteinization process.
(3) The process of decolorization
Based on the comparison of four decolorization methods, hydrogen peroxide oxidation method, live carbon attaching method, reverse micelle solution method and macroporous resin adsorption method, macroporous resin adsorption method was proved to have the most effective for the decolorization of polysaccharide. Through the experiments of static adsorption, desorption and dynamic adsorption, the relative parameters of adsorption kinetics and thermodynamics are obtained. The result showed that LSA-700B resin adsorption was chosen the suitable method to determine the optimal adsorption condition, which is the sample usage of 2 BV, the current velocity of 2.0 BV/h, the sample mass concentration of 4.0 mg/mL and the adsorption temperature of 30℃. The decolorazation rate was 90.11% whilethe polysaccharide retention rate was 90.75%.
(4) The characterisation of polysaccharides purified from Aralia chinensis
The APS-1-1 and UAPS-1-1 were got by the purification of DEAE-Cellulose column and sephadex G-100 with different solvents, respectively. The structural characterisation of APS-1-1 and UAPS-1-1 were studied. The APS-1-1 was a kind of water soluble polysaccharide with the average molecular weight of 1.28×106 Dal. The unit of monosaccharide for APS-1-1 was identified as rhamnose, arabinose and galactose with a ratio of 1:3.92:6.11, and its sugar chain was manily composed of 1→4 linkage. The UAPS-1-1 was a kind of water soluble polysaccharide with the average molecular weight of 4.28×105 Dal.Also, the unit of monosaccharide for UAPS-1-1 was identified as rhamnose, arabinose and galactose with a ratio of 1:1.19:1.25, and the sugar chain was manily composed of 1→4 linkage.
(5) The purification of flavonoids from Aralia chinensis
Macroporous resin adsorption was chosen to purify the flavonoids from Aralia chinensis. Through the detailed experiments of static adsorption, desorption and dynamic adsorption, the relative parameters of adsorption kinetics and thermodynamics were obtained. The result showed that the resin as the most suitable resin could effectively be used from the separation and purification of the flavonoids. The optimization condition was the sample concentration of 8.0 mg/mL, a flow rate of 2 BV/h and the pH value of 5.0 when 5 Bv ethnol (80%) as eluant was used. The final purity of polyphenol reached 30.87%.
(6) The biologic activity of the polysaccharides and the flavonoids from Aralia chinensis
The results showed that both the polysaccharides and flavonoids from Aralia chinensis had the cleaning capability of superoxide radical, hydroxy radical and DPPH. Especially the the cleaning capability of DPPH or superoxide radical had the satisfied scavenging effect.
Further by the study of in vitro antibacterial experiments of the representative bacteria, Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae, Pseudomonas aeruginosa or rhinitis, the result showed that the flavonoids extracted from Aralia chinensis have the stronger inhibition activity than that of the polysaccharides from Aralia chinensis.
(1)飞天蜈蚣七多糖提取工艺研究
分别对飞天蜈蚣七多糖的热水浸提法、超声辅助法两种提取工艺进行了研究,并优化了提取工艺参数。热水浸提法优化工艺参数：液料比10(mL/g),提取温度95℃,提取时间1.9h,提取次数2次,在上述工艺条件下,多糖提取率3.16%；超声辅助法优化工艺参数：超声时间32min,温度84℃,超声功率900W,液料比20(mL/g),提取次数2次;在上述工艺条件下,多糖提取率3.99%；试验结果表明超声辅助法有明显缩短提取时间、增加提取率的作用。
(2)飞天蜈蚣七多糖脱蛋白工艺研究
通过对4种多糖脱蛋白方法的比较试验表明,木瓜蛋白酶-Sevage法是最为理想的飞天蜈蚣七多糖脱蛋白方法。通过优化试验得到工艺条件为：酶用量2.7%,酶解温度60℃,酶解时间2h,pH=7,结合3次Sevage法操作,在此工艺条件下,多糖蛋白脱除率92.68%,多糖保留率86.91%。
(3)飞天蜈蚣七多糖脱色工艺研究
通过对活性炭法、双氧水法、反胶束溶液法、大孔树脂法四种脱色方法对比研究,得到了一条大孔树脂吸附法脱除飞天蜈蚣七多糖色素的工艺路线,并求得了飞天蜈蚣七多糖色素的吸附动力学、热力学等相关参数。通过静态、动态吸附和解吸试验表明：LSA-700B树脂是脱除飞天蜈蚣七多糖色素的理想树脂。试验结果表明该法最佳工艺条件为：上柱流速2BV/h,上样量2.OBV,上样浓度4mg/mL,温度为30℃,在此工艺条件下,多糖色素脱除率90.11%,多糖保留率90.75%。
(4)飞天蜈蚣七多糖结构分析
通过分级纯化试验,分别得到了水提纯化多糖(APS-1-1)和超声纯化多糖(UAPS-1-1)。结构解析研究结果表明：APS-1-1和UAPS-1-1均为均一多糖,结构十分相似。其中APS-1-1的分子量为1.28×106gmol-1,单糖组成为鼠李糖、阿拉伯糖及半乳糖,单糖的摩尔比为1：3.92：6.11,部分结构是(1→4)Gal的主链；UAPS-1-1的分子量为4.28×105Dal,单糖组成为鼠李糖、阿拉伯糖及半乳糖,单糖的摩尔比为1：1.19：1.25,部分结构是(1→4)Gal的主链。
(5)飞天蜈蚣七总黄酮纯化工艺
采用大孔树脂吸附法对飞天蜈蚣七脱脂液中的总黄酮的分离纯化进行了研究,得到了一条大孔树脂吸附法分离纯化飞天蜈蚣七总黄酮的工艺路线和吸附动力学及热力学的相关参数。通过静态、动态吸附和解吸试验表明：LX-38树脂是分离纯化总黄酮的理想树脂,纯化的优化条件为：上样液浓度为8.0mg/mL,流速为2BV/h, pH为5.5;以80%的乙醇为洗脱剂,用5BV的乙醇洗脱；纯化后纯度为30.87%。
(6)飞天蜈蚣七多糖和总黄酮的体外活性试验
试验结果表明：飞天蜈蚣七多糖和总黄酮均具有较好的体外抗氧化活性,其中多糖对DPPH、超氧阴离子(O2-)的清除效果较好；总黄酮对DPPH、超氧阴离子(O2-)及羟基自由基(·OH)的清除效果较好。
飞天蜈蚣七总黄酮在体外对肺炎链球菌、大肠埃希菌、铜绿假单胞菌以及金黄色葡萄球菌4种菌株具有较强的抑制活性；而飞天蜈蚣七多糖对上述四种菌株均无抑菌活性。
本课题的研究结果为飞天蜈蚣七多糖、总黄酮开发为药品和功能食品提供了一定的技术支撑和理论依据。
The thesis is aimed to develop valuable materials from the herbal resource of Province of Shaanxi, the Aralia chinensis. This thesis focused on the systematic study of the processes of the polysaccharide, extraction, purification and decolorization. Furthermore the sepeartion and purification of the flavonoids from the degreasing liquid were studied; Finally, the biologic activitites of the obtained polysaccharide and flavonoids were tested. The detailed achievements are as follows:
(1) The extraction of polysaccharide
Aralia chinensis polysaccharide was obtained by using hot water extraction and ultrasonic wave extraction. On the basis of mono-factor tests, the method of response surface analysis(RSA) was carried out to determine the optimal condition for the process of the extraction. The optimal process condition using hot water extraction was, the weight ratio of liquid and solid (10/1, water/dry weight), the extraction temperature of 95℃, the extraction time of 1.9 h and the extraction times of 2 times. The extraction rate of polysaccharide was 3.16% on the above mentioned condition. For the ultrasonic wave extraction, the optimal condition was:the extraction time of 32min, the extraction temperature of 84℃, the ultrasonic extraction power of 900 W, the weight ratio of liquid and solid (20/1, water/dry weight) and the extraction times of 2 times.Under the optimized condition, the extraction rate of polysaccharide was 3.99%. The comparison result shows that the ultrasonic method was more time-saving and the higher extraction rate of polysaccharide could be obtained.
(2) The separation process of protein
Through comparing the four methods of protein removal, papain-Sevage method was choosed as the optimal method. The optimal condition was the substrate concentration 2.7% based on papain, the separation temperature of 60℃, the hydrolysis time of 2 h, the pH value of 7.0 and the repeat times of 3 times. The amount of protein removal was 92.68% while the content of polysaccharide remained 86.91% after the deproteinization process.
(3) The process of decolorization
Based on the comparison of four decolorization methods, hydrogen peroxide oxidation method, live carbon attaching method, reverse micelle solution method and macroporous resin adsorption method, macroporous resin adsorption method was proved to have the most effective for the decolorization of polysaccharide. Through the experiments of static adsorption, desorption and dynamic adsorption, the relative parameters of adsorption kinetics and thermodynamics are obtained. The result showed that LSA-700B resin adsorption was chosen the suitable method to determine the optimal adsorption condition, which is the sample usage of 2 BV, the current velocity of 2.0 BV/h, the sample mass concentration of 4.0 mg/mL and the adsorption temperature of 30℃. The decolorazation rate was 90.11% whilethe polysaccharide retention rate was 90.75%.
(4) The characterisation of polysaccharides purified from Aralia chinensis
The APS-1-1 and UAPS-1-1 were got by the purification of DEAE-Cellulose column and sephadex G-100 with different solvents, respectively. The structural characterisation of APS-1-1 and UAPS-1-1 were studied. The APS-1-1 was a kind of water soluble polysaccharide with the average molecular weight of 1.28×106 Dal. The unit of monosaccharide for APS-1-1 was identified as rhamnose, arabinose and galactose with a ratio of 1:3.92:6.11, and its sugar chain was manily composed of 1→4 linkage. The UAPS-1-1 was a kind of water soluble polysaccharide with the average molecular weight of 4.28×105 Dal.Also, the unit of monosaccharide for UAPS-1-1 was identified as rhamnose, arabinose and galactose with a ratio of 1:1.19:1.25, and the sugar chain was manily composed of 1→4 linkage.
(5) The purification of flavonoids from Aralia chinensis
Macroporous resin adsorption was chosen to purify the flavonoids from Aralia chinensis. Through the detailed experiments of static adsorption, desorption and dynamic adsorption, the relative parameters of adsorption kinetics and thermodynamics were obtained. The result showed that the resin as the most suitable resin could effectively be used from the separation and purification of the flavonoids. The optimization condition was the sample concentration of 8.0 mg/mL, a flow rate of 2 BV/h and the pH value of 5.0 when 5 Bv ethnol (80%) as eluant was used. The final purity of polyphenol reached 30.87%.
(6) The biologic activity of the polysaccharides and the flavonoids from Aralia chinensis
The results showed that both the polysaccharides and flavonoids from Aralia chinensis had the cleaning capability of superoxide radical, hydroxy radical and DPPH. Especially the the cleaning capability of DPPH or superoxide radical had the satisfied scavenging effect.
Further by the study of in vitro antibacterial experiments of the representative bacteria, Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae, Pseudomonas aeruginosa or rhinitis, the result showed that the flavonoids extracted from Aralia chinensis have the stronger inhibition activity than that of the polysaccharides from Aralia chinensis.
引文
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