苜蓿总黄酮的提取方法、药效以及多种豆科牧草次生代谢产物的研究
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摘要
苜蓿、小冠花、百脉根、红豆草、鹰嘴紫云英、白三叶、黄花草木樨等优质豆科牧草含有丰富的次生代谢产物。本文以这几种豆科牧草为试验材料,对其总黄酮和总皂苷的积累规律进行了研究。同时为充分利用苜蓿中的黄酮类物质,提高苜蓿的经济附加值,分别采用微波和超声波辅助技术提取苜蓿总黄酮,对影响提取量的乙醇浓度、固液比、作用时间、微波功率、超声波温度、提取次数等进行了单因素试验,并在此基础上对各因素的互作效应进行了4因素3水平的正交试验。从而确定了最佳提取工艺,用于生产中提取苜蓿总黄酮。采用柱层析等多种分离手段对苜蓿活性成分进行了分离。并对苜蓿粗提液的药效进行了研究。为建立科学合理的牧刈制度,高效提取天然活性成分,为综合利用优质牧草提供指导依据。研究结果表明:
微波辅助技术提取苜蓿总黄酮优选条件为:40%乙醇、1:30固液比,在500W功率下作用60s。这个工艺提取条件温和,温度不超过50℃;节能,耗电量只有0.008千瓦时;省时,提取时间只有超声波法的1/9,冷浸法的1/540;节约有机溶剂,同时无噪音污染。
小冠花、百脉根、红豆草、紫云英、草木樨、白三叶6种豆科牧草的总黄酮含量普遍高于甘农3号、新疆大叶、低纤维苜蓿和白花苜蓿;而总皂苷含量整个生育时期均低于苜蓿4个品种。且这几种豆科牧草茎、叶、花、全株中总黄酮和总皂苷含量有极显著差异(P<0.01),在整个生育时期,均为叶片中含量多于茎秆。
小冠花、百脉根、红豆草、紫云英、草木樨、白三叶6种豆科牧草可作为提取总黄酮的优质材料;由于其总皂苷含量较低,不会造成家畜臌胀病,可在地上部生物量最高时刈割,用于青饲或调制干草。苜蓿总皂苷含量丰富,是提取皂苷产品的优质材料。
对苜蓿中的天然活性物质进行分离,乙酸乙酯层主要含有2-甲叉-3-胆甾烷醇和3,5-二烯豆甾烷两种活性物质。氯仿层主要含有维生素D3活性物质。正丁醇层所含有活性物质种类较多,有6种天然活性成分,分别为芳樟醇、香芹醇、苦杏仁苷、氧化石竹烯、乙酸维生素A和邻苯二甲酸二丁酯。
苜蓿提取液具有增强小鼠机体非特异性免疫功能和特异性免疫(体液免疫)功能的作用;体外具有抗菌作用,对革兰氏阴性菌和革兰氏阳性菌均有直接的抑制作用;对血脂具有双向调节作用,低剂量时具有降脂作用,减少了机体产生冠心病的危险,而高剂量时增加了冠心病发生的机率;苜蓿提取液具有抗氧化作用,显著升高机体肝和肾组织的SOD活性,降低MDA的含量。
Legumes, including Astragalus sinicus, Onobrychis viciaefolia, Lotus corniculatus, Coronilla varia, Melilotus officinalis and Medicago sativa, which were abundant in secondary metabolites, were used as raw materials to study the proper procedure for making full use of the total flavoniod and increase its economic additional value. The aim of this study was to find out the optimal extraction condition using microwave-assisted and utralsonic-assisted extraction technics respectively, including one-factor experiments, which took into account ethanol concentration, microwave power , ratio of solid to liquid, treatment time, treatment temperature, and orthogonal experiments of four factors and three levels based on the results of one-factor experiments. Column chromatography and other methods were utilized to separate natural bioactive substances from Medicago sativa. At the same time the pharmacodynamics of the crude liquid extracted from Medicago sativa and the law of accumulation of flavoniod and saponin in these legumes were studied as well for the purposes of providing the theoretic foundation to set up scientific grazing- -mowing system and to extract natural bioactive substances more efficiently and utilize excellent legumes comprehensively .The results were showed as follows:
The parameters of the optimized extraction condition were 40% of ethanol concentration, 1:30 of the ratio of solid to liquid, 500w of the microwave power, 60 seconds of the radiation time.
Contents of the total extracted flavoniods of Onobrychis viciaefolia, Lotus corniculatus, Coronilla varia, Melilotus officinalis, Trifolium repens and Astragalus cicer were more than that of Medicago sativa var.Gannong No.3, Medicago sagiva cv. Xiniang Daye, low fiber alfalfa and white flower alfalfa. While the total extracted saponin contents of six legumes were less than that of the four alfalfas in the whole growth period.
Contents of the total extracted flavoniods and saponins in these legume had significant differences in stems, leaves and flowers (p<0.01). All the speices had the same trend that more flavonoids and saponins in leaves than in stems.
Onobrychis viciaefolia, Lotus corniculatus, Coronilla varia, Melilotus officinalis, Trifolium repens and Astragalus cicer could serve as the excellent materials for flavoniod extraction and also as succulence or hay because of their low saponins contents, they could be mowed when the aboveground parts reached the highest yield.
After separation the natural active subatances from Medicago sativa, there were Cholestan-3-ol,2-methylene and stigmastan-3,5-diene in ethyl acetate absolute Vitamin d3 in CH3Cl absolute, and six kinds of natural active substances such as linalool oxide, carveol, D-amygdalin, retinol acetate and ethylhexyl ester dibutyl phthalate in N-butanol absolute.
Liquid parts extracted from Medicago sativa could strengthen nonspecific and specific immunity of mice, which shown the direct effects to restrain bacterial, and bidirectional regulation to blood fat, and this indicated that low-dose could decrease blood fat and the rate of incidence of CHD (coronary heart disease), but high-dose increased the risk of CHD, had anti-oxidative activity, which can increase SOD activity and reduce content of MDA of livers and kidney.
微波辅助技术提取苜蓿总黄酮优选条件为:40%乙醇、1:30固液比,在500W功率下作用60s。这个工艺提取条件温和,温度不超过50℃;节能,耗电量只有0.008千瓦时;省时,提取时间只有超声波法的1/9,冷浸法的1/540;节约有机溶剂,同时无噪音污染。
小冠花、百脉根、红豆草、紫云英、草木樨、白三叶6种豆科牧草的总黄酮含量普遍高于甘农3号、新疆大叶、低纤维苜蓿和白花苜蓿;而总皂苷含量整个生育时期均低于苜蓿4个品种。且这几种豆科牧草茎、叶、花、全株中总黄酮和总皂苷含量有极显著差异(P<0.01),在整个生育时期,均为叶片中含量多于茎秆。
小冠花、百脉根、红豆草、紫云英、草木樨、白三叶6种豆科牧草可作为提取总黄酮的优质材料;由于其总皂苷含量较低,不会造成家畜臌胀病,可在地上部生物量最高时刈割,用于青饲或调制干草。苜蓿总皂苷含量丰富,是提取皂苷产品的优质材料。
对苜蓿中的天然活性物质进行分离,乙酸乙酯层主要含有2-甲叉-3-胆甾烷醇和3,5-二烯豆甾烷两种活性物质。氯仿层主要含有维生素D3活性物质。正丁醇层所含有活性物质种类较多,有6种天然活性成分,分别为芳樟醇、香芹醇、苦杏仁苷、氧化石竹烯、乙酸维生素A和邻苯二甲酸二丁酯。
苜蓿提取液具有增强小鼠机体非特异性免疫功能和特异性免疫(体液免疫)功能的作用;体外具有抗菌作用,对革兰氏阴性菌和革兰氏阳性菌均有直接的抑制作用;对血脂具有双向调节作用,低剂量时具有降脂作用,减少了机体产生冠心病的危险,而高剂量时增加了冠心病发生的机率;苜蓿提取液具有抗氧化作用,显著升高机体肝和肾组织的SOD活性,降低MDA的含量。
Legumes, including Astragalus sinicus, Onobrychis viciaefolia, Lotus corniculatus, Coronilla varia, Melilotus officinalis and Medicago sativa, which were abundant in secondary metabolites, were used as raw materials to study the proper procedure for making full use of the total flavoniod and increase its economic additional value. The aim of this study was to find out the optimal extraction condition using microwave-assisted and utralsonic-assisted extraction technics respectively, including one-factor experiments, which took into account ethanol concentration, microwave power , ratio of solid to liquid, treatment time, treatment temperature, and orthogonal experiments of four factors and three levels based on the results of one-factor experiments. Column chromatography and other methods were utilized to separate natural bioactive substances from Medicago sativa. At the same time the pharmacodynamics of the crude liquid extracted from Medicago sativa and the law of accumulation of flavoniod and saponin in these legumes were studied as well for the purposes of providing the theoretic foundation to set up scientific grazing- -mowing system and to extract natural bioactive substances more efficiently and utilize excellent legumes comprehensively .The results were showed as follows:
The parameters of the optimized extraction condition were 40% of ethanol concentration, 1:30 of the ratio of solid to liquid, 500w of the microwave power, 60 seconds of the radiation time.
Contents of the total extracted flavoniods of Onobrychis viciaefolia, Lotus corniculatus, Coronilla varia, Melilotus officinalis, Trifolium repens and Astragalus cicer were more than that of Medicago sativa var.Gannong No.3, Medicago sagiva cv. Xiniang Daye, low fiber alfalfa and white flower alfalfa. While the total extracted saponin contents of six legumes were less than that of the four alfalfas in the whole growth period.
Contents of the total extracted flavoniods and saponins in these legume had significant differences in stems, leaves and flowers (p<0.01). All the speices had the same trend that more flavonoids and saponins in leaves than in stems.
Onobrychis viciaefolia, Lotus corniculatus, Coronilla varia, Melilotus officinalis, Trifolium repens and Astragalus cicer could serve as the excellent materials for flavoniod extraction and also as succulence or hay because of their low saponins contents, they could be mowed when the aboveground parts reached the highest yield.
After separation the natural active subatances from Medicago sativa, there were Cholestan-3-ol,2-methylene and stigmastan-3,5-diene in ethyl acetate absolute Vitamin d3 in CH3Cl absolute, and six kinds of natural active substances such as linalool oxide, carveol, D-amygdalin, retinol acetate and ethylhexyl ester dibutyl phthalate in N-butanol absolute.
Liquid parts extracted from Medicago sativa could strengthen nonspecific and specific immunity of mice, which shown the direct effects to restrain bacterial, and bidirectional regulation to blood fat, and this indicated that low-dose could decrease blood fat and the rate of incidence of CHD (coronary heart disease), but high-dose increased the risk of CHD, had anti-oxidative activity, which can increase SOD activity and reduce content of MDA of livers and kidney.
引文
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