葱白有效成分组群的成分类型及降脂作用研究
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摘要
葱白是百合科(Liliaceae)植物分葱Allium fistulosum L. var. caespitosum Makino.的鳞茎。葱属植物中主要含有甾体及皂苷类化合物、黄酮类化合物、多糖类、挥发油、含氮化合物等成分,与这些化合物相关产生了抗菌、抗肿瘤、防治心血管疾病等药理作用[1-2]。前期的研究将含硫化合物、不饱和脂肪酸类化合物、甾体类化合物和黄酮类化合物定为葱白超临界CO_2萃取物(搏心通原料药)中的有效成分,本课题主要探讨对葱白提取物中有效成分的富集和药效作用的研究,并进一步扩大到对葱白中极性成分的研究方面。
本研究在考虑富集葱白超临界CO_2萃取物中挥发性成分时采用了一种适合于热敏性化合物富集分离的方法—分子蒸馏技术,对馏分采用了GC-MS分析方法。分子蒸馏所得馏分在外观性状上有的呈明显淡黄色油状,有的则是深绿色固体状,不同于葱白超临界提取物所呈现的油状半固体;GC-MS分析结果也显示了该技术对其中化合物的富集效果,特别是脂肪酸类成分和邻苯二甲酸酯类成分,但是没有达到预期的富集含硫化合物的效果。
采用经典的尿素包合富集不饱和脂肪酸的方法,对葱白超临界萃取中不饱和脂肪酸类成分进行富集,对富集产物的GC结果分析得出,富集产物中饱和脂肪酸类成分种类及含量均明显减少,不饱和脂肪酸类成分的含量则明显升高,能达到80%以上。
对葱白提取物中另一大类成分—甾体类化合物采用了溶剂重结晶的方法,即先对葱白提取物进行皂化去除其中的脂肪酸类成分,然后再采用丙酮分步重结晶的方法得到富集产物。最后富集产物为淡黄色固体,通过紫外分析方法对其含量测定,结果显示,其中甾体类成分含量可达到50%。
本实验还以葱白经过微波干燥后所得葱粉为原料,采用溶剂加热回流提取及有机溶剂顺次萃取的方法,制备得到正丁醇部位,并对该部位中的皂苷类成分含量进行了测定,经过萃取富集得到的该部位中皂苷类成分含量不低于20%。
药理研究方面,采用经典的给实验动物喂食高脂饲料造高脂血症模型的方法,结果显示该造模也是非常成功的。实验过程中通过检测各组高脂血症大鼠肝指数、血清血脂指标TC、TG、LDL-c、HDL-c,肝功能指标GOT、GPT,抗氧化损伤指标MDA、SOD和炎症因子CRP、IL-6等指标考察了各组药物降脂作用,初步探讨降脂作用发生机制。结果显示,正丁醇部位给药组和葱白提取物给药组在降低血脂TC、LDL-c水平和升高HDL-c方面有显著疗效,与高脂模型组比较P<0.01。不饱和脂肪酸组和甾体化合物组在该方面与模型组比较P<0.05,效果也比较明显。反应肝功能的几个指标中,除不饱和脂肪酸组血清GOT的水平与高脂模型组比较P<0.01外,其他指标各组药物与模型组比较结果均为P<0.05。抗氧化损伤的检测指标结果显示正丁醇部位抗氧化损伤的作用最强,葱白超临界萃取物其次。炎症因子考察中显示,不饱和脂肪酸组减轻炎症反应的作用最明显,血清CRP和IL-6与模型组比较分别为P<0.01和P<0.05。
本研究特色在于首次将分子蒸馏的方法引入到对葱白超临界CO_2萃取物中化合物的富集中来,结果也表明了该方法对葱白提取物中脂肪酸特别是不饱和脂肪酸有着良好的富集作用。本实验还首次将葱白中不同类型的化合物分别进行富集,然后进行药效学的研究,更明确地对葱白超临界CO_2萃取物中的药效学成分的确定提供了依据。最后还将对葱白的药效研究延伸到正丁醇部位这一极性部位,而且该部位对高脂血症大鼠有着显著的降脂作用,为更加全面研究应用葱白提供了一定的支持。
Fistular onion stalk is the fresh bulb of Allium fistulosum L. var. caespitosum Makino., belonging to the Liliaceae family. Almost Allium species are the source of potential medical agents, useful for the prevention or treatment of many diseases, including cancer, cardiovascular disease, antibacterial, and so on. Many of these biological effects are related to the volatile sulfur compounds, steroids, saponins, volatile oils, nitrogen compounds, polysaccharide and flavonoids[1-2]. Sulfur compounds, steroidal, unsaturated fatty acids compounds, and flavonoids were confirmation as the effective ingredient of the Fistular onion stalk supercritical CO_2 extract (the crude drug of BoXinTong) in the preliminary studies. Therefor this research mainly focuses on the enrichment and pharmacodynamics of effective composition in the extract of Fistular onion stalk, and expand study to the polarity composition from the Fistular onion stalk.
Molecular distillation which is suitable for enriching and separating the heat-sensitive compounds was used when enrich the volatile organic compounds in the Fistular onion stalk supercritical CO_2 extract, and GC-MS was adopted to analysis the fractions. In the appearance, some fractions is yellowish oil, while others are dark green solid, which is different from oily semi-solid belonging to the extract. The GC-MS result also show that this technology can enrich some compounds, especially fatty acid ingredients and pathalic acid ester composition, but did not reach the expected results in the part of the enrichment of sulfur compounds.
Using the classical urea complexation method to enrich the unsaturated fatty acid in the Fistular onion stalk, the GC result show that the content and kinds of saturated fatty acid in the enrichment product reduced obviously, while the content of unsaturated fatty acid increased significantly, which can achieve 80%.
The another major component in Fistular onion stalk is the steroidal compound, which was enriched by using the method of solvent recrystallizate. Removing of the fatty acid composition by saponification was the first step, and then applies pyruvate to carry out step-by-step recrystallizate to get enrichment product fainally that was the buff solid. The content of the steroidal can achieve 50% in this product by using UV to analysis.
The polar fractions in the Fistular onion stalk have also been studied. Fistular onion stalk was dried by microwave drying, and then adopted the method of solvent heating reflux extraction and organic solvent extraction followed to obtain the n-butanol parts, the content of saponins in which part is not lower than 20%.
In pharmacology, the hyperlipdemia model was made by using the classical method of feeding the experimental animals with fat-rich diet, and the results show the model was copied successfully. Liver index, the concentration of the TC and TG, LDL-c, HDL-c, in the serum, liver function index of GPT and GOT, antioxidant damage index of MDA and SOD, proinflammatory factor of CRP and IL-6 were tested to study the hypocholesterolaemia effect and investigate the mechanism of each drug preliminarily. The result showed that the treatment group of n-butanol parts and Fistular onion stalk extract have obvious curative effects in the part of lowering blood lipid level of TC, LDL-c as well as increasing LDH-c level compared with the model group(P<0.01), and in this respect the effect of Unsaturated fatty acid group and steroidal compounds group were also obvious compared with the model group(P<0.05). Unsaturated fatty acid group showed the best effect in the indicators associated with liver function and n-butanol parts group showed the strongest capability about antioxidant injury, while the Fistular onion stalk extract group was secondly. Unsaturated fatty acid group can reduce inflammation response obviously compared with model group when statistical the concentration of serum CRP(P<0.01) and IL-6(P<0.05).
The feature about this paper is that the method of molecular distillation introduced to enrich the compounds in the Fistular onion stalk supercritical CO_2 extract for the first time, and the results also show that this method is effective in enrichment especially to the unsaturated fatty acids. In this study, the different types of compounds in the Fistular onion stalk supercritical CO_2 extract were enriched respectively for the first time, and then the pharmacodynamic study of this compounds provided the more clearly basis to determinate Pharmacodynamic composition in the Fistular onion stalk supercritical CO_2 extract. Finally, the hypolipidemic effect of the butanol extract that is the polar fractions in the Fistular onion stalk was also studied, which provides the support for the comprehensve reseach and applications of the Fistular onion stalk.
本研究在考虑富集葱白超临界CO_2萃取物中挥发性成分时采用了一种适合于热敏性化合物富集分离的方法—分子蒸馏技术,对馏分采用了GC-MS分析方法。分子蒸馏所得馏分在外观性状上有的呈明显淡黄色油状,有的则是深绿色固体状,不同于葱白超临界提取物所呈现的油状半固体;GC-MS分析结果也显示了该技术对其中化合物的富集效果,特别是脂肪酸类成分和邻苯二甲酸酯类成分,但是没有达到预期的富集含硫化合物的效果。
采用经典的尿素包合富集不饱和脂肪酸的方法,对葱白超临界萃取中不饱和脂肪酸类成分进行富集,对富集产物的GC结果分析得出,富集产物中饱和脂肪酸类成分种类及含量均明显减少,不饱和脂肪酸类成分的含量则明显升高,能达到80%以上。
对葱白提取物中另一大类成分—甾体类化合物采用了溶剂重结晶的方法,即先对葱白提取物进行皂化去除其中的脂肪酸类成分,然后再采用丙酮分步重结晶的方法得到富集产物。最后富集产物为淡黄色固体,通过紫外分析方法对其含量测定,结果显示,其中甾体类成分含量可达到50%。
本实验还以葱白经过微波干燥后所得葱粉为原料,采用溶剂加热回流提取及有机溶剂顺次萃取的方法,制备得到正丁醇部位,并对该部位中的皂苷类成分含量进行了测定,经过萃取富集得到的该部位中皂苷类成分含量不低于20%。
药理研究方面,采用经典的给实验动物喂食高脂饲料造高脂血症模型的方法,结果显示该造模也是非常成功的。实验过程中通过检测各组高脂血症大鼠肝指数、血清血脂指标TC、TG、LDL-c、HDL-c,肝功能指标GOT、GPT,抗氧化损伤指标MDA、SOD和炎症因子CRP、IL-6等指标考察了各组药物降脂作用,初步探讨降脂作用发生机制。结果显示,正丁醇部位给药组和葱白提取物给药组在降低血脂TC、LDL-c水平和升高HDL-c方面有显著疗效,与高脂模型组比较P<0.01。不饱和脂肪酸组和甾体化合物组在该方面与模型组比较P<0.05,效果也比较明显。反应肝功能的几个指标中,除不饱和脂肪酸组血清GOT的水平与高脂模型组比较P<0.01外,其他指标各组药物与模型组比较结果均为P<0.05。抗氧化损伤的检测指标结果显示正丁醇部位抗氧化损伤的作用最强,葱白超临界萃取物其次。炎症因子考察中显示,不饱和脂肪酸组减轻炎症反应的作用最明显,血清CRP和IL-6与模型组比较分别为P<0.01和P<0.05。
本研究特色在于首次将分子蒸馏的方法引入到对葱白超临界CO_2萃取物中化合物的富集中来,结果也表明了该方法对葱白提取物中脂肪酸特别是不饱和脂肪酸有着良好的富集作用。本实验还首次将葱白中不同类型的化合物分别进行富集,然后进行药效学的研究,更明确地对葱白超临界CO_2萃取物中的药效学成分的确定提供了依据。最后还将对葱白的药效研究延伸到正丁醇部位这一极性部位,而且该部位对高脂血症大鼠有着显著的降脂作用,为更加全面研究应用葱白提供了一定的支持。
Fistular onion stalk is the fresh bulb of Allium fistulosum L. var. caespitosum Makino., belonging to the Liliaceae family. Almost Allium species are the source of potential medical agents, useful for the prevention or treatment of many diseases, including cancer, cardiovascular disease, antibacterial, and so on. Many of these biological effects are related to the volatile sulfur compounds, steroids, saponins, volatile oils, nitrogen compounds, polysaccharide and flavonoids[1-2]. Sulfur compounds, steroidal, unsaturated fatty acids compounds, and flavonoids were confirmation as the effective ingredient of the Fistular onion stalk supercritical CO_2 extract (the crude drug of BoXinTong) in the preliminary studies. Therefor this research mainly focuses on the enrichment and pharmacodynamics of effective composition in the extract of Fistular onion stalk, and expand study to the polarity composition from the Fistular onion stalk.
Molecular distillation which is suitable for enriching and separating the heat-sensitive compounds was used when enrich the volatile organic compounds in the Fistular onion stalk supercritical CO_2 extract, and GC-MS was adopted to analysis the fractions. In the appearance, some fractions is yellowish oil, while others are dark green solid, which is different from oily semi-solid belonging to the extract. The GC-MS result also show that this technology can enrich some compounds, especially fatty acid ingredients and pathalic acid ester composition, but did not reach the expected results in the part of the enrichment of sulfur compounds.
Using the classical urea complexation method to enrich the unsaturated fatty acid in the Fistular onion stalk, the GC result show that the content and kinds of saturated fatty acid in the enrichment product reduced obviously, while the content of unsaturated fatty acid increased significantly, which can achieve 80%.
The another major component in Fistular onion stalk is the steroidal compound, which was enriched by using the method of solvent recrystallizate. Removing of the fatty acid composition by saponification was the first step, and then applies pyruvate to carry out step-by-step recrystallizate to get enrichment product fainally that was the buff solid. The content of the steroidal can achieve 50% in this product by using UV to analysis.
The polar fractions in the Fistular onion stalk have also been studied. Fistular onion stalk was dried by microwave drying, and then adopted the method of solvent heating reflux extraction and organic solvent extraction followed to obtain the n-butanol parts, the content of saponins in which part is not lower than 20%.
In pharmacology, the hyperlipdemia model was made by using the classical method of feeding the experimental animals with fat-rich diet, and the results show the model was copied successfully. Liver index, the concentration of the TC and TG, LDL-c, HDL-c, in the serum, liver function index of GPT and GOT, antioxidant damage index of MDA and SOD, proinflammatory factor of CRP and IL-6 were tested to study the hypocholesterolaemia effect and investigate the mechanism of each drug preliminarily. The result showed that the treatment group of n-butanol parts and Fistular onion stalk extract have obvious curative effects in the part of lowering blood lipid level of TC, LDL-c as well as increasing LDH-c level compared with the model group(P<0.01), and in this respect the effect of Unsaturated fatty acid group and steroidal compounds group were also obvious compared with the model group(P<0.05). Unsaturated fatty acid group showed the best effect in the indicators associated with liver function and n-butanol parts group showed the strongest capability about antioxidant injury, while the Fistular onion stalk extract group was secondly. Unsaturated fatty acid group can reduce inflammation response obviously compared with model group when statistical the concentration of serum CRP(P<0.01) and IL-6(P<0.05).
The feature about this paper is that the method of molecular distillation introduced to enrich the compounds in the Fistular onion stalk supercritical CO_2 extract for the first time, and the results also show that this method is effective in enrichment especially to the unsaturated fatty acids. In this study, the different types of compounds in the Fistular onion stalk supercritical CO_2 extract were enriched respectively for the first time, and then the pharmacodynamic study of this compounds provided the more clearly basis to determinate Pharmacodynamic composition in the Fistular onion stalk supercritical CO_2 extract. Finally, the hypolipidemic effect of the butanol extract that is the polar fractions in the Fistular onion stalk was also studied, which provides the support for the comprehensve reseach and applications of the Fistular onion stalk.
引文
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