蜈蚣提取物制备及药理活性研究
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摘要
蜈蚣是重要的中药材,传统以干燥蜈蚣全虫入药,具有抗惊厥、抗肿瘤、抗炎镇痛、抗衰老等多种生理功能。本论文以新鲜蜈蚣为原料,研究了蜈蚣醇提物的最佳提取条件,并且对蜈蚣醇提物及水提物在抗癫痫、抑菌两方面的活性进行研究比较,此外对醇提物、水提物提取时剩余沉淀部分采用碱提、水提两种方法进行粗多糖的提取分离,并得出水提法最佳提取条件。主要结论如下:
(1)以乙醇浓度、提取时间、固液比三个因素为影响因子设计正交实验。综合考虑各个因素对醇提物得率、蛋白质含量两个指标的影响,最终确定了蜈蚣醇提物制备条件:乙醇浓度为60%,提取时间为2.5h,固液比为1:40。
(2)蜈蚣水提物能够明显延长硝酸士的宁诱发小鼠的惊厥潜伏期,同时降低了死亡率,提高抗惊率。潜伏期高达(372±146.9)s,抗惊率达到50%,与对照组比较均有显著性差异(P＜0.01)。蜈蚣醇提物没有明显抗惊厥作用。
(3)采用滤纸片、平板孔穴扩散法,测定蜈蚣醇提物及水提物对大肠杆菌、金黄色葡萄球菌、铜绿假单胞菌、枯草杆菌黑色变种芽孢、白色念珠球菌、黑曲霉菌几种常见菌的抑制作用。结果表明蜈蚣醇提物、水提物对各供试细菌均有不同程度的抑制作用,对真菌抑制作用较弱,且醇提物抑菌效果明显强于水提物。用二倍稀释法确定醇提物对大肠杆菌、金黄色葡萄球菌、铜绿假单胞菌的最小抑菌浓度,分别为250mg/mL、125mg/mL、62.5mg/mL。
(4)采用热水浸提、碱液提取两种不同的方法对蜈蚣粗多糖进行提取,粗多糖的含量分别为8.87%、8.45%。以提取温度、提取时间、固液比为影响因子作正交实验设计,确定了热水浸提的最佳实验条件:提取温度为95℃,提取时间为4h,固液比为1:10。
Centipede is an important Chinese herbal medicine,traditional using the dry full-worm,and it has many physiological functions,such as anticonvulsant, anti-tumor and anti-inflammatory analgesic,anti-aging and so on.The extractions from fresh centipede were systematicalls studied in this dissertation.Meanwhile,investigation was made to choose the best conditions of alcohol extraction from centipede,alcohol and water extracts of centipede were compared to study on pharmacological effect.Two methods were employed in extraction process of polysaccharides from the materials in order to select the extraction.Main conclusions are as follows:
(1)Orthogonal experimental design was adopted to observe effect of ethanol concentration,extraction time and solid-liquid ratio,with the amount of the alcohol extracts and the content of protein as the detecting index.In the end determine the preparation conditions of centipede alcohol extracts as follow:the ethanol concentration 60%,time 2.5h,solid-liquid ratio 1:40.
(2)The water extract could prolong the convulsionary delitescence of mice caused by strychnine nitrate,increase the anticonvulsant rate and decrease mortality.The latent period of convulsion was reached(372±146.9) s,the anticonvulsant rate was 50%,which had signified statistic difference compared with control group(P<0.01).The alcohol extract of centipede did not have anticonvulsive effect.
(3)Escherichia coli,Staphylococcus aureus,Bacillus subtilis var.niger spore,Candida albicans,Aspergillus niger and Pseudomonas Aeruginosa were chosen to investigate the antibacterial activity of the alcohol and water extracts by stiletto and filter-disk diffusion method.The results showed that antibacterial activity was observed both of alcohol and water extracts,and the former performed better than the later.The minimal inhibition concentration (MIC)of ethanol extracts was assayed by use of agar dilution method,The MIC of ethanol extracts to Escherichia coli,Staphylococcus,Pseudomonas were 250 mg/mL,125mg/mL,62.5mg/mL.
(4)Crud polysaccharide was extracted from centipede using hot water extraction and alkaline-solution,the content of crud polysaccharide respective is 8.87%,8.45%.As result,hot water extraction is best method.The optimum conditions for the water extraction were obtained by using orthogonal design. The optimum conditions are:extraction temperature 95℃,time 4 h, solid-liquid 1:10.
(1)以乙醇浓度、提取时间、固液比三个因素为影响因子设计正交实验。综合考虑各个因素对醇提物得率、蛋白质含量两个指标的影响,最终确定了蜈蚣醇提物制备条件:乙醇浓度为60%,提取时间为2.5h,固液比为1:40。
(2)蜈蚣水提物能够明显延长硝酸士的宁诱发小鼠的惊厥潜伏期,同时降低了死亡率,提高抗惊率。潜伏期高达(372±146.9)s,抗惊率达到50%,与对照组比较均有显著性差异(P＜0.01)。蜈蚣醇提物没有明显抗惊厥作用。
(3)采用滤纸片、平板孔穴扩散法,测定蜈蚣醇提物及水提物对大肠杆菌、金黄色葡萄球菌、铜绿假单胞菌、枯草杆菌黑色变种芽孢、白色念珠球菌、黑曲霉菌几种常见菌的抑制作用。结果表明蜈蚣醇提物、水提物对各供试细菌均有不同程度的抑制作用,对真菌抑制作用较弱,且醇提物抑菌效果明显强于水提物。用二倍稀释法确定醇提物对大肠杆菌、金黄色葡萄球菌、铜绿假单胞菌的最小抑菌浓度,分别为250mg/mL、125mg/mL、62.5mg/mL。
(4)采用热水浸提、碱液提取两种不同的方法对蜈蚣粗多糖进行提取,粗多糖的含量分别为8.87%、8.45%。以提取温度、提取时间、固液比为影响因子作正交实验设计,确定了热水浸提的最佳实验条件:提取温度为95℃,提取时间为4h,固液比为1:10。
Centipede is an important Chinese herbal medicine,traditional using the dry full-worm,and it has many physiological functions,such as anticonvulsant, anti-tumor and anti-inflammatory analgesic,anti-aging and so on.The extractions from fresh centipede were systematicalls studied in this dissertation.Meanwhile,investigation was made to choose the best conditions of alcohol extraction from centipede,alcohol and water extracts of centipede were compared to study on pharmacological effect.Two methods were employed in extraction process of polysaccharides from the materials in order to select the extraction.Main conclusions are as follows:
(1)Orthogonal experimental design was adopted to observe effect of ethanol concentration,extraction time and solid-liquid ratio,with the amount of the alcohol extracts and the content of protein as the detecting index.In the end determine the preparation conditions of centipede alcohol extracts as follow:the ethanol concentration 60%,time 2.5h,solid-liquid ratio 1:40.
(2)The water extract could prolong the convulsionary delitescence of mice caused by strychnine nitrate,increase the anticonvulsant rate and decrease mortality.The latent period of convulsion was reached(372±146.9) s,the anticonvulsant rate was 50%,which had signified statistic difference compared with control group(P<0.01).The alcohol extract of centipede did not have anticonvulsive effect.
(3)Escherichia coli,Staphylococcus aureus,Bacillus subtilis var.niger spore,Candida albicans,Aspergillus niger and Pseudomonas Aeruginosa were chosen to investigate the antibacterial activity of the alcohol and water extracts by stiletto and filter-disk diffusion method.The results showed that antibacterial activity was observed both of alcohol and water extracts,and the former performed better than the later.The minimal inhibition concentration (MIC)of ethanol extracts was assayed by use of agar dilution method,The MIC of ethanol extracts to Escherichia coli,Staphylococcus,Pseudomonas were 250 mg/mL,125mg/mL,62.5mg/mL.
(4)Crud polysaccharide was extracted from centipede using hot water extraction and alkaline-solution,the content of crud polysaccharide respective is 8.87%,8.45%.As result,hot water extraction is best method.The optimum conditions for the water extraction were obtained by using orthogonal design. The optimum conditions are:extraction temperature 95℃,time 4 h, solid-liquid 1:10.
引文
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