紫蛇尾（Ophiopholis mirabilis）皂苷的制备及其生物活性研究
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摘要
本论文以黄渤海交界处长岛海域的紫蛇尾作为实验材料,按照国标法测定了紫蛇尾主要营养成分,采用正交实验法优化了紫蛇尾皂苷的提取工艺,并对紫蛇尾皂苷理化性质进行了测定,研究了紫蛇尾皂苷的提取、分离纯化,以及紫蛇尾皂苷的部分生物活性,包括:抗氧化活性,抑菌活性以及抗肿瘤活性,为紫蛇尾资源的开发利用提供必要的理论依据。
通过对采自长岛海域的紫蛇尾(整体)的营养成分(水分、灰分、蛋白质、总糖、粗脂肪、脂肪酸、氨基酸、无机元素)分析,结果显示紫蛇尾水分含量4.09%,灰分含量25.71%,蛋白质含量18.27%,总糖含量3.53%,粗脂肪含量1.74%。紫蛇尾营养元素丰富,其中Ca含量最高,其次是Mg、Fe。脂肪酸中不饱和脂肪酸占72.61%,尤其是富含人体必需的多不饱和脂肪酸EPA和α亚麻酸,并且必需氨基酸指数(EAAI)为25.90。可以预见,紫蛇尾具有广阔的潜在利用价值,可进一步考虑开发成海洋功能食品或新型动物性饲料。
采用L9(34)正交实验法,以皂苷产率为指标,研究提取温度、提取时间、料液比、提取次数4个因素对提取工艺的影响。以正交实验的实验结果为条件建立多元线性模型,所得到的统计参数分别为R = 0.914,F = 10.585,并用所建立的线性模型预测两个实验条件下的结果,结果可信,说明紫蛇尾皂苷的产率和提取工艺相关因素有显著的相关关系。
采用传统的溶剂浸提法得到紫蛇尾皂苷粗品,测定紫蛇尾皂苷的常规理化性质,包括:颜色反应、熔点、溶血指数、紫外特征吸收光谱和红外光谱。依次用水、20%乙醇,40%乙醇、60%乙醇、80%乙醇经过大孔树脂AB-8除去多糖和无机盐,收集40%乙醇洗脱部分,反复经过硅胶柱层析、Sephadex LH-20凝胶柱层析和反向硅胶柱层析分离纯化样品。
采用微弱发光仪测定紫尾皂苷的抗氧化活性,结果表明,在一定浓度范围内,紫蛇尾皂苷抗氧化能力随着浓度增大呈蛇线性增强,紫蛇尾皂苷抑制过氧化氢的能力最强,抑制超氧阴离子的能力次之,抑制羟基的能力最弱。紫蛇尾皂苷具有显著的抗氧化活性,可以考虑将紫蛇尾皂苷开发成为新型的抗氧化药剂。
采用滤纸片法测定紫蛇尾皂苷的抑菌能力,采用改进的琼脂稀释法测定紫蛇尾皂苷的最低抑菌浓度,结果表明粗皂苷经过大孔树脂AB-8富集40%乙醇洗脱部位抑菌活性比粗皂苷显著增强。经过初步硅胶柱层析后,氯仿部分抑菌活性最强,其次是氯仿:甲醇:水= 9:1:0.1部分和氯仿:甲醇:水= 8:2:0.2部分。大肠杆菌、枯草杆菌的最低抑菌浓度(MIC)是0.0443 mg/mL,是粗皂苷经大孔树脂40%乙醇洗脱部分。金黄色葡萄球菌、产气杆菌、变形杆菌、青霉、毛霉、黄曲霉、啤酒酵母的最低抑菌浓度MIC值是0.0148 mg/mL,是氯仿条带1部分。各部分对面包酵母均无抑制作用。
采用SRB法测定紫蛇尾粗皂苷对人肺癌NCI-H460细胞和肝癌HepG2细胞的抑制能力,结果表明,给药剂1000mg/L时,抑制率分别达到38.03%和34.57%,但是给药剂量较低时抑制率下降幅度明显,没有显著的抑制效果。
In this paper, Ophiopholis mirabilis from the Changdao Archipelago was used as the experimental material. The major nutritional components of Ophiopholis mirabilis was determined.The extraction process of ophiurasaponin was optimized by the orthogonal experiment. The physical and chemical properties of ophiurasaponin were determined. We aiso studied on the extraction, purification and part of the biological activity of ophiurasaponin,including antioxidant activity, antimicrobial activity and antitumor activity.These studies provided the necessary theoretical basis for the development and utilization of Ophiopholis mirabilis.
We determined the major nutritional components of Ophiopholis mirabilis(overall), including moisture, ash, protein, total sugar,rude fat, fatty acids, amino acids and inorganic elements. The concentration of moisture, ash, protein, total sugar and rude fat were 4.09%, 25.71%, 18.27%, 3.53% and 1.74%, respectively. The concentration of Ca, Mg, Fe were high among the inorganic elements. Unsaturated fatty acids was accounted for 72.61% of total fatty acids.There were especially rich in essential polyunsaturated fatty acids,such as EPA andαlinolenic acid.And the essential amino acid index (EAAI) was 25.90. Ophiopholis mirabilis had broad potential value,and it can be further considered the development of new functional food or animal feed.
Orthogonal experiment has been used to investigate the extraction process of ophiurasaponin. The extraction temperature, extraction time, ratio of the material to water and times of extraction were selected as variable parameters. Their different effects on the yield of ophiurasaponin that extracted from Ophiopholis mirabilis were studied by the L9(34)experimental design method. In addition, a multiple linear regression(MLR) model, in which the yield of ophiurasaponin was as the dependent and the experiment conditions of the orthogonal experiment was as the independent, was firstly constructed to predict the yield of ophiurasaponin of two experiment conditions in the orthogonal experiment. The statistic parameters were R = 0.914, F = 10.585. And the MLR model was used to predict yields of ophiurasaponin of two experiments. The predicted results were in good agreement with the experimental values, indicating that the model can be used to predict the yield of ophiurasaponin.
The crude ophiurasaponin was obtained by the traditional solvent extraction.We determined the physical and chemical properties of ophiurasaponin , including color reaction, melting point, hemolytic index, UV absorption spectra and infrared spectra.Polysaccharides and inorganic salts were removed through the AB-8 macroporous resin followed by water, 20% ethanol, 40% ethanol, 60% ethanol, 80% ethanol. Some 40% ethanol elution was through silica gel column chromatography, Sephadex LH-20 silica gel column chromatography, and reverse column chromatography.
Ultra-Weak Luminescence Analyzer was used to detect the antioxidant activity of ophiurasaponin.The results showed that the antioxidation capabilities increased linearly with the increasing of the concentration of ophiurasaponin in a certain range. The result showed that ophiurasaponin from ophiopholis mirabilis had strongest inhibition to hydroxyl, weakest to hydroxyl radicals, and mediate to superoxide. Ophiurasaponin from ophiopholis mirabilis had significant antioxidant activity, it may be considered to develop into a new type of antioxidant agents.
Using circular filter paper method , the antibacterial activity of ophiurasaponin was detected. The minimum inhibitory concentration (MIC) of ophiurasaponin was determined by modified agar dilution method. The results showed that the antibacterial activity of 40% ethanol elution was enhanced than the crude ophiurasaponin. After the initial silica gel column chromatography, the part of the chloroform had the strongest antibacterial activity, followed by the part of chloroform: methanol: water = 9:1:0.1 and chloroform: methanol: water = 8:2:0.2 . The MIC of Escherichia coli and Bacillus subtilis was 0.0443mg/mL,and it was the 40% ethanol elution through macroporous resin.The MIC of Staphylococcus aureus, Aerobacter aerogenes, Proteus, Penicillium, Mucor, Aspergillus, Saccharomyces cerevisiae was 0.0148mg/mL,and it was the first strip of chloroform. It had no inhibitory effect on Saccharomyces cerevisiae Hansen.
Cytotoxic effect of human liver cancer cell NCI-H460 and human lung cancer cell HepG2 were assayed using SRB method in vitro.The results showed that the inhibition rates were 38.03% and 34.57% when the drug reached 1000mg/L.The inhibition rate decreased rapidly when the adminitration was low.In short, Ophiurasaponin had no significant inhibition of tumor cells.
通过对采自长岛海域的紫蛇尾(整体)的营养成分(水分、灰分、蛋白质、总糖、粗脂肪、脂肪酸、氨基酸、无机元素)分析,结果显示紫蛇尾水分含量4.09%,灰分含量25.71%,蛋白质含量18.27%,总糖含量3.53%,粗脂肪含量1.74%。紫蛇尾营养元素丰富,其中Ca含量最高,其次是Mg、Fe。脂肪酸中不饱和脂肪酸占72.61%,尤其是富含人体必需的多不饱和脂肪酸EPA和α亚麻酸,并且必需氨基酸指数(EAAI)为25.90。可以预见,紫蛇尾具有广阔的潜在利用价值,可进一步考虑开发成海洋功能食品或新型动物性饲料。
采用L9(34)正交实验法,以皂苷产率为指标,研究提取温度、提取时间、料液比、提取次数4个因素对提取工艺的影响。以正交实验的实验结果为条件建立多元线性模型,所得到的统计参数分别为R = 0.914,F = 10.585,并用所建立的线性模型预测两个实验条件下的结果,结果可信,说明紫蛇尾皂苷的产率和提取工艺相关因素有显著的相关关系。
采用传统的溶剂浸提法得到紫蛇尾皂苷粗品,测定紫蛇尾皂苷的常规理化性质,包括:颜色反应、熔点、溶血指数、紫外特征吸收光谱和红外光谱。依次用水、20%乙醇,40%乙醇、60%乙醇、80%乙醇经过大孔树脂AB-8除去多糖和无机盐,收集40%乙醇洗脱部分,反复经过硅胶柱层析、Sephadex LH-20凝胶柱层析和反向硅胶柱层析分离纯化样品。
采用微弱发光仪测定紫尾皂苷的抗氧化活性,结果表明,在一定浓度范围内,紫蛇尾皂苷抗氧化能力随着浓度增大呈蛇线性增强,紫蛇尾皂苷抑制过氧化氢的能力最强,抑制超氧阴离子的能力次之,抑制羟基的能力最弱。紫蛇尾皂苷具有显著的抗氧化活性,可以考虑将紫蛇尾皂苷开发成为新型的抗氧化药剂。
采用滤纸片法测定紫蛇尾皂苷的抑菌能力,采用改进的琼脂稀释法测定紫蛇尾皂苷的最低抑菌浓度,结果表明粗皂苷经过大孔树脂AB-8富集40%乙醇洗脱部位抑菌活性比粗皂苷显著增强。经过初步硅胶柱层析后,氯仿部分抑菌活性最强,其次是氯仿:甲醇:水= 9:1:0.1部分和氯仿:甲醇:水= 8:2:0.2部分。大肠杆菌、枯草杆菌的最低抑菌浓度(MIC)是0.0443 mg/mL,是粗皂苷经大孔树脂40%乙醇洗脱部分。金黄色葡萄球菌、产气杆菌、变形杆菌、青霉、毛霉、黄曲霉、啤酒酵母的最低抑菌浓度MIC值是0.0148 mg/mL,是氯仿条带1部分。各部分对面包酵母均无抑制作用。
采用SRB法测定紫蛇尾粗皂苷对人肺癌NCI-H460细胞和肝癌HepG2细胞的抑制能力,结果表明,给药剂1000mg/L时,抑制率分别达到38.03%和34.57%,但是给药剂量较低时抑制率下降幅度明显,没有显著的抑制效果。
In this paper, Ophiopholis mirabilis from the Changdao Archipelago was used as the experimental material. The major nutritional components of Ophiopholis mirabilis was determined.The extraction process of ophiurasaponin was optimized by the orthogonal experiment. The physical and chemical properties of ophiurasaponin were determined. We aiso studied on the extraction, purification and part of the biological activity of ophiurasaponin,including antioxidant activity, antimicrobial activity and antitumor activity.These studies provided the necessary theoretical basis for the development and utilization of Ophiopholis mirabilis.
We determined the major nutritional components of Ophiopholis mirabilis(overall), including moisture, ash, protein, total sugar,rude fat, fatty acids, amino acids and inorganic elements. The concentration of moisture, ash, protein, total sugar and rude fat were 4.09%, 25.71%, 18.27%, 3.53% and 1.74%, respectively. The concentration of Ca, Mg, Fe were high among the inorganic elements. Unsaturated fatty acids was accounted for 72.61% of total fatty acids.There were especially rich in essential polyunsaturated fatty acids,such as EPA andαlinolenic acid.And the essential amino acid index (EAAI) was 25.90. Ophiopholis mirabilis had broad potential value,and it can be further considered the development of new functional food or animal feed.
Orthogonal experiment has been used to investigate the extraction process of ophiurasaponin. The extraction temperature, extraction time, ratio of the material to water and times of extraction were selected as variable parameters. Their different effects on the yield of ophiurasaponin that extracted from Ophiopholis mirabilis were studied by the L9(34)experimental design method. In addition, a multiple linear regression(MLR) model, in which the yield of ophiurasaponin was as the dependent and the experiment conditions of the orthogonal experiment was as the independent, was firstly constructed to predict the yield of ophiurasaponin of two experiment conditions in the orthogonal experiment. The statistic parameters were R = 0.914, F = 10.585. And the MLR model was used to predict yields of ophiurasaponin of two experiments. The predicted results were in good agreement with the experimental values, indicating that the model can be used to predict the yield of ophiurasaponin.
The crude ophiurasaponin was obtained by the traditional solvent extraction.We determined the physical and chemical properties of ophiurasaponin , including color reaction, melting point, hemolytic index, UV absorption spectra and infrared spectra.Polysaccharides and inorganic salts were removed through the AB-8 macroporous resin followed by water, 20% ethanol, 40% ethanol, 60% ethanol, 80% ethanol. Some 40% ethanol elution was through silica gel column chromatography, Sephadex LH-20 silica gel column chromatography, and reverse column chromatography.
Ultra-Weak Luminescence Analyzer was used to detect the antioxidant activity of ophiurasaponin.The results showed that the antioxidation capabilities increased linearly with the increasing of the concentration of ophiurasaponin in a certain range. The result showed that ophiurasaponin from ophiopholis mirabilis had strongest inhibition to hydroxyl, weakest to hydroxyl radicals, and mediate to superoxide. Ophiurasaponin from ophiopholis mirabilis had significant antioxidant activity, it may be considered to develop into a new type of antioxidant agents.
Using circular filter paper method , the antibacterial activity of ophiurasaponin was detected. The minimum inhibitory concentration (MIC) of ophiurasaponin was determined by modified agar dilution method. The results showed that the antibacterial activity of 40% ethanol elution was enhanced than the crude ophiurasaponin. After the initial silica gel column chromatography, the part of the chloroform had the strongest antibacterial activity, followed by the part of chloroform: methanol: water = 9:1:0.1 and chloroform: methanol: water = 8:2:0.2 . The MIC of Escherichia coli and Bacillus subtilis was 0.0443mg/mL,and it was the 40% ethanol elution through macroporous resin.The MIC of Staphylococcus aureus, Aerobacter aerogenes, Proteus, Penicillium, Mucor, Aspergillus, Saccharomyces cerevisiae was 0.0148mg/mL,and it was the first strip of chloroform. It had no inhibitory effect on Saccharomyces cerevisiae Hansen.
Cytotoxic effect of human liver cancer cell NCI-H460 and human lung cancer cell HepG2 were assayed using SRB method in vitro.The results showed that the inhibition rates were 38.03% and 34.57% when the drug reached 1000mg/L.The inhibition rate decreased rapidly when the adminitration was low.In short, Ophiurasaponin had no significant inhibition of tumor cells.
引文
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