卵叶娃儿藤生物碱的提取及其活性功能研究
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摘要
卵叶娃儿藤是我国传统的有毒药用植物,在我国医学中很早就已经临床使用,现代医学研究发现,卵叶娃儿藤中的生物碱成分具有抗肿瘤作用,因此对其生物碱类化合物的研究具有重要的科学价值,为卵叶娃儿藤的药理活性,药效试验研究及天然保健和治疗药物的开发提供理论与物质基础。现将主要研究结果概述如下;
1.应用现代食品分析技术,对卵叶娃儿藤的基本理化成分进行了分析,结果为(g/100g,干重);水分含量为9.44;灰分含量为6.30;总糖含量为2.67;粗纤维含量为17.03;蛋白质含量为14.20;脂肪含量为11.32。对卵叶娃儿藤SFE-CO_2萃取物采用气-质连用(GC-MS)技术进行成分分析,主要成分为Hexadecanoicacid,ethyl ester(十六酸乙酯)、n-Hexadecanoic acid(十六酸)、Hexadecanoicacidmethyl ester(十六酸甲酯)、2-Nitro-4-(trifluoromethyl)phenol(2-硝基-4-(三氯甲基)-苯酚)、8-Methoxy-2-methylquinoline(8-甲氧基-2-甲基喹啉)、1-(12-methyl-1-oxotetradecyl)-1-Pyrrolidine(12-甲基-1-十四烷酰基)-吡咯烷、1-Phenyl-1-azaspiro[4,5]decane(1-苯基-1-氮杂螺[4,5]庚烷)等。
2.将溴酚蓝酸性染料比色法用于卵叶娃儿藤总生物碱的测定。通过对比色条件进行优化得到最佳条件;加入pH值为4.2的磷酸氢二钠-柠檬酸缓冲溶液4.0mL,溴酚蓝染料1.5mL,用12mL氯仿萃取。结果表明,该测定方法精密度高,在60min内显色稳定,操作简单,是一种快速测定卵叶娃儿藤总生物碱的有效方法。
3.对溶剂法、微波辅助萃取法和超临界流体萃取法提取卵叶娃儿藤总生物碱的工艺分别进行了研究。
确立了溶剂法最佳工艺条件为;乙醇浓度90%、固液比1;25、提取温度90℃、提取2次、每次2h。卵叶娃儿藤总生物碱的提取率可达0.386%。各提取因素对总生物碱提取率的影响程度由大到小依次为;温度＞乙醇浓度＞固液比＞提取次数。
首次将超临界CO_2萃取技术用于卵叶娃儿藤生物碱的提取研究。通过考察夹带剂(乙醇)浓度、萃取温度、压力和萃取时间等影响因素,得出总生物碱的最佳提取工艺参数分别为;80%乙醇作为夹带剂、在温度50℃、压力25MPa的条件下提取90分钟。超临界提取各因素对总生物碱提取率的影响大小顺序依次为;提取温度＞提取时间＞夹带剂浓度＞提取压力。卵叶娃儿藤总生物碱的提取率为0.249%,低于溶剂法提取,但杂质含量低。
确立了微波辅助提取法最佳工艺条件为;乙醇浓度90%、固液比1;20、提取温度90℃、提取2次、每次15分钟。卵叶娃儿藤总生物碱的提取率可达0.436%。各提取因素对总生物碱提取率的影响程度由大到小依次为;温度＞乙醇浓度＞固液比＞提取时间。微波辅助提取法产率高于溶剂法提取和超临界流体萃取。
4.利用现代分析技术,建立了LC-MS分析卵叶娃儿藤中生物碱成分的定性方法。为以后在没有标准对照品的情况下进行分析、研究及定性检测植物样品中的生物碱成分提供了科学依据。
5.采用硅胶柱色谱纯化了卵叶娃儿藤总生物碱。得到两个化合物,经过对其UV、IR、ESI-MS、EI-MS、~1H-NMR和~(13)C-NMR色谱数据进行分析,鉴定出两种新的化合物,均是首次从卵叶娃儿藤中得到。
6.采用MTT法分析化合物1和化合物2抑制肿瘤细胞增殖作用,细胞毒实验表明化合物1和化合物2作用48h可显著抑制体外培养的人宫颈癌Hela细胞的生长,剂量-效应呈正相关,半数抑制浓度(IC_(50))分别为98μg/mL和218μg/mL。结果表明化合物1和化合物2具有明显抑制Hela肿瘤细胞增殖的作用。采用圆滤纸片法考察卵叶娃儿藤总生物碱的抑菌作用,通过卵叶娃儿藤总生物碱对大肠埃希氏杆菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcusaureus)、枯草芽孢杆菌(Bacillus subtilis)、产气杆菌(Aerobacter aerogenus)、普通变形杆菌(Proteus vulgaris);啤酒酵母(Saccharomy cescerevisiae)的作用表明,卵叶娃儿藤总生物碱对这6种菌具有很好的抑制作用。
Tylophora ovata(Lindl.)Hook.ex Steud belongs to Asclepiadaceae.It was a Chinese traditional toxic medicine.The alkaloid components of Tylophora ovata (Lindl.)Hook.ex Steud possessed very strong antitumor activitiesaccording to literature.It has played an important role as an ancient medicine for centuries. Particularly today the medicinal use of Tylophora ovata(Lindl.)Hook.ex Steud was widespread and growing.A wide array of therapeutic effects of Tylophora ovata (Lindl.)Hook.ex Steud such as antimicrobial,anticancer,have been reported.It was well known that Tylophora ovata(Lindl.)Hook.ex Steud contains alkaloids such as tylophoridicine A,tylophorinine,O-methyl tylophorinidine and tylophorinidine, etc.All of them were said to have most of the medical effects of Tylophora ovata (Lindl.)Hook.ex Steud.The main studies and results in the paper were mentioned as the following;
1.The data of nutrition and contents of nutrients in Tylophora ovata(Lindl.) Hook.ex Steud.The results were;(g/100 g,dry mass)water,9.44,flash,6.30,total sugar,2.67;fibre,17.03,protein,14.02,fat,11.32.GC-MS was used to study many components in the extracts with SFE-CO_2 for the first time.The results showed that Hexadecanoic acid,ethyl ester,n-Hexadecanoic acid,Hexadecanoic acidmethyl ester, 2-Nitro-4-(trifluoromethyl)phenol,8-Methoxy-2-methylquinoline,1-(12-methyl-1-oxo tetradecyl)-1-Pyrrolidine and 1-Phenyl-1-azaspiro[4,5]decane were the main components.
2.The total alkaloids in Tylophora ovata(Lindl.)Hook.ex Steud.were determined by Bromophenol Blue acid dye colorimetry method.The suitable conditions for colorimetry were obtained;1.5 mL of Bromophenol Blue acid dye,2 mL of CHCL_3 and 4.0 mL of Na_2HPO_3-citric acid at pH 4.2.The color of the treated samples was stable within 60 min.The results showed that the developed method with high precision could be simple,correct and reproducible for the determination of the contents of total alkaloids in Tylophora ovata(Lindl.)Hook.ex Steud.
3.The solvent extraction technology,microwave assisted extraction technology and the CO_2 supercritical fluid extraction technology of total alkaloids in Tylophora ovata(Lindl.)Hook.ex Steud.were optimized by orthogonal experiments respectively.
The experiment results showed that the optimal experimental parameters of the solvent extraction technology are 90% ethanol,90℃of the extraction temperature, 1;25 of the ratio of solid/liquid,extracted for twice,2 h each time.The yield of the total alkaloids were extracted from Tylophora ovata(Lindl.)Hook.ex Steud exceeds 0.386% on the optimal condition.
SFE-CO_2 was used to extract alkaloid from Tylophora ovata(Lindl.)Hook.ex Steud for the first time.In this paper,the effect of working parameters on the extraction process was studied respectively,including extracting pressure,extracting temperature,extracting period and modifiers(alcohol)concentration for the ratio of total alkaloids.The optimal experimental parameters of the CO_2 supercritical fluid extraction technology were 50℃of the extraction temperature,25 MPa of the extraction pressure,80% of the ethanol as modifier,extraction for 90 min.The results showed that the yield of the total alkaloids extracted from Tylophora ovata(Lindl.)Hook.ex Steud was 0.249%,which was lower than the yield by the solvent extraction technology but the content of the impurities was also lower.
The experiment results showed that the optimal experimental parameters of microwave assisted extraction technology were 90% ethanol,90℃of the extraction temperature,1;20 of the ratio of solid/liquid,extracted twice,15 min each time.The yield of the total alkaloids were extracted from Tylophora ovata(Lindl.)Hook.ex Steud exceeds 0.436% on the optimal condition.,which was higher than the yield by the solvent extraction technology and the CO_2 supercritical fluid extraction technology.
4.A LC-MS method was established for qualitative analysis of the total alkaloids that extracted from Tylophora ovata(Lindl.)Hook.ex Steud It indicated that in the future the similar qualitative analysis and research can be carried out by the same method without standard sample.
5.Silica gel column chromatography was used to isolate and purify the components from Tylophora ovata(Lindl.)Hook.ex Steud.UV,IR,ESI-MS,EI-MS, ~1H-NMR and ~(13)C-NMR were used to identify these components.The results showed four sterols had been isolated and purified and their structures were identified as component(1)and component(2).All the Compounds were isolated from Tylophora ovata(Lindl.)Hook.ex Steud for the first time.
6.The growth of cells was inhibited when Hela cells cultured in vitro were treated with the component(1)and component(2)from Tylophora ovata (Lindl.)Hook.ex Steud.for 48 h.The dose and effect appeared positive correlation. 50% inhibiting concentration(IC_(50))were 98μg/mL and 218μg/mL by the MTT assay.Dosedependently component(1)and component(2)exhibited good antiproliferation activity to Hela.The bacteriostasis activity of the total alkaloids from Tylophora ovata(Lindl.)Hook.ex Steud.at different concentrations was evaluated by through the filter paper method.The results showed that the total alkaloids from Tylophora ovata(Lindl.)Hook.ex Steud exhibited good bacteriostasis activity to Escherichia coli,Staphylococcus aureus,Bacillus subtilis,Aerobacter aerogenus, Proteus vulgaris,Saccharomy cescerevisiae.
1.应用现代食品分析技术,对卵叶娃儿藤的基本理化成分进行了分析,结果为(g/100g,干重);水分含量为9.44;灰分含量为6.30;总糖含量为2.67;粗纤维含量为17.03;蛋白质含量为14.20;脂肪含量为11.32。对卵叶娃儿藤SFE-CO_2萃取物采用气-质连用(GC-MS)技术进行成分分析,主要成分为Hexadecanoicacid,ethyl ester(十六酸乙酯)、n-Hexadecanoic acid(十六酸)、Hexadecanoicacidmethyl ester(十六酸甲酯)、2-Nitro-4-(trifluoromethyl)phenol(2-硝基-4-(三氯甲基)-苯酚)、8-Methoxy-2-methylquinoline(8-甲氧基-2-甲基喹啉)、1-(12-methyl-1-oxotetradecyl)-1-Pyrrolidine(12-甲基-1-十四烷酰基)-吡咯烷、1-Phenyl-1-azaspiro[4,5]decane(1-苯基-1-氮杂螺[4,5]庚烷)等。
2.将溴酚蓝酸性染料比色法用于卵叶娃儿藤总生物碱的测定。通过对比色条件进行优化得到最佳条件;加入pH值为4.2的磷酸氢二钠-柠檬酸缓冲溶液4.0mL,溴酚蓝染料1.5mL,用12mL氯仿萃取。结果表明,该测定方法精密度高,在60min内显色稳定,操作简单,是一种快速测定卵叶娃儿藤总生物碱的有效方法。
3.对溶剂法、微波辅助萃取法和超临界流体萃取法提取卵叶娃儿藤总生物碱的工艺分别进行了研究。
确立了溶剂法最佳工艺条件为;乙醇浓度90%、固液比1;25、提取温度90℃、提取2次、每次2h。卵叶娃儿藤总生物碱的提取率可达0.386%。各提取因素对总生物碱提取率的影响程度由大到小依次为;温度＞乙醇浓度＞固液比＞提取次数。
首次将超临界CO_2萃取技术用于卵叶娃儿藤生物碱的提取研究。通过考察夹带剂(乙醇)浓度、萃取温度、压力和萃取时间等影响因素,得出总生物碱的最佳提取工艺参数分别为;80%乙醇作为夹带剂、在温度50℃、压力25MPa的条件下提取90分钟。超临界提取各因素对总生物碱提取率的影响大小顺序依次为;提取温度＞提取时间＞夹带剂浓度＞提取压力。卵叶娃儿藤总生物碱的提取率为0.249%,低于溶剂法提取,但杂质含量低。
确立了微波辅助提取法最佳工艺条件为;乙醇浓度90%、固液比1;20、提取温度90℃、提取2次、每次15分钟。卵叶娃儿藤总生物碱的提取率可达0.436%。各提取因素对总生物碱提取率的影响程度由大到小依次为;温度＞乙醇浓度＞固液比＞提取时间。微波辅助提取法产率高于溶剂法提取和超临界流体萃取。
4.利用现代分析技术,建立了LC-MS分析卵叶娃儿藤中生物碱成分的定性方法。为以后在没有标准对照品的情况下进行分析、研究及定性检测植物样品中的生物碱成分提供了科学依据。
5.采用硅胶柱色谱纯化了卵叶娃儿藤总生物碱。得到两个化合物,经过对其UV、IR、ESI-MS、EI-MS、~1H-NMR和~(13)C-NMR色谱数据进行分析,鉴定出两种新的化合物,均是首次从卵叶娃儿藤中得到。
6.采用MTT法分析化合物1和化合物2抑制肿瘤细胞增殖作用,细胞毒实验表明化合物1和化合物2作用48h可显著抑制体外培养的人宫颈癌Hela细胞的生长,剂量-效应呈正相关,半数抑制浓度(IC_(50))分别为98μg/mL和218μg/mL。结果表明化合物1和化合物2具有明显抑制Hela肿瘤细胞增殖的作用。采用圆滤纸片法考察卵叶娃儿藤总生物碱的抑菌作用,通过卵叶娃儿藤总生物碱对大肠埃希氏杆菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcusaureus)、枯草芽孢杆菌(Bacillus subtilis)、产气杆菌(Aerobacter aerogenus)、普通变形杆菌(Proteus vulgaris);啤酒酵母(Saccharomy cescerevisiae)的作用表明,卵叶娃儿藤总生物碱对这6种菌具有很好的抑制作用。
Tylophora ovata(Lindl.)Hook.ex Steud belongs to Asclepiadaceae.It was a Chinese traditional toxic medicine.The alkaloid components of Tylophora ovata (Lindl.)Hook.ex Steud possessed very strong antitumor activitiesaccording to literature.It has played an important role as an ancient medicine for centuries. Particularly today the medicinal use of Tylophora ovata(Lindl.)Hook.ex Steud was widespread and growing.A wide array of therapeutic effects of Tylophora ovata (Lindl.)Hook.ex Steud such as antimicrobial,anticancer,have been reported.It was well known that Tylophora ovata(Lindl.)Hook.ex Steud contains alkaloids such as tylophoridicine A,tylophorinine,O-methyl tylophorinidine and tylophorinidine, etc.All of them were said to have most of the medical effects of Tylophora ovata (Lindl.)Hook.ex Steud.The main studies and results in the paper were mentioned as the following;
1.The data of nutrition and contents of nutrients in Tylophora ovata(Lindl.) Hook.ex Steud.The results were;(g/100 g,dry mass)water,9.44,flash,6.30,total sugar,2.67;fibre,17.03,protein,14.02,fat,11.32.GC-MS was used to study many components in the extracts with SFE-CO_2 for the first time.The results showed that Hexadecanoic acid,ethyl ester,n-Hexadecanoic acid,Hexadecanoic acidmethyl ester, 2-Nitro-4-(trifluoromethyl)phenol,8-Methoxy-2-methylquinoline,1-(12-methyl-1-oxo tetradecyl)-1-Pyrrolidine and 1-Phenyl-1-azaspiro[4,5]decane were the main components.
2.The total alkaloids in Tylophora ovata(Lindl.)Hook.ex Steud.were determined by Bromophenol Blue acid dye colorimetry method.The suitable conditions for colorimetry were obtained;1.5 mL of Bromophenol Blue acid dye,2 mL of CHCL_3 and 4.0 mL of Na_2HPO_3-citric acid at pH 4.2.The color of the treated samples was stable within 60 min.The results showed that the developed method with high precision could be simple,correct and reproducible for the determination of the contents of total alkaloids in Tylophora ovata(Lindl.)Hook.ex Steud.
3.The solvent extraction technology,microwave assisted extraction technology and the CO_2 supercritical fluid extraction technology of total alkaloids in Tylophora ovata(Lindl.)Hook.ex Steud.were optimized by orthogonal experiments respectively.
The experiment results showed that the optimal experimental parameters of the solvent extraction technology are 90% ethanol,90℃of the extraction temperature, 1;25 of the ratio of solid/liquid,extracted for twice,2 h each time.The yield of the total alkaloids were extracted from Tylophora ovata(Lindl.)Hook.ex Steud exceeds 0.386% on the optimal condition.
SFE-CO_2 was used to extract alkaloid from Tylophora ovata(Lindl.)Hook.ex Steud for the first time.In this paper,the effect of working parameters on the extraction process was studied respectively,including extracting pressure,extracting temperature,extracting period and modifiers(alcohol)concentration for the ratio of total alkaloids.The optimal experimental parameters of the CO_2 supercritical fluid extraction technology were 50℃of the extraction temperature,25 MPa of the extraction pressure,80% of the ethanol as modifier,extraction for 90 min.The results showed that the yield of the total alkaloids extracted from Tylophora ovata(Lindl.)Hook.ex Steud was 0.249%,which was lower than the yield by the solvent extraction technology but the content of the impurities was also lower.
The experiment results showed that the optimal experimental parameters of microwave assisted extraction technology were 90% ethanol,90℃of the extraction temperature,1;20 of the ratio of solid/liquid,extracted twice,15 min each time.The yield of the total alkaloids were extracted from Tylophora ovata(Lindl.)Hook.ex Steud exceeds 0.436% on the optimal condition.,which was higher than the yield by the solvent extraction technology and the CO_2 supercritical fluid extraction technology.
4.A LC-MS method was established for qualitative analysis of the total alkaloids that extracted from Tylophora ovata(Lindl.)Hook.ex Steud It indicated that in the future the similar qualitative analysis and research can be carried out by the same method without standard sample.
5.Silica gel column chromatography was used to isolate and purify the components from Tylophora ovata(Lindl.)Hook.ex Steud.UV,IR,ESI-MS,EI-MS, ~1H-NMR and ~(13)C-NMR were used to identify these components.The results showed four sterols had been isolated and purified and their structures were identified as component(1)and component(2).All the Compounds were isolated from Tylophora ovata(Lindl.)Hook.ex Steud for the first time.
6.The growth of cells was inhibited when Hela cells cultured in vitro were treated with the component(1)and component(2)from Tylophora ovata (Lindl.)Hook.ex Steud.for 48 h.The dose and effect appeared positive correlation. 50% inhibiting concentration(IC_(50))were 98μg/mL and 218μg/mL by the MTT assay.Dosedependently component(1)and component(2)exhibited good antiproliferation activity to Hela.The bacteriostasis activity of the total alkaloids from Tylophora ovata(Lindl.)Hook.ex Steud.at different concentrations was evaluated by through the filter paper method.The results showed that the total alkaloids from Tylophora ovata(Lindl.)Hook.ex Steud exhibited good bacteriostasis activity to Escherichia coli,Staphylococcus aureus,Bacillus subtilis,Aerobacter aerogenus, Proteus vulgaris,Saccharomy cescerevisiae.
引文
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