荷叶生物碱的提取分离纯化
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摘要
荷叶为卫生部批准的“可用于保健食品的物品”,现代临床广泛将荷叶应用于肥胖症及高血脂症,并取得了较好的疗效。药理研究显示荷叶生物碱类具有明显降脂减肥、抑制胆固醇、抗有丝分裂、抑菌和止痉挛等药理活性。目前有关荷叶生物碱的分离和制备的研究较少,本文主要报道了荷叶生物碱的提取工艺的优化、分离纯化进行了一系列的研究,为荷叶生物碱的进一步深入研究奠定了基础。现将主要研究结果报告如下:
一荷叶生物碱的提取工艺
采用热水提取法从荷叶中提取生物碱,对影响荷叶生物碱提取的四个主要因素――提取时间、提取温度、pH、固液比,在进行单因素实验的基础上,设计了L_(16)(4~4)正交实验,以生物碱的提取率为指标,并通过极差、方差分析对这些因素进行了统计分析,并用人工神经网络对生物碱的提取工艺进行优化。确立了荷叶生物碱热水提取工艺:pH值2.5,提取温度85℃,提取时间1.75h,固液比1:35。
二荷叶生物碱薄层层析鉴别
荷叶生物碱在硅胶薄层板上用溶剂展开体系三氯甲烷:甲醇:二乙胺(90:9:1,V/V)展开,使荷叶中生物碱达到了较好的分离效果,此条件也可用于检测分离纯化效果。
三高速逆流色谱法(HSCCC)分离纯化荷叶中的生物碱
首次采用HSCCC成功分离了荷叶中的生物碱化合物。选择三氯甲烷:甲醇:pH4.00的水(4:3:2,v/v)为溶剂系统进行分离纯化,得到了三种组分,含量分别为94.13%、89.68%、92.25%。四高效液相色谱法(HPLC)分离纯化及定量分析
建立了荷叶碱的高效液相色谱法(HPLC)定量分析,并利用制备型高效液相色谱对荷叶生物碱进行单体分离,分别得到了含量为98.78%、97.63%、99.99%的三种单体生物碱化合物。
研究表明,采用人工神经网络和传统的正交实验相结合的分析方法建立了荷叶生物碱的提取工艺。薄层层析法对荷叶碱粗提物进行鉴别,高速逆流色谱和高效液相色谱两种不同方法都成功制备荷叶生物碱单体。
A traditional Chinese herb called lotus leaf is approved as‘material that can be used in healthy foods’by Ministry of Health, P. R. China.
In the present study, there were few paper about the separation and preparation of lotus leaf alkaloids. In this paper, the technology for extraction of alkaloid from lotus leaf was studied. Quantitative analysis of nuciferine by HPLC was established here. Two methods-high speed countercurrent chromatography and high performance liquid chromatography for separation and purification of alkaloids were applied. The main results were described as follows:
1. The technology for extraction of alkaloid
The technology for extraction of alkaloid from lotus leaf was studied. Through combination of artificial neural network (ANN) with traditional orthogonal design, a new method for analyzing, processing and optimizing the test data was put forward. The optimum condition for extraction was summarized, namely, pH value 2.5,extracting temp. 85℃, duration of extraction 1.75h and solid to liquid ratio 1:35. The satisfactory results confirmed the reasonableness and practicability.
2. Alkaloids of lotus leaf were identified by TLC
Using thin layer chromatography(TLC) for identifying the alkaloid in the extract demonstrated that the kinds of alkaloid changed with the extract solvent. Chloroform-methanol- lignocaine amine (90:9:1)mixture as a mobile phase. The nuciferine in the lotus leaf were found.
3. Separation and purification of alkaloids from Lotus Leaf by high speed countercurrent chromatography
High speed countercurrent chromatography (HSCCC) was applied to the separation of alkaloids from Lotus Leaf the first time. High speed countercurrent chromatography with a two-phase solvent system composed of Chloroform:methanol:pH4.0 water(4:3:2,volume ratio). Five collections were separated. The results were verified by TLC, in which three components exhibited orange-red colour with bismuth potassium iodide reagent. HSCCC is a convenient, feasible and effective technique for the separation and purification of active components in Chinese herbal medicines.
4. Separation and purification alkaloids from Lotus Leaf by high performance liquid chromatography. And found the method of HPLC analytical of nuciferine.
A method using preparative high performance liquid chromatography (HPLC) to isolate four individual alkaloids from lotus leaf was investigated. A preparative C18 column was employed in the procedure. First, by isocratic elution with methanol:water:lignocaine amine (75:25:0.0125,V/V) as mobile phase at a flow rate of 8 ml/min, two mixture (part1, part2) were obtained. Then, by isocratic elution with acetonitrile:water:lignocaine amin(e55:45:0.0125,60:60:0.0125)as mobile phase at a flow rate of 8 ml/min for part1 and part2, respectively, three substance were obtained. HPLC analytical results showed that the purity of each alkaloid was over 97%. Under the chromatographic condition, every peak was completely baseline separated.
In a word, the technology for extraction of alkaloid from lotus leaf was founded. High speed countercurrent chromatography (HSCCC) and performance liquid chromatography (HPLC) are two efficient methods for separation and preparation of alkaloid.
一荷叶生物碱的提取工艺
采用热水提取法从荷叶中提取生物碱,对影响荷叶生物碱提取的四个主要因素――提取时间、提取温度、pH、固液比,在进行单因素实验的基础上,设计了L_(16)(4~4)正交实验,以生物碱的提取率为指标,并通过极差、方差分析对这些因素进行了统计分析,并用人工神经网络对生物碱的提取工艺进行优化。确立了荷叶生物碱热水提取工艺:pH值2.5,提取温度85℃,提取时间1.75h,固液比1:35。
二荷叶生物碱薄层层析鉴别
荷叶生物碱在硅胶薄层板上用溶剂展开体系三氯甲烷:甲醇:二乙胺(90:9:1,V/V)展开,使荷叶中生物碱达到了较好的分离效果,此条件也可用于检测分离纯化效果。
三高速逆流色谱法(HSCCC)分离纯化荷叶中的生物碱
首次采用HSCCC成功分离了荷叶中的生物碱化合物。选择三氯甲烷:甲醇:pH4.00的水(4:3:2,v/v)为溶剂系统进行分离纯化,得到了三种组分,含量分别为94.13%、89.68%、92.25%。四高效液相色谱法(HPLC)分离纯化及定量分析
建立了荷叶碱的高效液相色谱法(HPLC)定量分析,并利用制备型高效液相色谱对荷叶生物碱进行单体分离,分别得到了含量为98.78%、97.63%、99.99%的三种单体生物碱化合物。
研究表明,采用人工神经网络和传统的正交实验相结合的分析方法建立了荷叶生物碱的提取工艺。薄层层析法对荷叶碱粗提物进行鉴别,高速逆流色谱和高效液相色谱两种不同方法都成功制备荷叶生物碱单体。
A traditional Chinese herb called lotus leaf is approved as‘material that can be used in healthy foods’by Ministry of Health, P. R. China.
In the present study, there were few paper about the separation and preparation of lotus leaf alkaloids. In this paper, the technology for extraction of alkaloid from lotus leaf was studied. Quantitative analysis of nuciferine by HPLC was established here. Two methods-high speed countercurrent chromatography and high performance liquid chromatography for separation and purification of alkaloids were applied. The main results were described as follows:
1. The technology for extraction of alkaloid
The technology for extraction of alkaloid from lotus leaf was studied. Through combination of artificial neural network (ANN) with traditional orthogonal design, a new method for analyzing, processing and optimizing the test data was put forward. The optimum condition for extraction was summarized, namely, pH value 2.5,extracting temp. 85℃, duration of extraction 1.75h and solid to liquid ratio 1:35. The satisfactory results confirmed the reasonableness and practicability.
2. Alkaloids of lotus leaf were identified by TLC
Using thin layer chromatography(TLC) for identifying the alkaloid in the extract demonstrated that the kinds of alkaloid changed with the extract solvent. Chloroform-methanol- lignocaine amine (90:9:1)mixture as a mobile phase. The nuciferine in the lotus leaf were found.
3. Separation and purification of alkaloids from Lotus Leaf by high speed countercurrent chromatography
High speed countercurrent chromatography (HSCCC) was applied to the separation of alkaloids from Lotus Leaf the first time. High speed countercurrent chromatography with a two-phase solvent system composed of Chloroform:methanol:pH4.0 water(4:3:2,volume ratio). Five collections were separated. The results were verified by TLC, in which three components exhibited orange-red colour with bismuth potassium iodide reagent. HSCCC is a convenient, feasible and effective technique for the separation and purification of active components in Chinese herbal medicines.
4. Separation and purification alkaloids from Lotus Leaf by high performance liquid chromatography. And found the method of HPLC analytical of nuciferine.
A method using preparative high performance liquid chromatography (HPLC) to isolate four individual alkaloids from lotus leaf was investigated. A preparative C18 column was employed in the procedure. First, by isocratic elution with methanol:water:lignocaine amine (75:25:0.0125,V/V) as mobile phase at a flow rate of 8 ml/min, two mixture (part1, part2) were obtained. Then, by isocratic elution with acetonitrile:water:lignocaine amin(e55:45:0.0125,60:60:0.0125)as mobile phase at a flow rate of 8 ml/min for part1 and part2, respectively, three substance were obtained. HPLC analytical results showed that the purity of each alkaloid was over 97%. Under the chromatographic condition, every peak was completely baseline separated.
In a word, the technology for extraction of alkaloid from lotus leaf was founded. High speed countercurrent chromatography (HSCCC) and performance liquid chromatography (HPLC) are two efficient methods for separation and preparation of alkaloid.
引文
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[2] 冯崇廉.我国加入 WTO 后中药产业的发展契机.中药材,2001,24(3):206-207
[3] FAN Ji-shan(范继善).The Additive of Practical Food(实用食品添加剂).Tianjing(天津):Tianjing Science and Technology Press(天津科学技术出版社),1993.10
[4] TAO Bo,CHEN Mu-ying(陶波,陈慕英).The general of study on lotus leaf as medicinal material(荷叶药用研究概况).Chinese Medication Information(中医药信息),2001,18(2):4
[5] JI Li-lian(纪丽莲).Study on extraction and activity of bacteriostatic ingredient in lotus leaf(荷叶中抑菌成分的提取及其抑菌活性的研究).Food Science(食品科学),1998,8:64
[6] 卢秀华,陈海波.中药现代化面临的机遇和挑战.实用医药杂志,2004,11(10):2029
[7] 余秋颖.中药现代化的研究与发展.沈阳医学院学报,2003,5(2):134-135
[8] 国家药典委员会编制.中华人民共和国药典.化学工业出版社,2005 版
[9] 王德培,赵谋明,吴晖.荷叶蜂蜜保健饮料工艺研究.食品与机械,2005,21(3).57-60
[10] 唐裕芳,张妙玲,刘忠义,曾虹燕.荷叶提取物对肉类防腐保鲜应用的展望.肉类工业, 2004(11).44-45
[11] 刘成梅,游海主编.天然产物有效成分的分离与应用(第一版).化学工业出版社,2003,pp.80-169
[12] 吴立军主编.天然药物化学(第四版).人民卫生出版社,2003,pp.352-395
[13] 徐剑.复方荷叶冲剂降脂作用研究中成药,1989 1(11):24～25
[14] 钱彦方.轻健胶囊的降脂减肥动物实验研究.实用中西医结合杂志,1994 7(10):592～595
[15] 万阜昌,黄道斋.荷丹片的降血脂作用中国实验方剂学杂志,1996.2(1):19～21
[16] 龚康敏等.荷叶合剂时小鼠及高脂血症大鼠血脂、体重及血液流变学的影响.中国中医药科技,1998 5(5):294～295
[17] 吴小南等.降脂中药煎剂对实验大鼠减肥降血脂作用的观察.中国中医药信息,1998 5(7):2O～21
[18] 唐裕芳,张妙玲,刘忠义,邓孝平.荷叶生物碱的提取及其抑菌活性研究. 广州食品工业科技,2004,20(2).51-52,39
[19] 李志诚等.荷叶化学成分研究.中草药,1996,27(增刊):50～52
[20] 聂 波,佟丽华,于 敏.荷叶化学成分的分析分离[J].黑龙江中药科学,2003,26(8):54
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