药食两用植物玛咖（Lepidium meyenii）的功效物质研究
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摘要
作为食物或药用植物,玛咖(Lepidium meyennii Walp.)几千年前就在秘鲁高原上广泛应用。玛咖具有很高的营养价值,而且玛咖生物碱、芥子油苷、异硫氰酸酯、玛咖酰胺和甾醇等可能是它在提高生育力、促进性欲、适应原性、促进荷尔蒙代谢合成及其影响荷尔蒙平衡作用的功效物质。但这些玛咖可能的功效物质与功效机理的对应关系还很模糊,主要原因之一就是这些物质在提取分离、组成成分、化学结构、含量检测等方面还缺乏深入的研究。
本论文围绕玛咖功效物质基础研究开展了系统的研究,得到如下研究结果:
玛咖营养成分研究中验证了玛咖具有高蛋白(9.22 %)、高支链氨基酸(20.14 %总氨基酸)、高果糖(5.32 %)等营养特征,并首次发现了玛咖中含有少量的牛磺酸成分(0.08 %)以及具有抗氧化活性的多糖成分(6.85 %)。通过预试鉴别发现玛咖中含有非常广泛的次生代谢物,其中非水溶性生物碱、甾醇、有机酸、挥发油含量较为突出。
玛咖干根中总生物碱含量可以采用滴定法快速检测(含量0.82 %),芥子油苷可以采用改良的氯化钯比色法快速检测,玛咖干根中芥子油苷含量为含量1.92 %,而商品化干粉中却仅为0.13 %。通过芥子油苷溶出-降解动力学曲线可以发现在传统的水提或者稀醇提取方法中,玛咖中芥子油苷都有一定程度的损失,但通过灭酶等工艺调整可以减小损失。
玛咖总生物碱提取分离工艺研究中发现,玛咖总生物碱的最佳薄层色谱分离条件为正丁醇-甲醇-水-乙二胺(38:10:12:1 v/v),而脱色可以采用D152大孔丙烯酸系弱酸性阳离子交换树脂,能有效脱除色素并得到88 %以上的生物碱回收率;玛咖生物碱J5采用液质联用分析发现其中含三种咪唑类的生物碱(lepidiline A、B、C),其中lepidiline C是新物质,而且发现这三类物质在心脏功能调节上具有研究潜力。
几种在外观和功效上相似的植物材料,包括玛咖(秘鲁产玛咖和云南会泽产玛咖)、萝卜、人参和西洋参,它们的生物碱提取物进行了红外光谱和薄层色谱分析,挥发油提取物则采用气质联用检测。通过比较这些材料的谱图,可以发现玛咖功效成分提取物具有独特的红外及其气相特征。其中红外光谱表现出酰胺基团、共轭双键以及苯基结构,气相色谱则表明玛咖挥发油以苯乙腈、苯甲醛、间甲氧基苯乙腈为其主要特征成分。这些红外指纹图谱、气相指纹图谱的建立可以用于玛咖及其玛咖产品的鉴别。
急性毒性学研究表明,玛咖干粉对小鼠的半数致死剂量LD50>10.00 g/kg.BW。Ames实验、小鼠骨髓微核实验与精子畸形实验均为阴性。在慢性毒性实验中(90天),除中、高剂量组雌性大鼠的AST、高剂量组雌性大鼠TCH显著低于对照组外,其它指标均无显著性差异。所有结果均表明玛咖具有较高的安全性。
抗疲劳实验中发现,玛咖能显著降低疲劳小鼠血清尿素的产生,增加小鼠肝糖原的储备量,对小鼠运动中血乳酸上升具有显著的抑制作用,并对小鼠运动后血乳酸消除具有显著的促进作用。免疫功效研究中,玛咖可以增强细胞免疫,但对体液免疫功能、非特异性免疫以及NK细胞活性都没有显著影响,但玛咖与西洋参总皂甙复配物能起到显著的增强免疫功效,而且玛咖在其中具有增效作用。
The Maca plant (Lepidium meyennii Walp.) has several thousend years history of use as a food and medicine crop in the highlands of Peru. Maca is a highly nutritious food source, and Maca alkaloids, glucosinolates, isothiocyanates, macamides and steroids are probably responsible for its aptitude to act as a fertility enhancer, aphrodisiac, adaptogen, anabolic and to influence hormonal balance. However, the Correspond Relation between the active material basis and functional mechanism are still quite indistinct, and it is widely agreed that one of the main reasons for this is the deficiency of in-depth study on the extraction, separation, chemical composition, chemical structure and content determining of the active material basis.
The study in my dissertation was carried out surrounding the active material basis of maca. The study result is:
The characteristic of high nutrition of Maca, such as high protein(9.22 %), high branch chain amino acid(20.14% total aa) and high fructose(5.32 %) , was confirmed in the study on nutritional components, and the small amount taurine(0.08 %) and the polysaccharide(6.85 %) with antioxidative activity were found for the first time. Many secondary metabolites of Maca were found using preparatory tests for composition identifying, and the contents of the non-water-solubility alkaloids, sterols, organic acids and essential oils were remarkable.
The content of total alkaloids in dried Maca (0.82 %) root can be determined using the titration method rapidly, and the content of glucosinolates in dried Maca root (1.92 %) and in commercial powder of Maca (0.13 %) can be determined using the palladium chloride method rapidly. From the dissolution-degradation kinetics of glucosinolates in Maca, it was found that glucosinolates were losing partially in the tradition process of extract by water or dilute ethanol-soluble, but the losing could cut down by enzyme inactivation and other technical-adjusting measures.
TLC solvent system for separating Maca total alkaloids is n-Butanol : ethanol : water : ethylenediamine(38:10:12:1 v/v), and the decoloration by D152 macroreticular weak acidic acrylic acid resin can wipe off the coloring matter effectually and the recuperation rate obtained is higher than 88 %. Three imidazole alkaloid (lepidiline A, B, C) in J5 was affirmed by LC-MS. Furthermore, lepidiline C is a new compound found in Maca, and lepidilines have the potential research on heart function regulation.
Natural plants with similar appearance or medicinal effects, including Maca(peru Maca and Huizhe Maca), radish, oriental ginseng and American ginseng were investigated. Their alkaloid extracts of the hypogeal parts were analyzed by FTIR and TLC. The essential oils were analyzed by GC/MS. Through comparison of the characteristics of their spectra and chromatograms, it was found that the functional ingredient extracts of Maca have unique FTIR and GC behaviors. The secondary amide group, conjugated carbon-carbon double bond and phenyl structure in FTIR and the characteristic peaks produced by the major essential oil components (Phenylacetonitrile, Benzaldehyde, 3-Methoxyphenylacetonitrile, etc) in GC/MS are distinct. These behaviors can be applied to the identification of Maca or Maca products in the market.
The mice′s LD50 of dried Maca root was found higher than 10.00 g/kg.BW in acute toxicity test, and the results of Ames test, mice bone marrow micronucleus test and mice sperm malformation test are of negative. The long term toxicity test indicated that the level of female AST in medium and high dose groups, TCH in high dose groups significantly decreased than in the control group, however the changes of other indexes were not significantly different between the Maca groups and control group (P>0.05). All results indicated that Maca had a higher food safety level.
The anti-fatigue effects of dry maca powder were proved to be remarkable through animal experiments, and in comparison with control groups, the urea level in blood serum of fatigued mice decreased, the glycogen level in liver increased, the raise of blood lactic acid level during movement was restrained, the blood lactic acid decrease of mice after movement was accelerated. In the study of the experiments on immune regulation, Maca was shown to enhance cellular immunity, but had no effect on humoral immunity, non-specific immunity and NKC activity. However, the mixture of Maca and American ginseng general ginsenosides can enhance immune function of mice significantly, and Maca was shown the synergistic action.
本论文围绕玛咖功效物质基础研究开展了系统的研究,得到如下研究结果:
玛咖营养成分研究中验证了玛咖具有高蛋白(9.22 %)、高支链氨基酸(20.14 %总氨基酸)、高果糖(5.32 %)等营养特征,并首次发现了玛咖中含有少量的牛磺酸成分(0.08 %)以及具有抗氧化活性的多糖成分(6.85 %)。通过预试鉴别发现玛咖中含有非常广泛的次生代谢物,其中非水溶性生物碱、甾醇、有机酸、挥发油含量较为突出。
玛咖干根中总生物碱含量可以采用滴定法快速检测(含量0.82 %),芥子油苷可以采用改良的氯化钯比色法快速检测,玛咖干根中芥子油苷含量为含量1.92 %,而商品化干粉中却仅为0.13 %。通过芥子油苷溶出-降解动力学曲线可以发现在传统的水提或者稀醇提取方法中,玛咖中芥子油苷都有一定程度的损失,但通过灭酶等工艺调整可以减小损失。
玛咖总生物碱提取分离工艺研究中发现,玛咖总生物碱的最佳薄层色谱分离条件为正丁醇-甲醇-水-乙二胺(38:10:12:1 v/v),而脱色可以采用D152大孔丙烯酸系弱酸性阳离子交换树脂,能有效脱除色素并得到88 %以上的生物碱回收率;玛咖生物碱J5采用液质联用分析发现其中含三种咪唑类的生物碱(lepidiline A、B、C),其中lepidiline C是新物质,而且发现这三类物质在心脏功能调节上具有研究潜力。
几种在外观和功效上相似的植物材料,包括玛咖(秘鲁产玛咖和云南会泽产玛咖)、萝卜、人参和西洋参,它们的生物碱提取物进行了红外光谱和薄层色谱分析,挥发油提取物则采用气质联用检测。通过比较这些材料的谱图,可以发现玛咖功效成分提取物具有独特的红外及其气相特征。其中红外光谱表现出酰胺基团、共轭双键以及苯基结构,气相色谱则表明玛咖挥发油以苯乙腈、苯甲醛、间甲氧基苯乙腈为其主要特征成分。这些红外指纹图谱、气相指纹图谱的建立可以用于玛咖及其玛咖产品的鉴别。
急性毒性学研究表明,玛咖干粉对小鼠的半数致死剂量LD50>10.00 g/kg.BW。Ames实验、小鼠骨髓微核实验与精子畸形实验均为阴性。在慢性毒性实验中(90天),除中、高剂量组雌性大鼠的AST、高剂量组雌性大鼠TCH显著低于对照组外,其它指标均无显著性差异。所有结果均表明玛咖具有较高的安全性。
抗疲劳实验中发现,玛咖能显著降低疲劳小鼠血清尿素的产生,增加小鼠肝糖原的储备量,对小鼠运动中血乳酸上升具有显著的抑制作用,并对小鼠运动后血乳酸消除具有显著的促进作用。免疫功效研究中,玛咖可以增强细胞免疫,但对体液免疫功能、非特异性免疫以及NK细胞活性都没有显著影响,但玛咖与西洋参总皂甙复配物能起到显著的增强免疫功效,而且玛咖在其中具有增效作用。
The Maca plant (Lepidium meyennii Walp.) has several thousend years history of use as a food and medicine crop in the highlands of Peru. Maca is a highly nutritious food source, and Maca alkaloids, glucosinolates, isothiocyanates, macamides and steroids are probably responsible for its aptitude to act as a fertility enhancer, aphrodisiac, adaptogen, anabolic and to influence hormonal balance. However, the Correspond Relation between the active material basis and functional mechanism are still quite indistinct, and it is widely agreed that one of the main reasons for this is the deficiency of in-depth study on the extraction, separation, chemical composition, chemical structure and content determining of the active material basis.
The study in my dissertation was carried out surrounding the active material basis of maca. The study result is:
The characteristic of high nutrition of Maca, such as high protein(9.22 %), high branch chain amino acid(20.14% total aa) and high fructose(5.32 %) , was confirmed in the study on nutritional components, and the small amount taurine(0.08 %) and the polysaccharide(6.85 %) with antioxidative activity were found for the first time. Many secondary metabolites of Maca were found using preparatory tests for composition identifying, and the contents of the non-water-solubility alkaloids, sterols, organic acids and essential oils were remarkable.
The content of total alkaloids in dried Maca (0.82 %) root can be determined using the titration method rapidly, and the content of glucosinolates in dried Maca root (1.92 %) and in commercial powder of Maca (0.13 %) can be determined using the palladium chloride method rapidly. From the dissolution-degradation kinetics of glucosinolates in Maca, it was found that glucosinolates were losing partially in the tradition process of extract by water or dilute ethanol-soluble, but the losing could cut down by enzyme inactivation and other technical-adjusting measures.
TLC solvent system for separating Maca total alkaloids is n-Butanol : ethanol : water : ethylenediamine(38:10:12:1 v/v), and the decoloration by D152 macroreticular weak acidic acrylic acid resin can wipe off the coloring matter effectually and the recuperation rate obtained is higher than 88 %. Three imidazole alkaloid (lepidiline A, B, C) in J5 was affirmed by LC-MS. Furthermore, lepidiline C is a new compound found in Maca, and lepidilines have the potential research on heart function regulation.
Natural plants with similar appearance or medicinal effects, including Maca(peru Maca and Huizhe Maca), radish, oriental ginseng and American ginseng were investigated. Their alkaloid extracts of the hypogeal parts were analyzed by FTIR and TLC. The essential oils were analyzed by GC/MS. Through comparison of the characteristics of their spectra and chromatograms, it was found that the functional ingredient extracts of Maca have unique FTIR and GC behaviors. The secondary amide group, conjugated carbon-carbon double bond and phenyl structure in FTIR and the characteristic peaks produced by the major essential oil components (Phenylacetonitrile, Benzaldehyde, 3-Methoxyphenylacetonitrile, etc) in GC/MS are distinct. These behaviors can be applied to the identification of Maca or Maca products in the market.
The mice′s LD50 of dried Maca root was found higher than 10.00 g/kg.BW in acute toxicity test, and the results of Ames test, mice bone marrow micronucleus test and mice sperm malformation test are of negative. The long term toxicity test indicated that the level of female AST in medium and high dose groups, TCH in high dose groups significantly decreased than in the control group, however the changes of other indexes were not significantly different between the Maca groups and control group (P>0.05). All results indicated that Maca had a higher food safety level.
The anti-fatigue effects of dry maca powder were proved to be remarkable through animal experiments, and in comparison with control groups, the urea level in blood serum of fatigued mice decreased, the glycogen level in liver increased, the raise of blood lactic acid level during movement was restrained, the blood lactic acid decrease of mice after movement was accelerated. In the study of the experiments on immune regulation, Maca was shown to enhance cellular immunity, but had no effect on humoral immunity, non-specific immunity and NKC activity. However, the mixture of Maca and American ginseng general ginsenosides can enhance immune function of mice significantly, and Maca was shown the synergistic action.
引文
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