| 题名 | 柴胡根系次生代谢物积累对干旱的响应与抗脂质过氧化保护机制 |
| 学位类别 | 博士 |
| 答辩日期 | 2009 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 陕西 |
| 导师 | 梁宗锁 |
| 学位专业 | 生态学 |
| 中文摘要 | 中药材的质量是中药实现现代化、产业化、国际化的关键。但由于药用植物的生长和次生代谢过程受自然环境、栽培管理措施等多种因素的影响，中药材中次生产物含量“稳定、可控”成为一个难点。探讨药用植物次生代谢产物积累变化的内在规律及其影响因子，探索符合药用植物次生代谢物积累内在规律的代谢调控机制，将促使中药材的质量控制建立在科学的理论之上，对于提高活性物质含量、保证药材质量、稳定临床疗效等具有重要意义。故需研究自然环境、栽培管理措施等因素与药用植物次生代谢产物种类和含量的相关性，确定影响次生产物积累的主要生态因子，并进一步揭示其生物合成机制。虽然阐明次生代谢途径是调控药用植物次生代谢产物合成积累的关键，然而由于其复杂性，一时难以达到令人满意的效果。但是，植物次生代谢产物能作为治疗人类疾病的药效成分，是人类利用了其某些理化特性；同为需氧生物，植物和人类有时面临着相同的问题，同时又有类似的解决方法。如果从某些植物次生产物在药理试验中的作用入手，联系其在植物体内的可能生理生化作用来探讨调控其在植物体内含量的途径，可能会为次生代谢产物的代谢调控提供新思路和方法。本项工作通过野外调查采样、大田和控制栽培及室内分析等手段，以植物次生代谢理论为基础，结合药理学、自由基生物学和自由基医学以及植物逆境生理生态学等学科的研究基础，研究了柴胡的主要药效成分（柴胡皂苷和挥发油）对环境条件的响应规律及其次生代谢的可能调控机理，取得了如下主要结论：1．通过建立的柴胡HPLC皂苷类成分指纹图谱，检出了18个特征峰作为定性鉴别的指标峰，能简便、快捷的鉴别不同种柴胡药材；发现11个批次柴胡的柴胡皂苷a（SSa）、柴胡皂苷d（SSd）、柴胡总皂苷含量及其指纹图谱中18个共有峰总峰面积相互之间存在极显著正相关关系，SSa或SSd均可作为柴胡中柴胡皂苷类成分的质控指标成分，提出建立柴胡药材质量综合评价体系的思路。2．通过施肥和环境因素分析，发现氮、钙和锰元素是影响SSa、SSd和柴胡总皂苷含量的主要矿质元素。施用氮磷肥对于促进SSa和SSd的积累具有重要作用，而且可在一定程度上减轻干旱胁迫对SSa和SSd总产量的不利影响。年平均气温、七月平均气温、无霜期和SSa、SSd、柴胡总皂苷含量之间存在显著或极显著的正相关关系，尤其较高的七月平均气温对柴胡皂苷积累具有重要促进作用。气温日较差是影响SSd和柴胡总皂苷含量和产量的主要气候因子，而平均风速是影响SSa含量和产量的主要气候因子，且均为负向作用。中度干旱显著提高柴胡根系柴胡皂苷含量。 3．柴胡根系具有较强的抵御水分亏缺的能力。SSa和SSd含量随干旱程度加重而上升，但在严重干旱条件下或复水后下降。而且从适宜水分条件到中度干旱条件，SSa和SSd含量的上升伴随着O2-含量和超氧化物歧化酶(SOD)，过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性的增强。值得注意的是，体外抗氧化试验表明柴胡根系甲醇提取液的抑制脂质过氧化能力与SSa、SSd含量正相关，其中与SSd含量的相关系数达到显著水平。由以上结果可以推测柴胡根系具有较强的抗氧化保护机制以适应干旱逆境，而且干旱诱导的柴胡根系SSa和SSd积累可能是受到活性氧的刺激，由于其较强的抑制脂质过氧化能力而减轻干旱造成的氧化伤害。另外，通过不同浓度外源H2O2直接处理柴胡幼苗根系，发现其可诱导柴胡皂苷含量升高，同时也促进了脂质过氧化以及SOD、APX活性增强。而在PEG导致的渗透胁迫条件下，施加外源AsA能降低MDA和H2O2含量，在一定程度上抑制脂质过氧化，但同时显著降低SSa和SSd含量。表明氧化胁迫条件利于柴胡皂苷积累，控制柴胡根系细胞氧化还原程度可在一定程度上对柴胡皂苷含量进行调控。4．柴胡根系的挥发性成分以醛类物质为主体，其相对含量之和占总挥发性物质的57.24％－91.30%。不同产地样品的挥发油组分和相对含量差异悬殊，证明挥发油组分和相对含量因环境变化较大。在干旱复水进程中，大部分共有组分在适宜水分条件下相对含量最高，随干旱进程而下降。在干旱条件下2-甲基丁醛、正戊醛、正己醛、正壬醛和2-戊基呋喃的相对含量较高。在干旱复水进程中诱导产生29种新挥发性成分，而且其中多种为具有抗虫抗菌活性的物质。在干旱复水进程中共有组分的相对含量变化与LOX活性、脂质过氧化程度没有显著相关性，但LOX对于干旱复水过程中新诱导产生的挥发性组分及其含量变化具有重要影响。 |
| 英文摘要 | The quality controlling of Traditional Chinese Medicine is the key to its modernization, industrialization, and internationalization. However, the modulation of plant secondary metabolite concentration is difficult because plants growth and secondary metabolism are affected both by natural environment and culture methods. Therefore, it is important for medically important compounds content enhancement, chinese medicinal materials quality assurance and clinic effect stabilization to investigate the inherent law and its influencing factors of plant secondary metabolism, and to explore metabolic modulation mechanism which match the inherent law of plant secondary metabolism. Although elucidating secondary metabolism pathway is the key to modulate secondary metabolites biosynthesis and accumulation in medical plants, the development is not satisfied as a consequence of its complication at present. It is noteworthy that certain characteristic of secondary metabolites in medicinal plants is responsible for its pharmacological effect. Being aerobic, plants and human beings are confronted with same trouble sometimes, and they have similar solution. It is possible to propose new idea and methods for plant secondary metabolism modulation if we start with the pharmacological effect of certain secondary metabolites in animal pharmacological experiment to investigate the way for concentration modulation in plant. Response of medical plant secondary metabolites to environment and their possible mechanism were investigated, exemplified by saikosaponin and essential oil, which are the principal bioactive compounds in Radix Bupleuri. The investigation is based on plant secondary metabolism basic theory and results of zoopharmacology, free radical biology, free radical medical, and plant stress physiological ecology, using field investigation, field and pot cultivation, artificial intelligence climate chamber cultivation, and indoor analysis. The main results as follows:1. Eighteen common peaks were determined as characteristic peaks to qualitatively differentiate different varieties quickly and easily in HPLC fingerprint of saponins established. There was remarkable positive correlation among content of SSa、SSd、total saikosaponin and total peak areas of 18 common peaks in the fingerprint of 11 samples of B. chinense. The method that the retention time of common peaks are served as qualitative index and the quantitative analyses are carried out based on the total peaks area of common peaks together with the content of SSa or SSd can properly be used for controlling the quality of B. chinense. A comprehensive qualitative evaluating system for Radix Bupleuri was proposed.2. Through fertilization and environmental analysis, we found that N, Ca, and Mn had markedly effect on SSa、SSd and total saikosaponin content. Fertilization with N and P had marked and positive impacts on the production of SSa and SSd, and the negative effect of water-stress on total SSa and SSd yield could be partly mitigated through the application of N and P fertilizer. It is possible to increase total SSa and SSd yield through the combined use of fertilizer and properly timed exposure to water-stress. The content of SSa, SSd, and total saikosaponin were positively correlative with annually mean temperature, mean temperature in July, and frost-free period. In particular, relatively high mean temperature in July may be responsible for saikosaponin accumulation. Diurnal temperature range markedly affected negatively the content and total yield of SSd and total saikosaponin, while mean wind speed had markedly negative influence on the content and total yield of SSa. Mild water-stress significantly increased the SSa and SSd content in B. chinense roots.3. B. chinense root appeared highly resistant to water deficit. Both SSa and SSd content increased with the progressive water deficit, however, decreased under severe drought conditions or after water recovery. And increased SSa and SSd content during drought were accompanied by enhanced O2- content and superoxide dismutase, catalase, ascorbate peroxidase activity until severe drought stress. Notably, in vitra antioxidant tests demonstrated that the lipid peroxidation inhibition capacity was positively corrected with the content of SSa and SSd, particularly significant at p=0.05 with SSd content. These results suggest that B. chinense roots exhibit effective antioxidative protection mechanism to withstand drought stress. And it could be speculated that drought-induced SSa and SSd accumulation in B. chinense roots may be stimulated via active oxygen species, and consequently involve in mitigating the oxidative damage due to its high anti-lipid peroxidation capacity. Exogenous application of H2O2 resulted in SSa and SSd accumulation, enhancement in lipid peroxidation and increase in SOD、APX activities. Under PEG mediated water stress conditions, the exogenous application of AsA decreased MDA and H2O2 content, alleviated lipid peoxidation, and decreased SSa and SSd content. The results suggest that oxidative stress is responsible for saikosaponin accumulation in B. chinense root, and saikosaponin content could be modulated through controlling the redox state in B. chinense roots cell to some extent.4. Aldehydes dominated in volatile oil of B. chinense root, with relative content ranging from 57.24%-91.30%. The chemical compounds and their relative content were different markedly in B. chinense samples originated from three regions. During drought-rewatering phase, the relative content of majority of common components peaked under well-watered conditions, and decreased with drought development. Pentanal, 2-methyl-Butanal, Hexanal, Nonanal, and 2-pentyl-Furan showed higher relative content under drought conditions. Drought and subsequent rewatering induced 29 new compounds of volatile oil, and some of those have insect-resistant and atibacterial activity. There was no significant linear relationship between the variation of relative content of common components in volatile oil and LOX activity, MDA content. Nevertheless, LOX showed important role in induction of new volatile compounds and their variation in relative content. |
| 公开日期 | 2011-07-01 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.iswc.ac.cn/handle/361005/4071]  |
| 专题 | 水土保持研究所_水保所知识产出(1956-2013) |
| 推荐引用方式 GB/T 7714 | . 柴胡根系次生代谢物积累对干旱的响应与抗脂质过氧化保护机制[D]. 陕西. 中国科学院研究生院. 2009. |
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