ECHINACEA PURPUREA: HAIRY ROOT CULTURES FOR CAFFEIC ACID DERIVATIVES PRODUCTION
文献类型：学位论文
| 作者 | Bi L(比兰) |
| 学位类别 | 博士 |
| 答辩日期 | 2008-01-28 |
| 授予单位 | 中国科学院过程工程研究所 |
| 授予地点 | 过程工程研究所 |
| 导师 | 刘春朝 |
| 关键词 | 紫锥菊 毛状根 菊苣酸 光 赤霉素 生物反应器 |
| 其他题名 | ECHINACEA PURPUREA: HAIRY ROOT CULTURES FOR CAFFEIC ACID DERIVATIVES PRODUCTION |
| 学位专业 | 生物化工 |
| 中文摘要 | 紫锥菊能够提高人体的免疫能力，具有重要的经济价值。咖啡酸衍生物 （Caffeic acid derivatives，CADs) 是紫锥菊中的主要药理活性物质，具有很强的抗氧 化作用。其中，菊苣酸具有抗艾滋病（HIV-1）的功效。紫锥菊植物化学以及药理方 面的研究已经报道的很多，但是，通过毛状根培养生产紫锥菊有用次生代谢产物的 研究很少报道。 通过发根农杆菌感染紫锥菊（Echinacea purpurea Moench）叶片外植体获得紫锥 菊毛状根，该毛状根具有合成CADs 的能力，特别是菊苣酸。对液体毛状根生长和 CADs 生物合成的动力学过程进行研究表明，在培养到40 天时生物量达到最高为 12.2 g/l，毛状根生长量增加与培养基电导率降低成线性关系，HPLC 测定结果表明 毛状根中菊苣酸含量为19.21 mg/g DW，咖啡酸含量为3.56 mg/g DW，绿原酸含量 为0.93 mg/g DW。 在持续光照培养下紫锥菊毛状根中菊苣酸、咖啡酸和绿原酸的含量分别为27.0 mg/g DW，6.3 mg/g DW 和 2.3 mg/g DW，光照同时促进了毛状根表皮中花青素的合 成，根的颜色变为紫红色，光照促进CADs 和花青素积累的原因与苯丙氨酸解氨酶 (phenylalanine ammonium lyase，PAL) 的活力增高相关。光下根的生长速率以及细胞 活力与黑暗中培养条件下没有显著差异。 光照提高了紫锥菊毛状根中丙二醛(malondialdehyde ， MDA) 和过氧化氢 (hydrogen peroxide ，H2O2) 的积累。紫红色根中超氧化物歧化酶（ superoxide dismutase，EC 1.15.1.1，SOD）、过氧化氢酶（catalase，EC 1.11.1.6，CAT）、抗 坏血酸过氧化物酶(ascorbate peroxidase ， EC 1.11.1.11 ， APx) 、过氧化物酶 (peroxidase，EC 1.11.1.7，POD)的活性和木质素(lignin，LN)、总CADs 的含量均比 较高。这表明，在光照下，毛状根具有较强的清除自由基能力。光照下或者黑暗培 养条件下毛状根中谷胱甘肽过氧化物酶（glutathione peroxidase，EC 1.15.1.1，GPx) 活性没有很大变化，光照条件下毛状根中内源NO 的含量升高。这些结果表明，在外源赤霉素（gibberellic acid，GA3）对光胁迫下紫锥菊毛状根的生长、次生代 谢产物积累和细胞抗氧化系统有不同的影响。GA3 浓度在0.01 到 0.1 μM 范围时，毛 状根的生物量显著增加；GA3 浓度为0.025 μM 时，菊苣酸、咖啡酸和绿原酸的含量 达到最高值，分别为36.2 mg/g DW、7.8 mg/g DW、7.9 mg/g DW。研究了不同浓度 GA3（0, 0.005, 0.025 和1.0 μM）处理下PAL、细胞活力、抗氧化酶的变化和根的形 态解剖学状态。结果表明，GA3 浓度为0.025 μM 时，PAL、抗氧化酶以及细胞活力 增强；GA3 浓度为0.005 和1.0 μM 时，具有抑制作用。适量浓度的GA3 使毛状根变 粗、颜色加深；高浓度或者低浓度GA3 处理下毛状根变细、颜色变白。抗氧化酶活 性的变化与CADs 的合成具有显著相关性，而且影响毛状根的生长状态。另外，结 果表明GA3 减轻了光照对毛状根的胁迫，同时改变了毛状根的生长状态。 用2 L 气升式生物反应器（工作体积为1.7 L，导流筒为网状）对紫锥菊毛状根 培养生产CADs 进行了研究。发现反应器网状导流筒的网格大小对毛状根生长和 CADs 合成有显著的影响。研究结果表明700 μm 孔径的导流筒对毛状根的生长和 CADs 合成最好。研究了不同通气量对毛状根生长和次生代谢产物积累的影响，结 果表明通气量为0.012 m3/h 时最好，生物量达到15.2 g/反应器，菊苣酸、咖啡酸和 绿原酸的含量分别达到16.4 mg/g DW、 3.1 mg/g DW 和0.59 mg/g DW。研究了超声 对气升式反应器中毛状根生长和CADs 合成的影响。结果表明，6 分钟的超声显著 提高了毛状根的生物量（21.8 g/反应器），但是CADs 的积累受到抑制。对超声作 用下PAL 活性和花青素、木质素含量进行测定，结果表明，超声提高了PAL 的活 性，促进了花青素、木质素的积累。PAL 是CADs、花青素和木质素合成的关键 酶，试验结果表明超声影响了PAL 以后的生物代谢途经，主要是促进了代谢流向花 青素和木质素合成的方向。本论文建立了紫锥菊毛状根培养生产CADs（特别是菊苣酸）的工艺，特别是光 照和GA3 促进CADs 合成的策略，并建立了气升式生物反应器培养紫锥菊毛状根生 产CADs 的有效方法，为通过生物技术规模化生产C本论文建立了紫锥菊毛状根培养生产CADs（特别是菊苣酸）的工艺，特别是光 照和GA3 促进CADs 合成的策略，并建立了气升式生物反应器培养紫锥菊毛状根生 产CADs 的有效方法，为通过生物技术规模化生产CADs 提供了基础。CADs 提供了基础。 |
| 英文摘要 | Echinacea products have gained considerable attention in the commercial area as general immune system enhancer. Among Echinacea species, the natural products with medicinal bioactivity are several caffeic acid derivatives (CADs), which are also strong antioxidants, and one of which, cichoric acid, possesses potential against HIV-1 integrase. Agrobacterium rhizogenes-mediated transformed hairy roots hold the potential for economically feasible biotechnological routes to the controlled biosynthesis of complex, plant-derived and natural molecules. A great deal of research work focused on the phytochemical characterization and pharmacological activity of Echinacea has been conducted; however, limited information is available regarding hairy root culture for consistent production of CADs. Inoculation of leaf explants of Echinacea purpurea (Moench) with Agrobacterium rhizogenes induced hairy roots with the capacity to produce biologically active caffeic acid derivatives (CADs), especially cichoric acid. The kinetics of growth, the uptake of macronutrients, and the accumulation of CADs were investigated in the cultured hairy roots for a 50-day period. Maximum biomass of 12.2 g/l was obtained at day 40, and mathematical relationship between hairy root growth and ionic conductivity was established during exponential phase. HPLC analyses of methanolic (0.1 % phosphoric acid; 70:30, v/v) extracts from hairy roots revealed the accumulation of CADs: cichoric acid (19.21 mg/g DW), caftaric acid (3.56 mg/g DW) and chlorogenic acid (0.93 mg/g DW).Continuous light exposure affected the magnitude of CADs biosynthesis in the hairy root cultures, and increased cichoric acid, caftaric acid and chlorogenic acid to 27.0 mg/g DW, 6.3 mg/g DW and 2.3 mg/g DW, respectively. Light-grown hairy roots also accumulated anthocyanins, which became visible in outer cell layer of the cortex as a ring of purple color. The growth rate and cell viability of the hairy root cultures in light did not show obvious difference in comparison with that in dark. The enhanced accumulation of CADs and anthocyanins was correlated to light-stimulated phenylalanine ammonium lyase (PAL) activity. Enhanced levels of malondialdehyde (MDA) and hydrogen peroxide (H2O2) in lightgrown purple hairy roots indicated that light caused oxidative stress. Antioxidants in root extracts from light- and dark-grown cultures were identified, and activities quantified using enzymatic and α, α-diphenyl-β-picrylhydrazyl (DPPHº) assays. Purple morphs in hairy roots held greater amounts of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APx, EC 1.11.1.11), lignin (LN), total CADs and peroxidase (POD, EC 1.11.1.7). They were considerably more effective at scavenging DPPHº radicals, and also showed enhanced differentiation. Light- and dark-grown cultures did not show significant differences for glutathione peroxidase (GPx, EC 1.15.1.1) levels. Significantly higher levels of nitric oxide (NO) also contributed to antioxidant pool in light-grown cultures. It shows that hairy root cultures could grow under continuous light stress and protect themselves from toxicity of reactive oxygen species (ROS) by altering CADs levels and antioxidant enzymes. Exogenously applied gibberellic acid (GA3) has shown diverse effects on the growth, secondary metabolism and antioxidative defense system of light-grown hairy roots. Significant increase in biomass was obtained with moderate concentrations of GA3 ranging from 0.01 to 0.1 μM, and the treatment with 0.025 μM produced the highest amount of cichoric acid (36.2 mg/g DW), caftaric acid (7.8 mg/g DW) and chlorogenic acid (7.9 mg/g DW). Four concentrations of GA3 (control, 0.005, 0.025 and 1.0 μM) were selected tofurther study their impact on PAL, cell viability, hairy root morphology and anatomy and antioxidant enzymes. All of the activities determined were enhanced in 0.025 μM GA3 treatment whereas the opposite was observed with 0.005 and 1.0 μM levels of GA3. GA3 also affected root morphogenesis and radially thick, dense purple roots were observed at moderate concentrations and thin off-white roots were observed at higher and lower concentrations. Fluctuations in antioxidative system leads to conclusion that growth and CADs production is interrelated to antioxidative enzymes and is responsible for diverse effects observed in morphology and anatomy. Moreover, it is inferred that higher concentration of GA3 alleviated light-induced stress and alter growth phase. The hairy roots were grown in 2-l airlift bioreactor (1.7-l of working volume) with mesh for scaled-up CADs production. Mesh size affected hairy root distribution, growth and CADs accumulation with mesh 40 (700 μm pore size) being optimum for biomass and CADs production. 0.012 m3/h of air flow rate was found to be the best to accumulate dry biomass (15.2 g/bioreactor) and CADs (cichoric acid 16.4 mg/g DW; caftaric acid 3.1 mg/g DW and chlorogenic acid 0.59 mg/g DW). Ultrasonication (US) of root cultures in the airlift bioreactor resulted in considerably higher biomass accumulation and 6 minute exposure produced dry biomass of 21.8 g/bioreactor. US exposure inhibited CADs accumulation, but enhanced PAL activity. Further investigations revealed enhancement of anthocyanins and lignin by US, which were also PAL-derived metabolites. This thesis reports establishment of hairy root cultures with the ability to produce CADs, especially cichoric acid. Systematic approach was adopted to enhance CADs by light and GA3 in Erlenmeyer flask system. Furthermore, this study provides an effective approach for feasible production of CADs in airlift bioreactor. This hairy root system is exemplary due to its genetic and biochemical stability, and provides evidence that biotechnological production of CADs in hairy roots is possible. |
| 语种 | 中文 |
| 公开日期 | 2013-09-16 |
| 页码 | 160 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1269]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | Bi L. ECHINACEA PURPUREA: HAIRY ROOT CULTURES FOR CAFFEIC ACID DERIVATIVES PRODUCTION[D]. 过程工程研究所. 中国科学院过程工程研究所. 2008. |
入库方式： OAI收割
来源：过程工程研究所
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