地黄自毒物质提取及其生物指标测定
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国家自然科学基金资助项目(30772729);福建省自然科学基金资助项目(2008J0051)


Extraction, bioassay, and chemical identification of Rehmannia glutinosa L. autotoxins
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    摘要:

    连作障碍在药用植物地黄(Rehmannia glutinosa L.) 栽培中尤其明显,同一地块上种植一茬地黄后,须经8-10a后方可再种\. 从怀地黄主产区河南省焦作地区采集种植1a地黄的茬后土壤,采用不同溶剂提取地黄土壤自毒物质并利用生物测试确定抑制率最强的土壤提取液作温室栽培实验以确定对植物生理指标的影响。利用高效液相色谱法 (HPLC) 对土壤提取液和根系分泌物进行比较, 并利用高效液相色谱、傅立叶转换红外光谱(FTIR)和电喷雾离子阱质谱(ESI-MS)技术鉴定地黄连作自毒物质的化学成分。生物测试结果表明水和甲醇提取的地黄茬后土壤自毒物质对地黄生长具有最强的抑制作用;在培养器中加入浓度0.5g/mL的水和甲醇提取液对地黄胚根的抑制率分别达17% 和 26%,当浓度增至5.0 g/mL 时,抑制率增加到70% 以上。盆栽结果显示甲醇提取的自毒物质导致地黄地上部叶绿素含量减少,根系活力下降,明显抑制了地黄体内超氧化物歧化酶、过氧化物歧化酶、过氧化物酶的活性,加剧了地黄膜质过氧化,以及引起地黄植体内生长素类激素含量下降。高效液相色谱法显示土壤提取液和根系分泌物具有相似图谱。电喷雾离子阱质谱分析共检测到6个特征物质:香草酸、D-甘露醇、二十六烷酸苯羟基乙酯、毛蕊花糖苷、β-谷甾醇和胡萝卜苷。

    Abstract:

    Obsticals for recropping are especially severe in the production of Rehmannia glutinosa, a Chinese medicinal herb. It taks 8 to 10 years before one can replant a Rehmannia glutinosa to the same field where the crop had been grown. In this study, five different solvents were used to extract potential autotoxins from the soil samples taken from a major Rehmannia glutinosa production area in Jiao-zuo,Henan Province after harvesting the crop. Bioassy was conducted to test the inhibiton rate (IR) of the five soil extracts on Rehmannia glutinosa radicle growth. Soil extract that had the greatest IR was selected for a greenhouse growth study and various physiological and biochemical parameters were measured. Finally, HPLC analysis was used to compare soil extracts with root exudates, and ESI-MS analysis was used to identify the chemical compositions of the soil extracts. Bioassy results demonstrated that water and methanol extracted soil autotoxins had the greatest IR; by adding 0.5 g/mL concentration of soil extracts to a germination study, the IR to Rehmannia glutinosa radicle growth was 17% and 26% for water and methanol extracts, respectively, and the IR reached above 70% when the concentration was increased to 5.0 g/mL. Greenhouse study exhibited that methanol extracted soil autotoxins had resulted in (1) decreased root activities and chlorophyll contents,(2) decreased activities of SOD, CAT, and POD, (3) enhanced membranaceous peroxidation, and (4) decreased growth hormones contents. HPLC analysis showed very similar chromatograms between the methanol extracts and the root exudates. ESI-MS analysis detected six characteristic substances: vanillic acid, D-mannitol, 2\[4′-hydroxyphenyl\] -ethyl hexacosanoate, verbascoside, β-sitosterol, and daucosterol.

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李振方,齐晓辉,李奇松,戴林泉,林茂兹,张重义,林文雄*.地黄自毒物质提取及其生物指标测定.生态学报,2010,30(10):2576~2584

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