【作者】 谭斌；

【导师】 周朴华； 曹庸；

【作者基本信息】 湖南农业大学 ， 作物学， 2008， 硕士

【摘要】 缬草（Valeriana.officinalis L）,系败酱科多年生草本植物,其根和根茎可入药。缬草具有镇静、安眠、抗惊厥及抗菌、抗病毒、抗肿瘤等药理作用,缬草主要含环烯醚萜、倍半萜、生物碱和黄酮四大类型化学成分,其中环烯醚萜、倍半萜的研究较为系统全面,而缬草黄酮还未见深入研究。黄酮类化合物作为植物体内非常重要的一类次生代谢产物,其具有抗癌抗肿瘤、抗氧化抗自由基、抗心脑血管疾病、抗菌和抗病毒及保护神经系统等多种生物学活性,是降血脂、治疗心血管疾病、清除体内自由基等药物的有效成分,是一类具有广阔开发前景的天然药物,而黄酮类有效成分的分离提取又是其研发和应用的关键。到目前为止,缬草属中最具重要价值之一的宽叶缬草叶（V.officinalis L.Var.Latifolia Miq）黄酮类物质研究未见报道。为了充分利用资源,走综合开发科学合理利用资源的道路,本研究对宽叶缬草叶、愈伤组织黄酮的提取、分离、纯化及成分进行了较系统研究,主要结果如下:1.建立以芦丁为标准品的亚硝酸钠-硝酸铝-氢氧化钠比色法测定宽叶缬草总黄酮含量。比较研究了三氯化铝和亚硝酸钠-硝酸铝-氢氧化钠比色法测定宽叶缬草中总黄酮含量;进行野生与人工栽培宽叶缬草总黄酮及缬草叶中总黄酮含量差异的研究;对比研究超声波提取和乙醇回流提取总黄酮得率。综合比较分析最后确立最优提取和测定方法为:以乙醇回流提取总黄酮,用亚硝酸钠-硝酸铝-氢氧化钠显色体系,在510nm波长下测定宽叶缬草总黄酮。2.采用L₉（3⁴）正交优化提取试验,方差分析结果表明,各因素对宽叶缬草黄酮提取效果的影响程度从高到低依次为:乙醇浓度＞提取温度＞料液比＞提取时间。故选择最佳的提取工艺参数为:乙醇浓度为40%,提取时间为120min,提取温度为80℃,料液比为1:30。3.通过对五种不同性质的树脂进行静态吸附效果比较,数据表明AB-8型大孔树脂和聚酰胺吸附树脂对缬草黄酮均有较好的吸附及解吸效果,但聚酰胺的吸附率较AB-8大孔树脂要高,综合比较最后选用聚酰胺对宽叶缬草提取物进行分离纯化,从中得到1个纯度较高的未知化合物。4.采用紫外扫描、高效薄层分离、反相高效液相色谱三结合,鉴别宽叶缬草中主要黄酮成分。反相高效液相色谱测定采用Kromasil C₁₈ 5μm（4.6mm id×250mm）色谱柱,以甲醇-0.2%冰醋酸为流动相,进行梯度洗脱,紫外检测波长为330nm,流速为1mL/min.柱温35℃。通过数据分析,初步判定宽叶缬草叶中存在有芦丁、槲皮素、山奈酚、芹菜素等黄酮类物质。该方法简便、快速、准确,可用于黄酮类物质的定性定量检测。5.通过比较不同生长期及营养部位总黄酮含量,宽叶缬草离体培养诱导的愈伤组织总黄酮含量比叶外植体总黄酮含量要低,但为用细胞工程技术研究缬草的次生代谢产物提供了方向。更多还原

【Abstract】 Valeriana officinalis L.belongs to the family of Valerianace,It was a herbal traditional Chinese medicine which was long used to treat illness such as disquiet, insomnia and aches of various kind.The modern pharmacological and clinical experiments have proved that Valeriana herb have the functions of sedation hypnosis, antisepsis,antivirusis protecting heart,anticarcinogenic and so on.However,there are fewer reports on the extraction,isolation,and purification of total flavonoid from V.officinalis L.varlatifolia Miq.The objective of present study is to offer a reliable theory basis for people to make full use of V.officinalis L.var.latifolia Miq.The aim of this study was to extract,isolate and to purify of total flavonoid in V.officinalis L.var.latifolia Miq.The results are as follows.1.The method（ultraviolet spectrophotometry） was established to determine the total flavonoids（the rutin was as the standard and the NaNO₂-Al（NO₃）₃-NaOH was as the sprayed regents）.The method of NaNO₂-Al（NO₃）₃-NaOH was compared with the method of AlCl₃.The content difference of the total flavonoid in V.officinalis L.var.latifolia Miq was between wild and man-made plant.The extraction rate of the total fiavonoids was compared between the ultraphonic method and ethanol method. The best extraction of total flavonoids in V.officinalis L.var.latifolia Miq was extracted with ethanol in recirculation.The best determination method of total flavonoids in V.officinalis L.var.latifolia Miq was used the NaNO₂-Al（NO₃）₃-NaOH as sprayed regents and detected at 510 nm.2.A method of extraction and purification total flavonoid was established by orthogonal experiment.The optimum condition was 40%ethanol in 120 min at 80℃and solid to liquid ratio 1:30.The result indicated that the order of the four factors was: ethanol concentration>extraction temperature>stock ratio>extraction time.3.Polyamide was selected as the medium for initial isolation of total flavonoid by comparing 5 kinds of macroporous adsorption resin’s adsorption and separation performance.An unknown compound with high content was obtained from V.officinalis L.var.l atifolia Miq.by polyamide chromatography.4.The identification of the components of total flavonoids was confirmed by UV,TLC and HPLC.The HPLC conditions was as follows:column,Kromasil C₁₈（4.6 mmid×250mm 5μm）;temperature,35℃;wavelength,330nm;mobile phase, methanol:0.2%glacial acetic acid;flow rate,1 ml/min.There were many kinds of total flavonoids in this plant including rutin,quercetin,kaempferide and pelargidenon. The method is convenient,fast and accurate.It can be used to identify and to determine the flavonoids compounds.5.Total flavonoids content of V.officinalis L.var.latifolia Miq.callus was lower than that of V.officinalis L.vat.latifolia Miq,while plant cell engineering was to offer a reliable theory basis for people to study secondary metabolites of V.officinalis L.var.latifolia Miq.更多还原