【作者】 王淑英；

【导师】 岳永德；

【作者基本信息】 中国林业科学研究院 ， 生态学， 2013， 博士

【摘要】 植物资源的化学利用是当前的研究热点。竹子是森林资源的重要组成部分，在我国分布广泛。竹子具有丰富的次生代谢产物，对竹类资源化学成分的研究和开发利用具有重要意义。牡竹属是我国重要的丛生竹属，为了全面研究牡竹属竹叶化学成分，揭示竹叶化学成分的生物活性，本文以牡竹属中具有药用价值的麻竹为研究对象，对其竹叶的化学成分进行分离、纯化和结构鉴定；建立了竹叶中香豆素类化合物高效液相色谱检测法；并对牡竹属10种竹叶的化学成分进行了比较研究，探讨同属的竹类植物化学成分的差异，为更好的利用牡竹属竹叶资源提供理论依据。主要研究结果如下：（1）采集麻竹竹叶，用95%乙醇浸提，石油醚、乙酸乙酯和正丁醇萃取。乙酸乙酯相经硅胶柱色谱、凝胶柱色谱、高压制备色谱及重结晶，得到17种单体化合物。通过理化性质、波谱数据分析、高效液相色谱法等方法，分别鉴定为牡荆苷、异牡荆苷、木犀草素、苜蓿素、7-甲氧基-苜蓿素、苜蓿素-4’-O-葡萄糖苷、苜蓿素-7-O-葡萄糖苷、芹菜素、芹菜素-7-O-葡萄糖苷、芹菜素-6-C-阿拉伯糖苷、芹菜素-7-O-葡萄糖-6’’-O-鼠李糖苷、对羟基苯甲醛、5-羟基-2-甲氧基-苯甲醛、黑麦草内酯、去氢催吐萝芙木醇、催吐萝芙木醇-9-O-葡萄糖苷和4,4’,9,9’-四羟基-3,3’,5,5’-四甲氧基-7,7’-单环氧木脂素，其中有11个黄酮、2个酚酸、3个萜类及1个木脂素类化合物。除牡荆苷外，其它16种化合物均首次在麻竹中分离得到。对获得的质量相对较大化合物15即4,4’,9,9’-四羟基-3,3’,5,5’-四甲氧基-7,7’-单环氧木脂素进行了体外抗肿瘤细胞活性测定，结果表明其对HeLa细胞增殖没有抑制作用。（2）建立了一种简便、快速、准确的同时测定13种黄酮类化合物含量的高效液相色谱法。结果表明，异荭草苷、荭草苷、牡荆苷、异牡荆苷、木犀草素、芹菜素-7-O-葡萄糖-6’’-O-鼠李糖苷、芹菜素-7-O-葡萄糖苷、苜蓿素-7-O-葡萄糖苷、6-反式-(2’’-O-α-鼠李糖基)乙烯基-5,7,3’,4’-四羟基黄酮、芹菜素、苜蓿素、Demethyltorosaflavone和7-甲氧基-苜蓿素在各自的线性范围内呈良好的线性关系，各标准品的LOD值在0.01~0.03mg/kg之间，LOQ值在0.02~0.34mg/kg之间。本试验仪器的精密度良好，且13种标准品较稳定。采用建立的方法对牡竹属10种竹种包括麻竹、龙竹、牡竹、黄竹、梁山慈竹、花吊丝竹、云南龙竹、福贡龙竹、勃氏甜龙竹和版纳甜龙竹竹叶黄酮含量进行分析，结果显示各竹种竹叶黄酮类化合物含量存在差异。异荭草苷、荭草苷、牡荆苷、异牡荆苷和芹菜素-7-O-葡萄糖-6’-O-鼠李糖苷在10种竹叶中含量普遍较高，其次是芹菜素-7-O-葡萄糖苷和苜蓿素-7-O-葡萄糖苷，木犀草素、6-反式-(2’-O-α-鼠李糖基)乙烯基-5,7,3’,4’-四羟基黄酮、芹菜素、苜蓿素、Demethyltorosaflavone和7-甲氧基-苜蓿素含量较小。（3）以70%乙醇为提取溶剂，超声波提取，高效液相色谱检测，首次建立了竹叶中12种香豆素类化合物同时检测方法。竹叶样品经70%乙醇超声提取3次，每次30min；Florisil SPE柱净化，5mL甲醇洗脱。结果表明，茵芋苷、东莨菪苷、东莨菪内酯、伞形酮、6,7-二甲氧基香豆素、香豆素、补骨脂素、花椒毒素、5,7-二甲氧基香豆素、茴芹内酯、欧前胡素和蛇床子素等12种香豆素的LOD值在0.19~0.85mg/kg之间，LOQ值在0.64~2.82mg/kg之间。在5~45mg/kg的添加浓度内，添加回收率在47.41~112.25%之间，RSD≤19.69%。采用建立的方法分析了牡竹属10种竹叶中香豆素类化合物含量，共检测到东莨菪苷、东莨菪内酯、伞形酮、香豆素和茴芹内酯5种香豆素，其中，东莨菪内酯为其共有成分，含量在5.26~37.84mg/kg之间。10种竹叶香豆素总含量在20.39~152.96mg/kg之间，福贡龙竹竹叶中的香豆素类化合物总含量最高，梁山慈竹竹叶中所含香豆素总含量最低。（4）对牡竹属10种竹种竹叶挥发油化学成分进行分析比较，发现10种竹叶挥发油中醛类、酮类和醇类化合物在各竹叶挥发油中相对含量较高，其次是烷烯烃类、酚类和酯类化合物，醚类、酸类、萘类、呋喃类及其它类化合物在各竹叶挥发油中相对含量较低。10种竹叶挥发油化学成分中含有18种共有成分，其中，反式-2-己烯醛是各竹种挥发油中的重要成分。（5）对牡竹属10种竹种竹叶中的蛋白质、多糖、叶绿素及矿质元素等化学成分含量进行比较研究，结果表明，蛋白质含量在12.90~22.12%之间，梁山慈竹含量最高，麻竹含量最低，黄竹和云南龙竹、花吊丝竹和版纳甜龙竹之间蛋白质含量不存在显著性差异，其它几种竹种蛋白质含量存在显著性差异。10种竹叶多糖含量在0.38~0.82mg/g之间，含量最高的竹种为龙竹，含量最低的为牡竹。10种竹叶叶绿素a的含量在1.69~3.82mg/g之间，叶绿素b的含量在0.79~1.60mg/g之间，叶绿素总含量在2.48~5.37mg/g之间，其中勃氏甜龙竹竹叶叶绿素a、叶绿素b和叶绿素总含量均较大。10种竹叶中含有丰富的矿质元素，其中K、Ca、Mg元素含量较高，其次是Mn、Fe、Al、Na、Zn元素，Co、As、Se、Mo、Hg和Pb在竹叶中含量很低。本文研究结果为麻竹药理活性研究奠定基础，为竹叶香豆素类化学成分的药用、食用等的开发利用提供科学依据，对牡竹属竹叶资源的化学利用具有重要的理论指导意义。更多还原

【Abstract】 The chemical utilization of plant resources has been a hot area of research currently.Bamboo is an important part of the forest resources and widely distributed in China. Bamboohas a wealth of secondary metabolites. It has great significance to research and utilize thechemical compositions of bamboo resources. Dendrocalamus is the important sympodialbamboo in our country. In order to comprehensive study the chemical composition ofDendrocalamus, reveal biological activity of the chemical composition of bamboo leaves, thechemical compositions of the leaves of Dendrocalamus latiflorus Munro were isolated,purified and identified. The HPLC method for determination coumarins of bamboo wasestablished. Ten kinds of Dendrocalamus bamboo leaves were analysed so as to explore thedifferences of the chemical compositions of the same genus. The research could provide thetheoretical guidance for utilization of bamboo resources of Dendrocalamus. The researchresults were as follows:(1) The leaves of Dendrocalamus latiflorus Munro were collected, extracted by95%ethanol and leached by petroleum ether, ethyl acetate and n-butyl alcohol respectively. Theextractions were isolated by column chromatography on silica gel, Sephadex LH-20, PrepHPLC and recrystallization. Seventeen compounds were obtained. Their structures wereelucidated on the basis of physicochemical constants and spectral analysis. They wereidentified as vitexin, isovitexin, luteolin, tricin,7-methoxy-tricin, tricin-4’-O-glucopyranoside,tricin-7-O-glucopyranoside, apigenin, apigenin-7-O-glucopyranoside, apigenin-6-C-arabinfuranoside, apigenin-7-O-glucopyranoside-6’’-O-rhamnoside, p-hydroxybenzaldehyde,5-hydroxy-2-methoxy-benzaldehyde, loliolide, dehydrovomifoliol, vomifoliol-9-O-glucopyranoside,4,4’,9,9’-tetrahydroxy-3,3’,5,5’-tetramethoxy-7,7’-monoepoxy lignan. Therewere11flavonoids,2phenolic acids,3terpenes and1lignans. Sixteen compounds were firstlyisolated from Dendrocalamus latiflorus Munro except vitexin. Meanwhile, the anti-tumor cellactivity of4,4’,9,9’-tetrahydroxy-3,3’,5,5’-tetramethoxy-7,7’-monoepoxy lignan the quality of which was relatively large was determinated and the results showed that it had no inhibitoryeffect on the proliferation of HeLa cells.(2) A HPLC method was developed for simultaneous analysis of thirteen flavonoidscompounds in bamboo leaves. The method was simple, rapid and accurate. The result showedthat The standard curves of isoorientin, orientin, vitexin, isovitexin, luteolin,apigenin-7-O-glucopyranoside-6’’-O-rhamnoside, apigenin-7-O-glucopyranoside, tricin-7-O-glucopyranoside,6-trans-(2’’-O-α-rhamnopyranosyl)ethenyl-5,7,3’,4’-tetrahydroxyflavone,apigenin, tricin, demethyltorosaflavone and7-methoxy-tricin in the corresponding ranges hadgood linear relation. LOD of them were at the range of0.01mg/kg to0.03mg/kg and LOQ ofthem were at the range of0.02mg/kg to0.34mg/kg. The instrument of the test was fine and thestandards were stable.Ten bamboo leaves of Dendrocalamus (Dendrocalamus latiflorus, D. giganteus, D.strictus, D. membranceus, D. farinosus, D.minor var. amoenus, D. yunnanicus, D. fugongensis,D. brandisii and D. hamiltonii) were detected using this method. The results showed that theflavonoids contents in different bamboo leaves exist differences. The contents of isoorientin,orientin, vitexin, isovitexin and apigenin-7-O-glucopyranoside-6’’-O-rhamnoside were high inthe ten bamboo leaves. Next were the contents of apigenin-7-O-glucopyranoside andtricin-7-O-glucopyranoside. The contents of luteolin,6-trans-(2’’-O-α-rhamnopyranosyl)ethenyl-5,7,3’,4’-tetrahydroxyflavone, apigenin, tricin, demethyltorosaflavone and7-methoxy-tricin were less.(3) A HPLC method was firstly developed for simultaneous analysis of twelve coumarincompounds in bamboo leaves. The samples were extracted with ethanol/water (70/30, v/v)under ultrasonic and purified through Florisil SPE. The result showed that LOD of skimin,scopolin, scopoletin, umbelliferone,6,7-dimethoxycoumarin, coumarin, psoralen, xanthotoxin,5,7-dimethoxycoumarin, pimpinellin, imperatorin and osthole were at the range of0.19mg/kgto0.85mg/kg and LOQ of them were at the range of0.64mg/kg to2.82mg/kg. The recoveryrates were at the range of47.41%to112.25%with addition quantities of5mg/kg to45mg/kg.The values of RSDs were less than19.69%. Ten bamboo leaves of Dendrocalamus were detected using this method. The resultshowed that five coumarins, namely scopolin, scopoletin, umbelliferone, coumarin andpimpinellin were found in the extracts of ten bamboo leaves. Scopoletin was the commoncomposition of the leaves and the contents were at the range of5.26mg/kg to37.84mg/kg. Theconcentrations of total coumarins were at the range of20.39mg/kg to152.96mg/kg. Themaximum concentration of total coumarins was found in D. fugongensis and the minimum wasin D. farinosus.(4) The chemical compositions of essential oils of the ten bamboo leaves ofDendrocalamus were analysised and compared. In the ten bamboo essential oils, the relativecontents of aldehydes, ketones alcohols were more larger, the relative contents of alkanesolefins, phenols, esters were second and the relative contents of ethers, acids, naphthalenes,furans and the others were low. There were eighteen common compounds in the ten bambooessential oils and (E)-2-Hexenal was the important composition of them.(5) The contents of major chemical compositions (protein, polysaccharides, chlorophylland mineral elements) of the ten bamboo leaves of Dendrocalamus were analysised andcompared. The result showed that contents of protein were at the range of12.90%to22.12%.The maximum content of protein was found in D. farinosus and the minimum content ofprotein was found in D. latiflorus. Protein contents of D. membranceus and D. yunnanicus hadlittle difference, the same as D.minor var. amoenus and D. hamiltonii. Protein contents of theother bamboo leaves had significant differences. Polysaccharides contents of ten bambooleaves were at the range of0.38mg/g to0.82mg/g. The maximum content of polysaccharideswas found in D. giganteus and the minimum content of polysaccharides was found in D.strictus. The contents of chlorophyll a of ten bamboo leaves were at the range of1.69mg/g to3.82mg/g, the contents of chlorophyll b were at the range of0.79mg/g to1.60mg/g and thecontents of total chlorophyll were at the range of2.48mg/g to5.37mg/g. Chlorophyll contentsof D. brandisii were more larger. Ten kinds of bamboo leaves were rich in mineral elements.The contents of K, Ca,mg were more larger, the contents of Mn, Fe, Al, Na, Zn were secondand the contents of Co, As, Se, Mo, Hg, Pb were very low. The results of the research established the foundation for clarification pharmacologicalactivity of the leaves of Dendrocalamus latiflorus Munro, provided scientific basis fordevelopment and utilization of coumarins of bamboo leaves. It had important theoreticalsignificance on the chemical utilization of bamboo leaves resources of Dendrocalamus.更多还原