【作者】 魏琦；

【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】 我国竹类资源丰富，竹叶含有多种具有生物活性及药理作用的化学成分，是我国传统中医药典籍中记载的药用植物资源，具有很好的开发利用价值。本文从竹叶化学成分基础研究的角度，重点研究了苦竹叶化学成分，并对部分化学成分进行抑菌及抗肿瘤生物活性测定；分析比较了苦竹属10种常见竹种竹叶黄酮类化合物、香豆素类化合物、挥发油、多糖等化学成分，为推进竹叶化学成分基础研究和促进竹叶资源的综合利用提供参考。主要研究结果如下：1.从苦竹（P. amarus (Keng) Keng f.）竹叶乙醇提取物乙酸乙酯相中共分离得到了16种化合物，包括3种酚酸类化合物、1种萜类化合物、1种香豆素类化合物、1种苯丙素类化合物及10种黄酮类化合物，分别为4-羟甲基-苯甲醛（1）、对羟基苯甲醛（2）、去氢催吐萝芙木醇（3）、7-羟基-香豆素（4）、反式香豆酸（5）、对羟基苯甲酸（6）、苜蓿素（7）、7-甲氧基-苜蓿素（8）、Demethyltorosaflavone（9）、6-反式-(2’’-O-α-鼠李糖基)乙烯基-5,7,3’,4’-四羟基黄酮（10）、木犀草素-6-C-洋地黄毒糖苷-4’-O-葡萄糖苷（11）、苜蓿素-7-O-葡萄糖苷（12）、芹菜素-6-C-阿拉伯糖苷（13）、苜蓿素-4’-O-葡萄糖苷（14）、牡荆苷（15）和异荭草苷-2’’-O-鼠李糖苷（16）。分离得到的16种化合物中，除化合物4，7及12外，其余化合物均为首次从苦竹叶中分离得到。通过HPLC与标准品对照，确定了7种化合物，均为黄酮类化合物，分别为槲皮素（17）、木犀草素（18）、异牡荆苷（19）、荭草苷（20）、异荭草苷（21）、异牡荆苷-2’’-O-鼠李糖苷（22）及木犀草素-6-C-阿拉伯糖苷（23）。2.以大肠杆菌、金黄色葡萄球菌及枯草芽孢杆菌为研究对象，采用滤纸片法，测定了苦竹叶乙醇提取物的各组分及分离得到的部分化合物的离体抑菌活性，并对未见活性报道的木犀草素-6-C-洋地黄毒糖苷-4’-O-葡萄糖苷进行了体内体外抗肿瘤活性测定。24h后25mg mL-1浓度下，反式香豆酸对大肠杆菌和金黄色葡萄球菌的离体抑菌作用最强，抑菌圈直径分别为20.23mm及18.35mm，乙酸乙酯相Fr.3对枯草芽孢杆菌的离体抑菌作用最强，抑菌圈直径为21.05mm。乙酸乙酯相的离体抑菌作用整体强于石油醚相和正丁醇相，水相抑菌作用不明显。Fr.3的离体抑菌作用在乙酸乙酯相8个组分中最强。木犀草素-6-C-洋地黄毒糖苷-4’-O-葡萄糖苷对子宫颈癌HeLa细胞、肝癌HepG2细胞及结肠癌HT-29细胞的体外抑制作用的IC50值分别为592mg/L、2058mg/L和1712mg/L。5mg/kg和10mg/kg中低剂量木犀草素-6-C-洋地黄毒糖苷-4’-O-葡萄糖苷对H22荷瘤鼠肝肿瘤有体内抑制作用，抑制率分别为2.9%和20.0%。3.建立了一种同时测定13种黄酮类化合物含量的高效液相色谱法。该方法简便、快速、准确，其精密度、稳定性及准确性良好，各黄酮标准品在线性范围内呈良好的线性关系。采用HPLC法分析比较了苦竹属（Pleioblastus Nakai）竹种苦竹（P. amarus (Keng)Keng f.）、川竹（P. simonii (Carr.) Nakai）、斑苦竹（P. maculatus (McClure) C. D. Chu et C.S. Chao）、高舌苦竹（P. altiligulatus S. L. Chen et S.Y. Chen）、宜兴苦竹（P. yixingensis S. L.Chen et S. Y. Chen）、垂枝苦竹（P. amarus (Keng) Keng f. var. pendulifolius S. Y. Chen）、实心苦竹（P. solidus S. Y. Chen）、衢县苦竹（P. juxianensis Wen, C. Y. Yao et S. Y. Chen）、丽水苦竹（P. maculosoides Wen）及杭州苦竹（P. amarus (Keng) Keng f. var. hangzhouensis S.L. Chen et S. Y. Chen）竹叶中13种黄酮类化合物的含量。苦竹属10种竹叶中共检测出12种黄酮类化合物，10种竹叶中均含有异荭草苷、荭草苷、牡荆苷、异牡荆苷、苜蓿素-7-O-葡萄糖苷、苜蓿素、7-甲氧基-苜蓿素，其中异荭草苷、荭草苷、牡荆苷、异牡荆苷及苜蓿素-7-O-葡萄糖苷含量普遍较高，丽水苦竹异牡荆苷含量突出，斑苦竹苜蓿素-7-O-葡萄糖苷含量突出，均在1300mg/kg以上。4.对10种苦竹属竹种竹叶中香豆素类化合物及挥发油进行了分析测定。（1）共检测出6种香豆素类化合物，斑苦竹叶茵芋苷含量最高且与其它竹种差异显著（P<0.05），为40.66mg/kg。川竹叶东莨菪内酯和6,7-二甲氧基香豆素含量最高且与其它竹种差异显著（P<0.05），为82.20mg/kg和39.68mg/kg。伞形酮在杭州苦竹中含量最高，为3.01mg/kg。高舌苦竹叶香豆素含量最高且与其它竹种差异显著（P<0.05），为15.75mg/kg。垂枝苦竹叶茴芹内酯含量最高，为4.46mg/kg。（2）从10种竹叶挥发油中共鉴定出挥发性成分195种，其化合物类型基本一致，但含量在竹种间有一定差异，醛类化合物相对含量最高。10种苦竹属竹叶挥发油共有成分有15种，分别为苯甲醛、2-正戊基呋喃、反式-2,4-庚二烯醛、壬醛、2,3-二氢-2,2,6-三甲基苯甲醛、β-环柠檬醛、2,6,6-三甲基-1-环己烯基乙醛、对乙烯基愈疮木酚、1,1,6-Trimethyl-1,2-dihydronaphthalene、香叶基丙酮、4-[2,2,6-三甲基-7-氧杂二环[4.1.0]庚-1-基]-3-丁烯-2-酮、十五烷、植酮、异植物醇和叶绿醇。5.对10种苦竹属竹种竹叶多糖、蛋白质、叶绿素及矿质元素进行了分析测定。（1）10种竹种竹叶多糖含量均在600mg/kg以上，苦竹竹叶多糖含量最高，为843.29mg/kg。（2）10种竹种竹叶蛋白质含量在127547.50~182244.02mg/kg之间。苦竹竹叶蛋白质含量最高，为182244.02mg/kg。（3）10种竹种竹叶叶绿素a的含量大于叶绿素b的含量。川竹竹叶叶绿素总含量和叶绿素a含量最高且与其它竹种差异显著（P<0.05），分别为3339.24mg/kg和2390.97mg/kg。实心苦竹竹叶叶绿素b含量最高，为949.54mg/kg。（4）检测的19种矿质元素中含量较高元素的总体趋势为：K＞Ca＞Mg＞Mn＞Fe＞Al＞Na＞Zn。K元素含量在8143~12695mg/kg之间，Ca元素含量在5671~16080mg/kg之间，Mg、Mn元素含量均在1000mg/kg以上，Fe元素含量在300mg/kg左右，Al元素含量在160mg/kg以上。对于重金属元素，Ag元素在10种竹种竹叶中均未检测出，Cd元素只在少数竹叶中检出且含量很少，在0.01~0.14mg/kg之间。竹叶中As、Hg、Pb等重金属元素含量较低。更多还原

【Abstract】 China owns the most abundant bamboo resources in the world, bamboo leaves have manykinds of chemical constituents with biological activity and pharmacological action. Bambooleaves, which is recorded in traditional Chinese medicine books as resources of medicinalplants, have high value of development and utilization. To promote the basic research andcomprehensive utilization of bamboo leaves, chemical components of bamboo leavese werestudied. The compounds from Pleioblastus amarus (keng) keng f. leaves were isolated andidentified, and the antibacterial activity and antitumor activity of some compounds were tested.Components such as flavonoids, coumarins, essential oils, polysaccharides, etc. in the leaves of10bamboo species in Pleioblastus Nakai were compared.The main results were as following:1.16compounds were isolated from ethyl acetate extracts of ethanol extracts from P.amarus leaves, the compounds included3phenolic acids,1terpenoid compounds,1coumarincompounds,1phenylpropanoid compounds and10flavonoids.16compounds were identifiedas4-hydroxymethyl-benzaldehyde(1),4-hydroxybenzaldehyde(2),(E)-4-hydroxy-3,5,5-trimethyl-4-(3-oxobut-1-enyl)cyclohex-2-enone(3),7-hydroxycoumarin(4), β-coumaric acid(5),4-hydroxy-benzoic acid(6), tricin(7),7-methoxytricin(8), demethyltorosaflavone(9),6-trans-(2’’-O-α-rhamnopyranosyl)ethenyl-5,7,3’,4’-tetrahydroxyflavone(10),5,7,3’-trihydroxy-6-C-β-D-digitoxopyranosyl-4’-O-β-D-glucopyranosyl flavonoside(11),tricin-7-O-glucoside(12), apigenin-6-C-arabinoside(13), tricin-4’-O-glucoside(14), vitexin(15)and isoorientin-2’’-O-rhamnose(16).16compounds isolated except the compounds4,7and12were isolated from P. amarus leaves for the first time.7flavonoids were identified bycomparing with standards by HPLC.7flavonoids were quercetin(17), luteolin(18),isovitexin(19), orientin(20), isoorientin(21), isovitexin-2’’-O-rhamnose(22) andluteolin-6-C-arabinoside(23). 2. In vitro antibacterial activities of ethanol extracts and some compounds from the P.amarus leaves against Escherichia coli, Staphyloccocus aureus and Bacillus subtilis andantitumor activity of5,7,3’-trihydroxy-6-C-β-D-digitoxopyranosyl-4’-O-β-D-glucopyranosylflavonoside were evaluated. At the concentration of25mg mL-1after24h, the results showedthat β-coumaric acid show strongest in vitro antibacterial activity against Escherichia coli andStaphyloccocus aureus, the diameter of inhibition zone were20.23mm and18.35mmrespectively; Fr.3from ethyl acetate extracts show strongest in vitro antibacterial activityagainst Bacillus subtilis, the diameter of inhibition zone was21.05mm. The in vitroantibacterial activity of the entirety of ethyl acetate extracts was stronger than that of petroleumether extracts and n-butanol extracts, the water extracts had no obvious antibacterial effect. Thein vitro antibacterial activity of Fr.3was strongest in8parts of ethyl acetate extracts.The IC50of in vitro antitumor activity of5,7,3’-trihydroxy-6-C-β-D-digitoxopyranosyl-4’-O-β-D-glucopyranosyl flavonoside against Hela cells, HepG2cells and HT-29cells were592mg/L,2058mg/L and1712mg/L respectively. The dose of5mg/kg and10mg/kg5,7,3’-trihydroxy-6-C-β-D-digitoxopyranosyl-4’-O-β-D-glucopyranosyl flavonoside had invivo antitumor effect on the liver tumours of H22mice with tumor, the inhibition rates were2.9%and20.0%respectively.3. A simple, rapid and accurate HPLC method was developed for simultaneous analysis of13flavonoids in baoboo leaves. The accuracy and precision of the method were good, and thestandard curves of13flavonoids in the corresponding ranges had good linear relation.13flavonoids in the leaves of P. amarus (Keng) Keng f., P. simonii (Carr.) Nakai, P.maculatus (McClure) C. D. Chu et C. S. Chao, P. altiligulatus S. L. Chen et S.Y. Chen, P.yixingensis S. L. Chen et S. Y. Chen, P. amarus (Keng) Keng f. var. pendulifolius S. Y. Chen, P.solidus S. Y. Chen, P. juxianensis Wen, C. Y. Yao et S. Y. Chen, P. maculosoides Wen and P.amarus (Keng) Keng f. var. hangzhouensis S. L. Chen et S. Y. Chen in Pleioblastus Nakai werestudied by HPLC. The results showed that12flavonoids were detected. Isoorientin, orientin,vitexin, isovitexin, tricin-7-O-glucoside, tricin,7-methoxytricin were detected in10bambooleaves. The contents of isoorientin, orientin, vitexin, isovitexin, tricin-7-O-glucoside were high in the10baoboo leaves. The contents of isovitexin in P. maculosoides and tricin-7-O-glucosidein P. maculatus were higher than others, the contents were more than1300mg/kg.4. Coumarins and essential oils in the leaves of10bamboo leaves were studied.(1)6coumarins were detected, P. maculatus leaves had the highest content of skimin of40.66mg/kg, and the content was significantly different from other species (P<0.05). P. simoniileaves had the highest contents of scopoletin and6,7-dimethoxycoumarin of82.20mg/kg and39.68mg/kg, and the contents were significantly different from other species (P<0.05). P.amarus var. hangzhouensis leaves had the highest content of umbelliferone of3.01mg/kg. P.altiligulatus leaves had the highest content of coumarin of15.75mg/kg, and the content wassignificantly different from other species (P<0.05). P. amarus var. pendulifolius leaves had thehighest content of pimpinellin of4.46mg/kg.(2). Essential oils from bamboo leaves of10species of Pleioblastus Nakai were studied.195volatile components were identified. The essential oils had basically the same compoundstypes, but the compounds contents had some differences between species, aldehydes had thehighest relative contents.15common components of essential oils from bamboo leaves of10species were benzaldehyde,2-pentylfuran, trans,trans-2,4-heptadienal,1-nonanal,3-Cyclohexadiene-1-carboxaldehyde,2,6,6-trimethyl-1,2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde,2,6,6-trimethyl-1-Cyclohexene-1-acetaldehyde,4-Hydroxy-3-methoxystyrene,1,1,6-Trimethyl-1,2-dihydronaphthalene, geranylacetone,4-[2,2,6-trimethyl-7-oxabicyclo[4.1.0]hept-1-yl]-3-Buten-2-one, pentadecane,2-Pentadecanone,6,10,14-trimethyl-, isophytoland (E)-3,7,11,15-Tetramethyl-2-hexadedcen-1-ol.5. Polysaccharides, protein, chlorophyll and mineral elements in the bamboo leaves of10species of Pleioblastus Nakai were studied.(1) The polysaccharides contents of leaves of test bamboo species were higher than600mg/kg. P. amarus leaves had the highest polysaccharides content of843.29mg/kg.(2) The protein contents of test bamboo leaves were127547.50~182244.02mg/kg. P.amarus leaves had the highest protein content of182244.02mg/kg. (3) The chlorophyll a contents of test bamboo leaves were higher than chlorophyll bcontents. P. simonii had the highest total chlorophyll content and chlorophyll a content of3339.24mg/kg and2390.97mg/kg, and the contents were significantly different from otherspecies (P<0.05). P. solidus leaves had the highest chlorophyll b content of949.54mg/kg.(4)19mineral elements were tested, the general trend of elements which had relativelyhigh contents was K＞Ca＞Mg＞Mn＞Fe＞Al＞Na＞Zn. The K contents were8143~12695mg/kg, the Ca contents were5671~16080mg/kg, the Mg and Mn contents werehigher than1000mg/kg, the Fe contents were about300mg/kg, the Al contents were higherthan160mg/kg. Ag was not detected in all test bamboo leaves. Cd was detected only inminority species, and the Cd contents were0.01~0.14mg/kg. The contents of As, Hg and Pbwere relatively low.更多还原