【作者】 李丽；

【导师】 肖培根；

【作者基本信息】 中国协和医科大学 ， 生药学， 2009， 博士

【摘要】 “苦丁茶”作为一类非茶类的代茶饮品,一种重要的药茶两用植物在我国应用已有两千年的历史。目前市场上苦丁茶有两大类主流品种,分别为冬青科的大叶冬青和苦丁茶冬青（大叶苦丁茶）;木犀科的粗壮女贞及其他几种植物（小叶苦丁茶）。藤黄科及紫草科的几种植物在少部分地区作为苦丁茶使用。由于其基源复杂、品种混乱,对苦丁茶的质量控制带来难题,从苦丁茶的资源利用到市场应用都具有盲目性。本文先后以小叶苦丁茶和紫草科苦丁茶为研究对象,对其质量控制方法及化学成分等方面进行系统研究。具体内容如下:1.首先,对小叶苦丁茶的主流品种粗壮女贞化学成分分离鉴定,为质量控制研究提供定性定量用的标准品,共分离了18个化合物,鉴定了其中的16个。这些化合物分别为:ligustaloside B（1）,ligustaloside B di-Me acetal（2）,ligustaloside A（3）,acteoside（4）,ligupurpuroside A（5）,isoacteoside（6）,ligupurpuroside B（7）,ligupurpuroside D（8）,osmanthuside B₆（9）,ligupurpuroside C（10）,3,4-dihydro xyphenyl（11）,lipedoside B-Ⅲa（12）,lipedoside B-Ⅲb（13）,glucoseesters of（E） -2,6-dimethyl-6-hydroxyocta-2,7-dienoic acid（14）,luteolin-7-O-β-D-glucopy ranoside（15）,rutin（16）。其中有8个化合物为首次从该种植物中分离得到,2个为首次从该属植物中分离得到。2.对收集到的小叶苦丁茶84个批次的样品进行水提取物的指纹图谱研究。分别建立了小叶苦丁茶老叶原植物（35个批次）和芽的商品（18个批次）HPLC指纹图谱共有模式,通过系统聚类分析可将老叶原植物分成3类,第Ⅲ类为除粗壮女贞外的非主流品种的小叶苦丁茶,利用第一类和第二类27个批次样品生成的对照图谱共有模式可进一步将非主流品种老叶样品区分开。同时将商品老叶导入指纹图谱共有模式中,可以确定其是否为主流品种粗壮女贞。通过对全国各地买的小叶苦丁茶18个批次的芽商品指纹图谱分析发现系统聚类分析和相似度评价均不能将不同产地及品种的芽商品区分开,样品色谱峰整体化学信息非常相似,因此我们选择它们共有的特征性成分作为市场上小叶苦丁茶质量评价的指标对其进行进一步质量控制研究。3.对小叶苦丁茶中的特征性成分苯乙醇苷及环烯醚萜苷类成分推导ESI-MS/MS裂解规律,将指纹图谱中所反映的化学成分信息通过各色谱峰的ESI-MS/MS碎片,结合文献检索及与标准品比对的方法,使得10个化学成分得到明确鉴定,另外推导了15个化合物的结构,其中有7个为未见文献报道的化合物。4.建立了HPLP-UV法测定84个批次原植物和商品中3种特征性苯乙醇苷类成分阿克苷（acteoside,1）、紫茎女贞苷A（ligupurpuroside A,2）、紫茎女贞苷B（ligupurpuroside B,3）的含量。结合指纹图谱的研究结果将各样品之间含量的差异与品种、产地、炮制方法及药用部位之间的关系进行探讨。5.对包括粗壮女贞、女贞、丽叶女贞在内的7个批次的样品挥发油进行GC-MS研究,共鉴定了65个化合物的结构。6.利用硅胶吸附柱色谱、ODS柱色谱、SephadexLH-20等色谱分离手段从紫草科苦丁茶厚壳树（Ehretia thyrsiflora）叶中分离得到35个单体化合物,通过理化性质分析和波谱解析鉴定了其中的27个,分别为槲皮素3-O-α-D-阿拉伯糖苷（quercetin-3-O-α-D-arabinoside,1）,山奈酚3-O-α-D-阿拉伯糖苷（kaempferol-3-O-α-D-arabinoside,2）,异槲皮素（quercetin-3-O-β-D-glucopyranoside,3）,金丝桃苷（hyperoside,4）,山奈酚3-O-β-D-半乳糖苷（kampferol-3-O-β-D-galactopyra noside,5）,山奈酚3-O-β-D-葡萄糖苷（kampferol-3-O-β-D-glucopyranoside,6）,kaempferol-3-O-arabinosylgalactoside（7）,quercetin-3-O-arabinosylgalactoside（8）,槲皮素（quercetin,9）,山奈酚（kampferol,10）,阿魏酸（trans-ferulic acid,11）,咖啡酸（caffeic acid,12）,对羟基苯甲酸（p-hydroxy benzoic acid,13）,迷迭香酸甲酯（methyl rosmarinate,14）,迷迭香酸（rosemarinic acid,15）,紫草酸B（lithospermic acid B,16）,丹参素（danshensu,17）,肉桂酸（cinnamic acid,18）,icariside E₅（19）,咖啡酸乙酯[（E）-ethyl caffeate,20],邻苯二甲酸二（2-乙基己）酯[di（2-ethylhexyl）phthalate,21],2-甲氧基苯甲酸辛酯（2-methoxyl benzoic acidoctyl ester,22）,邻苯二甲酸十八烷基酯[di（octadecyl） phthalate,23],β-谷甾醇（β-Sitosterol,24）,胡萝卜苷（daucosterol,25）,尿囊素（allantoin,26）,1-十四烯酸甘油酯（tetradecenoic acid,2,3-dihydroxypropyl ester,27）。所有化合物均为首次从该植物中分离得到,化合物1-13,16-18,20-23,26,27为首次从该属植物中分离得到。7.首次建立了HPLC法测定厚壳树叶中包括5种黄酮苷和2种酚酸在内的7种成分含量的方法,该方法简便快捷,分离度和重现性均较好,同时将该方法应用于紫草科7个批次样品含量测定。为药用植物资源利用提供依据。8.对厚壳树化学成分研究结果结合国内外文献调研,初步探讨了厚壳树属植物在紫草科中的亲缘关系及分类学地位。更多还原

【Abstract】 "Ku-Ding-Cha" is a traditional Chinese tea used mainly in south of China for almost 2,000 years.Although different species used in different places of China are all called "Ku-Ding-Cha".Large leaves Ku-Ding-Cha which belongs to family Aquifoliaceae and small leaves Ku-Ding-Cha which belongs to family Oleaceae are two main species used in the markets of all the country.Several plants of family Guttiferae and Boraginaceae are also used as Ku-Ding-Cha in sparing places of China.In the present study,the chemical constituents in the Ligustrum robustum;quality control method of small leaves Ku-Ding-Cha;chemical constituents and preliminary pharmacophylogenetic study of Boraginaceae Ku-Ding-Cha were studied.Sixteen compounds were isolated and identified in L.robustum,they were ligustaloside B（1）,ligustaloside B di-Me acetal（2）,ligustaloside A（3）,acteoside（4）, ligupurpuroside A（5）,isoacteoside（6）,ligupurpuroside B（7）,ligupurpuroside D（8）, osmanthuside B₆（9）,ligupurpuroside C（10）,3,4-dihydroxyphenyl（11）,lipedoside B-Ⅲa（12）,lipedoside B-Ⅲb（13）,glucose esters of（E）-2,6-dimethyl-6-hy droxyocta-2,7-dienoic acid（14）,luteolin-7-O-β-D-glucopyranoside（15）,rutin（16）.Eight of them were first isolated from this species;two of them were first isolated from this genus.84 samples including raw materials and the commercial products were collected from the tea markets or from their habitats around China and the quality control methods were developed.The HPLC fingerprint used for the quality control of small leaves Ku-Ding-Cha was established.After investigating chromatography and extracting condition,two mutual patterns were established from 35 old leaves original plants and 18 buds of products, respectively.The data were used for Hierarchical Cluster analysis and similarity evaluation.It was indicated that 35 old leaves samples were identified into three types, the identified plant L.robustum were all in the typeⅠand typeⅡ.Through the mutual pattern established the alternative species can be classified out of the main species of L. robustum.The investigation of the mutual pattern of the bud products in the market indicated although the same sample of the buds maybe belong to different species,the global chemical characters of all the bud products are very similar.The quality control can be evaluated through the common characters existence in all the bud products.The MS/MS fragments pattern of phenylethanoid glycosides and iridoid glycosides were analyzed and the typical samples were detected by LC-ESI/MS under positive and negative ion mode.The major constituents in L.robustum were analyzed through comparing with the standard compounds,fragment pattern and looked up in the literature.A HPLC-UV method was established to simultaneously quantification of 3 major phenylethanoid glycosides:acteoside（1）,ligupurpuroside A（2） and ligupurpuroside B（3） in 84 batches samples.It is considered to be suitable for quality control of the tea products and be helpful to classify the species the small leaves Ku-Ding-Cha.The chemical compositions of essential oils from 7 batches of small leaves have been studied by GC-MS method,65 compounds were identified and quantified.35 compounds were isolated from the leaves of Ehretia thyrsiflora with silica gel, ODS,sephadex-LH-20,et al.Among them,27 compounds were elucidated on the basis of chemical and spectral evidence.They were quercetin-3-O-α-D-arabinoside（1）, kaempferol-3-O-α-D-arabinoside（2）,quercetin-3-O-β-D-glucopyranoside（3）, hyperoside（4）,kampferol-3-O-β-D-galactopyranoside（5）,kampferol-3-O-β-D-gl ucopyranoside（6）,kaempferol-3-O-arabinosylgalactoside（7）,quercetin-3-O-arabin osylgalactoside（8）,quercetin（9）,kampferol（10）,trans-ferulic acid（11）,caffeic acid （12）,p-hydroxy benzoic acid（13）,methyl rosmarinate（14）,rosemarinic acid（15）, lithospermic acid B（16）,danshensu（17）,ciunamic acid（18）,icariside E₅（19）,（E）-ethyl caffeate（20）,di（2-ethylhexyl） phthalate（21）,2-methoxyl benzoic acid octyl ester（22）, di（octadecyl） phthalate（23）,β-Sitosterol（24）,daucosterol（25）,allantoin（26）, [tetradecenoic acid,2,3-dihydroxypropyl ester（27）],respectively.All of the compounds were isolated from this species for the first time.Compounds 1-13,16-18,20-23,26,27 were isolated from this genus for the first time.A RP-HPLC method was firstly established for determination of 7 major polyphenols（quercetin 3-O-arabinosylgalactoside,isoquercetrin,quercetin-3-O-α-D-arabinoside, astragalin,kaempferol-3-O-α-D-arabinoside,caffeic acid and rosmarinic acid） in E.thyrsiflora.All the compounds were analyzed within 35 min.Validation statistics showed the sensitivity,precision,repeatability,stability and the recovery of the method were good.In the same time,the method was used to evaluate the content of these seven compounds in other species of family Boraginaceae which provided the evidences of resource utilization.Through the literature and chemical investigation of E.thyrsiflora,we discussed the systematic position of the genus Ehretia and the subfamily Ehretiaceae in the family Boraginaceae which has been doubtful and controversial for a long time.更多还原