运用含有偶氮苯的自组装单层膜在光化学条件下可逆地控制细胞黏附

细胞生物学研究的一个重要方向是动态地控制细胞在基底上的黏附。最近，随着表面化学的研究深入，尤其是对烷基硫醇在金基底上形成自组装单层膜（self-assembled monolayers, SAMs）这一体系的研究，使得人们能在分子水平的表面上控制细胞黏附。精氨酸-甘氨酸-天冬氨酸（arginine-glycine-aspartate, RGD）序列首先是从细胞外基质蛋白中分离出来的，能够识别并非共价结合细胞膜表面的整合素受体，从而促进细胞黏附。以前的一些工作已经证实，将含有RGD的肽链连接到SAMs表面之后，能够生物特异性地黏附动物细胞。已有的手段比如光照、电压、加热、微电极、微流控以及表面纳米形貌的梯度变化，都不能真正实现可逆地控制细胞黏附，原因是这些方法所用的化学有限；这些方法也不能得到完全抗拒细胞黏附的表面，原因是这些方法产生的表面缺陷等不完整。用两种不同波长的光（紫外光和可见光）照射偶氮苯，偶氮苯会发生可逆的光致异构变化，因此，偶氮苯的光致异构性质可以用来可逆地控制细胞在表面黏附。运用含有偶氮苯的混合SAMs，偶氮苯的末端连接GRGDS肽，混合SAMs中是以末端为六聚乙二醇的硫醇为背景，该SAMs修饰而成的表面能够黏附或者抗拒细胞黏附，其表面黏附性质取决于SAMs中偶氮苯的构象。该方法提供了一种在分子水平的表面上我们所了解到的唯一能可逆控制细胞黏附的方法，该方法需要用到的光源来自于标准荧光显微镜所配置的汞灯。 为了实现在金基底表面可逆的控制细胞黏附，我们合成了如下三个化合物： 由于化合物1的溶解性很差，几乎在所有溶剂里都不溶，所以不能直接用化合物1制备SAMs；化合物2能高效地抗拒细胞的黏附；化合物3的偶氮苯末端是活化酯，能够连接GRGDS肽，从而控制细胞黏附。 将化合物2和化合物3以一定的比例均匀混合在金基底表面形成SAMs，然后将GRGDS肽连接到偶氮苯(反式)的末端（通过GRGDS肽的甘氨酸上的伯胺基与偶氮苯末端的活化酯反应），从而得到细胞黏附的表面。用紫外光照射该细胞黏附表面5-10小时，随着偶氮苯的构象由反式变为顺式，偶氮苯末端的GRGDS肽淹没在化合物2的六聚乙二醇中，得到抗拒细胞黏附的惰性表面。再用可见光照射该惰性表面1个小时，随着偶氮苯的构象由顺式变为反式，原先埋没在六聚乙二醇中的GRGDS肽伸展至单层膜的末端，又得到了细胞黏附的表面。因此，该表面能完全可逆地控制细胞在金表面黏附。 An important area in cell biology is the dynamic control of cell adhesion on substrates. Recent advancements in surface chemistry, in particular, self-assembled monolayers (SAMs) of alkanethiols on gold substrates, have permitted unprecedented control of cell adhesion via molecularly defined surfaces. The tri-peptide sequence arginine-glycine-aspartate (RGD), initially isolated from the extracellular matrix (ECM) proteins, can recognize and non-covalently bind with integrin receptors on cell membranes to promote cell adhesion. Some previous work has demonstrated that RGD peptide grafted on SAMs can allow bio-specific adhesion of mammalian cells that mimic natural adhesion. Existing technologies such as light, voltage, heat, microelectrodes, microfluidic systems and surface gradient of nanotopography, either cannot realize fully reversible control of cell adhesion, due to the limitation in the chemistry used, or cannot yield a surface completely resistant against cell adhesion, due to the imperfection of surfaces. Azobenzenes undergo reversible photo-induced isomerization rapidly at two different wavelengths of light (UV and visible light), it therefore potentially allows the reversible control of cell adhesion on a surface. By using a mixed SAMs presenting azobenzene groups terminated in GRGDS peptides in a background of hexa(ethylene glycol) groups, the surface can either accommodate or resist cell adhesion depending on the conformation of the azobenzene embedded in SAMs. This method provides the only means we know to control cell adhesion reversibly on a molecularly well-defined surface by using light generated by a mercury lamp equipped on standard fluorescence microscopes. To realize the reversible control of cell adhesion on gold surface, we synthesized three kinds of compounds as following, We found that it was difficult to obtain SAMs directly from compound 1 because of its poor solubility in almost all kinds of solvents; compound 2 can resist cell adhesion efficiently; compound 3 presents an azobenzene terminated with NHS-activated ester, which can couple with a GRGDS peptide to control cell adhesion. After coating a gold surface with compound 2 and 3 in appropriate ratios to form a SAM followed by coupling the GRGDS peptides with NHS-activated esters at the end of azobenzene (E configuration) resulted in a cell-adhesive SAM. Irradiating this cell-adhesive SAM with UV light for 5-10 h converted the E configuration of azobenzene into the Z form, the GRGDS peptides becoming masked in the PEG, resulting in a cell-resistant surface. These SAM could again support cell adhesion as a result of the conformational switch of azobenzene from Z to E with the irradiation of visible light for 1 h. This surface, therefore, allows completely reversible control of cell adhesion on a gold surface.

尼泊尔水东哥、水麻、木姜冬青和青荚叶的化学成分研究

活性筛选中发现尼泊尔水东哥 (Saurauia napaulensis DC.) 树皮95%乙醇提取物具有α-淀粉酶抑制活性、水麻(Debregeasia orientalis) 枝叶95%乙醇提取物显示血管紧张素转化酶(ACE)抑制活性、青荚叶(Helwingia japonica (Thunb.) Dieter.) 95%乙醇提取物的中小极性部分显示蛋白酪氨酸磷酸酯酶(PTP)1B抑制活性。为全面了解它们的成分及相关活性成份，主要运用硅胶柱层析方法从这三个植物分离得到39个化合物，通过波谱分析或与已知品对照的方法对其进行了鉴定。对木姜冬青(Ilex litseaefolia Hu et Tang)的成分做了进一步的研究，取得了如下结果。 1. 从尼泊尔水东哥树皮的95%乙醇提取物分离并鉴定12个化合物： auranamide、aurantiamide benzoate、齐墩果酸、β-谷甾醇、β-胡萝卜甙、乌苏酸、2α,3α-二羟基-12-烯-28-乌苏酸、2α,3β,24-三羟基-12-烯-28-乌苏酸、(2S,3S,4R,10E)-2-[(2'R)-2' -hydroxytetracosanoylamino] -10-octadecene -1,3,4-triol、 2α,3α,24-三羟基-12-烯-28-齐墩果酸、2α,3β-二羟基-12-烯-28-乌苏酸和2α,3α,24-三羟基-12-烯-28-乌苏酸。 2. 从水麻枝叶的95%乙醇提取物分离并鉴定了18个化合物：棕榈酸、二十烷酸、二十烷酸甲酯、β-谷甾醇、Monogynol A、桦木酸、Hederagenin、β-胡萝卜甙、18αH-19(29)-烯-3-酮-乌苏烷、3，4-开环-20(30)-烯-乌苏烷-3-酸、Pomolic acid，表儿茶素、儿茶素、槲皮素、槲皮素-3-O-β-D-吡喃葡萄糖苷、紫丁香苷、紫丁香酚苷和山萘酚-3-O-芸香糖。儿茶素、槲皮素和槲皮素-3-O-β-D-吡喃葡萄糖苷为具有ACE抑制活性的成分。 3. 从木姜冬青95%乙醇提取物的乙酸乙酯部分分离并鉴定了5个化合物： 2-O-β-D-吡喃葡萄糖-6,2´-二羟基-4,4´-二香草酰氧甲基-1,1´-二苯醚（冬青苷）和四个已知化合物：七叶内酯、香草酸、3,4-二甲氧基苯乙酸和vanilloylcalleryanin。冬青苷为新化合物。 4. 从青荚叶95%乙醇提取物的中小极性部分分离并鉴定了9个化合物：β-谷甾醇、β-胡萝卜苷、羽扇豆醇、桦木醇、桦木酸、棕榈酸甘油酯、桂皮酸、6αH-4-烯-3-酮-豆甾醇和6βH-4-烯-3-酮-豆甾醇。 5. 对1985-2006年间天然二苯醚类化合物及活性研究进展进行综述. The in vitro test indicated that the 95% ethanolic extract of the barks of Saurauia napaulensis DC showed α-amylase inhibitory activity, the 95% ethanolic extract of the whole plants of Debregeasia. orientalis showed angiotensin converting enzyme (ACE) inhibitory activity and some fractions of the 95% ethanolic extract of the aerial parts of Helwingia japonica showed protein tyrosine phosphatase (PTP)1B inhibitory activity. In order to investigate components and active compounds of the three plants, they were chemically studied mainly using. Thirty-nine compounds were isolated predominantly by column chromatography identified by spectral methods or comparing them with authentic samples. Further investigation of Ilex litseaefolia Hu et Tang was carried out. Major results are as follows: 1. Twelve compounds were isolation from the 95% ethanolic extract of the barks of S. napaulensis DC. They were identified as auranamide, aurantiamide benzoate, oleanolic acid, β-sitosterol, β-daucosterol, ursolic acid, 2α,3α-dihydroxyurs-12-en-28-oic acid, 2α,3β,24-trihydroxyurs-12-en-28-oic acid, (2S,3S,4R,10E)-2-[(2'R)-2'-hydroxytetracosanoyl amino]-10-octadecene-1,3,4-triol, 2α,3α,24 -trihydroxyolean-12-en-28-oic acid, 2α,3β-dihydroxyurs-12-en-28-oic acid, and 2α,3α,24-trihydroxyurs-12-ene-28-oic acid, respectively, by spectral methods or comparing them with authentic samples. 2. Eighteen compounds were isolation from the 95% ethanolic extract of the whole plants of D. orientalis. They were identified as palmitic acid, henicosanoic acid, henicosanoic acid methyl ester, β-sitosterol, monogynol, betulinic acid, hederagenin, β-daucosterol, 18αH-urs-20(30)-en-3-one, 3,4-seco-urs-20(30)-en-3-oic acid, pomolic acid, (-)-epicatechin, (+)-catechin, quercetin, quercetin 3-O-β-D-glucopyranoside, syringin, syringiaresinol digloside and kaempferol-3-O-rutinose. (+)-Catechin, quercetin and quercetin 3-O-β-D-glucopyranoside were the ACE inhibitory active components. 3. Further phytochemical investigation of the ethyl acetate parts of 95% ethanolic extract of the whole plant of I. litseaefolia afforded 2-O-β-D-glucopyranose-4,4´-di-vanilloyloxymethyl-2,6´-dihydroxy-1,1´-diphenyl ether (ilexiside), esculetin, vanillic acid, 3,4-dimethoxybenzylacetic acid and vanilloylcalleryanin. Ilexiside was new compound. 4. Nine compounds were isolation from the 95% ethanolic extract of the whole plant of H. japonica: β-sitosterol, β-daucosterol, lupeol, betulin, betulinic acid, glycerol monopalmitate, cinnamic acid, stignast-4-en-6β-3-one and stignast-4-en-6α-3-one 5.Diphenyl ether compounds from nature between 1985-2006 were summarized.

几种中药材的化学成分及其定性定量检测方法研究

本论文由四部分组成。第一部分报道了佛手参提取物的化学成分研究，建立了活性成分含量测定的高效液相测定和指纹图谱研究，采用液质联用技术鉴定了主要色谱峰；第二部分报道了丹参及其复方制剂的特征图谱研究；第三部分探讨了两面针生物碱的电喷雾质谱裂解规律，并采用液质联用技术分离鉴定了提取物中的多种生物碱。第四部分概述了液质联用在药物代谢研究中的运用。 第一部分包括第一、第二和第三章。第一章针对佛手参（Gymnadeniaconopsea）块茎的甲醇提取物，采用大孔树脂和反相硅胶柱层析等各种分离方法，共分离鉴定出4 个化合物，通过波谱分析将它们的结构确定为dactylorhin B (1)、loroglossin (2)、dactylorhin A (3)和militarine (4)。这4 个化合物均是首次从佛手参中分离得到的琥珀酸葡萄糖苷类成分。第二章采用高效液相色谱法对西藏、四川、河北、青海和尼泊尔等不同地区产的十个佛手参样品进行腺嘌呤核苷和对羟基苯甲醇的定量分析，结果表明这2 个成份可视为佛手参的特征成分，但也注意到产地不同该2 个特征成分的含量也有所不同。第三章采用标准中药指纹图谱相似度计算软件，以10 个佛手参样品HPLC 图谱的平均值为相似性评价对照模板，对10 个样品进行了相似度评价，并经液质联用分析指认了7 个共有峰，分别为腺嘌呤核苷(1)、对羟基苯甲醇(2)、对羟基苯甲醛(3) 、dactylorhin B(4) 、loroglossin(5)、dactylorhin A(6)和militarine(7)。 第二部分包括第四、第五、第六和第七章。第四章运用电喷雾质谱检测了对照药材和五个不同产地的丹参药材中脂溶性和水溶性成分，系统地探讨了多种成分的电喷雾质谱规律，并以对照药材为标准建立了特征指纹图谱。五个产地的药II材通过与对照药材相对比，采用聚类分析的方法，得到了定性的鉴别与判断。并采用液质联用技术对丹参药材提取液中的化学成份进行分析，推测了九个特征峰，并对六样品的液相色谱图进行了聚类分析。第五章探讨了三七皂苷的电喷雾质谱电离和裂解规律，并采用电喷雾质谱法对三七标准药材，血通片中的皂苷成分进行了分析。第六章运用电喷雾质谱研究复方丹参片提取液的特征图谱，并和单味药材丹参和三七的特征图谱进行了对比研究。并运用HPLC-ESI MSn 分析鉴定了复方丹参片提取液中的化学成分，推测了12 个色谱峰。第七章总结了电喷雾质谱和液质联用技术在丹参药材，三七药材及复方丹参制剂中的运用的优势和局限性。 第三部分（第八章）研究了两面针生物碱中二氢白屈菜红碱(1)、二氢两面针碱(2)、8-酮基二氢白屈菜红碱(3)、8-丙酮基二氢两面针碱(4)、两面针碱(5)、和1,3-二(8-二氢两面针碱)丙酮(6)等六个苯并菲啶型生物碱的电喷雾质谱裂解规律，其中二氢两面针碱和二氢白屈菜红碱，8-丙酮基二氢两面针碱和8-酮基二氢白屈菜红碱是两对二个甲氧基分别在C-9 和C-10，C-10 和C-11 的同分异构体。实验结果表明，在相同的碰撞能下，这类位置异构体的ESI MS2 质谱二级碎片离子的相对峰度存在很大差异，这可以用于区分该类同分异构体，采用液-质联用可以对两面针的总生物碱提取物中的这些同分异构体加于区分。同时在本实验采用的液相色谱条件下，多种生物碱得到较好的分离，通过和对照品的保留时间，紫外吸收光谱及电喷雾质谱图对照，鉴定了11 个主要色谱峰。 第四部分（第九章）对液质联用技术在药物代谢中的运用进行了综述。 This dissertation consisted of four sections. The first two sections elaborated thephytochemical investigation of the rhizomes Gymnadenia conopsea R. Br., methoddevelopment for rapid identifying and qutifying the chemical condtituent of thistibetant medicine, and the chemical fingerprint analysis of rhizomes of G. conopsea,Salviae miltiorrhiza and P. notoginseng. The third section studied the fragmentationmechanism of six alkaloids from Zanthoxylum nitidium and method development forrapid identifying varieties of alkaloids from the extract of this herbal medicine. Thefourth section reviewed HPLC- MS method in drug metabolism studies. The first section consisted of chapters 1, 2, 3. Chapter 1 elaborated the phytochemicalinvestigation of Gymnadenia conopsea R. Br. Four succinate derivative esters wereisolated from the methanol extract of the rhizomes of G. conopsea through repeatedcolumn chromatography on normal and reversed phase silica gel, their structures weredetermined by ESI-MS, 1D and 2D NMR evidence. They were firstly discoveredfrom this species. In chapter 2, a high-performance liquid chromatography.diodearray detection (HPLC-DAD) method has been firstly developed for quantitation oftwo characteristic constituents, adenosine and 4-hydroxybenzyl alcohol, from theextract of rhizomes of G. conopsea. All 10 samples of G. conopsea contained differentamount of adenosine and 4-hydroxybenzyl alcohol. Adenosine and the4-hydroxybenzyl alcohol can be applied in identification and quality control for theroots of G. conopsea. In chapter 3, a high-performance liquid chromatography.diodearray detection.tandem mass spectrometry (HPLC-DAD-MSn) method has been firstly developed for chemical fingerprint analysis of rhizomes of G. conopsea andrapid identification of major compounds in the fingerprints. Comparing the UV andMS spectra with those of authentic compounds, seven main peaks in the fingerprintswere identified as adenosine, 4-hydroxybenzyl alcohol, 4-hydroxybenzyl aldehyde,dactylorhin B, loroglossin, dactylorhin A and militarine. The Computer AidedSimilarity Evaluation System for Chromatographic Fingerprint of TraditionalChinese Medicine (CASES) was employed to evaluate the similarities of 10 samplesof the rhizomes of G. conopsea collected from Sichuan, Qinghai and Hebei provincesand Tibet autonomous region of China, and Nepal. These samples from differentsources had similar chemical fingerprints to each other. The second section consisted of chapters 4, 5, 6 and 7. In chapter 4，both thecharacteristic spectra of liposoluble tanshinones and aqueous-soluble salvianolic acidswere established by the electrospray ionization mass spectrometry (ESI MS)technique and the differences between standard and crude rhizomes of Salviaemiltiorrhiza Bge. from 5 sources were analyzed. The law of electrospray ion trap mass(ESI ITMS) of typical tanshinones and salvianolic acids is studied.The analysis of the chemical constituent of rhizomes of Salviae miltiorrhiza Bge. byliquid chromatography coupled with mass spectrum (LC/MS) technique wasestablished，and the distances among standard herb and crude herb from 5 sourceswere calculated by clustering analysis. According the DAD spectra and MS2 data，9tanshinones could be speculated. In chapter 5, the character spectra of total saponinsin P. notoginseng extracts were established by ESI ITMS and selective ion monitoring(SIM) technology. The law of notoginsenosides by ESI MS2 was studied. In chapter 6,the characteristic spectra of Compound Danshen Tablet established and compared byESI-MS and HPLC/DAD/MS, 6 known tanshinones and 3 saponins were speculated.In chapter 7, the advantage and disadvantage of the strategy, using the ESI ITMS andLC/MS techniques for study of characteristic spetra of danshen and Compound Danshen Tablet, were summerized. The third section (chapter 8) studied the fragmentation mechanism of six alkaloids,dihydronitidine, dihydrochelerythrine, 8-acetonyl dihydronitidine,8-acetonyldrochelerythrine, nitidine and 1,3-bis (8-dihydronitidinyl)-acetone, by ESIMSn. Tandem mass spectrometry experiments indicated that different substitutionsites of the methoxyl groups at C-9 and C-10 or at C-10 and C-11 determined thedifferent abundances of the MS2 fragmentation ions using the same collision energy.According to the different abundances of MS2 product ions, positional isomericbenzo[c] phenanthridine alkaloids can be differentiated. Moreover, ten constituents inthe crude alkaloids extract from the roots of Zanthoxylum nitidium were rapidlyidentified by high-performance liquid chromatography coupled with tandem massspectrometry (HPLC-MSn), through comparing the retention times and ESI MSn spectra with the authentic standards. The fourth section (chapter 9) is a review on HPLC-MS method development in drug metabolism studies.

五枫藤、小驳骨和川西茶藨的化学成分研究

八月瓜属植物五枫藤(Holboellia latifolia Wall.)和驳骨草属植物小驳骨(Gendarussa vulgaris Nees)均为药用植物, 前者化学成分研究不深入, 后者的化学成分未见报道。川西茶藨(Ribes takare D. Don)为茶藨子属植物, 没有化学成分的报道。本论文对三个植物的化学成分和活性成分进行了研究, 主要通过色谱方法分离得到了48 个化合物, 采用波谱分析或与已知标准品对照等手段鉴定了它们的结构, 其中有1 个新的原小檗碱类化合物和3 个新的联苯类化合物,发现了具有细胞毒活性和α-葡萄糖苷酶抑制活性的化合物。1、从五枫藤地上部分的95%乙醇提取物中分离得到了12 个化合物: 五加苷K (1)、hederagenin 3-O- α-L-rhamnopyranosyl-(1→2)- α-L-arabinopyranoside (2)、β-萘乙酸(3) 、3-O-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-α-L-arabinopyranosyl oleanolic acid 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (4) 、3-O- α-L-rhamnopyranosyl-(1→2)-O- β- D-glucopyranosyl-(1→2)- α-L-arabinopyranosyl oleanolic acid (5) 、3-O-( β-D-glucopyranosiduronic acid)-oleanolic acid 28-O- β-D-glucopyranoside (6)、lup-20(29)-en-3-one (7)、lupeol (8)、β-谷甾醇(9)、齐墩果酸(10)、乌苏酸(11)、β-胡萝卜苷(12)。化合物1 对Lu-06、N-04 和Bre-04 癌细胞株的GI50 分别是0.77µg/mL、1.26 µg/mL 和1.55 µg/mL, 化合物2 对N-04 癌细胞株的GI50 为2.44 µg/mL。2、从小驳骨地上部分的95%乙醇提取物中分离得到了1 个原小檗碱类新化合物13-hydroxyl gusanlung A (25), β-谷甾醇(9)、齐敦果酸(10)、β-胡萝卜苷(12)、棕榈酸(1-)甘油酯(13)、棕榈酸(14)、阿苯哒唑(15)、阿苯哒唑砜(16)、阿苯哒唑亚砜(17)、aurantiamide acetate (18)、华良姜素(19)、芫花素(20)、(-)-丁香树酯醇(21)、gusanlung B (22) 、eupteleasaponinsⅤ acetate (23)、gusanlungA (24)、刺五加苷E (26)、岩白菜素(27)、咖啡酸(28)。化合物25 对肝癌细胞株(HepG2) 的GI50 为2.08 µg/mL。3、从川西茶藨地上部分的95%乙醇提取物中分离鉴定了22 个化合物: β-谷甾醇(9) 、β- 胡萝卜苷(12) 、O-acetyloleanolic aldehyde (29),4,7,8-trimethoxy-2,3-methylenedioxydibenzofuran (30) 、3', 5-dimethoxy-3, 4-methylenedioxybiphenyl (31) 、桦木醇(32) 、6,7-dimethoxy-1-methyl-3,4-dihydroquinolin-2-one (33)、3'-hydroxy-5-methoxy-3,4-methylenedioxybiphenyl (34) 、7-hydroxy-4,8-dimethoxy-2,3-methylenedioxydibenzofuran (35)、桦木醛(36)、没食子酸(37) 、6β- 羟基-4- 烯-3- 酮- 豆甾醇(38) 、5α, 8α-epidioxy-(22E,24R)-ergosta-6, 22-dien-3β-ol (39)、verrucofortine (40)、6-methoxycalpogoniumisoflavone A (41)、2-羟基二苯甲酮(42)、桦木酸(43), 3, 5-二甲氧基苯甲酸-4-O-β-D-吡喃葡萄糖苷(44)、洋芹素(45)、刺槐素(46)、水杨酸(47)、洋芹素-5-O- β-D-葡萄糖苷(48), 化合物30、31 和35 为新的联苯化合物。化合物30的α-葡萄糖苷酶抑制率为10.2% (1.00 mg/mL); 化合物35 的抑制率为17.2% (1.00mg/mL)。4、综述了1960 年以来原小檗碱类化合物药理活性研究进展。 Plants Holboellia latifolia Wall and Gendarussa vulgaris Nees, are used as folkmedicine. Ribes takare D. Don belongs to the genus Ribes. The three plants have notbeen chemically studied in detail. Chemical and bioactive study of three plants led tothe isolation of 48 compounds by chromatography. Their structures were elucidatedon the basis of spectroscopic evidence or comparison with authentic samples. Amongthe 48 componds isolated one protoberberine alkaloid and three biphenyls are newones. Cytotoxic and α-glucosidase inhibitory compounds had been found.1. Twelve compounds were isolated from the 95% ethanol extract of the aerial partof H. latifolia Wall. They were characterized as fellow: eleutheroside K (1),hederagenin-3-O- α-L-rhamnopyranosyl-(1→2)- α-L-arabinopyranoside (2),2-naphthyl acetic acid (3),3-O-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-α-L-arabinopyranosyl oleanolic acid 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester (4), 3-O- α-L-rhamnopyranosyl-(1→2)-O- β- D-glucopyranosyl-(1→2)- α-L-arabinopyranosyl oleanolic acid (5),3-O-( β-D-glucopyranosiduronic acid)-oleanolic acid-28-O- β-D-glucopyranoside (6),lup-20(29)-en-3-one (7), lupeol (8), β-sitosterol (9), oleanolic acid (10), ursolicacid (11), and β-daucosterol (12). Compound 1 showed moderate cytotoxicity againstLu-06 (GI50, 0.77 µg/mL), N-04 (GI50, 1.26 µg/mL) and Bre0-4 (GI50=1.55 µg/mL)and compound 2 showed moderate cytotoxicity against N-04 (GI50=2.44 µg/mL).2. A new protoberberine alkaloid, 13-hydroxyl gusanlung A (25), was isolated fromthe aerial part of Gendarussa vulgaris Nees, together with β-sitosterol (9), oleanolicacid (10), β-daucosterol (12), glycerol monopalmitate (13), palmific acid (14),albendazole (15), albendazole sulphone (16), albendazole sufloxide (17), aurantiamideacetate (18), kumatakenin (19), genkwanin (20), (-)-syringaresinol (21), gusanlung B(22), eupteleasaponinsⅤ acetate (23), gusanlung A (24), eleutheroside E (26),bergenin (27) and caffeic acid (28). Compound 25 showed cytotoxicity against HepG2 cells (GI50, 2.08 µg/mL).3. Phytochemical study of the Ribes takare D. Don led to the isolation of three newbiphenyls, 4,7,8-trimethoxy-2,3-methylenedioxydibenzofuran (30), 3', 5-dimethoxy-3,4-methylenedioxybiphenyl (31) and 7-hydroxy-4,8-dimethoxy-2,3-methylenedioxydibenzofuran (35), along with nineteenknown compounds, β-sitosterol (9), β-daucosterol (12), O-acetyloleanolic aldehyde(29), betulin (32), 6,7-dimethoxy-1-methyl-3,4-dihydroquinolin-2-one (33),3'-hydroxy-5-methoxy-3, 4-methylenedioxybiphenyl (34), betulinic aldehyde (36),gallic acid (37), stigmast-4-en-6β-ol-3-one (38), 5α, 8α-epidioxy-(22E, 24R)-ergosta-6,22-dien-3β-ol (39), verrucofortine (40), 6-methoxycalpogonium isoflavone A (41),2-hydroxybenzophenone (42), betulinic acid (43), 3,5-dimethoxygallic acid-4-O- β-D-glucopryranoside (44), apigenin (45), acacetin (46), salicylic acid (47) andapigenin-5-O- β-D-glucopryranoside (48). α-Glucosidase inhibitory rates ofcompound 30 and 35 were respectively 10.2% and 17.2% at a concentration of 1.00 mg/mL).4. Pharmacological activities of protoberberines were summarized.

余甘子、细叶草乌及土荆皮化学成分研究

本论文由三部分共6 章组成。第一部分报道了余甘子、细叶草乌和土荆皮等三种药用植物的化学成分研究成果；第二部分报道了细叶草乌和土荆皮中分离得到的化合物的活性测试，以及这两种植物的质谱分析；第三部分概述了土荆皮的研究现状。第一部分包括1－3 章。在第1 章、第2 章和第3 章中分别报道了余甘子(Phyllanthus emblica L.) 、细叶草乌(Aconitum richardsonianum var.pseudosessiliflorum) 和土荆皮( pseudolarix kaempferi) 的化学成分。采用正、反相硅胶柱层析等各种分离方法，从余甘子中共分离出10 个化合物，其中1 个为新化合物，另外还有2 个为首次从该植物中分离得到。细叶草乌的化学成分研究尚未见报道，我们从该植物中共分离出15 个化合物，其中6 个为二萜生物碱，9个为非生物碱成分。从土荆皮中分离得到16 个化合物，其中8 个二萜、1 个三萜和7 个其它类型化合物，其中有4 个化合物为首次在该植物中分离得到；从土荆皮挥发油中分离鉴定出了22 个化合物，占挥发油总量的90%。第二部分包括4－5 章。第4 章报道了从细叶草乌和土荆皮中分离得到的13个化合物的药理活性研究，结果显示，展花乌头宁和土荆乙酸葡萄糖苷等表现出较高的组织蛋白酶K 抑制活性；土荆乙酸葡萄糖苷表现出较高的组织蛋白酶B抑制活性；8-去乙酰滇乌碱表现出较高的蛋白质酪氨酸磷酸酶抑制活性。第5 章报道了细叶草乌和土荆皮总浸膏的质谱( ESI-MS ) 分析，研究结果表明，ESI-MS 法可以简单快速地检测这两种植物的主要成分；通过ESI-MS2 分析初步探讨了一些化合物的裂解规律，尝试质谱在其结构测定中的具体应用。第三部分为第6 章。从化学成分、药理、构效关系、主要成分的定量分析、及其合成研究等方面概述了土荆皮的研究进展。 This dissertation consists of three parts. The first part elaborate thephytochemical investigation of three medicinal plants, Phyllanthus emblica L.,Aconitum richardsonianum var. pseudosessiliflorum and pseudolarix kaempferi. Thesecond part reported the bioassay of 13 constituents from Aconitum richardsonianumvar. pseudosessiliflorum and pseudolarix kaempferi, and ESI-MS analysis of these twoplant . The third part is a review on the research progress of pseudolarix kaempferi.The first part is composed of three chapters. Chapters 1-3 focus on the isolationand identification of chemical constituents from Phyllanthus emblica L., Aconitumrichardsonianum var. pseudosessiliflorum and pseudolarix kaempferi. 10 compoundsincluding a new tannin were isolated from the fruits of Phyllanthus emblica by repeatcolumn chromatography over normal and reversed phase silica gel, 2 of them werefirstly reported in this plant. The chemical constituents of Aconitum richardsonianumvar. pseudosessiliflorum never reported before, 15 compounds including 6 diterpenealkaloids were isolated and identified from the roots of this plant. 16 compoundsincluding 8 diterpenes , 1 triterpene and 7 other compounds were isolated from the bark of pseudolarix kaempferi, among them, 4 compounds were firstly reported fromthe EtOH extracts of this plant, and 22 compounds were identified from its essentialoil, representing 90% of the total essential oil.The second part includes chapters 4 and 5. Chapter 4 reported thepharmacological activities of 13 compounds isolated from Aconitum richardsonianumvar. pseudosessiliflorum and pseudolarix kaempferi. Results demonstrated that chasmanine and pseudolaric acid B-β-D-glucoside exhibit relatively high anti-Cathepsin K activities; pseudolaric acid B-β-D-glucoside exhibits relatively highanti-Cathepsin B activity; 8-deacetyl-yunaconitine exhibits relatively high anti-PTP1Bactivity. Chapter 5 reported the ESI-MS analysis of extractions from Aconitumrichardsonianum var. pseudosessiliflorum and pseudolarix kaempferi, it was showedthat ESI-MS can be used as an useful tool in analyzing the major constituents of thesetwo plant much quick and easy, in addition, the fragmentation rules of somecompounds were discussed, in order to find some applications of ESI-MS2 method in their structure determination.The third part is a review on the research progress of pseudolarix kaempferi,including the chemical constituents, pharmacology, structure-activity relationship(SAP), quantitative analysis and synthesis of the major constituents.

低极性化合物的电喷雾质谱检测方法及几种中藏药材的活性成分研究

本学位论文首先报道了为解决低极性化合物的电喷雾质谱（ESI-MS）分析难题而建立的一种衍生化分析方法；然后从色谱-质谱联用分析、分离纯化和结构鉴定等方面分别报道了几种中藏药材的活性成分研究。论文由下述六章组成： 第一章报道了盐酸羟胺衍生化方法在电喷雾质谱 (ESI-MS) 分析中的应用。该方法利用盐酸羟胺和羰基成肟的快速反应，建立了针对三萜酮等含酮或醛羰基低极性化合物的ESI-MS 信号增强技术。此方法不仅可应用于增强羰基化合物的ESI-MS 质谱信号，还可检测化合物中羰基的个数以及辨别涉及羰基官能团的同分异构体。此外，通过简单的氧化反应，还可将该方法拓展到三萜醇、甾醇等含羟基的低极性化合物，增强它们的ESI-MS 信号。对比已报道的相关ESI-MS 增强质谱信号的衍生化方法，此方法有经济、实用、快速和简便的显著特点。 第二章是关于野生羌活及其栽培品种化学成分的色谱-质谱联用分析。对不同产地野生羌活生长过程中活性成分的动态变化、野生羌活不同形态部位和人工栽培羌活中的活性成分含量进行了HPLC 定量分析。结果表明主要活性成分羌活醇和异欧前胡素都随生长期存在规律性变化，羌活不同形态部位中的活性成分含量也有明显不同。这些实验结果有些较好地印证了传统中医的用药理论，有些也对羌活的传统使用方法提出了新的建议。 第三章介绍了几种传统中藏药材的色谱-质谱联用及串联质谱分析。通过GC-MS 方法，从藏药材长花党参挥发油中共分离鉴定出45 个化合物；利用HPLC方法测定了该藏药材中的主要化学成分——木犀草素的含量（0.7%）；利用串联质谱技术，对西番莲和射干中的主要成分进行了快速鉴定，从西番莲中鉴定了4个黄酮碳苷；从不同产地的射干和川射干中鉴定了8 个主要异黄酮成分，其中包括一个未见报道的化合物。 第四章的内容为藏药材石莲叶点地梅的活性成分研究。从植物石莲叶点地梅(Androsace integra (Maxim.) Hand.-Mazz.) 乙醇提取物的正丁醇萃取部分共分离和鉴定了6 个化合物，利用MS 和NMR 等现代波谱学技术阐明了它们的结构：其中包括4 个三萜类化合物：分别是androsacin (1)、 ardisiacrispin A (2) 、saxifragifolin A (3) 和20(29)-lupen-3-one (4)；一个神经酰胺：4-羟基-Δ8,9(Z)-鞘氨醇-2'-羟基正二十四碳酸酰胺(5)；一个甾体类化合物：胡萝卜苷(6)。化合物1为新的13,28-epoxy-oleanane 型三萜皂苷，在其结构表征的过程中，采用LC-MS 进行糖分析，获得了值得推广的好结果。通过活性筛选发现化合物1~3 对HepG2肝癌细胞表现出不同程度的抑制活性，其中化合物2 活性最好，其IG50 为1.65μg/mL。 第五章是关于一些传统中藏药材的农药活性筛选。利用Syngenta 公司的活性筛选平台对68 种传统中藏药材醇提物进行了抗菌和除草的生物源农药活性筛选。结果表明所筛选的68 种植物提取物中，共有14 种样品表现出明显的除草/杀虫活性，其中水母雪莲花、松萝和茯神木等植物提取物还具有多种生物活性。活性成分还有待进一步追踪分离、纯化和结构鉴定。 第六章为文献综述，概述了羌活药材的研究进展。对羌活属及药用羌活植物从分类学、本草学、品质评价、人工栽培、化学成分及药理作用等方面进行了文献归纳和总结。 In this dissertation, an electrospray ionization mass spectrometry (ESI-MS) signal enhancement method, as well as the work of bioactive components study, HPLC-MS/MS application, bioassay screening, chromatograph separation and structure identification of the metabolites in several medicinal herbs have been reported. First chapter expounded a rapid, simple ESI-MS sensitivity enhancement method for detecting carbonyl groups in natural products has been developed by using hydroxylamine hydrochloride (NH2OH·HCl) as a derivatization reagent. We use the oxime formed during the derivatization reactions and its Beckmann rearrangement intermediates as a means of detecting the carbonyl groups originally present in these triterpenoids. In comparison with other derivatization methods in the literature, this method is simple, specific and can be used to detect carbonyl groups in triterpenoids which have low polarity and are poorly or non-ionizable. Moreover, it can also be used to detect hydroxyl groups by using the Dess-Martin periodinane (DMP) to convert primary and secondary hydroxyls into carbonyl groups. Chapter 2 reported an HPLC-MS method for analyzing the main bioactive compounds in both wild and cultured Notopterygium incisum. The results indicated that the main bioactive compounds varied through different seasons regularly, and in different commercial parts of this herb the content of these compounds also differed from each other. The quantitative analysis results showed that in the traditional commercial parts, the content of main chemical constitutes in Silkworm Notopterygium, Bamboo Notopterygium and Irregular-nodal Notopterygium are higher than that in Striped Notopterygium. This result is tally with the traditionally concept that the quality of Notopterygium, Bamboo Notopterygium and Irregular-nodal Notopterygium are better than that of Striped Notopterygium, which means that the quality of rhizomes is better than main roots. The chemical constituents of cultured N. incisum is reported for the first time in this dissertation and the analysis results showed some growth curves of chemical constituents in this plant, but still left some questions unanswered. Chapter 3 discussed the GC/LC-MS analysis of the traditional Chinese medicines Codonopsis thalictrifolis, Passiflora incarnate, Belamcanda chinensis and Passiflora incarnate. The main constituent, luteolin was isolated and identified from the traditional Tibet medicine of C. thalictrifolis. The quantitative analysis by HPLC has revealed that the content of luteolin in this herb is 0.7%. GC-MS was employed to analyzed chemical constituents of the essential oil from the flower of C. thalictrifolis. More than 60 peaks were detected and 45 of them were identified by comparing their spectra with that of the standards in the database and literatures. ESI-MS/MS was used to analyze the n-butanol extract of Passiflora incarnate. Based on the information of pseudo molecular ions and fragment ions of the glycosides, four major flavone-C-glycosides have been detected and identified as 7-methoxyluteolin-6-C-β-D-glucopyranoside, vitexin, swertisin and orientin. The isoflavone compounds in theextracts of three samples of B. chinensis collected in Gansu, Sichuan and Hunan, and the extract of Iris tectorum collected in Sichuan were analyzed by using TOF-HRMS and IT-MS. From the extracts of these herbs, a new isoflavone, identified as 5’,5,6,7-tetrahydroxy-3’4’-dimethoxyl isoflavon, and 7 known ones have been identified by analyzing the fragmentation patterns and their molecular formulas given by HRMS and the tandem mass spectrometry acquired by IT-MS. Chapter 4 elucidated the isolation and identification of a new triterpene saponin, androsacin (1), along with five known compounds (2-6) were isolated from the whole plants of Androsace integra (Maxim.) Hand.-Mazz., an herb used in traditional Chinese and Tibetan medicine. The chemical structure of the new compound was established as 3β-O-{β-D-glucopyranosyl-(1→4)-O-β-D-xylopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→4)-[O-β-D-glucopyranosyl-(1→2)]-α-L-arabinopyranosyl}-16α-hydroxy-13β,28-epoxy-olean-30-al by analyzing its MS, 1D- and 2D-NMR spectra. Compound 2 was cytotoxic toward HepG2 cancer cell with the GI50 value of 1.65 μg/mL. Chapter 5 described the biogenic pesticide activity screening of 68 traditional Chinese and Tibetan medicine extractions. The intention of this study is to explore bioactive natural compounds from these traditional medicinal herbs for biogenic insecticides use. Based on Syngenta’s bioassay, 14 extractions of these traditional medicines showed pesticide activities, and some of them had multi-activities on antibacterial and insecticidal. Chapter 6 is a review on the chemical and bioactivity research progress of Notopterygium incisum and N. forbesii.

Facile synthesis of ginsenoside Ro

Two concise synthetic routes, being different in the glycosylation sequence, toward ginsenoside Ro (1) are developed. These syntheses feature the elaboration of the glucuronide residue at a later stage via the TEMPO-mediated selective oxidation and the installation of AZMB as a benzoylic neighboring participating group capable of being selectively removed afterward.

石油污染土壤生物修复过程中微生物生态研究

The analysis on microbiological ecology for four types of oil contaminates soils showed that the bacteria utilizing the oil as carbon sources increase,wheras the fugi become less .Zooloea and Bacillu are the dominant bacteria ; Mocor and Cunninghamella ,and Fursarium are the dominant fungi streptomyces take the superiority among the actinomyces.The anaiysis on esterase activity showed that the microbes above mentioned have abilies of degrading esters. The biodeg radationrates are 55.45%,56.74%,38.37% and 45.19%respectively,after 53 days,the biodegradation rate can be increased by 12.6% when the dominant microbes are added.

Effect of acetylation on the properties of corn starch

Acetylated corn starches with different degrees of substitution (DS 0.85, DS 1.78, DS 2.89) were synthesized by the reaction of corn starch with acetic anhydride in the presence of acetic acid under varying reaction temperatures. The product was characterized by FTIR spectroscopy, H-1 NMR, X-ray diffraction and contact angle measurement. Acid-base titration and H-1 NMR methods were employed to determine the degree of substitution of product. FTIR spectroscopic analysis showed that the characteristic absorption intensities of esterified starch increased with increase in the degree of substitution, and the characterized peak of hydroxyl group almost disappeared in the spectrum of DS 2.89 acetylated starch. The detailed chemical microstructure of native starch and acetylated starch was confirmed by H-1 NMR, C-13 NMR and C-13-(1) H-1 COSY spectra.

Rare-Earth Metal Bis(alkyl)s Supported by a Quinolinyl Anilido-Imine Ligand: Synthesis and Catalysis on Living Polymerization of epsilon-Caprolactone

The tridentate ligand N-(2-((2,6-diisopropylphenylimino)methyl)phenyl)quinolin-8-amine (HL) was prepared. Treatment of HL with 1 equiv of Ln(CH2SiMe3)(3)(THF)(2) afforded the corresponding rare-earth metal bis(alkyl) complexes LLn(CH2SiMe3)(2)(THF)(n) (Ln = Sc, n = 0 (1); Y, n = 1 (2); Lu, n = 0 (3)) in high yields. Variable-temperature H-1 NMR spectral analysis showed that these complexes were fluxional at room temperature. Complexes 1 and 3 were THF-free, where the metal center adopted a square-pyramidal geometry, while in 2 the metal center generated a distorted octahedral geometry owing to the coordination of a THF molecule.

Enolic Schiff base aluminum complexes and their catalytic stereoselective polymerization of racemic lactide

A series of enolic Schiff base aluminum(III) complexes LAIR (where L = NNOO-tetradentate enolic Schiff base ligand) containing ligands that differ in their steric and electronic properties were synthesized. Their single crystals showed that these complexes are five -coordinated around the aluminum center. Their coordination geometries are between square pyramidal and trigonal bipyramidal. Their catalytic properties in the solution polymerization of racemic lactide (rac-LA) were examined. The modifications in the auxiliary ligand exhibited a dramatic influence on the catalytic performance.

Grown-like macrocycle zinc complex derived from beta-diketone ligand for the polymerization of rac-lactide

Enolic Schiff base zinc (II) complex 1 was synthesized. XRD revealed 1 was a novel crown-like macrocycle structure consisted of hexanuclear units of (LZnEt)(6) via the coordination chelation between the Zn atom and adjacent amine nitrogen atom. Further reaction of 1 with one equivalent 2-propanol at RT produced Zn-alkoxide 2 by in situ alcoholysis. Complex 2 was used as an initiator to polymerize rac-lactide in a controlled manner to give heterotactic enriched polylactide. Factors that influenced the polymerization such as the polymerization time and the temperature as well as the monomer concentration were discussed in detail in this paper.

Novel aliphatic poly(ester-carhonate) with pendant allyl ester groups and its folic acid functionalization

A series of novel poly(ester-carbonate)s bearing pendant allyl ester groups P(LA-co-MAC)s were prepared by ring-opening copolymerization Of L-lactide (LA) and 5-methyl-5-allyloxycarbonyl-1,3-dioxan-2-one (MAC) with diethyl zinc (ZnEt2) as initiator. NMR analysis investigated the microstructure of the copolymer. DSC results indicated that the copolymers displayed a single glass-transition temperature (T-g), which was indicative of a random copolymer, and the Tg decreased with increasing carbonate content in the copolymer.

Esterification of acetic acid with N-butanol over the catalysts of surface hydrophobicity and lipophobicity

A series of silica-supported silicotungstic acid catalysts (H4SiW12O40, abbreviated as HSiW), modified with various loadings of Teflon (HSiW/SiO2-Teflon), were prepared by impregnation method. The surface properties of the catalysts were studied by means of XRD, BET, NH3-TPD and the Drop Shape Analyzer (DSA) measurements. Both the surface hydrophobicity and the surface lipophobicity of HSiW/SiO2-Teflon catalysts are enhanced by means of the addition of Teflon.

Chiral Salan Aluminium Ethyl Complexes and Their Application in Lactide Polymerization

Synthetic routes to aluminium ethyl complexes supported by chiral tetradentate phenoxyamine (salan-type) ligands [Al(OC6H2(R-6-R-4)CH2)(2){CH3N(C6H10)NCH3}-C2H5] 7: R = H ; 5, 8: R = Cl; 6, 9: R = CH3) are reported. Enantiornerically pure salan ligands 1-3 with (R,R) configurations at their cyclohexane rings afforded the complexes 4, 5, and 6 as mixtures of two diastereoisomers (a and b). Each diastereoisomer a was, as determined by X-ray analysis, monomeric with a five-coordinated aluminium central core in the solid state, adopting a cis-(O,O) and cis-(Me,Me) ligand geometry. From the results of variable-temperature (VT) H-1 NMR in the temperature range of 220-335 K, H-1-H-1 NOESY at 220 K, and diffusion-ordered spectroscopy (DOSY), it is concluded that each diastereoisomer b is also monomeric with a five-coordinated aluminium central core.