几种植物药材的色谱－质谱联用分析

本论文由四章组成，第一、二、三章为实验论文，分别报道了中药羌活、菊花、全缘叶绿绒蒿的化学成分的高效液相色谱(HPLC)和液相色谱-质谱(LC-MS)联用分析以及挥发油的气相色谱-质谱(GC-MS)联用分析。第四章概述了重要藏药材化学成分的研究进展。 第一章首先对28批不同产地的羌活药材进行了HPLC分析，建立了羌活的指纹图谱。结果表明，不同产地羌活的化学成分基本相似，但是各组分在含量上存在较大差异。其次，对羌活的主要化学组分包括紫花前胡苷、紫花前胡素、6'-O-反式阿魏酸紫花前胡苷、茴香酸对羟基苯乙酯、羌活醇和异欧前胡素进行了定量分析。此外，针对同一产地不同采集时间的羌活挥发油进行系统分析，结果表明它们的化学成分基本相似，主要含有a－蒎烯、b－蒎烯、柠檬烯和龙脑乙酸酯等，只是各组分含量有所变化，这说明采集药材时要注意采集时间。 第二章分别报道了不同产地不同品种菊花非挥发性成分的液相色谱－二极管阵列检测－串联质谱(LC-PDA-MSn)分析和挥发性成分的气相色谱－质谱(GC-MS)联用分析比较。首先通过液相色谱－质谱－串联质谱对各色谱峰进行定性分析，通过与标准品对照，以及UV和MSn提供的结构信息，结合文献报道共鉴定了11个化学成分，包括绿原酸和10个黄酮化合物，并比较了不同品种菊花的化学成分相同之处和不同之处。另外，对七种不同品种不同产地的菊花挥发性成分通过GC-MS分析表明其主要挥发性成分为单萜类、倍半萜类化合物，共有成分樟脑、龙脑和龙脑乙酸酯等，各成分在不同挥发油中的含量变化明显。 第三章为藏药全缘叶绿绒蒿不同部位挥发油成分的气相色谱－质谱(GC-MS)联用分析，比较其挥发油化学成分及其含量变化的异同点。研究结果表明，全缘叶绿绒蒿花精油的化学成分明显多于全草部位，且两者主要成分有较大的差别。 第四章综述了青藏高原重要藏药材化学成分的研究进展。分别对藏药的资源特色和110多种常用重要藏药材的化学成分的研究情况以及藏药未来发展思路进行了阐述，以期对相关的研究提供一些信息。 This dissertation consists of four parts. The first part reports studies on the fingerprint of Notopterygium incisum and N. forbesii by HPLC-PDA-MSn, and on the constitutents of essential oil by GC-MS. The second part elaborates the chemical constitutents of Chrysanthemum L. by LC-MS and GC-MS analysis. The third part reports the chemical compositions of the essential oil from the different parts of Meconopsis integrifolia. The fourth part reviews on the progress of the studies on the chemical constitutents in Tibetan medicines. The first chapter is about HPLC analysis of a traditional Chinese herbal medicine Qiang-huo (Notopterygium incisum and N. forbesii ). Firstly, based on analyzing and contrasting the relative retention time and relative paek area in chromatographic fingerprint, the HPLC chromatographic fingerprint of Notopterygium incisum was established, which can used as a scientific basement for the quality evalution of this herb. Secondly, quantitative analysis were performed on the main chemical constitutents of Notopterygium incisum and N. forbesii including nodakenin, nodakenetin, 6’-O-trans-feruloylnodakenin, p-hydroxypenethylanisate, notopterol and isoimperatorin. The results indicated that the contents were variable related to different growth regions. Lastly, the essential oil of Notopterygium incisum collected in different harvest times is analyzed by GC-MS. The second chapter is about HPLC-MS and GC-MS analysis of several species of Chrysanthemum L. Firstly, eleven compounds including chlorogenic acid and ten flavone compounds were identified in the methanol extract of Chrysanthemum morifolium Ramat. from different regions by HPLC-MS analysis. Secondly, the essential oil of seven different species of Chrysanthemum L.were extracted by steam distillation, and its compositions were isolated and identified by GC-MS. The main active constitutents such as camphor, borneol and bornyl acetate were detected, but the relative content varied notably. The third chapter is about GC-MS analysis of the essential oil from different parts of Meconopsis integrifolia. It indicated great difference of the chemical compositions of their oil in the flowers and residual overground part. The last chapter is a review of the research progress of the Tibetan medicines, which includes their features and their main chemical constitutents.

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.

两种银莲花属植物的化学成分研究

本学位论文由5 章组成。第1 章报道了两头尖中三萜皂苷类化合物的全扫描电喷雾多级质谱分析及银莲花素A 的电喷雾质谱裂解规律；第2 和3 章报道了两种银莲花属药用植物化学成分的研究结果；第4 章报道了银莲花素A 的化学结构修饰及其对一种蛋白酪氨酸磷酸酯酶（PTP-1B）的抑制活性；第5 章综述了电喷雾质谱在皂苷类化合物结构鉴定中的应用进展。 第1 章报道了运用全扫描电喷雾多级质谱对两头尖中三萜皂苷类化合物的快速定性检测，共检测出18 个准分子离子峰，根据多级质谱数据并结合文献报道，对其中的15 个准分子离子峰进行了归属，并区分了一些同分异构体；更有意思的是，发现了3 个未见文献报道的三萜皂苷类化合物。根据它们的多级质谱数据，对其结构分别进行了初步解析。本章同时对银莲花素A 特殊的质谱裂解途径通过衍生物制备及其质谱分析进行了确认。 第2 和3 章，分别报道了两头尖和打破碗花花中水溶性多糖苷的分离纯化和结构鉴定。采用D101 大孔树脂和反复硅胶柱层析等分离手段，从两头尖水溶性部分分离得到8 个三萜皂苷类化合物，通过红外、电喷雾质谱和核磁共振等现代谱学方法，并结合传统的化学手段鉴定了它们的结构，其中4 个化合物为新化合物，分别命名为多被银莲花皂苷19、多被银莲花皂苷20、多被银莲花皂苷21和多被银莲花皂苷22。从打破碗花花水溶性部位分离得到6 个三萜皂苷类化合物，通过现代谱学手段，并结合传统的化学方法对它们进行了鉴定，其中1 个为新化合物，命名为打破碗花花苷H。 第4 章报道了以银莲花素A 及其同系物为先导化合物，进行化学结构修饰并对修饰产物进行广泛的生物活性筛选，发现在银莲花素A 及其同系物的结构中引入一些酸性基团后，其生物活性发生了变化，一些修饰产物显示出很强的PTP-1B 抑制作用，提示这些化合物有可能用于治疗II 型糖尿病。 第5 章综述了电喷雾多级质谱用于皂苷类化合物结构鉴定的研究进展。 This dissertation composes of five chapters. The first chapter elaborates the detection and qualification of the triterpenoidal saponins in Anemone raddeana by positive and negative full scan ESI-MSn. This part also reports the special fragmentation pathway of Raddeanin A by ESI-MS/MS. The second and third chapters present the phytochemical investigation of two medicinal plants from Anemone. The fourth part dwells on the structure modification of Raddeanin A and their inhibitory activity against PTP-1B. The last part is a review on the progress in the application of ESI-MSn in the structure identification of saponins. The first chapter reports the application of full scan ESI-MSn for fast analysis of triterpenoid saponins in Anemone raddeana. Eighteen quasi- molecular ion peaks were detected in the positive full scan ESI-MS and fifteen of them were identified by analysis of their tandem mass spectral data in the negative ion mode. Several isomers were differentiated. More interesting, three unreported triterpenoid saponins in this medicinal plant were detected and their structures were deduced according to the dissociation pathway of the known triterpenoidal saponins. This chapter also confirms the special fragmentation pathway of Raddeanin A by its derivative and the mass spectral analysis. The second and third chapters expatiate on the isolation and identification of the chemical constituents from A. raddeana and A. hupehensis. Eight compounds were isolated from the roots and stems of A. raddeana by methanol extraction and repeated column chromatography (including D101 and silica gel), and their structures were determined on the basis of IR, ESI-MS, NMR and chemical methods (including acid hydrolysis and alkaline saponification). Among them, four are new triterpenoid saponins and named as Raddeanoside R19, Raddeanoside R20, Raddeanoside R21 and Raddeanoside R22. Six compounds were isolated from the whole plants of A. hupehensis by the same methods as above, and their structures were also determined with the same way. One of them was confirmed to be new triterpenoid saponins and named as hupehensis saponin H. In the fourth chapter, in order to look for new active compounds, the structure of Raddeanin A and its analogs were modified. It was found that the modified products exhibited obvious inhibitory activity against PTP-1B when several acid groups were introduced. The fifth chapter summarizes the progress on the application of ESI-MSn in the structure identification of saponins.

环糊精衍生物的设计、合成及其对环辛烯光异构化研究

环糊精(Cyclodextrins, CDs)经化学修饰后可以得到各种类型的衍生物，不仅可以扩展其原有的键合能力，而且还可以改变其选择性，是当代超分子化学的一个研究热点。环糊精第二面的仲羟基比第一面的伯羟基有着更好的催化性能，第二面的选择性修饰将产生更多有价值的衍生物，可用于催化、酶模拟、手性识别等方面。 取代苯甲酰基修饰环糊精对顺式环辛烯(cis-cyclooctene)光异构化反应有非常重要的影响作用，苯环上取代基的性质和取代位置与产物的%ee值和对映体构型之间存在某种内在联系。有目的地选择适宜取代基，设计、合成新型环糊精光增感剂, 有可能按预定目的得到更高%ee值的反式环辛烯；同时，取代苯甲酰基修饰环糊精对cis-cyclooctene光异构化的增感机理有待于进一步阐明。 本论文工作对环糊精的化学修饰以及超分子体系对cis-cyclooctene不对称光异构化反应方面的进展进行了调研。合成了一系列单-6-位取代苯甲酰基修饰环糊精，用于cis-cyclooctene光异构化增感反应，并用圆二色光谱滴定法研究这些环糊精衍生物与cis-cyclooctene的相互作用，以探索光增感反应机理。在此基础上，探讨了环糊精第二面的选择性修饰方法。内容主要包括： 1. 简要介绍了超分子化学的概况，并对环糊精的选择性修饰方法和超分子体系对cis-cyclooctene不对称光异构化反应的主要成果和最新进展进行了评述。 2. 合成了12种单-6-O-(取代苯甲酰基)-β-环糊精，其中10种为新化合物。采用紫外光谱、红外光谱、核磁共振波谱以及质谱等手段对化合物的结构进行了表征。 3. 探索了直接选择性修饰环糊精第二面的便捷新方法。用取代苯甲酰咪唑酯为酰化试剂，0.2M碳酸盐缓冲溶液(pH=9.9)作催化剂，能够有效地活化2-位仲羟基，对环糊精第二面进行选择性修饰，此方法既简便又经济；同时，发现取代苯甲酰基能够在β-CD第二面的2-位、3-位羟基间相互迁移。 4. 用单-6-O-(取代苯甲酰基)-β-环糊精作光增感剂，对cis-cyclooctene光异构化反应进行研究。实验结果证明：取代苯甲酰基上的取代基性质、位置、长度对反应的对映选择性有很大影响；此外，反应体系溶剂极性对产物的%ee值和对映体构型也有重大影响。用单-6-O-(3-甲氧苯甲酰基)-β-CD作增感剂，cis-cyclooctene光异构化反应产物(R)-trans-cyclooctene的对映选择性为45.8%ee，是到目前为止取得的最好对映选择性。 5. 采用圆二色光谱滴定法研究环糊精衍生物与cis-cyclooctene的相互作用，计算包结物的平衡常数，研究包结物的相对稳定性，为探索光增感反应机理提供基础。我们猜测：电子效应对cis-cyclooctene光异构化反应的影响，可能比取代基位置对反应的影响更大，借助电子效应有希望获得更高的%ee值。 Cyclodextrins can be subjected to diverse modifications to give a wide variety of cyclodextrin derivatives, which could not only extend their original molecular binding ability, but also alter their molecular selectivity. Therefore, cyclodextrin chemistry is currently a significant topic in supramolecular chemistry. The more open secondary hydroxyl side of CDs is stated to be catalytically very important, modifications of this face are believed to produce valuable derivatives for catalysis, enzyme mimic, chiral discrimination, etc. Mono-6-O-(substituted benzoyl)-β-CDs as novel supramolecular photosensitizing hosts have recently excited considerable attention in photochirogenesis. The supramolecular photosenstization of cis-cyclooctene mediated by them gave chiral trans-cyclooctene, enantiomeric excess of which was critically affected by the substituent introduced to the sensitizer moiety. In order to enhance the photoenantiodifferentiating ability, and elucidate the origin mechanisms of substituent-dependent enantioselectivity, in this work a series of mono-6-O-(substituted benzoyl)-β-CDs have been synthesized, and applied for enantiodifferentiating photoisomerization of cis-cyclooctene. The major contents are as follows: 1. The general aspects of supramolecular chemistry were descibed briefly. The new progress and important achievements on methods of selective modification of cyclodextrin and supramolecular enantiodifferentiating photoisomerization of cis-cyclooctene were reviewed. 2. Twelve mono-6-O-(substituted benzoyl)-β-CDs including ten novel compounds have been synthesized. Their structures have been characterized by using UV-vis, IR, NMR and MS methods. 3. A new convenient strategy for direct acylation of β-cyclodextrin on the secondary hydroxyl face was achieved by using the combination of N-benzoylimidazole and carbonate buffer in DMF, and the acyl migration between the C-2 and C-3 hydroxyl groups of β-cyclodextrin was found. 4. Experiments using mono-6-O-(substituted benzoyl)-β-CDs as chiral sensitizing hosts for mediating the enantiodifferentiating photoisomerization of cis-cyclooctene, were carried out. The results indicate that enantiomeric excess was critically affected, or even switched in sign, by the substituent introduced to the sensitizer moiety, and polarity of solvent. Using mono-6-O-(3-methoxybenzoyl)-β-CD as chiral sensitizing host, (R)-trans-cyclooctene was obtained in up to 45.8% enantiomeric excess, which is the highest value ever reported for supramolecular photochirogenesis with analogous hosts. 5. The conformational variation of these modified CDs and their complexation behaviors with cis-cyclooctene were examined by circular dichroism spectroscopy in water-methanol mixed solvents, which reveal that the orientation of chromophore was highly sensitive to the type, position and length of the introduced substituents. In the end, the complex stability constants(Ks) were calculated, and the mechanisms of reaction were discussed. Maybe, electronic effects are more important than positions of substituents for mediating the enantiodifferentiating photoisomerization of cis-cyclooctene.

无指盘臭蛙皮肤抗菌肽Odorgrin A和Odorgrin C基因表达载体的构建及其在毕赤酵母中的表达

研究背景与目的：近二十年来，抗生素的广泛使用以及一些不当应用导致临床上出现大量的耐药性病原菌，所以不易产生耐药性的抗菌肽就成为目前研究的热点。本课题组此前的研究表明无指盘臭蛙（Odorrana grahami）皮肤抗菌肽具有广谱抗菌活性，但对真核细胞没有毒性，因此有成为新型药物的潜力。本研究采用毕赤酵母真核表达系统来生物合成抗菌肽Odorgrin A和Odorgrin C，为大量获取抗菌肽资源提供技术支撑。 方法：依照Odorgrin A和C的氨基酸序列、采用酵母偏爱密码子分别设计并化学合成了相应的目的基因序列。目的片段从合成质粒上用Xho Ι和EcoR Ι双酶切下后，与经同样限制酶完全酶切pPIC9K载体所获得的两个大片段直接连接，并转化至大肠杆菌DH5α。用PCR扩增、酶切及测序检测，鉴定正确的重组质粒。提取大量表达载体pPIC9K - Odo A和C并使之线性化后经电击法分别转化毕赤酵母（Pichia pastoris）GS115宿主菌，用营养缺陷型筛选、遗传霉素抗性筛选、PCR扩增和测序检测，鉴定并筛选出对G418具高抗性的Odorgrin A和C重组酵母菌。用甲醇对之进行诱导表达，SDS - PAGE电泳及反相层析检测表达产物，并做抑菌活性检测。 成果：PCR扩增、酶切及测序等结果表明表达载体pPIC9K - Odo A和C构建成功。营养缺陷型筛选、遗传霉素抗性筛选、PCR扩增和测序等证实pPIC9K - Odo A和C已整合入酵母基因组中。SDS - PAGE电泳及反相层析结果表明抗菌肽Odorgrin A和C成功地获得了分泌表达。而抑菌活性实验则检测到部分阳性克隆菌诱导分泌表达的抗菌肽Odorgrin A和C都对测试菌的生长具有较高（>94%）的抑制率。 结论：无指盘臭蛙皮肤抗菌肽Odorgrin A和Odorgrin C基因的表达载体都构建成功，并且都在毕赤酵母系统中获得了成功表达。 Background ＆ Objective: In the recent twenty years, a lot of pathogenic bacteria have come forth in clinic with durable trait derived from making use of and abusing the traditional antibiotics. Therefore, studying antimicrobial peptides, not be easy to be invalidated by durable bacteria, are becomimg popular and important. The skin antimicrobial peptides of Odorrana grahami with broad spectrum antibacterial activity and no toxicity to eukaryotic cell, discovered by previous research work of our workgroup, are looked forward to being potential medication. Pichia pastoris expressional system was used for biosynthesis antimicrobial peptides Odorgrin A and Odorgrin C in this study, for producing abundant antimicrobial peptides. Methods: The foreign fragments which included Odorgrin A or Odorgrin C gene according to their amino acid sequence respectively were synthesized based on the biased codon usage of yeast. The DNA fragments, obtained from the plasmids containing them by digested with Xho Ι and EcoR Ι, were directly ligated with the two bigger fragments obtained from the vector pPIC9K by digested with the same restriction enzymes. And then they were transformed into Escherichia coli DH5α to be selected and amplified positive colonies. The recombinants were testified by using PCR amplification, enzymes digestion and sequencing of the foreign fragment. After the expressional vector pPIC9K - Odo A and pPIC9K - Odo C were linearized, they were transformed into Pichia pastoris GS115 strain by the electroporation. Then the positive colonies which were of the highest geneticin resistant were selected through auxotrophic screening, genetic resistant screening, PCR amplification and sequencing of the inserted fragment. Methanol was used to induce the recombinant yeasts to express the foreign gene. SDS-PAGE electrophoresis, reversed phase chromatography and antibacterial activity experiment were used to testify the expressional products. Results: The evidences of PCR, enzymes digestion and sequence analysis confirmed that the expressional vector pPIC9K - Odo A and pPIC9K - Odo C have been constructed correctly. The results of auxotrophic screening, of genetic resistant screening, of PCR and sequencing of the foreign fragment showed that Odorgrin A and Odorgrin C gene have been homologous integrated with the Pichia pastoris genome. And it was also testified that antimicrobial peptides Odorgrin A and Odorgrin C have been expressed successfully by using SDS - PAGE electrophoresis, reversed phase chromatography and antibacterial activity experiment. Conclusion: The expressional vector of the skin antimicrobial peptides Odorgrin A and Odorgrin C gene of Odorrana grahami have been constructed correctly and both of the genes have been expressed successfully in Pichia pastoris system in this study.

影响沼气甲基产孢弧菌生长及其甲烷单加氧酶活性的若干因素研究

本文叙述了影响甲烷氧化细菌沼气甲基产孢弧菌81Z菌株生长和甲烷单加氧酶(MMO)活性的若干因素。沼气甲基产孢弧菌81Z菌株细胞生长被高浓度PO43-(>8mM),NH4+([NH4cl]>500mg/l)抑制；[CuSO4·5H2O]在0～4mg/l范围内。生长随[Cu2+]升高而加强，低[Cu2+](0.1mg/l CuSO4·5H2O)培养基中，添加Cocl2·6H2O(0.238mg/l)；促进菌体细胞生长。发酵罐分批培养过程中，生长延迟期过后，沼气甲基产孢弧菌81Z菌株细胞MMO比活很快达到最高，并稳定至对数生长中后期，随即急剧下降至初始水平。发现沼气甲基产孢弧菌81Z细胞中存在一种MMO活性，它不同于已报道过的两种MMO,MMOL最适PH6.2～6.4，4℃相对稳定，其产生不受培养基中[Cu2+]调控能与甲醇-甲醇脱氢酶系统相偶联，在无细胞抽提液中其活性被400μM[Cu2+]抑制。在低[Cu2+]发酵罐培养条件下，沼气甲基产孢弧菌81Z菌株产生可溶性MMC，其最适PH7.0，4℃不稳定，可被DE-52分离为三组分：A、B、C。为了获得沼气甲基产孢弧菌81Z细胞MMO的最佳催化活性，①采用高[Cu2+]培养基进行发酵罐培养，收集对数生长中期的细胞；②选择反应缓冲液PH6.3；③反应体系中添加5mM甲醇或甲酸是有效的方法。在本研究所采取过的最佳条件下，测得MMO活性为15.9nmol/min·mg干细胞，是以前报道的该菌株活性0.97nmol/min·mg干细胞的十六倍。Some factors which influence growth and MMO activity of Methylosporovibrio methanica 81Z were described. The growth of Methylosporovibrio methanica 81Z is inhibited by high concentration of PO43-(8mM)or NH4+(500mg/lNH4cl). The growth of Methylosporovibrio methanica 81Z increased with rising of copper concentration up to 4mg/l CuSO4·5H2O. At low copper concentration(0.1mg/lCuSO4·5H2O),adding Cocl2·6H2O(0.238mg/l)could enhance the growth of Methylosporovibrio methanica 81Z.With batch culture of Methylosporovibrio methanica 81Z in a fermentor, after lag phase, the activity of MMO reached the highest level rapidly and steady until later log phase, then falled to initial level.MMOL activity differenct from that of two types of MMO reported before was found from Methylosporovibrio methanica 81Z with optimum PH value from 6.2 to 6.4 and relative stabilty at 4℃. Synthsis of the MMOL was not regulated by copper concentaration in medium. Its activity could couple with methane-l-methanoldehydrogenase system, and in cell-free extract, were inhibited by 400μm copper ion. At low copper concentration(0.1mg/lCuSO4·5H2O) and in a fermentor, Methylosporovibrio methanica 81Z could syntheis soluble MMO similar to solble MMO reported before by Palton and Patel. Its optimum PH value was 7.0. It was unstable at 4℃. It could be resoluted into three components: A, B, and C. It was effentive for obtaining the maxtmum MMO with Methylosporovibrio methanica 81Z that (1) to keep high copper concentration(4mg/lCuSO4·5H2O) in a fermentor and harvest cell at middlel lag phase;(2) to choose 6.3 as the PH value of reaction buffer;(3)and to add 5mM methanol or formate into reaction system. In this dy, the MMO activity of cells of Methylosporovibrio methanica 81Z was reached 15.9 nmol/min.mg, dry weight, sixteen times as high as the value(0.97nmol/min.mg, dry weight) reported with the same strain.

一种新奇的甲烷氧化细菌——能兼性营养和厌氧生长的沼气甲基弯菌

经过细心的条件选择实现了无甲烷条件下甲烷氧化菌沼气甲基弯菌81Z(methylosinus methanica 81Z)利用C2化合物的生长，同时 发现二碳代谢中间产物甘氨酸的胞外积累及对生长的抑制作用。又在此基础上从81Z原种中富集得到一株菌81Z-A，兼性生长能力大 幅度提高，而且除乙酸外又能利用丙酮酸、苹果酸、柠檬酸、葡萄糖而生长。对细胞氧化各种有机底物时氧吸收的测定及酶分析结 果发现了在其它甲烷氧化细菌中未曾发现的异柠檬酸裂解酶和苹果酸酶的存在，表明81Z除了具有通常II型菌的碳代谢途径外，具 有特殊的补偿代谢途径——乙醛酸支路以及从乙酸生糖的回补途径。因此推证其兼性生长的能力是固有的，从而说明了甲烷氧化菌 的专一性概念没有普遍意义。说明了81Z还能在含有二碳的培养基中厌氧生长，包括细胞的分裂和增值行为。虽然这种厌氧生长还 很弱，但至少可以说明它不是严格好氧的，这对于传统的关于甲烷氧化菌的严格好氧的概念是一个冲击。81Z正常条件下是利用甲 烷而生长的，当供给它乙酸、乙醛酸和丝氨酸时能促进含C-C键有机物氧化的活性，而对甲烷单加氧酶和其它C2化合物的氧化有抑 制或阻遏作用，对碳同化的丝氨酸途径的关键酶羟基丙酮酸还原酶有阻抑作用。同时也证明了81Z的甲烷单加氧酶和甲醇氧化活性 可被甲烷、甲醇所诱导，而因甲酸而降低。The growth of Methylosinus methanica 81Z on C2-compounds without methane was realized by selecting suitable conditions. The intermediate product Gly from its C2 metabolism was found to accumulate out cells and inhibit its growth. 81Z-A, which was obtained from 81Z by richening, could grow on C2- compounds rapidly. It can even grow on pyruvate, malate, citrate and glucose. The results of measurements of oxygen consumption by cell suspensions in the presence of various organic compounds and the results of enzyme assays of detected activities of isocitrate lyaze and malic enzyme in cell extracts which were not found in other methantrophs showed that 81Z possesses not only the regular carbon metabalic pathways but also some peculiar anaplerotic pathways--the glyoxylate cycle and the gluconeogenic pathway from acetate. As a consequence of these studies, its ability of facultative growth is inherent. Therefore, the concept of obligate dependence on C2- compounds of methanotrophs is not of universal significance. The ability of 81Z's growth(including desintegration and proliferation behaviour) on C2-compounds anaerobically was also demonstrated. Despite of the weakness of this growth, at least it could be said that 81Z is not strictly aerobic. This is a strike to the traditonal concept about the strictly aerobic action of methanotrophs. Regularly, 81Z grows on methane. The presence of acetate, glyoxylate and serine could increaze its ability of oxidizing the organic coumpounds containing C-C ponds. In contrast, they could inhibit the activities of MMO and other C2-compounds oxidation, they also repressed the key enzyme hydroxypyruvate reductase of the serine-pathway for carbon assimilation. At the some time, it was testified that the activities of MMO and methanol oxidation were inducible by methane or methanol and were lower in the presence of formate.

A quick isolation method for mutants with high lipid yield in oleaginous yeast

A novel method has been developed to easily isolate the mutants with high lipid yield after irradiating oleaginous yeast cells with carbon ions of energy of 80 MeV/u. Pre-selection of the mutants after ion irradiation was performed with culture medium in which the concentration of cerulenin, a potent inhibitor of fatty acid synthetase, was at 8.96 mu mol/l. Afterwards, lipid concentration in the fermentation broth of the pre-selected colonies was estimated by the sulfo-phospho-vanillin reaction instead of the conventional methanol-chloroform extraction. Two mutants with high lipid yield have been successfully selected out by the combined method. This easy and simple method is much less time-consuming but very efficient in the mutant isolation, and it has demonstrated great potential on mutation breeding in oleaginous microorganism.

Biosorption of uranium by chemically modified Rhodotorula glutinis

The present paper reports the biosorption of uranium onto chemically modified yeast cells, Rhodotorula glutinis, in order to study the role played by various functional groups in the cell wall. Esterification of the carboxyl groups and methylation of the amino groups present in the cells were carried out by methanol and formaldehyde treatment, respectively. The uranium sorption capacity increased 31% for the methanol-treated biomass and 11% for the formaldehyde-treated biomass at an initial uranium concentration of 140 mg/L The enhancement of uranium sorption capacity was investigated by Fourier transform infrared (FTIR) spectroscopy analysis, with amino and carboxyl groups were determined to be the important functional groups involved in uranium binding. The biosorption isotherms of uranium onto the raw and chemically modified biomass were also investigated with varying uranium concentrations. Langmuir and Freundlich models were well able to explain the sorption equilibrium data with satisfactory correlation coefficients higher than 0.9. (C) 2010 Elsevier Ltd. All rights reserved.

Performance comparison of low-temperature direct alcohol fuel cells with different anode catalysts

Low-temperature polymer electrolyte membrane fuel cells directly fed by methanol and ethanol were investigated employing carbon supported Pt, PtSn and PtRu as anode catalysts, respectively. Employing Pt/C as anode catalyst, both direct methanol fuel cell (DMFC) and direct ethanol fuel cell (DEFC) showed poor performances even in presence of high Pt loading on anode. It was found that the addition of Ru or Sn to the Pt dramatically enhances the electro-oxidation of both methanol and ethanol. It was also found that the single cell adopting PtRu/C as anode shows better DMFC performance, while PtSn/C catalyst shows better DEFC performance. The single fuel cell using PtSn/C as anode catalyst at 90degreesC shows similar power densities whenever fueled by methanol or ethanol. The cyclic voltammetry (CV) and single fuel cell tests indicated that PtRu is more suitable for DMFC while PtSn is more suitable for DEFC. (C) 2003 Elsevier B.V. All rights reserved.

Pt-based anode catalysts for direct ethanol fuel cells

In the present work, several carbon supported PtSn and PtSnRu catalysts were prepared with different atomic ratios and tested in direct ethanol fuel cells (DEFC) operated at lower temperature (T=90 degreesC). XRD and TEM results indicate that all of these catalysts consist of uniform nano-sized particles of narrow distribution and the average particle sizes are always less than 3.0 nm. As the content of Sn increases, the Pt lattice parameter becomes longer. Single direct ethanol fuel cell tests were used to evaluate the performance of carbon supported PtSn catalysts for ethanol electro-oxidation. It was found that the addition of Sn can enhance the activity towards ethanol electro-oxidation. It is also found that a single DEFC of Pt/Sn atomic ratioless than or equal to2, "Pt1Sn1/C, Pt3Sn2/C, and Pt2Sn1/C" shows better performance than those with Pt3Sn1/C and Pt4Sn1/C. But even adopting the least active PtSn catalyst, Pt4Sn1/C, the DEFC also exhibits higher performance than that with the commercial Pt1Ru1/C, which is dominatingly used in PEMFC at present as anode catalyst for both methanol electro-oxidation and CO-tolerance. At 90 degreesC, the DEFC exhibits the best performance when Pt2Sn1/C is adopted as anode catalysts. This distinct difference in DEFC performance between the catalysts examined here is attributed to the so-called bifunctional mechanism and to the electronic interaction between Pt and Sn. It is thought that -OHads, Surface Pt active sites and the ohmic effect of PtSn/C catalyst determines the electro-oxidation activity of PtSn catalysts with different Pt/Sn ratios. (C) 2004 Elsevier B.V. All rights reserved.

An improved palladium-based DMFCs cathode catalyst

A novel carbon-supported palladium-rich Pd3Pt1/C catalyst prepared by a modified polyol process showed a better cell performance than Pt/C in direct methanol fuel cells, which may be attributed to palladium's inactivity to methanol electro-oxidation while exhibiting good performance to oxygen reduction reaction.

Study of an electroosmotic pump for liquid delivery and its application in capillary column liquid chromatography

A packed-bed electroosmotic pump (EOP) was constructed and evaluated. The EOP consisted of three capillary columns packed in parallel, a gas-releasing device, Pt electrodes and a high-voltage power supply. The EOP could generate output pressure above 5.0 MPa and constant flow rate in the range of nl/min to a few mul/min for pure water, pure methanol, 2 mM potassium dihydrogenphosphate buffer, the buffer-methanol mixture and the pure water-methanol mixture at applied potentials less than 20 W The composition of solvent before/after pumping was quantitatively determined by using a gas chromatograph equipped with both flame ionization detector and thermal conductivity detector. It was found that there were no apparent changes in composition and relative concentrations after pumping process for a methanol-ethanol-acetonitrile mixture and a methanol-water mixture. Theoretical aspect of the EOP was discussed in detail. An capillary HPLC system consisting of the EOP, an injection valve, a 15 cm x 320 mum i.d., 5 mum Spherigel C(18) stainless steel analytical column, and an on-column UV detector was connected to evaluate the performance of the EOP. A comparative study was also carried out with a mechanical capillary HPLC pump on the same system. The results demonstrated that the reproducibility of flow rate and the pulsation-free flow property of the EOP are superior to that of mechanical pump in capillary HPLC application. (C) 2004 Elsevier B.V. All rights reserved.

Chemisorption and reaction characteristics of methyl radicals on Cu(110)

Methyl radicals are generated by pyrolysis of azomethane, and the condition for achieving neat adsorption on Cu(110) is described for studying their chemisorption and reaction characteristics. The radical-surface system is examined by X-ray photoemission spectroscopy, ultraviolet photoemission spectroscopy, temperature-programmed desorption, low-energy electron diffraction (LEED), and high-resolution electron energy loss spectroscopy under ultrahigh vacuum conditions. It is observed that a small fraction of impinging CH3 radicals decompose into methylene possibly on surface defect sites. This type of CH2 radical has no apparent effect on CH3(ads) surface chemistry initiated by dehydrogenation to form active CH2(ads) followed by chain reactions to yield high-mass alkyl products. All thermal desorption products, such as H-2, CH4, C2H4, C2H6, and C3H6, are detected with a single desorption peak near 475 K. The product yields increase with surface coverage until saturation corresponding to 0.50 monolayer of CH3(ads). The mass distribution is, however, invariant with initial CH3(ads) coverage, and all desorbed species exhibit first-order reaction kinetics. LEED measurement reveals a c(2 x 2) adsorbate structure independent of the amount of gaseous exposure. This strongly suggests that the radicals aggregate into close-packed two-dimensional islands at any exposure. The islanding behavior can be correlated with the reaction kinetics and is deemed to be essential for the chain propagation reactions. Some relevant aspects of the CH3/Cu(111) system are also presented. The new results are compared with those of prior studies employing methyl halides as radical sources. Major differences are found in the product distribution and desorption kinetics, and these are attributed to the influence of surface halogen atoms present in those earlier investigations.

Oxidative activation of light alkanes on dense ionic oxygen conducting membranes

The oxidative dehydrogenation of ethane to ethylene (ODHE) has been studied in a catalytic membrane reactor (CMR) using a dense mixed ionic oxygen and electronic conducting perovskite membrane Ba0.5Sr0.5Co0.8Fe0.2O3-&. At 1080K, an ethylene yield of 66% was obtained with the bare membrane. After Pd cluster deposition, the ethylene yield reached 76% at 1050K. Ni cluster deposition led to a decrease of ethane conversion compared to the bare membrane without changing ethylene selectivity.