Waterborne exposure to PFOS causes disruption of the hypothalamus-pituitary-thyroid axis in zebrafish larvae

Thyroid hormones (THs) play an important role in the normal development and physiological functions in fish. Environmental chemicals may adversely affect thyroid function by disturbing gene transcription. Perfluorooctane sulfonate (PFOS), a persistent compound, is widely distributed in the aquatic environment and wildlife. In the present study, we investigated whether PFOS could disrupt the hypothalamic-pituitary-thyroid (HPT) axis. Zebrafish embryos were exposed to various concentrations of PFOS (0, 100, 200 and 400 mu g L-1) and gene expression patterns were examined 15 d post-fertilization. The expression of several genes in the HIPT system, i.e., corticotropin-releasing factor (CRF), thyroid-stimulating hormone (TSH), sodium/iodide symporter (NIS), thyroglobulin (TG), thyroid peroxidase (TPO), transthyretin (TTR), ioclothyronine deiodinases (Dio1 and Dio2) and thyroid receptor (TR alpha and TR beta), was quantitatively measured using real-time PCR. The gene expression levels of CRF and TSH were significantly up-regulated and down-regulated, respectively, upon exposure to 200 and 400 mu g L-1 PFOS. A significant increase in NIS and Diol gene expression was observed at 200 mu g L-1 PFOS exposure, while TG gene expression was down-regulated at 200 and 400 mu g L-1 PFOS exposure. TTR gene expression was down-regulated in a concentration-dependent manner. Up-regulation and down-regulation of TR alpha and TR beta gene expression, respectively, was observed upon exposure to PFOS. The whole body thyroxine (T-4) content remained unchanged, whereas triiodothyronine (T-3) levels were significantly increased, which could directly reflect disrupted thyroid hormone status after PFOS exposure. The overall results indicated that PFOS exposure could alter gene expression in the HPT axis and that mechanisms of disruption of thyroid status by PFOS could occur at several steps in the synthesis, regulation, and action of thyroid hormones. (C) 2009 Elsevier Ltd. All rights reserved.

Biodegradation of dibenzofuran by Janibacter terrae strain XJ-1

The dibenzofuran (DF)-degrading bacterium, Janibacter terrae strain XJ-1, was isolated from sediment from East Lake in Wuhan, China. This strain grows aerobically on DF as the sole source of carbon and energy; it has a doubling time of 12 hours at 30 degrees C; and it almost completely degraded 100 mg/L-1 DF in 5 days, producing 2,2',3-trihydroxybiphenyl, salicylic acid, gentisic acid, and other metabolites. The dbdA (DF dioxygenase) gene cluster in the strain is almost identical to that on a large plasmid in Terrabacter sp. YK3. Unlike Janibacter sp. strain YY-1, XJ-1 accumulates gentisic acid rather than catechol as a final product of DF degradation.

A colorimetric assay for screening microcystin class compounds in aquatic systems

Secondary metabolites produced by water-blooming cyanobacteria in eutrophic waters include some potent hepatotoxins, These compounds also have tumour-promoting properties, attributable to their inhibition and activation of protein phosphatases and kinases respectively. The inhibitory effect of these toxins on protein phosphatases have been employed in a commonly used radiometric assay, involving the use of a P-32-labeled substrate, for the detection and quantitation of these compounds. This paper investigates and describes a colorimetric method in which the activity of protein phosphatase 2A is determined by measuring the rate of colour production from the release of yellow p-nitrophenol using p-nitrophenyl phosphate as the substrate. Results of this study suggest that the colorimetric protein phosphatase inhibition assay is a simple, inexpensive tool for screening substances that may have tumour-promoting characteristics in aquatic systems. The detection limit of the colorimetric method is comparable to the radiometric assay. (C) 1998 Elsevier Science Ltd. All rights reserved.

Waterborne exposure to PFOS causes disruption of the hypothalamus-pituitary-thyroid axis in zebrafish larvae

Thyroid hormones (THs) play an important role in the normal development and physiological functions in fish. Environmental chemicals may adversely affect thyroid function by disturbing gene transcription. Perfluorooctane sulfonate (PFOS), a persistent compound, is widely distributed in the aquatic environment and wildlife. In the present study, we investigated whether PFOS could disrupt the hypothalamic– pituitary–thyroid (HPT) axis. Zebrafish embryos were exposed to various concentrations of PFOS (0, 100, 200 and 400 lg L 1) and gene expression patterns were examined 15 d post-fertilization. The expression of several genes in the HPT system, i.e., corticotropin-releasing factor (CRF), thyroid-stimulating hormone (TSH), sodium/iodide symporter (NIS), thyroglobulin (TG), thyroid peroxidase (TPO), transthyretin (TTR), iodothyronine deiodinases (Dio1 and Dio2) and thyroid receptor (TRa and TRb), was quantitatively measured using real-time PCR. The gene expression levels of CRF and TSH were significantly up-regulated and down-regulated, respectively, upon exposure to 200 and 400 lg L 1 PFOS. A significant increase in NIS and Dio1 gene expression was observed at 200 lg L 1 PFOS exposure, while TG gene expression was down-regulated at 200 and 400 lg L 1 PFOS exposure. TTR gene expression was down-regulated in a concentration-dependent manner. Up-regulation and down-regulation of TRa and TRb gene expression, respectively, was observed upon exposure to PFOS. The whole body thyroxine (T4) content remained unchanged, whereas triiodothyronine (T3) levels were significantly increased, which could directly reflect disrupted thyroid hormone status after PFOS exposure. The overall results indicated that PFOS exposure could alter gene expression in the HPT axis and that mechanisms of disruption of thyroid status by PFOS could occur at several steps in the synthesis, regulation, and action of thyroid hormones.

苯并（a）芘的微生物降解及其酶蛋白应答研究

本文以苯并(a)芘为目标污染物，探讨了母体化合物BaP及其次生代谢产物的连续降解的方法、降解过程和微生物的酶蛋白应答，并运用种子（小麦、白菜和萝卜）根伸长生长实验，考查了BaP的不同降解时期次生代谢产物造成的复合污染整体效应，旨在探讨BaP及其次生代谢产物的降解影响因素，减少其环境累积，为BaP污染环境的全面修复提供实验依据和理论基础。 在实验条件下，运用HPLC鉴定出真菌FZSY-1降解BaP的同时生成了三个次生代谢产物BP1,6-quinone, BP7,8-diol 和 3-OHBP；同时鉴定出真菌FZSY-2降解BaP的同时生成了两个代谢产物BP1,6-quinone 和 3-OHBP。 驯化微生物与氧化剂（KMnO4）的耦合降解系统对BaP及其代谢产物的连续降解效果好于单纯微生物降解。三个次生代谢产物中，BP1,6-quinone在环境中最易累积。同时提出了微生物与氧化剂协同的作用可以有效促进环境中持久有机污染物（尤其是高浓度，小面积污染）的连续降解。对于FZSY-1与氧化剂（KMnO4）耦合降解BaP，在TW80存在下，与对照（未加TW80）相比，在降解的前期（3天取样），BaP及其代谢产物的降解相对滞后；而在降解的后期（12天取样），BaP及其代谢产物的降解高于对照。 在不同BaP浓度下，检测了四种酶，C120、C230、CAT和PPO。三株细菌的CAT酶活与BaP的浓度无关；三株细菌的C230酶活都比较高；三株细菌的PPO酶活均较低。加入共代谢底物(琥珀酸钠)后，与对照(未加入共代谢底物)相比，C120、C230酶活明显提高。 以BaP以及FZSY-1（BZSY-2）降解BaP不同时期的复合降解产物（BaP，M6，M12，CK）为目标污染物，它们对小麦种子根伸长的抑制作用顺序为：M6﹥BaP﹥M12﹥CK。BaP，M6，M12，CK对白菜和萝卜种子根伸长的抑制作用顺序和小麦相同；同一目标污染物（M6）对这几种供试种子（小麦、白菜和萝卜）根伸长的抑制作用顺序为小麦﹥白菜﹥萝卜；三种植物种子根伸长抑制作用均表现为：真菌的M6﹥细菌的M6，真菌的M12﹥细菌的M12。

CO2浓度升高对三种树木叶片化学和舞毒蛾幼虫取食及生长的影响

探讨全球气候变化的生物学和生态学效应是当今生态学中的热点，研究大气CO2浓度升高对植物-昆虫相互作用关系的影响具有重要的理论和实践意义。本文使用开顶式气室（Open-top chamber，OTC）在野外条件下研究了CO2浓度升高对三种树木（小青杨、白桦和蒙古栎）叶片化学成分含量的影响，以及树木叶片品质变化对一种广食性森林昆虫（舞毒蛾）幼虫取食、生长发育和取食偏嗜性的影响。得出如下结果：（1）CO2浓度升高对3个受试树种叶片中的营养成分及次生代谢物含量均有显著影响，总体表现为氮含量降低，而碳氮比、非结构性碳水化合物、总酚和缩合丹宁含量增加。叶片中的化学成分含量可随时间发生显著变化，不同树种、甚至同一树种不同冠层高度的叶片对CO2浓度升高的响应强度也是不同的。叶片的干物质含量和比叶重对CO2浓度升高的响应不显著。（2）室内非选择性取食实验、室内选择性取食实验以及上树取食饲养方式下的多龄期取食实验，均发现高浓度CO2处理组内舞毒蛾幼虫的生长发育受到显著抑制。但对四龄舞毒蛾幼虫所进行的短期生物测定并未发现不同CO2浓度处理下幼虫的生长发育速率、对食物的取食率和转化率等昆虫营养指标存在显著差异。（3）叶片品质的降低是导致舞毒蛾幼虫生长发育受抑制的主要原因。但是总体上，CO2浓度升高导致的叶片品质变化并未显著影响幼虫的取食率和取食量。（4）舞毒蛾幼虫对不同叶片种类表现出清晰的取食选择性，这种选择性在其幼龄期就可表现出来。幼虫对小青杨上层叶片有最显著的偏嗜性，对蒙古栎下层叶片有最明显的拒食性。但是CO2浓度升高导致的叶片品质变化对舞毒蛾幼虫的取食选择性和寄主偏嗜行为并未产生显著影响。（5）检测出高浓度CO2处理组内舞毒蛾幼虫虫粪中含有浓度更高的植物次生代谢物质（总酚和缩合单宁），这很可能是昆虫整体生长发育受抑制的重要原因之一。

吸水链霉菌、放线菌Sp.253和金色毛壳菌的次生代谢产物研究

链霉菌是十分重要的一类放线菌，绝大多数的抗生素都由该类细菌产生。毛壳属真菌是一类重要的丝状真菌，从中也发现有很多结构新颖、活性独特的活性物质。因此本论文对两株链霉菌的活性成分及一株金毛壳菌的次生代谢产物进行了研究。 1．从吸水链霉菌（Streptomyces hygroscopicus 1.358）液态发酵产物（乙酸乙酯提取物）中分离得到3个化合物，通过波谱方法鉴定为RK955A (1)、Nigericin（2）、Elaiophylin（3）。以青霉素耐药-金黄色葡萄球菌作为指示菌的抗菌活性测定表明，三者均具有较强抗菌活性。 2．通过抗肿瘤体外活性筛选模型筛选得到得到一株链霉属土壤放线菌，从中分离得到六个化合物：苯乙酰胺（4）、苯丙酰胺（5）、肉桂酰胺（6）、3-（N-（甲酰胺基）乙酰基）吲哚（7）、鸟苷磷酸（8）、鸟苷（9）。 3．从金色毛壳菌（Chaetomium aureus）的固态培养物中分离得到13个化合物，利用波谱方法将其鉴定为：金毛壳菌素A（10）、金毛壳菌素B（11）、Eugenetin（12）、Eugenitol（13）、Chaetoquadrin A（14）、Chaetoquadrin B（15）、Chaetoquadrin G（16）、Chaetoquadrin H（17）、Chaetochromin A（18）、Sterigmatocystin（19）、O-methylsterigmatocystin（20）、3β-羟基-麦角甾-5，7，22-三烯（21）和过氧麦角甾醇（22）。 4．综述了聚醚类抗生素的结构、生物合成、生物活性及作用机理。 The genus Streptomyces (Actinomycetes) is an important group of microbe. Most antibiotics known nowdays are discovered from species of Streptomyces. The fungi of the genus Chaetomium have attracted much attention because various kinds of secondary metabolites with diverse bioactivities have been found from them. Thus, the bioactive compounds from two strains of Streptomyces and the secondary metabolites of Chaetomium aureus were investigated. 1. Three compounds were isolated from the ethyl acetate extract of the fermentation broth of Streptomyces hygroscopicus. They are identified to be elaiophylin (1), nigericin (2), and antibiotic RK955A (3) on the basis of their spectroscopic data. Compounds 1-3 possess antibacterial activities against Staphyloccocus aureus. 2. It was found that the extract of the fermented broth of a strain of Actinomycetes could inhibit some tumor cel lines. Separation of the bioactive fraction led to the isolation of six compounds. They were characterized to be phenylacetamide (4), phenylpropylamide (5), trans-cinnamamide (6), 3- (N- (formylmethyl) acetamide) indole (7), guanylicacid (8), and guanosine (9). 3. From the fermented broth of Chaetomium aureus, 13 compounds were isolated for the first time. They were determined to be chaetomiumycin A (10), chaetomiumycin B (11), eugenetin (12), eugenitol (13), chaetoquadrin A (14), chaetoquadrin B (15), chaetoquadrin G (16), chaetoquadrin H (17), chaetochromin A (18), sterigmatocystin (19), O-methylsterigmatocystin (20), 3β-hydroxyergosta-5, 7, 22-triene (21) and peroxy-ergosterol (22). Compounds 10 and 11 are new ones. 4. Structure, biosynthesis, biological activity, and mechanisms of polyether antibiotics were reviewed.

水菖蒲的化学成分研究和忍冬属植物的色谱分析

本论文由三章组成。第一章阐述了藏药水菖蒲的化学成分研究，共分离鉴定了39个化学成分，其中6个为新化合物。第二章报道了几种忍冬属植物的HPLC、HPLC-MS、GC分析以及抑菌活性、重金属含量测定结果。第三章概述了菖蒲属植物的研究进展。 第一章报道了水菖蒲(Acorus calamus L.)化学成分的分离纯化与结构鉴定。采用正、反相硅胶柱层析等分离方法，从水菖蒲的根中共分离出41个化合物，通过红外、质谱、核磁共振及X-ray单晶衍射等波谱方法和模拟计算方法鉴定了其中39个化合物的结构，主要为倍半萜、苯丙素、甾体类化合物。其中含有5个新的倍半萜类化合物和1系列新的甾体皂苷衍生物。经波谱分析将它们的结构鉴定为 1b, 7a(H)-cadinane-4a, 6a, 10a-triol (1), (2R,6R,7S,9S)-1(10), 4-cadinadiene-2, 9-diol (2), 1a, 5b-guaiane-10a-O-ethyl-4b, 6b-diol (7), 6b, 7b(H)-cadinane-1a, 4a, 10a-triol (13)，(1R,4R,6S,10R)-1-hydroxy-7(11)-cadinen-5, 8-dione (14)， 4′-O-正n碳酰基-3-O- β-D-葡萄糖基谷甾醇(n=14, 16, 18, 22) (15)。 第二章包括四个部分。第一部分报道了忍冬属三种植物40个样品的HPLC测定和对主要活性成分绿原酸的定量分析结果，以及运用HPLC-MS技术对色谱图中8个峰进行指认。在此基础上，考察了种植和采收多个因素对绿原酸含量的影响。第二部分报道了忍冬属三种植物27个样品的GC分析，根据样品的挥发性成分的保留时间对不同样品进行了定性比较，并考察了花期及海拔高度对植物挥发性成分的影响。第三、四部分分别阐述了灰毡毛忍冬和红腺忍冬的体外抑菌活性研究和重金属含量测定结果。 第三章全面系统地概述了菖蒲属植物的化学成分和药理活性研究进展。 This dissertation is composed by three chapters. The first chapter elaborates the phytochemical investigation of Acorus calamus L. Thirty-nine compounds including six new compounds were isolated and identified. The second chapter reports the research on genus Lonicera by HPLC, HPLC-MS and GC. Antifungal activity and heavy metals measurement of genus Lonicera were reported. The third chapter is a review about the research progress on the plant family of Acorus. The first chapter focuses on the isolation and identification of chemical constituents from Acorus calamus L.. Forty-one compounds were isolated from the root of Acorus calamus L. by repeat column chromatography over normal and reversed phase silica gel, the structure of thirty-nine compounds was identified by spectroscopic methods and computational methods, including IR, MS, NMR and X-ray. Those compounds mainly belonged to sesquiterpene, phenylpropanoid and steroid. Among them, five are new sesquiterpenes and one series are new steroid glycoside derivatives. Their structure were suggested as 1b, 7a(H)-cadinane-4a, 6a, 10a-triol (1), (2R,6R,7S,9S)-1(10), 4-cadinadiene-2, 9-diol (2), 1a, 5b-guaiane-10a-O-ethyl-4b, 6b- diol (7), 6b, 7b(H)-cadinane-1a, 4a, 10a-triol (13), (1R,4R,6S,10R)-1-hydroxy-7(11)- cadinen-5, 8-dione (14), 4′-O-carbonyl-3-O-β-D-glucosyl-sitosterol (carbonyl = tetradecanoyl, hexadecanoyl, octadecyl, docosanoyl) (15). The second chapter consists of four parts. The first part reports the HPLC analysis of forty samples of the genus Lonicera, and the quantitative investigation of chlorogenic acid in these samples by HPLC analysis. Relationship between the content of chlorogenic acid in different samples and their planting conditions and harvesting time were discussed. Furthermore, eight compounds were identified or tentatively characterized based on their mass spectra and UV spectra profiles. The second part is about qualitative analysis of the volatile constituent in twenty-seven samples of genus Lonicera by GC. The effect of planting altitude and harvesting time on the volatile constituent was also investigated. The third and fourth parts describe the antifungal activity and content of some kinds of heavy metals of L. macranthoides Hand.-Mazz. and L. hypoglauca Miq.. The third chaspter is a review about the research progress of the plant family of Acorus.

五种毛壳霉属和曲霉属真菌代谢产物及细胞毒活性

毛壳霉属(Chaetomium)和曲霉属(Aspergillus)真菌产生多种具有生物活性的化合物。为系统阐明两属微生物的次生代谢物，对三种毛壳霉、两种曲霉真菌分别进行固态发酵，以色谱和波谱技术研究发酵物中的成分，分离鉴定了51个化合物，其中23个为新化合物，测试了部分化合物对肿瘤细胞的活性。 1、从螺卷毛壳霉（C. cochloides）固态发酵物中分离鉴定了11个化合物，3个新化合物为螺卷毛壳霉素A~C（1~3）。化合物1、3及dethio-tetra (methylthio) chetomin（4）对Bre-04、Lu-04和N-04细胞株生长抑制的GI50值为0.05~7.0 μg/mL。 2、从印度毛壳霉（C. indicum）固态发酵物中鉴定的三个异喹啉生物碱印度毛壳霉素A~C（12~14）代表两类骨架新颖的异喹啉生物碱。 3、从巴西毛壳霉（C. brasiliense）固态发酵物中鉴定了11个化合物，其中Mollicellins I~J（15~16）、2-Hydroxymethyl-6-methylmethyleugenin（19）为新化合物。化合物16和Mollicellin H（18）对Bre-04、Lu-04、N-04细胞株生长抑制的GI50值在2.5~8.6 μg/mL。 4、从土曲霉（A. terreus）固态发酵物中鉴定了18个化合物。5个为新化合物为Terretonin A~D（24~27）和Asterrelenin（28），24~27为二倍半萜化合物，28为吲哚生物碱。 5、从杂色曲霉(A. versicolor）固态发酵物中鉴定了16个化合物。9个新的化合物Brevianamides K~N (40~43)、Averins A~C (44~46)和Glyanphenines A~B (47~48)代表三种类型的生物碱。 6、综述了1997－2007年间新的二倍半萜的研究进展。 The fungi of the genera Chaetomium and Aspergillus produce various secondary metabolites with biological activities. In order to systematically study the secondary metabolites, the solid-state fermented rice culture of three species of Chaetomium and two of Aspergillus were chemically studied. By the means of chromatograhy and spectroscopy, 55 compounds were isolated and identified, among of them 23 were new ones. The biological activities of some compounds were investigated. 1. From the fungus C. cochliodes, three new epipolythiodioxopiperazines, chaetocochins A-C (1-3) were isolated, together with 8 known ones (4-11). Compounds 1, 3 and 4 showed growth inhibitory effects against cancer cell lines Bre-04, Lu-04 and N-04 with GI50 values from 0.05 to 7.0 μg/mL. 2. Three novel isoquinolines Chaetoindicins A-C (12-14) were isolated and identified from the fungus C. indicum. Chaetoindicin A, Chaetoindicins B-C represented two classes of novel carbon skeletons. 3. Three new compounds, Mollicellins I-J (15-16), and 2-hydroxymethyl-6-methylmethyleugenin (19), were isolated from C. brasiliense. Compound 16 and Mollicellin H (18) showed growth inhibitory effects against cancer cell lines Bre-04, Lu-04 and N-04 with GI50 values from 2.5 to 8.6 μg/mL. 4. Eighteen compounds were isolated from the fungus A. terreus. Terretonin A-D（24 - 27）and Asterrelenin（28） are new compounds belonging to sesterterpoids and indole-ralated alkaloid, respectively. 5. From the fungus A. versicolor, sixteen secondary metabolites, including nine new ones, Brevianamides K-N (40-43), Averins A-C (44-46), and Glyanphenines A-B (47-48), were isolated and identified. Brevianamides K-N (40-43), Averins A-C (44-46), and Glyanphenines A-B (47-48) represented three classes of alkaloids. 6. New sesterterpenes and their bioactivities reported from 1997 to 2007 were summarized.

钮子瓜和袋花忍冬的化学成分研究

钮子瓜（Zehneria maysorensis Arn.）是一种常用的中草药，其性味苦、凉，主要功效为清热利湿、散风止痛，主治膀胱炎、头痛。体外活性筛选实验表明，袋花忍冬（Lonicera saccata Rehd.）95%乙醇提取物的乙酸乙酯部分对血管紧张素转化酶显示较强的抑制活性。为明确钮子瓜的药用物质基础和袋花忍冬中具有ACE抑制活性的成分，首次对两个植物的成分进行了研究。 1. 从钮子瓜95%乙醇提取物中主要通过色谱方法首次分离了14个化合物，通过波谱方法鉴定为(2S,3S,4R,10E)-2-[(2R)-2-羟基二十四烷酰基氨基]-10-十八烷-1,3,4-三醇（1）、(2S,3S,4R)-2-二十四烷酰基氨基-十八烷-1,3,4-三醇 （2）、胡萝卜苷（3）、swertish （4）、苯甲酸（5）、水杨酸（6）、loliolide （7）、胸腺嘧啶（8）、尿嘧啶（9）、(23Z)-9,19-环阿尔廷-23-烯-3β,25-二醇（10）、(20S,22E,24R)-5α,8α-表二氧-麦角甾-6,22-二烯-3β-醇（11）、十六烷酸 1-甘油酯（12）、大豆脑苷Ⅰ（13）和(22E,24S)-24-甲基-5α-胆甾-7,22-二烯-3β,5α,6β-三醇（14）。其中化合物4为一黄酮碳苷，具有旋转异构现象，有止痛作用；化合物6具有抗炎、镇痛、减热的活性，它们可能是钮子瓜药用物质基础的一部分。 2. 从袋花忍冬95%乙醇提取物中首次分离并鉴定了16个已知化合物：胡萝卜苷（3）、(20S,22E,24R)-5α,8α-表二氧-麦角甾-6,22-二烯-3β-醇（11）、十六烷酸 1-甘油酯（12）、E-p-coumaryl behenate (15)、谷甾醇（16）、2,6-dihydroxyhumula-3(12), 7(13),9(E)-triene (17)、环阿尔廷-25-烯-3β,24ξ-二醇 (18)、二十四烷酸 (19)、2,4-二羟基-3,6-二甲基苯甲酸甲酯 (20)、乌苏酸 (21)、柚皮素 (22)、木犀草素 (23)、柏双黄酮（24）咖啡酸 (25)、洋芹素（26）和木犀草素-7-O-β-D-葡萄糖苷 (27)。其中木犀草素（23）和咖啡酸（25）含量较高，它们为抑制ACE活性的成分。 3．综述了黄酮碳苷的旋转异构现象。 Zehneria maysorensis is a folk medicine for the treatment of cystitis and headache. The ethyl acetate soluble fraction of the 95% ethanol extract of Lonicera saccata showed obvious ACE inhibitory activity in vitro. To reveal their active constitutents, they were subjected to chemically study. From the 95% ethanol extract of the whole plants of Zehneria maysroensis fourteen compounds were isolated for the first time. On the basis of spectral data and/or by comparison with authentic samples, they were characterized to be (2S,3S,4R,10E)-2-[(2R)-2-hydroxytetracosanoylamino]-10-octadecene-1,3,4-triol (1), (2S,3S,4R)-2-tetracosanoylamino-1,3,4-octadecanetriol (2), daucosterol (3), swertish (4), benzoic acid (5), salicylic acid (6), loliolide (7), thymine (8), uracil (9), (23Z)-9,19-cycloart-23-ene-3β,25-diol (10), (20S,22E,24R)-5α,8α-epidioxy-ergosta- 6,22-diene-3β-ol (11), 2,3-dihydroxypropyl hexadecoate (12), soya-cerebroside (13) and (22E,24S)-24-methyl-5α-cholesta-7,22-diene-3β,5α,6β-triol (14). Compound 4, a C-glycosylflavone, showed a very interesting rotational isomerism. Compounds 4 and 6 may be the active constituents of Zehneria maysorensis considering their sedative and anti-inflammation activity, respectively. From the whole plants of Lonicera saccata, sixteen compounds were isolated for the first time. On the basis of spectral data and/or by comparison with authentic samples, they were identified to be daucosterol (3), (20S,22E,24R)-5α,8α-epidioxy- ergosta-6,22-diene-3β-ol (11), 2,3-dihydroxypropyl hexadecoate (12), E-p-coumaryl behenate (15), β-sitosterol (16), 2,6-dihydroxyhumula-3(12),7(13),9(E)-triene (17), cycloart-25-ene-3β,24ξ-diol (18), tetracosanoic acid (19), methyl 2,4-dihydroxy- 3,6-dimethylbenzoate (20), ursolic acid (21), naringenin (22), luteolin (23), cupressuflavone (24), caffeic acid (25), apigenin (26) and luteolin-7-O-β-D- glucopyranoside (27). Luteolin (23) and caffeic acid (25) were the ACE inhibitory active constituents. Rotational isomerism for C-glycosylflavonoid was reviewed.

Aspergillus sp136抗细菌耐药活性成分研究

基于广谱抗细菌耐药性这一思路，本研究中心建立了一套抗细菌耐药性化合物的筛选方法。由此从3000多种西南地区特殊生境的微生物和植物样品提取物中筛选获得17个抗细菌耐药性活性样品。对其中一株来自峨嵋山土样的微生物（Aspergillus sp136）进行了深入研究。通过TLC自显影等方法从其发酵产物中追踪分离得到抗耐药有效成分，并鉴定为烟曲霉酸。 采用多种方法对烟曲霉酸的体外抗细菌耐药活性进行评价。在平板扩散法中，烟曲霉酸表现出对青霉素（β-内酰氨抗生素）的协同抗耐药能力，其活性大约3倍于克拉维酸。在MIC的测试实验中，烟曲霉酸表现出对青霉素（β-内酰氨抗生素）以及非β-内酰氨抗生素如红霉素、四环素、氯霉素、链霉素、卡那霉素、庆大霉素的抗耐药能力。在棋盘格杀菌以及时间致死曲线的研究中，烟曲霉酸也表现出对青霉素、红霉素、四环素的协同抗细菌耐药活性。 在广泛的活性筛选中发现烟曲霉酸对LDLR基因具有上调活性，表明烟曲霉酸可能具有降血脂的活性。 在研究中发现，同空白对照相比，烟曲霉酸使耐药菌（Bacillus cereus NCPF63509）细胞外β-内酰胺酶酶活大幅度下降，而细胞内β-内酰胺酶酶活仅略有上升，这表明烟曲霉酸对β-内酰胺酶分泌过程具有抑制作用。 综述了β-内酰胺酶的研究进展。 A two-step agar diffusion method was established to screen wide spectrum synergistic antibacterial agents. By using this method, 17 active samples against antibiotic resistance were discovered from more than 3000 plants and microbes, which were collected from southwest china. One isolate Aspergillus sp136 collected from E-mei mountain area was selected for further studies. From the metabolites of this strain, a synergistic antibacterial compound was isolated by bioautographic TLC assay-guided fractionation and identified as helvolic acid. The synergistic effect of helvolic acid was confirmed by several methods in vitro. The synergistic effect of helvolic acid with penicillin (β-lactam antibiotics) was about 3 times as that of clavulanic acid with penicillin in agar diffusion assay. In MIC studies, helvolic acid exhibited synergistic effects with β-lactam antibiotics such as penicillin and non β-lactam antibiotics such as erythromycin, tetracycline, kanamycin, streptomycin and gentamycin. In checkerboard and time-kill studies, helvolic acid also exhibited synergistic effects with penicillin, erythromycin and tetracycline. In general screen of bioactivities, helvolic acid upregulate LDLR gene, which was indirectly determined by the activity of fluorescent enzyme. Therefore, helvolic acid might have the ability to lower lipid in blood. Compared with blank control, the extracellular β-lactamase activity decrease significantly and the intracellular β-lactamase activity increase slightly in Bacillus cereus NCPF63509 in the presence of helvolic acid, indicating that the secretion of β-lactamase was inhibited by helvolic acid. The research of β-lactamase was reviewed.

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.

厚朴中α-葡萄糖苷酶抑制活性成分研究

本论文由三章组成。 第一章是关于厚朴中具有α-葡萄糖苷酶抑制活性成分的研究。凹叶厚朴的乙醇提取物显示了较强的α-葡萄糖苷酶抑制活性。为了确定其活性成分，在活性测试的指导下，通过溶剂萃取、树脂吸附和反复硅胶柱层析等分离方法从凹叶厚朴乙醇提取物中分离得到6 个生物碱，并用质谱和核磁共振等波谱方法分别鉴定为：木兰箭毒碱，木兰花碱，鹅掌楸碱，蕃荔枝碱，罗默碱和Lysicamine。应用小肠α-葡萄糖苷酶模型测定了它们对α-葡萄糖苷酶的抑制作用。其中，番荔枝碱和木兰箭毒碱对α-葡萄糖苷酶相对抑制活性最好，分别为60%和62%；其它四个生物碱成分对α-葡萄糖苷酶的抑制活性几乎相当，鹅掌楸碱为46%，罗默碱为51%，Lysicamine 为49%，木兰花碱为51%。 第二章报道了厚朴酚的衍生物及其对α-葡萄糖苷酶的抑制活性。根据糖苷酶抑制剂的结构特点，设计合成了一系列厚朴酚的衍生物。厚朴酚经过Mannich 反应和环氧化及开环反应制备了一系列衍生物，经活性测试发现衍生物活性与取代基关系较大，其中5,5′-diallyl-3-((bis(2-hydroxyethyl)amino)methyl)biphenyl-2,2′-diol 的抑制活性最高，为72%。 第三章综述了厚朴的化学成分及药理活性两个方面的研究进展。 The dissertation consists of three chapters. The first chapter is about the study on the constituents with α-glycosidase inhibitory activity from Magnolia officinalis. The EtOH extracts of M. officinalis Rehd. et Wils showed good inhibitory activity against α-Glucosidase. In order to determine the active compounds, bio-assay was used to guide the isolation. Six known alkaloids were isolated by solvent extraction and repeated silica gel column chromatography, and their structures were identified as liriodenine, anonaine, roemerine, lysicamine, magnoflorine and magnocurarine by spectroscopic methods. The inhibitory activity against α-Glucosidase of these alkaloids was measured with alvine screening model of α-glucosidase. Among them, lysicamine and liriodenine have the best inhibitory activity at 60% and 62%, respectively. The other four alkaloids have close inhibitory activity, from 46% to 51%. The second chapter is about the derivation of magnolol and the inhibitory a ctivity of the derivatives. Seven derivatives of magnolol were prepared by Manni-ch reaction, epoxidation followed by ring-opening reaction. Biological activity as say indicated the inhibitory activity was related to substituting groups. Among them, 5,5′-diallyl-3-((bis(2-hydroxyethyl)amino)methyl)biphenyl-2,2′-diol had the highest activity at 72%. The third chapter is a review on the progress of M. officinalis including chemical constituents and pharmacological activity.