两株海藻内生真菌次生代谢产物及其生物活性研究

海洋微生物拥有丰富多样的次生代谢途径，其中海洋生物内生真菌次生代谢产物研究日益受到天然产物化学界的重视。本论文以菌丝体生物量、发酵产物重量、抗菌与细胞毒活性、薄层色谱分析结果以及高效液相色谱分析结果等为评价依据对采自青岛沿海的13株海藻内生真菌在四种液体培养基上的静置发酵产物进行了综合评价，并从中选择了黑曲霉Aspergillus niger EN-13（分离自褐藻囊藻Colpomenia sinuosa）和杂色曲霉A. versicolor EN-7（分离自褐藻鼠尾藻Sargassum thunbergii）两株真菌进行了30升规模发酵（分别采用GPYM培养基和PDB培养）和化学成分的研究，对分离得到的大部分化合物进行了初步的生物活性筛选。 发酵提取物采用常规的硅胶柱层析、反相硅胶柱层析，凝胶Sephadex LH-20柱层析、制备薄层层析、半制备高效液相色谱以及重结晶等分离手段，得到单体化合物。利用各种现代波谱技术(IR、UV、EI-MS、FAB-MS、HR-ESI-MS、1H-NMR、13C-NMR、DEPT、1H-1H COSY、HSQC、HMBC等)并结合化学方法从两种菌株发酵提取物中鉴定了55个化合物的结构。其中从菌株A. niger EN-13分离鉴定了31个化合物，发现9个新化合物，包括2个鞘酯类化合物（AN-1~2）、3个萘并-γ-吡喃酮类化合物（AN-3~5）、3个苯乙基取代的α-吡喃酮类化合物（AN-17, AN-19~20）和1个甾体Diels-Alder加成产物（AN-21），另有1个新的天然环二肽（AN-27）被分离鉴定；从菌株A. versicolor EN-7分离鉴定了24个化合物，发现2个新化合物，为蒽醌AV-12与AV-17，另外，从前一菌株（A. niger EN-13）中鉴定的2个新鞘酯类化合物（AN-1~2）在A. versicolor EN-7中也被再次分离到。 对大部分单体化合物进行了抗菌活性、DPPH自由基清除活性和细胞毒活性测试。结果显示新化合物AN-1、AN-5和AN-20具有弱或中等强度的抑制白色念珠菌生长的活性，AN-4、AN-5、AN-21显示了弱或中等强度的抑制黑曲霉生长的活性，AV-12、AV-17显示了弱的抑制大肠杆菌生长的活性。在DPPH自由基清除活性筛选中，AN-5显示了中等强度的活性，其EC50为109.3 mM，与阳性对照BHT相近（EC50为81.8 mM）。其它部分已知化合物在抗菌和DPPH自由基清除活性的筛选中也显示了弱或中等强度的活性。在针对人肝癌细胞株SMMC-7721和人肺腺癌细胞株A549的体外细胞毒活性筛选中，所测样品均未显示显著活性。

An efficient combination of supercritical fluid extraction and high-speed counter-current chromatography to extract and purify homoisoflavonoids from Ophiopogon japonicus (Thunb.) Ker-Gawler

Supercritical fluid extraction (SFE) was used to extract homoisoflavonoids from Ophiopogon japonicus (Thunb.) Ker-Gawler. The optimization of parameters was carried out using an orthogonal test L-9 (3)(4) including pressure, temperature, dynamic extraction time and the amount of modifier. The process was then scaled up by 100 times with a preparative SFE system under the optimized conditions of 25 MPa, 55 degrees C, 4.0 h and 25% methanol as a modifier. Then crude extracts were separated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane/ethyl acetate/methanol/ACN/water (1.8:1.0:1.0:1.2:1.0 v/v). There three homoisoflavonoidal compounds including methylophiopogonanone A 6-aldehydo-isoophiopogonone A, and 6-formyl-isoophiopogonanone A, were successfully isolated and purified in one step. The collected fractions were analyzed by HPLC. In each operation, 140 mg crude extracts was separated and yielded 15.3 mg of methylophiopogonanone A (96.9% purity), 4.1 mg of 6-aldehydo-isoophiopogonone A (98.3% purity) and 13.5 mg of 6-formyl-isoophiopogonanone A (97.3% purity) respectively. The chemical structure of the three homoisoflavonoids are identified by means of ESI-MS and NMR analysis.

单糖衍生物的电喷雾质谱裂解规律研究

以1-（2-萘基）-3-甲基-5-吡唑啉酮（NMP）作单糖标识剂，经在线串联的LC—ESI—MS建立了单糖衍生物的电喷雾质谱裂解方法。衍生物在质谱裂解中糖类化合物特有的规范信息。借助糖类化合物在ESI-MS条件下表现出的分子离子峰m／z[M＋H]~＋，及在ESI—MS／MS条件下呈现出的特征碎片离子峰m／z 473，可有效地确定出单糖类化合物的组成。尽管一些脂肪醛和芳香醛也能同时被标识，然而在质谱条件下不产生m／z473的特征碎片离子峰，且它们的洗脱远在糖类组分之后，因此不干扰糖类化合物的分离和结构确定。通过建立的LC—ESI—MS方法，对水解蜂花粉中的单糖进行了分析。结果表明：水解的蜂花粉中含甘露糖（Man）、半乳糖醛酸（GalUA）、葡萄糖醛酸（GlcUA）、鼠李糖（Rha）、葡萄糖（Glc）、半乳糖（Gal）、阿拉伯糖（Ara）、木糖（Xyl）和岩藻糖（Fuc）。本方法为环境样品中单糖类化合物的确定提供了准确、可靠的技术手段。

2-（11H-苯[a]咔唑）乙基氯甲酸酯柱前衍生化用于氨基酸的高效液相色谱分析

利用新型荧光试剂2-（11H-苯[a]咔唑）乙基氯甲酸酯（BCEC-Cl）作为柱前衍生化试剂,在HypersilBDS C_(18)（200mm×4.6mm,5μm）反相色谱柱上,采用梯度洗脱对20种氨基酸衍生物进行了分离检测。在乙腈与Na_2B_4O_7缓冲液中,室温下BCEC-Cl与氨基酸反应5min可实现完全衍生。检测的激发和发射波长分别为λ_(ex)=279nm,λ_(em)=380nm。采用柱后质谱电喷雾离子源（ESI source）正离子模式,实现了水解牛血清白蛋白中氨基酸和油菜蜂花粉中氨基酸的定性定量检测。荧光定性检测的线性回归系数均大于0.9990,检出限为1.49~19.74fmol（S/N=3）。

基于多目标权重分析的草地适宜放牧率探讨

依据生态系统服务的理念，首次发展了生态系统服务指数（ESI），试图通过对生态系统服务功能的综合考虑，提出科学的适宜放牧率的评价方法。本研究利用美国北达科他州立大学中部草原研究站17年长期放牧试验数据，选取植物多样性Shannon-Wiener指数、地上净初级生产力、土壤表层含水量和单位面积家畜增重4个指标，通过对不同指标分别赋予不同的权重，计算不同管理目标下ESI及其稳定性，并对单目标管理与多目标管理进行了比较研究。结果表明，对于北美混合普列里（Prairie）草地，围封不利用或建立自然保护区，虽然生态系统比较稳定，但既不能有效的提高植物多样性、初级生产力和土壤水分含量等生态功能，又没有畜产品产出；而在重牧或极重牧处理下虽然获得了较大的畜产品生产，但导致了草地生态系统的退化和较大的系统不稳定性。因此，这2种管理方式在实践中都是不可取的。应用生态系统服务指数综合考虑，认为应该权衡各项生态功能和生产功能，此时轻牧或中牧是最适宜的。因此，ESI的建立避免了单项指标的评价偏差，使得适宜放牧率的确定更加合理。

大鼠端脑中神经递质的荧光衍生化高效液相色谱质谱联用法研究

以1,2-苯并-3,4-二氢咔唑-9-乙基氯甲酸酯(BCEOC)作为柱前荧光衍生试剂,在Hypersil BDS C_(18)(4.6 mm×100 mm,5 μm)反相色谱柱上,荧光检测波长为390 nm(激发波长为333 nm)下,采用梯度洗脱实现了5种神经递质标准品(谷氨酸、γ-氨基丁酸、多巴胺、5-羟色胺、5-羟吲哚乙酸)衍生物的同时分离检测.5种组分的线性范围为24 fmol～200 pmol,线性回归系数均大于0.9997;检出限为4.0～12.6 fmol(S/N=3∶1).在柱后在线质谱电喷雾电离源(ESI Source)正离子模式下实现了各组分的快速质谱鉴定,对其二级质谱裂解碎片峰进行了归属,并以5-羟色胺衍生物为例进行了质谱裂解机理解析.对A、B两组(A:安静对照组,B:运动训练组)4种取样状态(安静、运动1 h、力竭即刻、力竭后恢复12 h)下的64只大鼠端脑组织中5种神经递质的定量测定结果表明,长期中等强度的运动训练能够有效提高大鼠的运动能力及耐力运动后疲劳恢复能力.方法的灵敏度高、重现性好,为大鼠端脑中氨基酸类和单胺类神经递质的同时测定建立了一种新方法.

Chemical Constituents of Nitraira tangutorum Seed from Qaidam Basin

Six compounds were isolated from the 75% ethanol extract of Nitraria tangutorum seed.On the basis of spectroscopic methods including 1H NMR,13C NMR and ESI-MS and comparison with literature,their structures were elucidated as daucosterol(1),4-hydroxypipecolic acid(2),quercetin(3),allantoin(4),1,2,3,4-tetrahydro-1-methyl-β-carboline-3-carboxylic acid(5) and L-tyrosine(6).Compounds 1,2,3,5 and 6 were isolated from Nitraria tangtorum for the first time.

大鼠血浆中痕量氨基酸的柱前衍生-高效液相色谱串联质谱测定

以1,2-苯并-3,4-二氢咔唑-9-乙基氯甲酸酯(BCEOC)作为柱前荧光衍生试剂,在Hypersil BDS C18 (4.6 ×200 mm, 5 μm)反相色谱柱上,荧光检测波长为390 nm(激发波长为333 nm),采用梯度洗脱实现了20种氨基酸标准品衍生物的分离检测.20种组分的线性范围为51.6 fmol～105.6 pmol,线性回归系数均大于0.9995;检出限为6.3～177.6 fmol (S/N=3:1).经柱后串联质谱电喷雾电离源(ESI Source)实现了各组分的质谱鉴定,并以酪氨酸(Tyr)衍生物为例进行了质谱裂解机理解析.对A、B、C三组(A:安静对照组;B:运动力竭即刻组;C:力竭恢复12 h 组) 24只大鼠血浆中氨基酸的定量测定结果表明,运动力竭即刻较安静状态大鼠血浆氨基酸含量明显增加;力竭恢复12 h 后血浆氨基酸水平基本恢复到运动前状态.方法的灵敏度高、重现性好,为大鼠血浆中氨基酸的测定提供了一种新方法

珠芽蓼全草化学成分研究

本文探讨了珠芽蓼全草的化学成分.我们利用硅胶柱多次层析分离和Sephadex LH-20纯化等方法分离得到5个化合物,经NMR,IR,HR-ESI-MS等技术及理化性质鉴定结构,5个化合物分别为β-谷甾醇(β-sitosterol,1)、胡萝卜苷(daucosterol,2)、槲皮素(quercetin,3)、6-O-没食子酰熊果苷(6-O-galloylarbutin,4)、蔗糖(sucrose,5).其中化合物3、4为首次从该植物中分离得到.

Application of 2-(11H-benzo[a] carbazol-11-yl) Ethyl Carbonochloridate as a Pro-column Derivatization Reagent of Amino Acid by High Performance Liquid Chromatography with Fluorescence Detection

On a reversed phase Hypersil BDS C-18 (200 mm x 4. 6 mm, 5 mu m) column, 20 amino acids, which were derivatized using 2-(11H-benzo [a] carbazol-11-yl) ethyl carbonochloridate (BCEC-Cl) as pre-column derivatization reagent, were separated in conjunction with a gradient elution. Optimum derivatization was obtained by reacting of amino acids with BCEC-Cl at room temperature for 5 min in the presence of sodium borate catalyst in acetonitrile solvent. The fluorescence excitation and emission wavelengths were 279 nm and 380 nm respectively. The identification of amino acid derivatives from hydrolyzed bovine serum albumin and bee pollen was carried out by post-column mass spectrometry with electrospray ion source in positive ion mode. Linear correlation coefficients of the amino acid derivatives were > 0.9990, and detection limits (at signal to noise of 3:1) were 1.49 - 19.74 fmol for the labeled amino acids.

孤南-三合村洼陷含油气系统研究

The development petroleum geology has made people from studying and studying and predicting in statically and respectively the pool-forming conditions of an area such as oil source bed, reservoir, overlying formation, migration, trap and preservation, etc. to regarding these conditions as well as roles of generation, reservation and accumulation as an integrated dynamic evolution development system to do study .Meanwhile apply various simulating means to try to predict from quantitative angle. Undoubtedly, the solution of these questions will accumulate exploration process, cut down exploration cost and obtain remarkable economic and social benefits. This paper which take sedimentology ,structural geology and petroleum geology as guides and take petroleum system theory as nucleus and carry out study thinking of beginning with static factor and integration of point and face as well as regarding dynamic state factor as factor and apply study methods of integration of geology, Lab research and numerical modeling proceed integrated dissect and systematic analysis to GuNan-SanHeCun depression. Also apply methods of integration of sequence stratigraphy, biostratigraphy, petrostratigraphy and seismic data to found the time-contour stratigraphic framework and reveal time-space distribution of depositional system and meantime clarify oil-source bed, reservoir and overlying distribution regular patterns. Also use basin analysis means to study precisely the depositional history, packed sequences and evolution. Meanwhile analyze systematically and totally the fracture sequence and fault quality and fault feature, study the structural form, activity JiCi and time-space juxtaposion as well as roles of fault in migration and accumulation of oil and gas of different rank and different quality fault. Simultaneously, utilize seismic, log, analysis testing data and reservoir geology theory to do systematic study and prediction to GuNan-SanHeCun reservoir, study the reservoir types macroscopic distribution and major controlling factors, reservoir rock, filler and porosity structural features as well as distribution of reservoir physical property in 3D space and do comprehensive study and prediction to major controlling and influential factors of reservoir. Furthermore, develop deepingly organic geochemistry comprehensive study, emphasis on two overlaps of oil source rock (ESI, ES3) organic geochemistry features, including types, maturity and spatial variations of organic matter to predict their source potential .Also apply biological marks to proceed oil-to-source correlation ,thereby establish bases for distribution of petroleum system. This study recover the oil generation history of oil source rocks, evaluate source and hydrocarbon discharge potential ,infer pool-forming stages and point out the accumulation direction as well as discover the forming relations of mature oil-source rock and oil reservoir and develop research to study dynamic features of petroleum system. Meanwhile use systematic view, integrate every feature and role of pool forming and the evolution history and pool-forming history, thereby lead people from static conditions such as oil source bed, reservoir, overlying formation, migration, trap and preservation to dynamically analyzing pool-forming process. Also divide GuNan-SanHeCun depression into two second petroleum system, firstly propose to divide second petroleum system according to fluid tress, structural axis and larger faults of cutting depression, and divide lower part of petroleum system into five secondary systems. Meanwhile establish layer analysis and quantitative prediction model of petroleum model, and do quantitative prediction to secondary petroleum system.

Identification of ginsenosides in roots of Panax ginseng by HPLC-APCI/MS

A new HPLC-APCI/MS method for the identification of ginsenosides has been developed. The analyses were performed on a reversed-phase C-18 column using a binary eluent (acetonitrile and water) under gradient conditions. Although APCI is a high-temperature evaporative process, HPLC-APCI/MS could effectively identify thermo-labile ginsenosides. The [M-H](-) ions and the thermal degradation ions of ginsenosides could be clearly observed under negative and positive ion conditions, respectively, and these were used to identify the molecular masses, the aglycone structures and the sugar groups of ginsenosides. APCI/MS can provide more explicit information than ESI/MS for identifying and distinguishing ginsenosides. Using the HPLC-APCI/MS method, 35 ginsenosides were identified in Panax ginseng. Copyright (C) 2005 John Wiley & Sons, Ltd.

Identification of phospholipid structures in human blood by direct-injection quadrupole-linear ion-trap mass spectrometry

Direct-injection electrospray ionization mass spectrometry in combination with information-dependent data acquisition (IDA), using a triple-quadrupole/linear ion trap combination, allows high-throughput qualitative analysis of complex phospholipid species from child whole blood. In the IDA experiments, scans to detect specific head groups (precursor ion or neutral loss scans) were used as survey scans to detect phospholipid classes. An enhanced resolution scan was then used to confirm the mass assignments, and the enhanced product ion scan was implemented as a dependent scan to determine the composition of each phospholipid class. These survey and dependent scans were performed sequentially and repeated for the entire duration of analysis, thus providing the maximum information from a single injection. In this way, 50 different phospholipids belonging to the phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylcholine and sphingomyelin classes were identified in child whole blood. Copyright (C) 2005 John Wiley & Sons, Ltd.