两种红树林植物海漆和海桑化学成分研究

本论文对两种红树林植物海漆 (Excoecaria agallocha L.) 和海桑（Sonneratia caseolaris L.）的化学成分进行了系统研究。 采用常规的硅胶柱层析、制备薄层层析、凝胶 Sephadex LH-20 柱层析、MCI柱层析、反相硅胶柱层析、半制备型 HPLC 以及重结晶等手段，从海漆 (Excoecaria agalloch L. ) 中分离得到 40 个化合物，从海桑（Sonneratia caseolaris L.）中分离得到 30 个化合物。利用各种现代波谱技术 (IR、UV、ESI-MS、EI-MS、1D-NMR、2D-NMR等) 及其化学物理性质，确定了海漆中 32 个化合物的结构，其中包括 1 个新的三萜天然产物以及 15 个首次从海漆中报道的化合物；确定了海桑中 27 个化合物的结构，其中包括 1 个新化合物和一个首次报道其碳谱数据的化合物。本文为首次报道海桑的化学成分研究。 对海漆和海桑粗提物及分离得到的部分化合物进行了抗肝癌细胞毒活性筛选，化合物S22表现出较强活性，其IC50为2.8 μg/mL；海漆和海桑粗提物及其它部分单体化合物只表现出微弱活性；阳性对照丝裂霉素C的IC50为1.1 μg/mL。 对分离得到的部分样品还进行了抗菌活性测试，各样品在测试浓度下对测试菌均未表现出明显的抗菌活性。 首次研究了海漆挥发性成分及其季节性变化。利用水蒸汽法提取了不同季节海漆的挥发性成分，通过GC-MS鉴定其化学组成，发现脂肪酸、二萜和倍半萜是海漆挥发性成分的主要组成，不同季节的挥发性成分差异较大。

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。

Experimental study on the impact of dinoflagellate Alexandrium species on populations of the rotifer Brachionus plicatilis

To investigate harmful effects of the dinoflagellate Alexandrium species on microzooplankton, the rotifer Brachionus plicatilis was chosen as an assay species, and tested with 10 strains of Alexandrium including one known non-PSP-producer (Alexandrium tamarense, AT-6). HPLC analysis confirmed the PSP-content of the various strains: Alexandrium lusitanicum, Alexandrium minutum and Alexandrium tamarense (ATHK, AT5-1, AT5-3, ATC102, ATC103) used in the experiment were PSP-producers. No PSP toxins were detected in the strains Alexandrium sp1, Alexandrium sp2. Exposing rotifer populations to the densities of 2000 cells ml(-1) of each of these 10 Alexandrium strains revealed that the (non-PSP) A. tarnarense (AT-6) and two other PSP-producing algae: A. lusitanicum, A. minutum, did not appear to adversely impact rotifer populations. Rotifers exposed to these three strains were able to maintain their population numbers, and in some cases, increase them. Although some increases in rotifer population growth following exposures to these three algal species were noted, the rate was less than for the non-exposed control rotifer groups. In contrast, the remaining seven algal strains (A. tamarense ATHK, AT5-1, AT5-3, ATC102, ATC103; also Alexandrium sp1 and Alexandrium sp2) all have adverse effects on the rotifers. Dosing rotifers with respective algal cell densities of 2000 cells ml-1 each, for Alexandrium spl, Alexandrium sp2, and A. tamarense strains ATHK and ATC103 showed mean lethal time (LT50) on rotifer populations of 21, 28, 29, and 36h, respectively. The remaining three species (A. tamarense strains AT5-1, AT5-3, ATC102) caused respective mean rotifer LT50S of 56, 56, and 71 h, compared to 160 h for the unexposed "starved control" rotifers. Experiments to determine ingestion rates for the rotifers, based on changes in their Chlorophyll a content, showed that the rotifers could feed on A. lusitanicum, A. minutum and A. tamarense strain AT-6, but could graze to little or no extent upon algal cells of the other seven strains. The effects on rotifers exposed to different cell densities, fractions, and growth phases of A. tamarense algal culture were respectively compared. It was found that only the whole algal cells had lethal effects, with strongest impact being shown by the early exponential growth phase of A. tamarense. The results indicate that some toxic mechanism(s), other than PSP and present in whole algal cells, might be responsible for the adverse effects on the exposed rotifers. (C) 2004 Elsevier B.V. All rights reserved.

Effects of the dinoflagellate Alexandrium tamarense on early development of the scallop Argopecten irradians concentricus

The effects of Alexandrium tamarense (strain ATHK) on early development of the bay scallop Argopecten irradians concentricus were studied under laboratory conditions. The algal culture was verified by HPLC to produce paralytic shellfish poisoning (PSP) at a level of 37.48 fmol/cell. Survival of the scallop larvae was not affected when they were grown with A. tamarense at concentrations of 500-10,000 cells/ml for 48 h. However, the activity of D-shape larvae was inhibited after 48-h exposure to A. tamarense at the algal cell density of 10,000 cells/ml. Scallop growth was inhibited significantly by A. tantarense during a 14-day exposure starting at the eye-spot larval stage. The size of juvenile scallops in the group of 10,000 cells/ml was only about 32% of that of the controls, although no obvious effect of A. tamarense was found on the rate of larval metamorphosis. All juvenile scallops survived in algal concentrations of 600-2400 cells/ml, however, attachment rates were significantly lower than control values after a 5-h exposure to A. tamarense at concentrations >600 cells/ml, while they were not obviously reduced after only 1 h of exposure. At concentrations >600 cells/ml, the climbing ability of juveniles was clearly reduced by exposure to A. tamarense after only 1 h. The climbing rate and height were only 55% and 45%, respectively, of those of the controls, when exposed to A. tantarense at a concentration of 600 cells/ml. The results indicated that A. tamarense blooms may have detrimental impacts on shellfish at early life stages, therefore, special attention should be paid to the toxic algal blooms in shellfish breeding area. (C) 2003 Elsevier Science B.V. All rights reserved.

A recent shellfish toxin investigation in China

A shellfish toxin investigation along the Chinese coast has recently been conducted using both HPLC and mouse assay methods. The results showed that DSP was widely distributed in different shellfish species in China. 26 out of 89 samples had DTX1 (dinophysistoxin-1) or OA (okadaic acid) but the DSP content in most shellfish samples did not reach the regulatory limit for human consumption adopted in many countries (20 mu g/100 g soft tissue). PSP was also found in 5 out of 96 samples along the coast. One sample, Chlamys nobilis from Hong Kong contained high levels of PSP (320 mu g STX equivalent/100 g soft tissue), compared to the regulatory limit (80 mu g STX equivalent/100 g soft tissue). After the recent outbreak of red tide in Hong Kong waters, three further shellfish samples were collected within 40 days to investigate the impacts of this event, It was shown that high levels of PSP continued to exist in Hong Kong waters. This report provides the first report of DSP and PSP distribution along the Chinese coast. (C) 1999 Elsevier Science Ltd, All rights reserved.

Investigation of Extration Method for Paralytic Shellfish Poisoning Toxins in Shellfish

Me optimal conditions were established for the extraction of paralytic shellfish poisoning toxins from gonad of Chlamys nobills using acetic acid and hydrochloric acid in the concentration range of 0.04-1.0 mol/L. A 10-g portion of gonad of Chlamys nobilis was extracted by boiling for 5 min with 1.0 mL acetic acid and hydrochloric acid in a 50-mL beaker. Meanwhile, a portion of gonad of Chlamys nobilis was extracted by sonication in the solution of 0.3 mol/L HAc + 0.2 mol/L HCl for a total period of 5-30 min. The raw extract was centrifuged at 3500 r/min for 5 min and the pH of supernatant was adjusted from 2.0 to 4.0 by 0.1 mol/L NaOH or 5 mol/L HCL After passing through a Millipore ultrafiltration membrane (10000 MW cut-off), ultrafiltrate was then analyzed by HPLC. The results showed that hydrochloric acid in the concentration range of 0.25-1.0 mol/L caused a significant decrease of N-sulfocarbarnoyl-11-hydroxysulfate toxin C1 (C1), C2 and gonyautoxin 5 (GTX5) and the concomitant increase of GTX2,3. However, the amount of the three unstable toxins did not show any change using the extraction with acetic acid. Under the same concentration of acetic acid (0.3 mol/L) and hydrochloric acid (0.2 mol/L), the amount of C1 in the ultrasonic extraction was obviously lower than the boiling one, while C2 showed slightly higher than the latter.

Transfer of paralytic shellfish toxins via marine food chains: A simulated experiment

Objective To study the transfer of paralytic shellfish toxins (PST) using four simulated marine food chains: dinoflagellate Alexandrium tamarense -> Arterriia Artemia salina -> Mysid shrimp Neomysis awatschensis; A. tamarense-N. awatschensis: A. taniarense A. salina -> Perch Lateolabrax japonicus; and A. tamarense -> L. japonicus. Methods The ingestion of A. tamarense, a producer of PST, by L. japonicus, N. awatschensis, and A. salina was first confirmed by microscopic observation of A. tamarense cells in the intestine samples of the three different organisms, and by the analysis of Chl.a levels iii the samples. Toxin accumulation in L. japonicus and N. awatschensis directly from the feeding on A. tamarense or indirectly ibrough the vector of A. salina was then studied. The toxicity of samples was measured using the AOAC mouse bioassay method, and the toxin content and profile of A. tamarense were analyzed by the HPLC method. Results Both A. salina and N. awatschensis could ingest A. tamarense cells. However, the ingestion capability of A. salina exceeded that of N. awatschensis. After the exposure to the culture of A. tamarense (2 000 cells(.)mL(-1)) for 70 minutes, the content of ChLa in A. salina and N. awatschensis reached 0.87 and 0.024 mu g-mg(-1), respectively. Besides, A. tamarense cells existed in the intestines of L. japonicus, N. awatschensis and A. salina by microscopic observation. Therefore, the three organisms could ingest A. tamarense cells directly. A. salina could accumulate high content of PST, and the toxicity of A. salina in samples collected on days 1, 4, and 5 of the experiment was 2.18, 2.6, and 2.1 MU(.)g(-1), respectively. All extracts from the samples could lead to death of tested mice within 7 minutes, and the toxin content in arternia sample collected on the 1st day was estimated to be 1.65x10(-5) pg STX equa Vindividual. Toxin accumulation in L. japonicus and N. awatschensis directly from the feeding on A. tamarense or indirectly froin the vector of A. salina was also studied. The mice injected with extracts from L. japonicus and N. awatschensis samples that accumulated PST either directly or indirectly showed PST intoxication symptoms, indicating that low levels of PST existed in these samples. Conclusion Paralytic shellfish toxins can be transferred to L. japonicus, N. awatschensis, and A. salina from A. taniarense directly or indirectly via the food chains.

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.

Crystallographic and NMR studies on sterols from green alga Chaetomorpha basiretorsa Setchell

Chemical investigation of the ethanol extract of the marine green alga Chaetomorpha basiretorsa Setchell led to the isolation of a new sterol stigmast-4,28-dien-3 alpha 6 beta-diol 1 in addition to the five known sterols of beta-lawsaritol 2, saringosterol 3, 24-hydroperoxy-24-vinyl - cholesterol 4, beta-stigmasterol 5, 29-hydroxystigmasta-5, 24(28) -dien-3 beta-ol 6. Compounds were isolated by normal phase silica gel and Sephadex LH - 20 gel colum chromatography, reverse phase HPLC and recrystalization. Their structures were elucidated by spectroscopic methods including MS, IR 1D/2D NMR and X-ray analysis. Cytotoxicity of compounds was screened by using the standard WIT method. All these compounds were isolated from the green alga Chaetomorpha basiretorsa Setchell for the first time and they were inactive (50% inhibitory concentration was greater than 10 mu g /cm(3)) against KB, Bel -7402, PC - 3M, Ketr 3 and MCF - 7 cell lines.

Strain H-2-419-4 of Haematococcus pluvialis induced by ethyl methanesulphonate and ultraviolet radiation

Two strains H-2-410 and H-2-419 were obtained from the chemically mutated survivors of wild Haematococcus pluvialis 2 by using ethyl methanesulphonate (EMS). Strains H2-410 and H2-419 showed a fast cell growth with 13% and 20% increase in biomass compared to wild type, respectively. Then H-2-419-4, a fast cell growth and high astaxanthin accumulation strain, was obtained by exposing the strain H2-419 to ultraviolet radiation (UV) further. The total biomass, the astaxanthin content per cell, astaxanthin production of H-2-4194 showed 68%, 28%, and 120% increase compared to wild H. pluvialis 2, respectively. HPLC (High Performance Liquid Chromatography) data showed also an obvious proportional variation of different carotenoid compositions in the extracts of H2-4194 and the wild type, although no peak of carotenoids appeared or disappeared. Therefore, the main compositions in strain H-2-419-4, like its wild one, were free of astaxanthin, monoester, and diester of astaxanthin. The asexual reproduction in survivors after exposed to UV was not synchronous, and different from the normal synchronous asexual reproduction as the mother cells were motile instead of non-motile. Interestingly, some survivors from UV irradiation produced many mini-spores (or gamete?), the spores moved away from the mother cell gradually 4 or 5 days later. This is quite similar to sexual reproduction described by Elliot in 1934. However, whether this was sexual reproduction remains questionable, as no mating process has been observed.

The chemical constituents from red alga Gymnogongrus flabelliformis Harv.

Eight compounds were isolated from red alga Gymnogongrus flabelliformis Harv. In normal phase silica gel, Sephadex LH-20 gel column chromatography, reverse phase HPLC, and recrystallization. Based on MS and 1D NMR spectroscopic data, their structures were determined as: stigmast-4-en-3-one (I), cholest-4-en-3-one (II), cholesterol (III), uracil (IV), uridine (V), adenosine (VI), succinic acid (VII), and 5-hydroxy-4-methyl-5-pentyl-2,5-dihydro-furan-2-on (VIII). All of them were obtained from this species for the first time. Cytotoxicity of these compounds was screened using standard MTT method, but all the compounds were inactive (IC50 > 10 mu g/ml).

Residues of enrofloxacin, furazolidone and their metabolites in Nile tilapia (Oreochromis niloticus)

The residues of enrofloxacin and its metabolite in Nile tilapia (Oreochromis niloticus) were studied after oral dose of 50 mg/kg for 7 days. To find the differences between Nile tilapia and Chinese shrimp (Penaeus chinensis), the residues of enrofloxacin in P chinensis were also studied under the same conditions. The results showed that enrofloxacin metabolized into ciprofloxacin in both Nile tilapia and P chinensis, the maximal concentration of enrofloxacin in muscle, liver and plasma of Nile tilapia were 3.61 mu g/g, 5.96 mu g/g, 1.25 mu g/ml respectively, and ciprofloxacin in muscle was 0.22 mu g/g. The maximal concentration of enrofloxacin and ciprofloxacin in P chinensis were 1.68 mu g/g and 0.07 mu g/g respectively. The predicted withdrawal time for Nile tilapia was 22 days, and P. chinensis was 12 days under our experiment conditions. The residues of fitrazolidone [3-(5-nitrofurfurylidenamino)-2-oxazolidinone] and its main metabolite 3-amina-2-oxazolidinone (AOZ) in Nile tilapia were first determined by HPLC/MS. Results showed that after oral dose of 30 mg/kg for 7 days, the maximum concentration of farazolidone in Nile tilapia was 413 mu g/kg after 6 h, whereas AOZ residue reached its maximum (31 mu g/kg) right after stopping treatment. In contrast to the high metabolic rate of furazolidone, AOZ was very difficult to eliminate in vivo, thus the withdrawal time of furazolidone in Nile tilapia was 22 days at least. (c) 2005 Elsevier B.V. All rights reserved.

Simultaneous Determination of Multi-Organotin Compounds in Seawater by Liquid-Liquid Extraction-High Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry

The hyphenated technique of high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS) was applied to the simultaneous determination of five organotin compounds (trimethyltin, dibutyltin, tributyltin, diphenyltin and triphenyltin) in seawater samples. Agilent TC-C18 column was used for the separation, the mobile phase of HPLC was CH3CN : H2O: CH3COOH=65 : 23 : 12 (phi), 0.05% TEA, and pH value was adjusted to 3.0 by diluent ammonia. The flow rate was 0.6 mL . min(-1). Five mixed organotin compounds in a mix standard solution from 100 to 0.5 mu g . L-1 were applied for the method assessment. The experimental results indicate that the correlation coefficient of calibration curves (R-2) for each organotin compound was over 0.998 and the detection limits of the five organotin compounds were lower than 3 ng . L-1. Different mixed organic solvents including dichloromethane or toluene were used for extraction of organotin and the extraction condition of organotin from seawater was optimized. The 100 mL seawater acidized by hydrochloric acid was extracted by 10 mL carbon dichloride (CH2Cl2) with 2% tropolone for 10 min twice. Extracted organic solvents were mixed And blown to one drop by nitrogen with the rate of 1.7 mL . min(-1), then 1 mL acetonitrile was added to the drop for redissolving the organotin compounds. Finally, the mixed redissolution was filtered by 0.22 mu m organic filter membrane before analysis. it was found that the only organotin compound in seawater was triphenyltin (TPHT) and the content was 53.2 ng . L-1. The recoveries test from the standard addition for diphenyltin (DPHT), dibutyltin (DBT), tributyltin (TBT) and triphenyltin (TPHT) were over 80%. However, the recovery for trimethyltin (TMT) was relatively low and the value was 50%. The reason might be attributed to the decomposition or adsorption of those compounds during the extraction procedure. Further study on this subject is in progress.

Speciation Analysis of Organotin Compounds in Shellfish by Hyphenated Technique of High Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry

The hyphenated technique of high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry(HPLC-ICP-MS) was applied to the simultaneous determination of five organotin compounds in the shellfish samples. Agilent TC-C-18 column was selected, mobile phase of the HPLC was CH3CN:H2O: CH3COOH = 65:23:12 (V/V), 0. 05% TEA, pH = 3.0 at flow rate 0.4 mL/min. Five mixed organotin standards from 100 mu g/L to 0. 5 mu g/L was used for the method evaluation. The experimental results indicate that the linearity (R-2) for each compound was over 0.998. The shellfish samples were treated by supersonic extraction with mobile phase for 30min. Four organotin compounds including dibutyltin (DBT), tributyltin (TBT), diphenyltin (DphT) and triphenyltin (TPhT) in shellfish samples were detected with method mentioned above. It was found that the domain compounds in the samples were tributyltin (TBT) and triphenyltin (TPhT). The recoveries test from the standard addition for trimethyltin (TMT tributyltin (TBT), and triphenyltin (TPhT) were, over 80%. However, the recoveries for diphenyltin (DPhT) and dibutyltin (DBT) were relatively low, 37.3% and 75.2% respectively. The reason might be attributed to the decomposition of those compounds during the extraction procedure. The further study on this subject is under the progress.

氮杂多环光致发光分子用于食品中脂肪胺的分析及质谱鉴定

对含氮类光致发光分子2-（11H-苯[a]咔唑）-异丙基氯甲酸酯进行柱前衍生处理，并经荧光检测对脂肪胺进行高效液相色谱（HPLC）分离和质谱定性。衍生物于λ_(ex)=279nm产生强烈荧光，发射波长为λ_(ex)=380nm。40℃下在pH9．0的硼酸缓冲液中，衍生反应3min后获得稳定的荧光产物。在Eclipse XDB-C_8色谱柱（4．6mm×150mm，5μm）上，采用梯度洗脱对12种脂肪胺衍生物进行了优化分离。采用大气压化学电离源（APCI Source）正离子模式进行质谱定性，实现了各种脂肪胺衍生物的快速、准确测定。该方法具有良好的重复性，多数脂肪胺的线性回归系数大于0．9994，检出限在2．73～23．16fmol水平。