2012-Marine Carotenoids and Oxidative Stress=FX=抗氧化

Molecules2013, 18, 6298-6310; doi:10.3390/molecules18066298OPEN ACCESSmoleculesISSN 1420-3049/journal/molecules ArticleFucoxanthin from Undaria pinnatifida: Photostability and Coextractive EffectsAnna Piovan 1,*, Roberta Seraglia 2, Bruno Bresin 3, Rosy Caniato 1 and Raffaella Filippini 11Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo 5, Padova 35131, Italy; E-Mails: rosy.caniato@unipd.it (R.C.);raffaella.filippini@unipd.it (R.F.)2CNR-ISTM, Corso Stati Uniti 4, Padova 35100, Italy; E-Mail: roberta.seraglia@r.it3 ARPA-FVG Regional Agency for Environmental of Friuli Venezia Giulia Region,Via delle Acque 28, Pordenone 33170, Italy; E-Mail: bdbres@alice.it*Author to whom correspondence should be addressed; E-Mail: anna.piovan@unipd.it;Tel.: +39-049-827-6266; Fax: +39-049-827-6260.Received: 11 April 2013; in revised form: 17 May 2013 / Accepted: 24 May 2013 /Published: 29 May 2013Abstract: Fucoxanthin is one of the most abundant carotenoids and possesses a number ofbeneficial medicinal qualities which include its anti-oxidant, anti-obesity and anti-cancerproperties. In this study, the photostability of fucoxanthin in extracts with differentchemical profiles was studied. The extracts were obtained from Undaria pinnatifida, aseaweed rich in this carotenoid, using conventional liquid solvent extraction proceduresand the QuEChERS method. All the extracts contained all-trans-fucoxanthin as the majorcompound. Conventional procedures produced a fucoxanthin purity of lower than 50%,whereas after liquid-liquid partition, PSA cleanup, and PSA and GCB cleanup(QuEChERS method) fucoxanthin purity increased to 70%, 86%, and 94%, respectively.Although in the acetone extract the initial content of fucoxanthin was the highest, resultsdemonstrate that coextractives play an important role in enhancing the rate ofphotodegradation. After light exposure, the conventional extracts lost around 90% of theinitial fucoxanthin content. On the other hand, the extracts obtained by the QuEChERSmethod showed significantly higher light stability than the conventional extracts. Theseresults suggest that the QuEChERS method could be used and further improved to obtainmore purified and stable extracts for fucoxanthin from U. pinnatifida.Keywords: fucoxanthin; Undaria; photostability; coextractives; QuEChERS1. IntroductionFucoxanthin is one of the most abundant carotenoids, and contributes to more than 10% of the estimated total production of carotenoids in Nature, especially in the marine environment [1]. Fucoxanthin is a pigment, along with chlorophylls and β-carotene, widely distributed in brown algae and diatoms [2]. It has an unusual structure with an allenic bond and 5,6-monoepoxide in its molecule (Figure 1).Figure 1. Fucoxanthin.Undaria pinnatifida, a brown seaweed better known as wakame, is a rich source of fucoxanthin [3]. Actually, U. pinnatifida is widely used as a human food in many countries especially Korea and Japan, and it is becoming increasingly popular in the European market, above all in the form of extracts used as food supplements. The numerous activities attributed to U. pinnatifida products are essentially linked to their fucoxanthin content [4]. There is indeed a growing evidence from in vitro and in vivo experiments suggesting that fucoxanthin has health promoting effects because of its strong anti-oxidant properties [5–8]. Fucoxanthin provides protective effects on liver, blood vessels of the brain, bones, skin and eyes. It has anti-obesity and anti-diabetic properties, and anti-inflammatory and anti-malarian effects. Moreover it is very effective in inhibiting cell growth and inducing apoptosis in human cancer cells. Particularly, the anti-adult T-cell leukemia effects, the induction of apoptosis in human leukemia cells, and the anti-obesitive effect of fucoxanthin are distinctly more potent than that of β-carotene and astaxanthin. The unique effects of fucoxanthin is due its characteristic chemical structure, and carotenoids without an allenic bond are not active [4,9,10]. Recently, it has also been demonstrated that pretreatment with fucoxanthin improves the chemotherapeutic efficacy of cisplatin by enhancing the inhibition of cell proliferation of human hepatoma HepG2. These results suggest that the combined treatment of fucoxanthin and cisplatin may provide a novel therapeutic approach to decrease cisplatin-induced drug resistance [11,12].Owing to these properties fucoxanthin has attracted considerable interest both as nutraceuticals and pharmaceuticals, and there is an increasing interest in the effects of this seaweed carotenoid as a functional supplement in human diets helping to enhance the nutritional profiles of foods such as pasta, beverages, cakes and spreads [13–16].Though chemical synthesis of fucoxanthin is possible, it is very expensive and therefore the viability of obtaining directly from brown seaweeds should not be overlooked [4]. Indeed there havebeen several studies which have focused on its extraction and purification from seaweeds. A crude oil, a mixture of carotenoids and polyphenols, was obtained from U. pinnatifida using supercritical carbon dioxide [17] and a method for its separation and purification from edible brown algae by microwave-assisted extraction coupled with high-speed countercurrent chromatography has been developed [18]. However, the most common way for fucoxanthin extraction is by liquid solvent extraction whereas the available commercial products are mainly constituted of extracts [19].Fucoxanthin is highly susceptible to degradation and this can lead to cis-trans isomerisation, oxidative cleavage and/or epoxidation of the backbone [20]. While the deleterious effects of heat, light, or coextractives on fucoxanthin have regularly been cited for almost half a century, the extent of the impact of these factors has rarely been determined [21–23]. In this work, we have studied the effects of coextractives present in different extracts on the photostability of the fucoxanthin.2. Results and DiscussionFrequent references are made in literature concerning procedures that impact on fucoxanthin stability [24–26]; however, the extent of these effects have not as yet been quantified. In this study, the photostability of the fucoxanthin in Undaria pinnatifida extracts with different chemical profiles was studied.We used fresh starting material and not exhaustive extraction procedures in order to prevent as far as possible any degradation event occurring during the sample processing steps. The extractions were performed by using three different solvents (methanol, acetone, acetonitrile) with different extraction power both versus fucoxanthin and other compounds present in U. pinnatifida. The extracts were obtained in an ultrasonic bath (conventional procedures) and with the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method [27]. The QuEChERS method is the most commonly applied prep method for the determination of pesticide residues from a variety of fruit and vegetables, fatty food matrixes like milk and eggs, and water [28]. This method involves an extraction with acetonitrile partitioned from the aqueous matrix using anhydrous magnesium sulphate (MgSO4) and sodium chloride (NaCl) (acetonitrile raw extract) followed by a dSPE cleanup with MgSO4and primary secondary amine (PSA) (cleanup) or a combination of PSA and graphitized carbon black (GCB) (additional cleanup). The use of the QuEChERS method allowed us to evaluate the fucoxanthin photodegradation of increasingly purified acetonitrile extracts.The identification of fucoxanthin in the different extracts was confirmed with an external calibration i.e., by comparing retention time, and UV-Vis and MS/MS spectra, respectively, of the samples to those of the standard. The quantitative analyses were performed by HPLC UV-Vis. The validation data of the method are shown in Table 1.Table 1. Regression curve data, detection limit, quantification limit and reproducibility.Regression curve data Detection limit Quantification limit Reproducibility (%RSD)y = 903598x + 26548 r2 = 0.9999(µg/mL)(µg/mL)intra-day inter-day 0.01120.035 <4<7The use of the QuEChERS method resulted in a visible cleanup of the extracts compared to the conventional procedures showing differences in color. The HPLC chromatograms of the extracts areshown in Figure 2. All the extracts contained all-trans-fucoxanthin as the major compound (RT = 5.5 min, a) and two other minor peaks (RT = 5.5–6 min, b and c) around the fucoxanthin peak, which showed similar UV-Vis spectra to fucoxanthin, as reported by Fung et al. [29]. These minor peaks were identified by MS/MS spectra as the cis-isomers of fucoxanthin. A broad and tailing peak was detected in all the conventional extracts and assigned to other coeluted matrix components; this peak was not present in the extracts obtained with the QuEChERS approach. It is worth noting that the acetonitrile raw extract (QuEChERS method) was obtained from an acetonitrile aqueous solution by salt-induced phase-separation. The different polarity of the solvent in presence of salts gave a greater matrix cleanup of the extract than with the conventional procedure with acetonitrile. The impurity content was determined as a percentage of the total area of all the peaks. The conventional procedures allowed for fucoxanthin purity lower than 50%, whereas in the acetonitrile raw extract (liquid-liquid partition), after PSA cleanup and improved cleanup (QuEChERS approach) fucoxanthin purity reached 70%, 86%, and 94% respectively.Figure 2.HPLC chromatograms (449 nm): (A) conventional extracts (acetone-black,acetonitrile-red, methanol-blue); (B) extracts obtained using QuEChERS method(acetonitrile raw extract-blue, PSA cleanup-red, GCB additional cleanup-black).Table 2 shows the fucoxanthin contents in the different extracts. Considering the QuEChERS method, the fucoxanthin amounts in the acetonitrile raw extract and after PSA cleanup were not significantly different (usin g Tukey’s multiple range test, p< 0.05). Applying the improve cleanup with PSA and GCB, GCB had a significant effect on the recovery of fucoxanthin and drove to itsdrastic lost: only one third of the fucoxanthin was recovered with GCB compared to the acetonitrile raw extract.Table 2. Fucoxanthin contents (µg/mL) in the conventional extracts (acetone, acetonitrile,methanol) and in the extracts obtained with the QuEChERS method (acetonitrile rawextract, PSA cleanup, GCB additional cleanup).Conventional procedures QuEChERS method acetone acetonitrile methanol MeCN raw extract PSA cleanup GCB additional cleanup 12.3 ± 1.10 e9.8 ± 0.99 c 1.0 ± 0.12 a10.5 ± 0.98 c,d10.6 ± 1.01 d 3.3 ± 0.29 bMeans ± SD. Numbers followed by the same lowercase letter did not differ statistically (Tukey test, p > 0.05).In order to evaluate the effects of coextractives on the stability of the fucoxanthin, all the extracts, fucoxanthin standard solutions and fucoxanthin standard solutions added of ascorbic acid were placed in direct daylight to compare the effects of coextractives and antioxidant ascorbic acid on fucoxanthin stability under light conditions.Figure 3. Fucoxanthin content after light exposure expressed as a percentage of the initialcontent in the conventional extracts (acetone, acetonitrile, methanol; green), in the extractsobtained with the QuEChERS method (acetonitrile raw extract, PSA, GCB; grey) and instandard solutions (with and without ascorbic acid; orange).Figure 3 shows the fucoxanthin content as percentage of the initial concentration after light exposure. Results here indicate that fucoxanthin is susceptible to photodegradation. Although Mise et al. [30] reported that pure fucoxanthin is unstable but, the fucoxanthin extracted from the alga is rendered stable by the coexisting antioxidants, our data clearly demonstrate that conventional extracts are the least stable, which could be related to the matrix effects. Indeed all the solutions had lost around 90% of their initial fucoxanthin contents. In the acetonitrile raw extract (QuEChERS method) the content of fucoxanthin was reduced to 40% of its initial concentration. A steady and significantincrease of the stability was observed after PSA cleanup and additional cleanup (PSA and GCB). The extract obtained after PSA cleanup retained around 60% of the initial concentration; in the extract obtained applying the additional cleanup the fucoxanthin was 70% of the initial concentration. The fucoxanthin standard solution and the fucoxanthin standard solution added of ascorbic acid retained around 60% and 80% of the initial concentration respectively.As shown by the HPLC chromatograms (Figures 4 and 5), the degradation pattern of fucoxanthin in the conventional extracts on the one hand, in the extracts obtained with the QuEChERS method and in standard solutions on the other hand was notably different. Light exposure of fucoxanthin standard solutions and extracts obtained with the QuEChERS method predominantly leads to the formation of cis-isomers, whereas different unidentified compounds, displaying a retention time between 4–5 and 5.5–8 min, are mainly formed in the conventional extracts. Again, the observed fucoxanthin loss was not mirrored by the appearance of comparable levels of isomers or degradation products as already reported [22].Fucoxanthin is subject to isomerization and oxidation which are recognized as important reactions causing carotenoid degradation [20]. The different pattern and rate of fucoxanthin degradation occurring in the conventional extracts most probably depend on other components present in solution able to catalyze the oxidation of fucoxanthin, producing different products.Figure 4.HPLC chromatograms (449 nm) obtained after light exposure of the extracts:(A) conventional extracts (acetone-black, acetonitrile-red, methanol-blue); (B) extractsobtained using QuEChERS method (acetonitrile raw extract-blue, PSA cleanup-red, GCBadditional cleanup-black.Figure 5.HPLC chromatograms (449 nm) of fucoxanthin standard solutions:(A) fucoxanthin standard solution-black, fucoxanthin standard solution with ascorbicacid-red; (B) after light exposure.The results suggest that the first stage of degradation is the isomerization of all-trans- to more oxidable cis-isomers. Indeed in the presence of ascorbic acid, standard fucoxanthin was subject to isomerization, but the further oxidation was significantly prevented by the antioxidant agent.On the basis of these results, a second set of experiments was carried out in order to evaluate a possible role of the water present in the conventional extracts and in the raw extract on the degradation rate. Moreover the fucoxanthin degradation rate in relation to its concentration was evaluated (Figure 6). No differences were observed in the degradation rate in pure acetonitrile solvent and in the acetonitrile/water mixture. On the contrary the results show that the degradation is faster at the higher fucoxanthin concentration. Indeed after only 30 min of light exposure the 10 µg/mL solutions had lost 20% of their initial content i.e., around 2 µg/mL, whereas the 5 µg/mL solutions had lost 15% of their initial content i.e., around 0.75 µg/mL. The contents of fucoxanthin after 5 h exposure were reduced by 80% and 55% respectively to around 2 µg/mL in all the solutions.These results support the statement that the observed different pattern and rate of fucoxanthin degradation in the extracts depend on coextractives present in solution rather than the presence of water or fucoxanthin concentration. Indeed the extracts obtained by conventional procedures with acetone and acetonitrile, and the acetonitrile raw extract and the extract obtained by PSA cleanup (QuEChERS method) had a similar initial fucoxanthin content but underwent different rates of degradation. Moreover, the extract obtained after additional cleanup (PSA and GCB) (3 µg/mL) having a fucoxanthin purity of 94% showed a degradation rate very similar to the standard solutions (5 µg/mL).Figure 6. Fucoxanthin content after light exposure expressed as percentage of the initialcontent in standard solutions (5 and 10 µg/mL) in pure acetonitrile solvent (MeCN) and inacetonitrile/water mixture (1:1, v/v) (MeCN + H2O).Table 3 shows the fucoxanthin content in the different extracts after light exposure. It needs to be pointed out that, although in the acetone extract (conventional procedure) the initial content of fucoxanthin was the highest (12.3 µg/mL), the coextractives played an important role in enhancing the rate of photodegradation. After only 90 min of light exposure, the content of fucoxanthin was reduced by 90% to 1.0 µg/mL. On the other hand, even if the extract obtained after PSA cleanup (QuEChERS method) had a lower fucoxanthin content (10.6 µg/mL), the extract showed significantly increased light stability since the fucoxanthin content after light exposure was decreased by 40% to 6.4 µg/mL. As pointed out above, there was no significant difference between the fucoxanthin degradation rate in the extract obtained after PSA cleanup and in standard solution, and the fucoxanthin content after light exposure was around 60% of the initial concentrations in both the samples.Table 3. Fucoxanthin content (µg/mL) in the conventional extracts (acetone, acetonitrile,methanol) and in the extracts obtained with the QuEChERS method (acetonitrile rawextract, PSA cleanup, GCB additional cleanup) after light exposure.Conventional procedures QuEChERS methodacetone acetonitrile methanol MeCN raw extract PSA cleanup GCB additional cleanup1.0 ± 0.07 b 1.0 ± 0.08 b0.1 ± 0.01 a 4.0 ± 0.30 d 6.4 ± 0.57 e2.3 ± 0.18 cMeans ± SD. Numbers followed by the same lowercase letter did not differ statistically (Tukey test, p > 0.05).3. Experimental3.1. MaterialsSolvents: methanol was purchased from Merck; acetonitrile and acetone from Fluka. Water was purified with a Milli-Q deionization unit (Millipore). All-trans-fucoxanthin with ≥95% purity was purchased from Sigma-Aldrich.QuEChERS materials were obtained from commercial suppliers. For the initial extraction step, QuEChERS Extraction SPE Kits (Agilent, Santa Clara, CA, USA) were used consisting of 50 mLplastic centrifuge tubes containing 4 g anhydrous magnesium sulphate (MgSO4) and 1 g sodium chloride (NaCl). For cleanup Q-sep™ QuEChERS dSPE Tubes for Extract Clean-Up (Restek) were used consisting of 2 mL mini-centrifuge tubes containing 150 mg anhydrous MgSO4, 25 mg primary secondary amine (PSA) sorbent, with and without 7.5 mg graphitized carbon black (GCB).Undaria pinnatifida samples were harvested from the lagoon of Venice (Italy) in June 2012.3.2. Standard Stock SolutionA stock solution of fucoxanthin was prepared in acetonitrile at a concentration of 1 mg/mL and stored at −20 °C in amber-colored vials to protect fucoxanthin from light. The working solutions were accurately diluted with acetonitrile just prior to use.3.3. Sample PreparationThe collected seaweed samples were washed with water three times. The cleaned samples were stored at −20 °C until use. Brown seaweed samples (about 500 g) made up of whole algae were homogenised with an Ultraturrax (Janke & Kunkel IKA Labortechnik, Staufen, Germany). Extractions were performed using conventional procedures and the QuEChERS approach.Conventional procedures: homogenised seaweed samples (10 g) were extracted with acetonitrile, methanol and acetone (10 mL) in an ultrasonic bath at room temperature (10 min).QuEChERS approach: the original method entailed the following steps: (a) weigh 10 g of thoroughly homogenized sample into a 50 mL centrifuge tube; (b) add 10 mL acetonitrile; (c) add 4 g anhydrous MgSO4and 1 g NaCl; shake vigorously for 1 min by hand; (d) centrifuge the tube at 3000 rpm for 1 min; (e) transfer 1 mL of the upper organic phase (raw extract) to a mini-centrifuge tube and subject it to a dispersive cleanup by mixing it with 150 mg anhydrous MgSO4, 25 mg PSA; shake by hand for 30 s; (f) centrifuge the tube at 3,000 rpm for 5 min; transfer the final extract in a vial for analysis. A first set of extracts was prepared according to the original QuEChERS method; in a second set dSPE was performed using a combination of PSA and GCB (additional cleanup).The extractions were performed in triplicates. The extracts were conveniently diluted before the analyses.3.4. Light Exposure ExperimentsIn a first set of experiments, freshly prepared 1 mL aliquots of extracts, and standard fucoxanthin solutions (10 µg/mL) with and without ascorbic acid (equimolar concentration) were placed in clear glass sample vials at room temperature in direct daylight (2,500 lux; 90 min).In a second set of experiments standard fucoxanthin solutions (5 and 10 µg/mL) in pure acetonitrile solvent and in acetonitrile/water mixture (1:1, v/v) were placed in clear glass sample vials at room temperature in direct daylight (2,500 lux). Samples were collected at 30 min intervals for 5 h. The experiments were performed in triplicates.3.5. HPLC UV-Vis Analysis3.5.1. MethodologyHPLC analysis was performed on ChromQuest (Thermoseparation, San Jose, CA, USA) pump P4000 equipped with a photodiode array detector UV6000. The data were recorded and processed using ChromQuest Chromathography Workstation. The separation was achieved with a reversed-phase analytical column, Gemini C6-Phenyl column (250 ×4, 60 mm i.d., 5 µm; Phenomenex, Torrance, CA, USA), using an isocratic elution, and the injection volume was 20 µL. The mobile phase was composed of methanol:water, 90:10; the flow rate was 1 mL/min. UV-Vis spectra were recorded in the 200–700 nm range; chromatograms were acquired at 449, 330 and 254 nm.3.5.2. Method ValidationThe linearity of the method was evaluated by using six calibration standard solutions over a range of 0.05–7 µg/mL. The calibration curve was established by plotting peak area ratios of calibration solutions vs the nominal concentrations of fucoxanthin. The linearity was determined using linear regression analysis. The limit of detection was defined as the lowest concentration level resulting in a peak area of three times the baseline noise (S/N > 3) in solvent. The limit of quantification was defined as the lowest concentration level resulting in a peak area of ten times the baseline noise (S/N > 10) in solvent. Measurements of the intra- and inter-day variability were utilised to determine the reproducibility of the method. Fucoxanthin standard solutions were analysed to determine the intra-day repeatability (examined in one day) and inter-day repeatability (determined on 3 different days). The relative standard deviation (RSD) was calculated as a measurement of method reproducibility (n = 3).3.6. HPLC MS/MS AnalysisThe HPLC conditions were those already described. A post-column split was employed to deliver approximately 250 µL/min to the electrospray interface. The ESI-MS analysis was performed using a LCQ Deca Ion Trap (Thermo Finnigan, San Jose, CA, USA), operating in the positive ion mode. Sheath gas and auxiliary gas flow rates were 60 and 40 (arbitrary units) respectively, spray voltage 4 kV and entrance capillary temperature 280 °C. Collision-induced dissociation spectra were obtained applying a supplementary RF voltage, 6 V, to the end caps of the ion trap. The data were recorded and processed using Xcalibur Workstation.3.7. Statistical AnalysisData were subjected to analysis of variance (ANOVA) to assess significant differences between the fucoxanthin content in the different extracts. Significance between the mean values was tested by Tukey's test at the confidence level of p≤ 0.05.4. ConclusionsIn this work it was demonstrated that coextractives play an important role in enhancing the rate of fucoxanthin photodegradation. After light exposure, the conventional extracts lost around 90% of theirinitial fucoxanthin concentrations, whereas there was no significant difference between the fucoxanthin degradation rate in the extract obtained after PSA cleanup (QuEChERS method) and in standard solution with 40% loss of fucoxanthin. To the best of our knowledge, this is the first report on the application of the QuEChERS method to obtain fucoxanthin extracts from U. pinnatifida. Compared to conventional extraction techniques, the QuEChERS method provided a progressive extract cleanup and extract stability.The promising results of fucoxanthin activity studies prompt the development of extraction and purification methods to obtain pure fucoxanthin. The above results suggest that the QuEChERS method could be used and further improved to obtain purified and stable extracts for fucoxanthin from U. pinnatifida.AcknowledgmentsThis work has been supported by the MIUR. The authors are grateful to Mara Marzocchi for her valuable suggestions and Emiliano Checchin (Selc) for his assistance in harvesting Undaria pinnatifida. Proofread by Savio de Souza.Conflict of InterestThe authors declare no conflict of interest.References1. Dembitsky, V.M.; Maoka, T. Allenic and cumulenic lipids. Prog. Lipid Res.2007, 46, 328–375.2. Takaichi, S. Carotenoids in algae: Distributions, biosyntheses and functions. Mar. Drugs 2011, 9,1101–1118.3. Mori, K.; Ooi, T.; Hiraoka, M.; Oka, N.; Hamada, H.; Tamura, M.; Kusumi, T. Fucoxanthin andits metabolites in edible brown algae cultivated in deep seawater. Mar. Drugs 2004, 2, 63–72.4. D’Orazio, N.; Gemello, E.; Gammone, M.A.; de Girolamo, M.; Ficoneri, C.; Riccioni, G.Fucoxantin: A treasure from the sea. Mar. Drugs 2012, 10, 604–616.5. 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关于沙棘作文英文版"英文，"Seabuckthorn, also known as Hippophae, is a remarkable plant that thrives in harsh environments, such as mountainsides and coastal areas. It has gained attention worldwide for its exceptional nutritional and medicinal properties.Seabuckthorn is rich in various vitamins, including vitamin C, vitamin E, and vitamin A. These vitamins play crucial roles in maintaining overall health and boosting the immune system. For example, vitamin C is essential for collagen production, which promotes healthy skin and wound healing. Vitamin E acts as an antioxidant, protecting cells from damage caused by free radicals. Vitamin A supports vision, immune function, and skin health.Not only is seabuckthorn packed with vitamins, but it also contains a wide range of antioxidants, such asflavonoids and carotenoids. These compounds help reduce inflammation, lower cholesterol levels, and prevent oxidative stress-related diseases like heart disease and cancer.Aside from its nutritional benefits, seabuckthorn has been used in traditional medicine for centuries. In Chinese medicine, it is believed to promote digestion, relieve cough and phlegm, and invigorate blood circulation. Moreover, seabuckthorn oil extracted from its berries is renowned for its skin-rejuvenating properties and is often used in cosmetic products.In addition to its health benefits, seabuckthorn plays a vital role in environmental conservation. Its deep root system helps prevent soil erosion, making it an excellent choice for stabilizing slopes and preventing landslides. Furthermore, seabuckthorn plants provide habitat and food for various wildlife species, contributing to biodiversity conservation.Overall, seabuckthorn is a versatile plant withnumerous benefits for human health and the environment. Its ability to thrive in challenging conditions and its rich nutritional content make it a valuable asset forsustainable agriculture and holistic healthcare practices."中文，"沙棘，又称沙柳，是一种在恶劣环境中茁壮成长的卓越植物，如山坡和沿海地区。

・论　文・中国黄海柄海鞘的化学成分李　亮1,2,王长云2,郭跃伟131中国科学院上海生命科学研究院药物研究所国家新药研究重点实验室,上海201203;2中国海洋大学医药学院海洋药物教育部重点实验室,青岛266003【摘　要】　目的:对采自中国黄海的柄海鞘(Styela clava )的化学成分进行研究,从中寻找有生物活性的次生代谢产物。

方法:用硅胶柱层析和凝胶柱层析对柄海鞘的丙酮提取物进行分离纯化,根据其化学性质,结合现代波谱技术(MS ,NMR 等),对得到的化合物进行结构鉴定。

结果:分离得到3个类胡萝卜素和1个芳香胺类化合物,其结构分别鉴定为mytiloxanthinone (1)、fucoxanthin (2)、all 2trans 2astaxanthin (3)和naphthalen 222yl 2phenyl 2amine (4)。

结论:这些化合物均系首次从柄海鞘中分离得到,并首次对化合物mytiloxanthinon (1)和fucoxanthin (2)的1H 和13C NMR 数据进行了全归属。

【关键词】　尾索动物;柄海鞘(Styela clava );类胡萝卜素;芳香胺【中图分类号】　R284 【文献标识码】　A 【文章编号】　167223651(2007)0620408205【收稿日期】　2007209230【基金项目】　国家海洋863计划(N o.2006AA609Z 412和N o.2007AA609Z 447)、基金委项目(N o.20721003,30730108,20572116)、中国科学院重点项目(grant K SCX22Y W 2R 218)资助课题、并得到教育部科技创新工程重大项目培育资金项目(N o.706038)、新世纪优秀人才支持计划(N o.NCET 20520600)部分资助【3通讯作者】　郭跃伟:教授,博导,T el :021*********,E 2mail :ywguo @ 海鞘是接近脊索动物门的高等无脊椎动物,属尾索动物门,是海洋环境特有的生物。

®ASTAXANTHINWhat is Solasta™ AstaxanthinSolasta is a standardized, natural astaxanthin extract produced from Haematococcus pluvialis. Solasta is an excellent source of astaxanthin for use in dietary supplement and personal care applications. Solasta is non-GMO, vegetarian, and produced in the USA. How is Solasta™ MadeNatural, non-genetically modified strains of Haematococcus pluvialis are cultivated in enclosed photobioreactors using purified water to produce algal biomass used in the production of Solasta. The algae are harvested by centrifugation and the astaxanthin is extracted using super-critical carbon dioxide to ensure the safety, quality, and consistency of Solasta. No pesticides, GMOs, or animal derived materials are used in the production of Solasta.Solasta™ QualitySolasta TM is manufactured in accordance with industry best practices and guidelines (current Good Manufacturing Practice) defined by the United States Food and Drug Administration (FDA). Solasta meets the strict quality standards set forth in the Astaxanthin Esters Monographs of the Food Chemical Codex1 and the US Pharmacopeia2. Solasta™ ApplicationsResearch has shown that algal astaxanthin has superior antioxidant activity compared to other well-known antioxidants3. Because of its unique chemical structure, dietary supplementation of astaxanthin may help protect the central nervous system4, eye5, skin6, joint7, and muscle8tissues against the effects of oxidation and inflammation. Topical application of algal astaxanthin has also been found to reduce the appearance of wrinkles and the size of age spots, and improved skin elasticity and moisture9.Introducing Solix AlgredientsSolix Algredients is a B2B supplier of algae-based, natural ingredients that benefit health-conscious consumers. The company is a recognized leader in algal cultivation and has demonstrated its technology at scale. Solix is applying its algae supply chain experience and expertise to bring Solasta™ Astaxanthin and other natural algal ingredients to market. Solix Algredients is headquartered in Fort Collins, Colorado with additional R&D resources in Russia and China.References1.Food Chemical Codex (2013) 8th ed., pp 89-94. Pharmacopeia (2015) 38th ed., pp 5892-5894.3.Shimidzu, et al. (1996). “Carotenoids as singlet oxygen quenchers in marine organisms.” FisheriesScience. 62(1):134-137.4.Guest & Grant (2012). “Effects of dietary derived antioxidants on the central nervous system.” Int. J. ofNutrition, Pharmacology, Neurological Diseases. 2(3):185-197.5.Kajita et al. (2009). “The effects of a dietary supplement containing astaxanthin on the accommodationfunction of the eye in middle-aged and older people.” Med Consult New Remedies. 46:89-93.6.Suganuma et al. (2010). “Astaxanthin attenuates the UVA-induced up-regulation of matrix-metalloproteinase-1 and skin fibroblast elastase in human dermal fibroblasts.” J. of Dermatological Science. 58(2):136-142.7.Kimble et al. (2013). “Astaxanthin Mediates Inflamma tion Biomarkers Associated with Arthritis inHuman Chondrosarcoma Cells Induced with Interleukin-1β.” Am. J. of Advanced Food Science and Technology. 2:37-51.8.Aoi et al. (2008). “Astaxanthin improves muscle lipid metabolism in exercise via inhibitory effec t ofoxidative CPT 1 modification.” Biochem. Biophys. Res. Commun. 366(4):892-897.9.Tominaga et al. (2012). “Cosmetic benefits of astaxanthin on human subjects.” Acta BiochimicaPolonica. 59(1):43-47.Product SpecificationsAstaxanthin Content (free basis) ≥ 5%, ≥ 10%Monoester ≥ 75%Diester ≥ 20%Free ≤ 5%Appearance Dark Red Viscous OilHeavy Metals ≤ 10 ppmLead – Pb ≤ 1 ppmArsenic – As ≤ 2 ppmCadmium – Cd ≤ 1 ppmMercury – Hg ≤ 1 ppmMicrobiologicalTotal Plate Count ≤ 1000 CFU/gYeast and Mold ≤ 100 CFU/gTotal Coliform ≤ 100 CFU/gPseudomonas aeruginosa Negative CFU/gStaphylococcus aureus Negative CFU/gEscherichia coli Negative CFU/gSalmonella Negative per 25gAsh ≤ 5%Moisture ≤ 1%Aflatoxin≤ 5 ppbPesticides ≤ 10 ppb for eachOther Ingredients:Safflower oil, mixed-tocopherols.Recommended Storage Conditions:Material is sensitive to light, heat, oxygen and moisture. Store in original sealed container at 4-10o C. Product Stability:Retest one year after date of manufacture.Packaging:2.5 Kg, 5 Kg, 10 Kg pails.All information in this brochure has been carefully compiled but no guarantee can be given of its applicability in any given situation because of the wide variation in conditions of use and regulatory requirements in various countries. Nothing in this information should be construed as a recommendation to use our products in violation of any patent or as a warranty (express or implied) of non-infringement of any patent rights. Prospective purchasers are advised to conduct their own tests and studies to determine the fitness of Solix Algredients’ prod ucts for their particular purposes and specific applications.。

Adopted:2 October 2012OECD GUIDELINE FOR THE TESTING OF CHEMICALSFish Short Term Reproduction AssayINTRODUCTION1. The need to develop and validate a fish assay capable of detecting endocrine active substances originates from the concerns that environmental levels of chemicals may cause adverse effects in both humans and wildlife due to the interaction of these chemicals with the endocrine system. In 1998, the OECD initiated a high-priority activity to revise existing guidelines and to develop new guidelines for the screening and testing of potential endocrine disrupters. One element of the activity was to develop a Test Guideline for the screening of substances active on the endocrine system of fish species. The Fish Short Term Reproduction Assay underwent an extensive validation programme consisting of inter-laboratory studies with selected chemicals to demonstrate the relevance and reliability of the assay for the detection of substances that impact reproduction in fish by various mechanisms including endocrine modalities (1, 2, 3, 4, 5). All endpoints of the Test Guideline have been validated on the fathead minnow, and a subset of endpoints have been validated in the Japanese medaka (i.e. vitellogenin and secondary sex characteristics) and the zebrafish (i.e. vitellogenin). The validation work has been peer-reviewed by a panel of experts nominated by the National Coordinators of the Test Guideline Programme (6) in part, and by an independent panel of experts commissioned by the United States Environmental Protection Agency (29). The assay is not designed to identify specific mechanisms of hormonal disruption because the test animals possess an intact hypothalamic-pituitary-gonadal (HPG) axis, which may respond to substances that impact on the HPG axis at different levels.2. This Test Guideline describes an in vivo screening assay where sexually mature male and spawning female fish are held together and exposed to a chemical during a limited part of their life-cycle (21 days). At termination of the 21-day exposure period, two biomarker endpoints are measured in males and females as indicators of endocrine activity of the test chemical; these endpoints are vitellogenin and secondary sexual characteristics. Vitellogenin is measured in fathead minnow, Japanese medaka and zebrafish, whereas secondary sex characteristics are measured in fathead minnow and Japanese medaka. Additionally, quantitative fecundity is monitored daily throughout the test. Gonads are also preserved and histopathology may be evaluated to assess the reproductive fitness of the test animals and to add to the weight of evidence of other endpoints3. This bioassay serves as an in vivo reproductive screening assay and its application should be seen in the context of the “OECD Conceptual Framework for the Testing and Assessment of Endocrine Disrupting Chemicals”. In this Conceptual Framework the Fish Short Term Reproduction Assay is © OECD, (2012)You are free to use this material for personal, non-commercial purposes without seeking prior consent from the OECD, provided the source is duly mentioned. Any commercial use of this material is subject to written permission from the OECD.proposed at Level 3 as an in vivo assay providing data about selected endocrine mechanism(s)/pathway(s) (30).INITIAL CONSIDERATIONS AND LIMITATIONS4. Vitellogenin is normally produced by the liver of female oviparous vertebrates in response to circulating endogenous oestrogen. It is a precursor of egg yolk proteins and, once produced in the liver, travels in the bloodstream to the ovary, where it is taken up and modified by developing eggs. Vitellogenin is almost undetectable in the plasma of immature female and male fish because they lack sufficient circulating oestrogen; however, the liver is capable of synthesizing and secreting vitellogenin in response to exogenous oestrogen stimulation.5. The measurement of vitellogenin serves for the detection of chemicals with various oestrogenic modes of action. The detection of oestrogenic chemicals is possible via the measurement of vitellogenin induction in male fish, and it has been abundantly documented in the scientific peer-reviewed literature (e.g., 7). Vitellogenin induction has also been demonstrated following exposure to aromatizable androgens (8, 9). A reduction in the circulating level of oestrogen in females, for instance through the inhibition of the arom atase converting the endogenous androgen to the natural oestrogen 17β-estradiol, causes a decrease in the vitellogenin level which is used to detect chemicals having aromatase inhibiting properties (10, 11). The biological relevance of the vitellogenin response following oestrogenic/aromatase inhibition is established and has been broadly documented. However, it is possible that production of VTG in females can also be affected by general toxicity and non-endocrine toxic modes of action, e.g. hepatotoxicity.6. Several measurement methods have been successfully developed and standardised for routine use. This is the case of species-specific Enzyme-Linked Immunosorbent Assay (ELISA) methods using immunochemistry for the quantification of vitellogenin produced in small blood or liver samples collected from individual fish (12, 13, 14, 15, 16, 17, 18). Fathead minnow blood, zebrafish blood or head/tail homogenate, and medaka liver are sampled for VTG measurement. In medaka, there is a good correlation between VTG measured from blood and from liver (19). Annex 6 provides the recommended procedures for sample collection for vitellogenin analysis. Kits for the measurement of vitellogenin are widely available; such kits should be based on a validated species-specific ELISA method.7. Secondary sex characteristics in male fish of certain species are externally visible, quantifiable and responsive to circulating levels of endogenous androgens; this is the case for the fathead minnow and the medaka - but not for zebrafish which does not possess quantifiable secondary sex characteristics. Females maintain the capacity to develop male secondary sex characteristics, when they are exposed to androgenic substances in water. Several studies are available in the scientific literature to document this type of response in fathead minnow (20) and medaka (21). A decrease in secondary sex characteristics in males should be interpreted with caution because of low statistical power, and should be based on expert judgement and weight of evidence. There are limitations to the use of zebrafish in this assay, due to the absence of quantifiable secondary sex characteristics responsive to androgenic acting substances.8. In the fathead minnow, the main indicator of exogenous androgenic exposure is the number of nuptial tubercles located on the snout of the female fish. In the medaka, the number of papillary processes constitutes the main marker of exogenous exposure to androgenic compounds in female fish. Annex 5a and Annex 5b indicate the recommended procedures to follow for the evaluation of sex characteristics in fathead minnow and in medaka, respectively.9. The 21-day fish assay includes the evaluation of quantitative egg production and preservation of gonads for optional histopathology examination. Some regulatory authorities may require this additional endpoint for a more complete evaluation of the reproductive fitness of the test animals, or in cases where2© OECD, (2012)vitellogenin and secondary sex characteristics did not respond to the chemical exposure. Although some endpoints may be highly diagnostic (e.g., vitellogenin induction in males and tubercle formation in females), not all endpoints (e.g., fecundity and gonad histopathology) in the assay are intended to unequivocally identify specific cellular mechanisms of action. Rather, the suite of endpoints, collectively, allows inferences to be made with regard to possible endocrine disturbances and thus provide guidance for further testing. Although not endocrine specific, fecundity, due to its demonstrated sensitivity across known endocrine active substances (5), is an important endpoint to include because when it and other endpoints are unaffected one is more confident that a compound is not likely endocrine active. However, when fecundity is affected it will contribute heavily in weight of evidence inferences. Guidance on data interpretation and acceptance of test results is provided further in this Guideline.10. Definitions used in this Test Guideline are given in Annex 1.PRINCIPLE OF THE TEST11. In the assay, male and female fish in a reproductive status are exposed together in test vessels. Their adult and reproductive status enables a clear differentiation of each sex, and thus a sex-related analysis of each endpoint, and ensures their sensitivity towards exogenous chemicals. At test termination, sex is confirmed by macroscopic examination of the gonads following ventral opening of the abdomen with scissors. An overview of the relevant bioassay conditions are provided in Annex 2. The assay is normally initiated with fish sampled from a population that is in spawning condition; senescent animals should not be used. Guidance on the age of fish and on the reproductive status is provided in the section on Selection of fish. The assay is conducted using three chemical exposure concentrations as well as a water control, and a solvent control if necessary. Two vessels or replicates per treatment are used for zebrafish (each vessel containing 5 males and 5 females). Four vessels or replicates per treatment are used for fathead minnow (each vessel containing 2 males and 4 females). This is to accommodate the territorial behaviour of male fathead minnow while maintaining sufficient power of the assay. Four vessels or replicates per treatment are used for medaka (each vessel containing 3 males and 3 females). The exposure is conducted for 21-days and sampling of fish is performed at day 21 of exposure. Quantitative fecundity is monitored daily.12. On sampling at day 21, all animals are killed humanely. Secondary sex characteristics are measured in fathead minnow and medaka (see Annex 5A and Annex 5B); blood samples are collected for determination of vitellogenin in zebrafish and fathead minnow, alternatively head/tail can be collected for the determination of vitellogenin in zebrafish (Annex 6); liver is collected for VTG analysis in medaka (Annex 6); gonads are fixed either in whole or dissected for potential histopathological evaluation (22).TEST ACCEPTANCE CRITERIA13. For the test results to be acceptable the following conditions apply:•the mortality in the water (or solvent) controls should not exceed 10 per cent at the end of the exposure period;•the dissolved oxygen concentration should be at least 60 per cent of the air saturation value (ASV) throughout the exposure period;•the water temperature should not differ by more than ± 1.5 °C between test vessels at any one time during the exposure period and be maintained within a range of 2°C within the temperatureranges specified for the test species (Annex 2);© OECD, (2012)3•evidence should be available to demonstrate that the concentrations of the test substance in solution have been satisfactorily maintained within ±20% of the mean measured values;•evidence that fish are actively spawning in all replicates prior to initiating chemical exposure and in control replicates during the test.DESCRIPTION OF THE METHODApparatus14. Normal laboratory equipment and especially the following:(a) oxygen and pH meters;(b) equipment for determination of water hardness and alkalinity;(c) adequate apparatus for temperature control and preferably continuous monitoring;(d) tanks made of chemically inert material and of a suitable capacity in relation to therecommended loading and stocking density (see Annex 2);(e) spawning substrate for fathead minnow and zebrafish, Annex 4 gives the necessary details.(f) suitably accurate balance (i.e. accurate to ± 0.5mg).Water15. Any water in which the test species shows suitable long-term survival and growth may be used as test water. It should be of constant quality during the period of the test. The pH of the water should be within the range 6.5 to 8.5, but during a given test it should be within a range of ± 0.5 pH units. In order to ensure that the dilution water will not unduly influence the test result (for example by complexion of test substance); samples should be taken at intervals for analysis. Measurements of heavy metals (e.g. Cu, Pb, Zn, Hg, Cd, and Ni), major anions and cations (e.g. Ca, Mg, Na, K, Cl, and SO4), pesticides (e.g. total organophosphorus and total organochlorine pesticides), total organic carbon and suspended solids should be made, for example, every three months where dilution water is known to be relatively constant in quality. If water quality has been demonstrated to be constant over at least one year, determinations can be less frequent and intervals extended (e.g. every six months). Some chemical characteristics of acceptable dilution water are listed in Annex 3.Test solutions16. Test solutions of the chosen concentrations are prepared by dilution of a stock solution. The stock solution should preferably be prepared by simply mixing or agitating the test substance in dilution water by using mechanical means (e.g. stirring or ultrasonication). Saturation columns (solubility columns) can be used for achieving a suitable concentrated stock solution. The use of a solvent carrier is not recommended. However, in case a solvent is necessary, a solvent control should be run in parallel, at the same solvent concentration as the chemical treatments. For difficult to test substances, a solvent may be technically the best solution; the OECD Guidance Document on aquatic toxicity testing of difficult substances and mixtures should be consulted (23). The choice of solvent will be determined by the chemical properties of the substance. The OECD Guidance Document recommends a maximum of 100µl/L, which should be observed. However a recent review (24) highlighted additional concerns when using solvents for endocrine activity testing. Therefore it is recommended that the solvent concentration, if necessary, is minimised wherever technically feasible (dependent on the physical-chemical properties of the test substance).4© OECD, (2012)17. A flow-through test system will be used. Such a system continually dispenses and dilutes a stock solution of the test substance (e.g. metering pump, proportional diluter, saturator system) in order to deliver a series of concentrations to the test chambers. The flow rates of stock solutions and dilution water should be checked at intervals, preferably daily, during the test and should not vary by more than 10% throughout the test. Care should be taken to avoid the use of low-grade plastic tubing or other materials that may contain biologically active substances. When selecting the material for the flow-through system, possible adsorption of the test substance to this material should be considered.Holding of fish18. Test fish should be selected from a laboratory population, preferably from a single stock, which has been acclimated for at least two weeks prior to the test under conditions of water quality and illumination similar to those used in the test. It is important that the loading rate and stocking density (for definitions, see Annex 1) be appropriate for the test species used (see Annex 2).19. Following a 48-hour settling-in period, mortalities are recorded and the following criteria applied:-mortalities of greater than 10% of population in seven days: reject the entire batch;-mortalities of between 5% and 10% of population: acclimation for seven additional days; if more than 5% mortality during second seven days, reject the entire batch;-mortalities of less than 5% of population in seven days: accept the batch.20. Fish should not receive treatment for disease during the acclimation period, in the pre-exposure period, or during the exposure period.Pre-exposure and selection of fish21. The one to two-week pre-exposure period is recommended with animals placed in vessels similar to the actual test. Fish should be fed ad libitum throughout the holding period and during the exposure phase. The exposure phase is started with sexually dimorphic adult fish from a laboratory supply of reproductively mature animals (e.g. with clear secondary sexual characteristics visible as far as fathead minnow and medaka are concerned), and actively spawning. For general guidance only (and not to be considered in isolation from observing the actual reproductive status of a given batch of fish), fathead minnows should be approximately 20 (±2) weeks of age, assuming they have been cultured at 25±2°C throughout their lifespan. Japanese medaka should be approximately 16 (±2) weeks of age, assuming they have been cultured at 25±2°C throughout their lifespan. Zebrafish should be approximately 16 (±2) weeks of age, assuming they have been cultured at 26±2°C throughout their lifespan. Egg production should be assessed daily during the pre-exposure phase. It is recommended that spawning be observed in all replicate tanks prior to inclusion in the exposure phase of the assay. Quantitative guidance on desirable daily egg production cannot be provided at this stage, but it is relatively common to observe average spawns of >10 eggs/female/day for each species. A randomized block design according to egg production output should be used to allocate replicates to the various experimental levels to ensure balanced distribution of replicates. TEST DESIGN22. Three concentrations of the test substance, one control (water) and, if needed, one solvent control are used. The data may be analyzed in order to determine statistically significant differences between treatment and control responses. These analyses will inform whether further longer term testing for© OECD, (2012)5adverse effects (namely, survival, development, growth and reproduction) is required for the chemical, rather than for use in risk assessment (25).23. For zebrafish, on day 21 of the experiment, males and females from each treatment level (5 males and 5 females in each of the two replicates) and from the control(s) are sampled for the measurement of vitellogenin. For medaka, on day 21 of the experiment, males and females from each treatment level (3 males and 3 females in each of the four replicates) and from the control(s) are sampled for the measurement of vitellogenin and secondary sex characteristics. For fathead minnow, on day 21 of exposure, males and females (2 males and 4 females in each of the four replicates) and from the control(s) are sampled for the measurement of vitellogenin and secondary sex characteristics. Quantitative assessment of fecundity is required, and gonadal tissues should be fixed in whole or in situ for potential histopathological evaluation, if required.Selection of test concentrations24. For the purposes of this test, the highest test concentration should be set by the maximum tolerated concentration (MTC) determined from a range finder or from other toxicity data, or 10 mg/L, or the maximum solubility in water, whichever is lowest. The MTC is defined as the highest test concentration of the chemical which results in less than 10% mortality. Using this approach assumes that there are existing empirical acute toxicity data or other toxicity data from which the MTC can be estimated. Estimating the MTC can be inexact and typically requires some professional judgment.25. Three test concentrations, spaced by a constant factor not exceeding 10, and a dilution-water control (and solvent control if necessary) are required. A range of spacing factors between 3.2 and 10 is recommended.PROCEDURESelection and weighing of test fish26. It is important to minimise variation in weight of the fish at the beginning of the assay. Suitable size ranges for the different species recommended for use in this test are given in Annex 2. For the whole batch of fish used in the test, the range in individual weights for male and female fish at the start of the test should be kept, if possible, within ± 20% of the arithmetic mean weight of the same sex. It is recommended to weigh a subsample of the fish stock before the test in order to estimate the mean weight.Conditions of exposureDuration27. The test duration is 21 days, following a pre-exposure period. The recommended pre-exposure period is one to two weeks.Feeding28. Fish should be fed ad libitum with an appropriate food (Annex 2) at a sufficient rate to maintain body condition. Care should be taken to avoid microbial growth and water turbidity. As a general guidance, the daily ration may be divided into two or three equal portions for multiple feeds per day, separated by at least three hours between each feed. A single larger ration is acceptable particularly for weekends. Food should be withheld from the fish for 12 hours prior to sampling/necropsy.6© OECD, (2012)29. Fish food should be evaluated for the presence of contaminants such as organochlorine pesticides, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs). Food with an elevated level of phytoestrogens that would compromise the response of the assay to known oestrogen agonist (e.g. 17beta estradiol) should be avoided.30. Uneaten food and faecal material should be removed from the test vessels at least twice weekly,e.g. by carefully cleaning the bottom of each tank using a siphon.Light and temperature31. The photoperiod and water temperature should be appropriate for the test species (see Annex 2).Frequency of analytical determinations and measurements32. Prior to initiation of the exposure period, proper function of the chemical delivery system should be ensured. All analytical methods needed should be established, including sufficient knowledge on the substance stability in the test system. During the test, the concentrations of the test substance are determined at regular intervals, as follows: the flow rates of diluent and toxicant stock solution should be checked preferably daily but as a minimum twice per week, and should not vary by more than 10% throughout the test. It is recommended that the actual test chemical concentrations be measured in all vessels at the start of the test and at weekly intervals thereafter.33. It is recommended that results be based on measured concentrations. However, if concentration of the test substance in solution has been satisfactorily maintained within ±20% of the nominal concentration throughout the test, then the results can either be based on nominal or measured values.34. Samples may need to be filtered (e.g. using a 0.45 µm pore size) or centrifuged. If needed, then centrifugation is the recommended procedure. However, if the test material does not adsorb to filters, filtration may also be acceptable.35. During the test, dissolved oxygen, temperature, and pH should be measured in all test vessels at least once per week. Total hardness and alkalinity should be measured in the controls and one vessel at the highest concentration at least once per week. Temperature should preferably be monitored continuously in at least one test vessel.Observations36. A number of general (e.g. survival) and biological responses (e.g. vitellogenin levels) are assessed over the course of the assay or at termination of the assay. The daily quantitative monitoring of fecundity is required. Measurement and evaluation of these endpoints and their utility are described below.Survival37. Fish should be examined daily during the test period and any mortality should be recorded and the dead fish removed as soon as possible. Dead fish should not be replaced in either the control or treatment vessels. Sex of fish that die during the test should be determined by macroscopic evaluation of the gonads.© OECD, (2012)7Behaviour and appearance38. Any abnormal behaviour (relative to controls) should be noted; this might include signs of general toxicity including hyperventilation, uncoordinated swimming, loss of equilibrium, and atypical quiescence or feeding. Additionally external abnormalities (such as haemorrhage, discoloration) should be noted. Such signs of toxicity should be considered carefully during data interpretation since they may indicate concentrations at which biomarkers of endocrine activity are not reliable. Such behavioural observations may also provide useful qualitative information to inform potential future fish testing requirements. For example, territorial aggressiveness in norm al m ales or m asculinised fem ales has been observed in fathead m innows under androgenic exposure; in zebrafish, the characteristic m ating and spawning behaviour after the dawn onset of light is reduced or hindered by oestrogenic or anti-androgenic exposure.39. Because some aspects of appearance (primarily colour) can change quickly with handling, it is important that qualitative observations be made prior to removal of animals from the test system. Experience to date with fathead minnows suggests that some endocrine active chemicals may initially induce changes in the following external characteristics: body colour (light or dark), coloration patterns (presence of vertical bands), and body shape (head and pectoral region). Therefore observations of physical appearance of the fish should be made over the course of the test, and at conclusion of the study Fecundity40. Daily quantitative observations of spawning should be recorded on a replicate basis. Egg production should be recorded as the number of eggs/surviving female/day on a replicate basis. Eggs will be removed daily from the test chambers. Spawning substrates should be placed in the test chamber for the fathead minnow and zebrafish to enable fish to spawn in normal conditions. Annex 4 gives further details of recommended spawning substrates for zebrafish (Annex 4A) and fathead minnow (Annex 4B). It is not considered necessary to provide spawning substrate for medaka.Humane killing of fish41. At day 21, i.e. at termination of the exposure, the fish should be euthanized with appropriate amounts of Tricaine (Tricaine methane sulfonate, Metacain, MS-222 (CAS.886-86-2), 100-500 mg/L buffered with 300 mg/L NaHCO3(sodium bicarbonate, CAS.144-55-8) to reduce mucous membrane irritation; blood or tissue is then sampled for vitellogenin determination, as explained in the vitellogenin section.Observation of secondary sex characteristics42. Some endocrine active chemicals may induce changes in specialized secondary sex characteristics (number of nuptial tubercles in male fathead minnow, papillary processes in male medaka). Notably, chemicals with certain modes of action may cause abnormal occurrence of secondary sex characteristic in animals of the opposite sex; for example, androgen receptor agonists, such as trenbolone, methyltestosterone and dihydrotestosterone, can cause female fathead minnows to develop pronounced nuptial tubercles or female medaka to develop papillary processes (11, 20, 21). It also has been reported that oestrogen receptor agonists can decrease nuptial tubercle numbers and size of the dorsal nape pad in adult males of fathead minnow (26, 27). Such gross morphological observations may provide useful qualitative and quantitative information to inform potential future fish testing requirements. The number and size of nuptial tubercles in fathead minnow and papillary processes in medaka can be quantified directly or more practically in preserved specimens. Recommended procedures for the evaluation of8© OECD, (2012)。

Anti-inflammatory effect of fucoxanthin derivatives isolated from Sargassum siliquastrum in lipopolysaccharide-stimulated RAW 264.7macrophageSoo-Jin Heo a ,Weon-Jong Yoon b ,Kil-Nam Kim c ,Chulhong Oh a ,Young-Ung Choi a ,Kon-Tak Yoon a ,Do-Hyung Kang a ,Zhong-Ji Qian d ,Il-Whan Choi e ,Won-Kyo Jung d ,⇑aGlobal Bioresources Research Center,Korea Institute of Ocean Science &Technology,Ansan 426-744,Republic of KoreabJeju Biodiversity Research Institute (JBRI)and Jeju Hi-Tech Industry Development Institute (HiDI),Jeju 697-943,Republic of Korea cMarine Bio Research Team,Korea Basic Science Institute (KBSI),Jeju 690–140,Republic of Korea dDepartment of Marine Life Science,Chosun University,Gwangju 501–759,Republic of Korea eDepartment of Microbiology,College of Medicine and Advanced Research Center for Multiple Myeloma,Inje University,Busan 614–735,Republic of Koreaa r t i c l e i n f o Article history:Received 21December 2011Accepted 15June 2012Available online 23June 2012Keywords:Anti-inflammationFucoxanthin derivatives Sargassum siliquastrum Lipopolysaccharide Macrophagea b s t r a c tIn this study,the anti-inflammatory effect of fucoxanthin (FX)derivatives,which was isolated from Sargassum siliquastrum were evaluated by examining their inhibitory effects on pro-inflammatory medi-ators in lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7cells.The FX derivatives were isolated from activity-guided chloroform fraction using inhibition of nitric oxide (NO)production and identified as 90-cis -(60R )fucoxnathin (FXA),and 13-cis and 130-cis -(60R )fucoxanthin complex (FXB)on the basis of a comparison of NMR spectroscopic data.Both FXA and FXB significantly inhibited the NO production and showed slightly reduce the PGE2production.However,FXB exhibited cytotoxicity at the whole tested concentration,therefore,the results of FXA was only illustrate for further experiments.FXA induced dose-dependent reduction in the inducible nitric oxide synthase (iNOS)and cyclooxygenase 2(COX-2)proteins as well as mRNA expression.In addition,FXA reduced the LPS-stimulated production and mRNA expressions of TNF-a and IL-6in a dose-dependent manner whereas IL-1b production do not inhibit by addition of FXA.Taken together,these findings indicate that the anti-inflammatory properties of FXA may be due to the inhibition of iNOS/NO pathway which associated with the attenuation of TNF-a and IL-6formation.Thus FXA may provide a potential therapeutic approach for inflammation related diseases.Ó2012Elsevier Ltd.All rights reserved.1.IntroductionInflammation is a physiological response of a body to stimuli,including infections and tissue injury,and protects a body from these inflammatory stimuli (Dung et al.,2009).Macrophage plays critical roles in immune reaction,allergy,and inflammation.These cells induce inflammatory reaction,and initiate and maintain spe-cific immune responses by releasing different types of cytokines (Lee et al.,2011;Poltorak et al.,1998).Macrophage activation by lipopolysaccharides (LPS),which are derived from gram-negative bacteria cell walls,results in the release of several inflammatory mediators including nitric oxide (NO),cyclooxygenase (COX)-2,interleukin (IL)-6,IL-1b ,and tumor necrosis factor (TNF)-a (Kanno et al.,2006).Over-expression of the inflammatory mediators in macrophage is involved in many inflammation related diseases,such as atherosclerosis,rheumatoid arthritis,chronic obstructive pulmonary disease,and autoimmune diabetes (Coker and Laurent,1998;Kern,2007;Schroder et al.,2006).Thus,inhibition of inflammatory mediators produced by macrophages is believed to be crucial for managing inflammatory diseases.Many investigators have,therefore,focused either on identifying anti-inflammatory agent from natural resources or on developing synthetic anti-inflammatory compounds (Kazłowska et al.,2010;Michelini et al.,2008;Mueller et al.,2010;Paulino et al.,2009;Prawan et al.,2009;Van et al.,2009).Carotenoids are natural pigments containing more than 600members,which synthesized by many microorganisms and plants,so animals have to obtain them from food resources (Cardozo et al.,2007;Quirós and Costa,2006).The carotenoids have recently at-tracted popular interest not only as a source of pigmentation but also for their beneficial effects on human health by functioning as antioxidant,which include a possible role in cancer preventation and enhancing immune responses (Kim et al.,2008).Among the carotenoids,fucoxanthin (FX)is one of the major carotenoid in brown algae,which has an unique structure featuring including an unusual allenic bond,conjugated carbonyl,epoxide,and acetyl group within its molecule.Many of the biological functions of FX have been previously characterized,including antioxidant,anti-obesity,antitumor,and UV-preventative activities (Heo and Jeon,2009;Kim et al.,2010a;Maeda et al.,2005;Yan et al.,1999).More recently,in a previous study we isolated FX from brown algae and0278-6915/$-see front matter Ó2012Elsevier Ltd.All rights reserved./10.1016/j.fct.2012.06.025Corresponding author.Tel.:+82622306657;fax:+82622306557.E-mail address:wkjung@chosun.ac.kr (W.-K.Jung).evaluated its potential anti-inflammatory activity(Heo et al.,2010; Kim et al.,2010b).However,the anti-inflammatory effects of FX derivatives have not yet been reported.Accordingly,the present study isolated the FX derivatives from Sargassum siliquastrum and their anti-inflammatory effect in lipopolysaccharide(LPS)-stimu-lated RAW264.7cells were investigated.2.Materials and methods2.1.MaterialsThe brown alga,Sargassum siliquastrum,was collected along the coast of Jeju Is-land,Korea,between October2009and March2010.The samples were washed three times with tap water to remove the salt,epiphytes,and sand attached to the surface,then carefully rinsed with fresh water,and maintained in a medical refrigerator atÀ20°C.Then,the frozen samples were lyophilized and homogenized with a grinder prior to extraction.2.2.Extraction and isolationThe powdered S.siliquastrum was extracted three times with80%aqueous methanol,and was evaporated under vacuum at40°C.The methanol extract was dissolved in distilled water and partitioned with hexane,chloroform ethyl acetate, and butanol.Since the chloroform fraction exhibited higher nitric oxide(NO)pro-duction inhibitory effects than that of other fractions,the chloroform fraction was fractionated by silica column chromatography with stepwise elution of chloro-form–methanol mixture(100:1?1:1)to separate active fractions in chloroform fraction.A combined active fraction was further subjected to a Sephadex LH-20 column saturated with100%methanol,and then purified by reversed-phase high performance liquid chromatography(HPLC)using a Waters HPLC system(Alliance 2690,NY,USA)equipped with a Waters996photodiode array detector and C18 column(J’sphere ODS-H80,150Â20mm,4l m,YMC Co.,Kyoto,Japan)by stepwise elution with methanol–water gradient(UV range:440nm,flow rate:0.8ml/min). Finally,the purified compounds were identified by comparing their1H and13C NMR data with literature(Haugan and Liaaen-Jensen,1994;Heo et al.,2010).The purity of compounds were>97%,based on the peak area of all components absorbed at each specific wavelength in HPLC analysis.The compounds were dissolved in dimethylsulfoxide(DMSO)and employed in experiments in which thefinal concen-tration of DMSO in culture medium was adjusted to<0.01%.2.3.Cell cultureThe murine macrophage cell line RAW264.7was purchased from the Korean Cell Line Bank(KCLB;Seoul,KOREA).RAW264.7cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM;GIBCO Inc.,NY,USA)supplemented with 100U/ml of penicillin,100l g/ml of streptomycin and10%fetal bovine serum (FBS;GIBCO Inc.,NY,USA).The cells were incubated in an atmosphere of5% CO2at37°C and were sub-cultured every3days.2.4.Determination of NO productionAfter pre-incubation of RAW264.7cells(1.5Â105cells/ml)with LPS(1l g/ml) plus samples at37°C for24h,the quantity of nitrite accumulated in the culture medium was measured as an indicator of NO production(Lee et al.,2007).Briefly, a100l l of cell culture medium was mixed with100l l of Griess reagent(1%sulfa-nilamide and0.1%naphthylethylenediamine dihydrochloride in2.5%phosphoric acid),the mixture was incubated at room temperature for10min,and the absor-bance at540nm was measured in a microplate reader(ThermoMax,CA,USA).Fresh culture medium was used as a blank in every experiment.ctic dehydrogenase(LDH)cytotoxicity assayRAW264.7cells(1.5Â105cells/ml)plated in96well plates were pre-incu-bated and then treated with LPS(1l g/ml)plus samples at37°C for24h.The med-ium was carefully removed from each well,and the LDH activity in the medium was determined using an LDH cytotoxicity detection kit(Promega,Madison,WI,USA). Briefly,a100l l of reaction mixture was added to each well,and the reaction was incubated for30min at room temperature in the dark.The absorbance of each well was measured at490nm using a microplate reader.2.6.Determination of prostaglandin E2(PGE2)productionSamples were diluted with DMEM before treatment.Cells were treated with LPS (1l g/ml)to allow cytokine production for24h.The PGE2concentration in the cul-ture medium was quantified using a competitive enzyme immunoassay kit(R&D Systems,Minneapolis,MN,USA)according to the manufacturer’s instructions. The production of PGE2was measured relative to that of control value.2.7.Measurement of pro-inflammatory cytokines(TNF-a,IL-1b,and IL-6)productionSamples solubilized with DMSO were diluted with DMEM before treatment.The inhibitory effect of samples on the pro-inflammatory cytokines(TNF-a,IL-1b,and IL-6)production from LPS(1l g/ml)treated RAW264.7cells was determined as de-scribed by Cho et al.(2000).Supernatants were used for pro-inflammatory cyto-kines assay using mouse ELISA kit(R&D Systems Inc.,MN,USA).2.8.RNA Isolation and RT-PCR analysisTotal RNA from LPS(1l g/ml)-treated RAW264.7cells was prepared with Tri-Reagent(MRC,Cincinnati,OH,USA),according to the manufacturers protocol. RNA was stored atÀ70°C until used.The reverse transcription of1l g RNA was carried out with M-MuLV reverse transcriptase(Promega,WI,USA),oligo dT-18 primer,deoxyribonucleotide triphosphates(dNTP,0.5l M)and1U RNase inhibitor. After this reaction cocktail was incubated at70°C for5min,25°C for5min,and 37°C for60min in series,M-MuLV reverse transcriptase was inactivated by heating at70°C for10min.Polymerase chain reaction(PCR)was performed in reaction buf-fer[cDNA, 1.25U Taq DNA polymerase(Promega,WI,USA),30-and50-primer (50l M each)and200mM dNTP in200mM Tris–HCl buffer(pH8.4)containing 500mM KCl and1–4mM MgCl2].The PCR was performed in a DNA gene cycler (Bio-Rad,HC,USA)with amplification by30cycles of94°C for45s(denaturing), 60–65°C for45s(annealing)and72°C for1min(primer extension).The PCR products were electrophoresed in1.2%agarose gels and stained with ethidium bromide.2.9.ImmunoblottingRAW264.7cells(1.0Â106cells/ml)were treated with LPS(1l g/ml)plus samples for24h,and cellular proteins were extracted from the cells.Protein concentrations were determined using a Bio-Rad protein assay kit(Bio-Rad,CA, USA)with bovine serum albumin(BSA)as a standard.Cell lysates(30–50l g) were electrophoresed in SDS–polyacrylamide gels(8–12%),and the separated proteins were transferred to PVDF membranes(Bio-Rad)for2h.The membranes were pre-incubated with blocking solution(5%skim milk in Tris buffered saline containing Tween-20)at room temperature for2h and then incubated with anti-mouse iNOS(1:1,000;Calbiochem,La Jolla,CA,USA)and anti-mouse COX-2 (1:1,000;BD Biosciences Pharmingen,San Jose,CA,USA)for2h at room temperature.After washing,the blots were incubated with horseradish peroxidase conjugated goat anti-mouse IgG secondary antibody(1:5,000;Amersham Pharmacia Biotech,Little Chalfont,UK)for30min.The bands were visualized on X-rayfilm using ECL detection reagent(Amersham Biosciences,Piscataway, NJ,USA).2.10.Statistical AnalysisAll the measurements were made in triplicate and all values were represented as means±standard error.The results were subjected to an analysis of the variance (ANOVA)using the Tukey test to analyze the difference.A value of p<0.05was con-sidered to indicate statistical significance.3.Results3.1.Inhibitory effect of NO production of S.siliquastrum extracts and cytotoxicityIn an effort to express the potential anti-inflammatory effect of S.siliquastrum in LPS-induced RAW264.7cells,we investigated inhibitory effect of80%methanol extracts of S.siliquastrum on NO production as well as its partitioned fraction with hexane, chloroform,ethyl acetate,and butanol,to detect bioactive com-pounds(Fig.1).Among those tested samples,chloroform fraction showed the highest level of inhibitory effect(87.5%)on NO produc-tion than that of other tested samples.Hexane fraction also showed higher inhibitory effect(66.3%)on NO production,whereas the other samples evidenced less than25%inhibitory activities. The cytotoxic effects of S.siliquastrum were assessed in the pres-ence or absence of LPS via an LDH assay.As shown in the line graph of Fig.1,only hexane fraction affect cell viability around15.3%, however,other tested samples did not influence the cytotoxicity of RAW264.7cells.Thus,the chloroform fraction was selected for additional experiments,owing to its higher inhibitory effect of NO production and cell viability.S.-J.Heo et al./Food and Chemical Toxicology50(2012)3336–334233373.2.Isolation of active compounds from S.siliquastrumThe chloroform fraction was subjected to silica gel and Sepha-dex LH-20column chromatography,due to its prominent NO inhibitory effect.Finally,the active compounds(Fig.2)of this frac-tion were isolated via HPLC and were identified as all-trans-(60R) fucoxanthin(FX),and its cis isomers such as90-cis-(60R)fucoxna-thin(FXA),and13-cis and130-cis-(60R)fucoxanthin complex (FXB)on the basis of a comparison of NMR spectroscopic data(data not shown)with previous literature(Haugan and Liaaen-Jensen, 1994;Heo and Jeon,2009).Their quantitative composition rate ob-tained as FX:FXA:FXB=82:8:10.However,in case of FX,we already demonstrate its anti-inflammatory activity in our previous study (Heo et al.,2010;Kim et al.,2010b).Thus,FXA and FXB were then employed in additional experiments.3.3.Effects of FX derivatives on LPS-induced NO production and cytotoxicityTo assess the effect of FX derivatives on LPS-induced NO pro-duction in RAW264.7cells,cells were treated with LPS(1l g/ml) for24h after treatment in the presence or absence of various con-centrations of FXA and FXB(15,30,and60l M)for1h.And then, the levels of NO production were measured using the Griess reaction.NO was produced by the treatment of LPS,which was inhibited as22.2,43.6,and65.3%by the addition of FXA at15, 30,and60l M,respectively,whereas LPS-induced NO production were decreased in a FXB dose-dependently(Fig.3,bar graph). The cytotoxic effects of FXA and FXB were assessed in the presence or absence of LPS via an LDH assay.FXA did not influence the cyto-toxicity of RAW264.7cells at the employed concentrations(15,30, and60l M)to inhibit NO.Although60l M of FXA showed4.3%of cytotoxic effect in RAW264.7cells it was not that much affect levels in this experiment.However,FXB showed cytotoxic effect at the whole tested concentrations(Fig.3,line graph).Thus,the inhibitory effects of FXA were deemed not to be attributable to cytotoxic effects.3.4.Effect of FX derivatives on LPS-induced PGE2productionThe inhibitory effects of FX derivatives on PGE2production in LPS-induced cells were measured via an ELISA assay.FXA inhibited LPS-induced PGE2production in a concentration-dependent man-ner as2.6,14.3,and25.6%at15,30,and60l M,respectively. Although FXA had an inhibitory effect on PGE2production,the ef-fect was not as strong as that exhibited in the inhibition of NO pro-duction.However,FXB did not affect to the inhibition of PGE2 production(Fig.4).3.5.Effects of FX derivatives on LPS-induced iNOS and COX-2protein and mRNA expressionsTo elucidate the mechanism involved in the inhibitions of NO and PGE2generation by FX derivatives in LPS-induced RAW264.7 cells,we further studied the effect of FX derivatives on iNOS and COX-2protein and gene expression by Western Blot and RT-PCR analysis(Fig.5).In unstimulated RAW264.7cells,the protein and mRNA expressions of iNOS and COX-2were undetectable. However,in response to LPS the expression of iNOS was markedly increased,and FXA significantly inhibited iNOS protein in a dose-dependent manner while FXA slightly inhibit the COX-2protein (Fig.5A).Under the same condition,the levels of iNOS and COX-2mRNA expression were correlated with their protein levels (Fig.5B).A similar patterns were observed when the effect of FXB on LPS-induced iNOS and COX-2protein and mRNA expression (data not shown).However,FXB had cytotoxicity,therefore,the levels of protein and mRNA expression are not that much cleared. In general,these results indicate that the reduced expressions of iNOS and COX-2by FXA were responsible for the inhibition of NO and PGE2production.3338S.-J.Heo et al./Food and Chemical Toxicology50(2012)3336–33423.6.Effects of FX derivatives on LPS-induced TNF-a,IL-6,and IL-1bSecretion of pro-inflammatory cytokines such as TNF-a,IL-6, and IL-1b was measured in culture supernatants form RAW264.7 cells that had been stimulated with LPS(1l g/ml),either alone, or in combination with different concentrations of FX derivatives for24h,and the cytokine levels were measured by ELISA(Fig.6). The treatment of RAW264.7cells with LPS alone resulted in signif-icant increases in cytokine production relative to the control group. However,pretreatment of RAW264.7cells with FXA significantly reduced TNF-a,IL-6,and IL-1b production relative to the LPS group,in a dose-dependent manner(Fig.6A-C).Especially,FXAS.-J.Heo et al./Food and Chemical Toxicology50(2012)3336–33423339was significantly inhibited the TNF-a,and IL-6production in LPS-induced macrophages,and the production rate was recorded as57.94and62.1%at60l M,respectively.Consistently,RT-PCR was performed to determine whether FXA reduce the expression of those cytokines at the mRNA levels.All the mRNA levels were in-creased by treatment of LPS,and these increases were significantly decreased in a concentration dependent manner by treatment with FXA(Fig.6D).A similar patterns were observed when the effect of FXB on LPS-induced pro-inflammatory cytokines and mRNA expression(data not shown).However,the production of pro-inflammatory cytokines and mRNA expression by FXB was cannot detect exact result due to its cytotoxicity.4.DiscussionMarine algae have been used as a traditional food and medicinal additives in oriental countries.Several studies have done with marine algae tofind their potential bioactivities and some of active compounds have been isolated as chromenes,chlorophylls,phloro-tannins,and carotenoids(Ferruzzi and Blakeslee,2007;Heo et al., 2009;Jang et al.,2005;Kim et al.,2010a).For centuries,dietary and medicinal phytochemicals have been used as anti-inflammatory remedies,but identifying their active compounds has only recently been initiated(Sarkar et al.,2008).Previous studies have revealed that LPS-stimulated inflammatory mediator production by macro-phages can be attenuated by carotenoid(Heo et al.,2010;Kim et al.,2010b).FX is one of the most common carotenoid isolated from brown algae and has a variety of pharmacological activities (Heo and Jeon,2009;Heo et al.,2010;Kim et al.,2010a,b;Maeda et al.,2005;Yan et al.,1999).Nevertheless,there is no information with respect to the molecular mechanisms underlying the anti-inflammatory effect of FX derivatives.In the present study,the chloroform fraction of S.siliquastrum was found to have potent anti-inflammatory effect as demonstrated by inhibition of NO pro-duction(Fig.1)and the activity-guided fractionation led to the iso-lation of FX and its derivatives(Fig.2).The structures of these compounds were identified spectroscopically and confirmed by comparing these data with previous literature(Haugan and Liaaen-Jensen,1994;Heo and Jeon,2009).In case of FXB,it was difficult to separate single form of13-cis-(60R)and130-cis-(60R) fucoxanthin,respectively.Thus,the complex of13-cis-(60R)and 130-cis-(60R)fucoxanthin was named as FXB.FX demonstrated its anti-inflammatory effect in previous reports(Heo et al.,2010;130 kDaof LPS-induced iNOS and COX-2protein(A)3340S.-J.Heo et al./Food and Chemical Toxicology50(2012)3336–3342Kim et al.,2010b),therefore,FXA and FXB were used to determine their anti-inflammatory activity using LPS-stimulated macrophage, a standard model for studying anti-inflammatory drugs.Macrophages produce NO and pro-inflammatory cytokines in response to bacterial LPS and the NO production can be controlled by selective pharmacological inhibition of distinct nitric oxide syn-thase isoforms(Southan and Szabo,1996).iNOS is one of three key enzymes generating NO from arginine.Basically,NO plays a pivotal role in many body functions,however,its over production espe-cially in macrophages can lead to cytotoxicity,inflammation,and autoimmune disorders(Liu and Hotchkiss,1995;Sarkar et al., 2008).Therefore,NO inhibitors are essential for prevention of inflammatory diseases.PGE2also has been implicated as important mediator in the processes of inflammation which produced by COX-2(Van et al.,2009).Thus,inhibition of PGE2and COX-2can provide an effective strategy for inhibiting the inflammation.In the present study,we compared the inhibitory effects of FXA and FXB on the LPS-stimulated pro-inflammatory molecules,including NO and PGE2,and found that FXA is a more potent inhibitor than FXB(Fig.3and4).To further investigate the mechanism underly-ing these inhibitions by FXA,the expression levels of iNOS and COX-2proteins and its mRNA levels were examined by Western Blot and RT-PCR,respectively.Here,we found that FXA down-reg-ulated iNOS and COX-2at both the protein and mRNA levels in a parallel concentration-dependent manner(Fig.5),which suggest that inhibitions of the release of NO and PGE2may be attributed to the expressional inhibitions of iNOS and COX-2followed by their suppression at the transcriptional level.In addition,these inhibi-tions were not due to the cytotoxicity of FXA as determined by LDH assay(Fig.2).However,treatment with FXB on RAW264.7 cells exhibited cytotoxicity and which affect expression of inflam-matory proteins,mRNA,and related cytokines(data not shown).The pro-inflammatory cytokines are known to play key roles in the induction or aggravation of inflammation in macrophages.This inflammation is activated upon appropriate extracellular stimula-tion by pathogens such as bacterial components including LPS through the Toll-like receptors(Kim et al.,2007;Moynagh, 2005).Since inflammation,particularly chronic inflammation,is a significant factor in disease processes,natural anti-inflammatory compounds may offer certain health benefits in the treatment of inflammatory diseases(Bailey et al.,1990).In the present study, we found that FXA reduced the LPS-stimulated production and mRNA expressions of TNF-a and IL-6in a dose-dependent manner (Fig.6).These two cytokines are known to act as pro-inflammatory mediators in vitro and in vivo.TNF-a is a potent activator of mac-rophages and can stimulate the production or expression of IL-6, IL-1b,PGE2,collagenase,and adhesion molecules.It is up-regulated in many inflammatory diseases including rheumatoid arthritis, septic shock,psoriasis,and cytotoxicity(Aggarwal and Natarajan, 1996;Brennan and McInners,2008).IL-6is a multifunctional cyto-kine with pro-/anti-inflammatory properties and it plays a major role in immune and inflammatory responses.In addition,over-expression of IL-6is involved in pathological conditions such as rheumatoid arthritis and fever(Van et al.,2009).Our results exhib-ited that FXA significantly inhibited the production of pro-inflam-matory cytokine TNF-a and IL-6in RAW264.7macrophages stimulated by LPS but showed less effect on IL-1b formation,sug-gesting that the inhibition of iNOS/NO pathway by FXA may be associated with the attenuation of TNF-a and IL-6formation and less mediated by enhancing IL-1b release.It has been reported that the trans isomers of carotenoids are more common in natural sources and are more stable as compared to their cis isomer(Nakazawa et al.,2009).Therefore,very little known about the biological characteristics of cis isomers and itsS.-J.Heo et al./Food and Chemical Toxicology50(2012)3336–33423341role in human health than that of trans isomers.Boileau et al. (1999)reported that lycopens showed different biological reactiv-ity by its cis and trans isomers.Nakazawa et al.(2009)also re-ported about stereoisomers of FX and their comparative cancer cell proliferation.Several reports showed that FX can convert to different isomer forms by which some kinds of isomerization fac-tors including light,thermal energy,chemical reactions and inter-action with biological molecules such as proteins(Bernhard et al., 1974;Englert et al.,1990;Haugan and Liaaen-Jensen,1994).FX showed potential anti-inflammatory effect and has non-cytotoxic-ity in our previous report(Heo et al.,2010).In the present study, we evaluate anti-inflammatory effect of FX derivatives as well as cytotoxicity.FX shows more effective anti-inflammatory potential then that of FXA especially in inhibition of NO and TNF-a produc-tion as well as exhibited non-cytotoxicity in tested concentrations. Taken together,we found that FX and its derivatives have anti-inflammatory activities,however,their cis isomer such as FXB induce cytotoxicity.Therefore,further studies are needed to demonstrate the mechanisms as to how FX and its derivatives show different levels of anti-inflammatory effect and how can control the cytotoxicity.In conclusion,the present study demonstrates that FXA is a po-tent inhibitor of NO,TNF-a and IL-6production at the transcrip-tional level in LPS-stimulated RAW264.7cells.The mechanism of inhibition of NO production seems to be due to down-regulation of iNOS protein and mRNA expression which might be associated with the attenuation of TNF-a and IL-6formation.Although the ex-act mechanisms regulating the anti-inflammatory activity of FXA are not yet fully known,however,thesefindings suggest that FXA appears to have the potential to prevent inflammatory dis-eases and may act as a modulator of macrophage activation.Conflict of InterestThe authors declare that there are no conflicts of interest. 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收稿日期：2013-08-23；修回日期：2013-11-18基金项目：吉林省科技厅科技引导计划国际科技合作项目（0130413037GH ）；吉林省科技发展计划项目（20110224）；吉林农业大学博士启动基金项目（201221）；科技部农业科技成果转化项目（2012GB2B100106）作者简介：崔焕忠（1973－），男，副教授，硕士，研究方向为动物营养免疫调控，huanzhongcui@163．com．通信作者：郑鑫（1965－），女，教授，博士，研究方向为动物营养免疫调控，zhengxinjilin@126．com．叶黄素生物学功能的研究进展崔焕忠，张辉，马思慧，杨欢，兰海楠，郑鑫（吉林农业大学动物科技学院，长春130118）中图分类号：S816．7文献标识码：A文章编号：1004-7034（2014）07－0050－03关键词：萜类化合物;叶黄素;类胡萝卜素;抗氧化活力;免疫调节;生物学功能摘要：叶黄素是自然界广泛存在的类胡萝卜素，它的抗氧化特性及免疫调节功能日益受到人们关注。

越来越多的研究表明，叶黄素可提高机体的细胞免疫和体液免疫，通过清除自由基、猝灭单线态氧和降低光化学敏感剂等作用发挥其抗氧化功能，在保护视觉、预防心血管疾病、糖尿病、肿瘤和癌症发生等方面同样具有较强功能，文章对叶黄素的各种生物学功能进行了简要综述。

叶黄素是类胡萝卜素中叶黄素类的一种，又名植物黄体素，为一种萜类化合物，在自然界中广泛存在，是构成玉米、蔬菜、水果、花卉等植物色素的主要组分。

人类首次在胡萝卜中发现叶黄素是19世纪初，目前发现许多植物中都含有叶黄素，其中万寿菊中含量十分丰富。

叶黄素在人与动物体内不能合成，只能从食物获得。

叶黄素分子有10个共轭双键，使其具有较强的抑制自由基能力［1］。

近年来，随着研究的不断深入，发现叶黄素具有多种生物学功能，如在提高机体抗氧化能力、增强免疫功能、保护视觉、减少癌症的发生和发展、降低心血管疾病发病率等方面发挥着独特的功能［2］。

Combination of fucoxanthin and conjugated linoleic acid attenuates bodyweight gain and improves lipid metabolism in high-fat diet-induced obese ratsXiaojie Hu a ,Yanmei Li b ,Chunhua Li b ,Yuanqing Fu a ,Fang Cai a ,Qi Chen a ,Duo Li a ,⇑a Department of Food Science and Nutrition,Zhejiang University,Hangzhou,China bGingko Research Institute,Beijing,Chinaa r t i c l e i n f o Article history:Received 10November 2011and in revised form 28December 2011Available online 26January 2012Keywords:AntiobesityConjugated linoleic acid FucoxanthinLipid metabolism White adipose tissue mRNA expressiona b s t r a c tThe present study investigated the effects of combined fucoxanthin (Fc)and conjugated linoleic acid (CLA)on high-fat diet-induced obese rats.Thirty five rats were divided into four groups,fed a high-fat diet (Control,15%fat,wt/wt),supplemented with low Fc (FCL,0.083mg/kg/bw),high Fc (FCH,0.167mg/kg/bw)and FCL (0.083mg/kg/bw)plus CLA (0.15g/kg/bw)(FCL +CLA)for 52d.Body weight and white adipose tissue (WAT)weight were significantly suppressed in FCL +CLA group than those in control group.WAT weight was also markedly attenuated in FCL and FCH groups.Accumulation of hepa-tic lipid droplets and the perirenal adipocyte size of FCL,FCH and FCL +CLA groups were diminished com-pared to control group.Serum total cholesterol level in FCH group,triacylglycerol and leptin levels in FCL,FCH and FCL +CLA groups,and glucose concentration in FCH and FCL +CLA groups were significantly decreased than those in control group.The mRNA expression of adiponectin,adipose triacylglycerol lipase,carnitine palmitoyltransferase 1A was remarkably up-regulated in FCL,FCH and FCL +CLA groups.These results suggest that Fc and FCL +CLA could reduce serum levels of triacylglycerol,glucose and lep-tin,and FCL +CLA could exert anti-obesity effects by regulating mRNA expression of enzymes related to lipid metabolism in WAT of diet-induced obesity rats.Ó2012Elsevier Inc.All rights reserved.IntroductionObesity,recognized as a major public health problem world-wide,is closely related to many chronic diseases in both humans and animals such as diabetes mellitus,cardiovascular disease,digestive disease,respiratory disease and various cancers [1–3].The excessive fat accumulation observed in obesity leads to the dys-regulation of adipocytokine production in white adipose tissue (WAT).Adipose tissue,the energy reserve organ,plays an important role in regulating energy metabolism in organisms [4].Adipocyte dysfunction is strongly associated with the development of obesity.It is accepted that specific regulation of gene expression in adipo-cytes is one of the most important targets for the intervention ofobesity.In addition,leptin and adiponectin,which is known to play an important role in maintaining insulin sensitivity and glucose homeostasis,is reduced in obese rats.Fucoxanthin (Fc),an edible seaweed carotenoid that is charac-terized by a unique structure including an allenic bond and 5,6-monoepoxide,differs from that of common carotenoids such as b -carotene and lycopene [5].Fc is mainly present in marine plants such as Undaria pinnatifida ,Sargassum fulvellum ,Laminaria japonica and Hizikia fusiformis [6].mRNA of tumor necrosis factor-alpha (TNF-a )and monocyte chemoattractant protein-1(MCP-1)is overexpressed in WAT of diabetic/obese KK-A y mice [7],the latter of which also induces the over-production of inflam-matory adipocytokines [8,9]and inhibits insulin-dependent glucose uptake,thus leading to insulin resistance [10].TNF-a and IL-6are important pro-inflammatory adipocytokines that influence insulin sensitivity [11,12].In addition,the expression of adiponec-tin,one of adipocytokines,correlates with insulin sensitivity.Remarkably,insulin resistance was completely reversed by a com-bination of physiological doses of adiponectin and leptin [13].Therefore,the suppressive effects of Fc on the development of obesity and diabetes may depend on changes in the production of adipocytokines in WAT.More recently,a crude mixture of conjugated linoleic acid (CLA)isomers has been shown to reduce body fat and enhance fat-free0003-9861/$-see front matter Ó2012Elsevier Inc.All rights reserved.doi:10.1016/j.abb.2012.01.011Abbreviations:ATGL,adipose triacylglycerol lipase;BAT,brown adipose tissue;CLA,conjugated linoleic acid;CPT1A,carnitine palmitoyltransferase 1A;DG,diacylglycerol;FA,fatty acid;Fc,fucoxanthin;HSL,hormone-sensitive lipase;LPL,lipoprotein lipase;MCP-1,monocyte chemoattractant protein-1;PPAR-c ,peroxi-some proliferator-activated receptor gamma;TC,total cholesterol;TG,triacylglyc-erol;TNF-a ,tumor necrosis factor-alpha;UCP,uncoupling protein;WAT,white adipose tissue.⇑Corresponding author.Address:Department of Food Science and Nutrition,Zhejiang University,866Yuhangtang Road,Hangzhou,Zhejiang 310058,China.Fax:+8657188982024.E-mail address:duoli@ (D.Li).mass in animals and humans[14,15].In addition,the treatment of CLA during adipocyte differentiation reduces lipid accumulation and inhibits the expression of peroxisome proliferator-activated receptor gamma(PPAR-c),which is a nuclear receptor that acti-vates genes involved in lipid storage and metabolism[16,17].Of the two major isomers of CLA(10,12and9,11isomers),the10, 12isomer is specifically responsible for the antiobesity effects [18–21].The potential mechanisms of CLA on weight loss include the regulation of energy metabolism,adipogenesis,inflammation, lipid metabolism and apoptosis[22].In the present study,we investigated the effects of Fc and FCL+CLA on body weight and adipose tissue weight,serum lipid profile,and obesity-related parameters in serum/plasma and gene expressions of lipid-regulating enzymes in perirenal WAT of diet-induced obesity rats.Materials and methodsMaterialsCLA was purchased from Cognis Chemicals Co.,Ltd.China.The Fc oil,which contains1%Fc,81.3%modified starch,17.2%seaweed crude extract,and0.5%natural vitamin E,was obtained from Beijing Gingko Group Biological Technology Co.,Ltd.,China. Animals and dietsThirty-five male Sprague Dawley(SD)rats,aged3week old, were obtained from Zhejiang University Laboratory Animal Center (Hangzhou,China).The rats were housed at23±1°C and at50% humidity with a12h light/12h dark cycle.After acclimation for 1week by feeding pellets of commercial chow,rats were randomly divided into four groups,fed a high-fat diet containing approxi-mately15%fat(wt/wt,Control,n=5),a high-fat diet plus 0.083mg/kg/bw Fc(FCL,n=10),a high-fat diet plus0.167mg/kg/ bw Fc(FCH,n=10)or a high-fat diet plus0.083mg/kg/bw Fc and 0.15g/kg/bw CLA(FCL+CLA,n=10).The formula of the high-fat diet comprises79%GB/T14924.9diet(General Administration of Quality Supervision,Inspection and Quarantine of the People’s Republic of China,AQSIQ,2001),10%lard,10%yolk powder and 1%cholesterol.The rats had free access to food and water ad libi-tum.Food intake and body weight were measured daily and twice a week,respectively.After feeding the control and experimental diets for45days, rats were starved for12h and sacrificed with decapitation.Blood was collected into EDTA-treated vacuum tubes.Plasma was obtained from blood samples after centrifugation at3000rpm for 10min at4°C.After collecting the blood,liver,white fat tissue from four regions(epididymal,perirenal,mesenteric and inguinal), and scapular brown fat were immediately removed,rinsed with a physiological saline solution,weighed,and then frozen in liquid nitrogen.All samples were stored atÀ70°C until analyzed. The study protocol was approved by the Ethics Committee of College of Biosystems Engineering and Food Science,Zhejiang University.Histological observation of liver and WATThe specific part of liver and perirenal WAT were removed from the rats,rinsed with saline andfixed in a buffer solution of10%for-malin.Sections offixed tissue specimens were processed for paraf-fin embedding,and4-l m sections were prepared and stained with hematoxylin-eosin and observed under the light microscopy (OLYMPUS BX41)with the magnifying power of100Âand200Â.Analysis of serum lipid profileConcentrations of serum total cholesterol(TC),triacylglycerol (TG),high-density lipoprotein-cholesterol(HDL-C),low-density lipoprotein-cholesterol(LDL-C),and glucose were analyzed on HIT-ACHI7020chemistry analyzer using colorimetric test supplied by Diasys Diagnostic Systems(Shanghai)Co.,Ltd.,China. Determination of serum/plasma insulin,leptin,ghrelin and obestatin levelsSerum insulin and leptin,and plasma ghrelin and obestatin con-centrations were analyzed by Rat Insulin(INS)ELISA kit,Rat Leptin ELISA kit,Rat Growth hormone releasing peptide-Ghrelin(GHRP-Ghrelin)ELISA kit and Rat Obestatin ELISA kit,respectively (Nanjing Jiancheng Technology Co.,LTD.,China).RNA extraction and quantitative real-time RT-PCR analysisTotal RNA was extracted from perirenal WAT using TRIZOL re-agent(Takara Biotechnology Co.,LTD.,China)according to the man-ufacture’s instructions.The concentrations of RNA samples were measured and quantified spectrophotometrically(Thermo Scien-tific NanoDrop2000c Spectrophotometer).Then,cDNA was synthe-sized from total RNA using the PrimeScript RT reagent kit(Takara Biotechnology Co.,LTD.,China).Real-time quantitative RT-PCR analysis was performed with an automated sequence detection sys-tem(BIO-RAD,CFX96).The mRNA expression of adiponectin,leptin, adipose triacylglycerol lipase(ATGL),hormone-sensitive lipase (HSL),lipoprotein lipase(LPL),carnitine palmitoyltransferase1A (CPT1A),PPAR c,and uncoupling protein2(UCP2)in WAT was mea-sured by quantitative real-time RT-PCR using SYBR green PCR re-agents(Takara Biotechnology Co.,LTD.,China).PCR rat primers were used for adiponetin,50-GGAAACTTGTGCAGGTTGGATG-30(for-ward),50-GGGTCACCCTTAGGACCAAGAA-30(reverse);leptin,50-TTCAAGCTGT GCCTATCCACAAAG-30(forward),50-TGAAGCCCGG-GAATGAAGTC-30(reverse);ATGL,50-TGACTCGAGTTTCGGATGGA-GA-30(forward),50-GAAATGCCGCCATCCA CATAG-30(reverse);HSL (lipe),50-CTGGAGTTAAGTGGGCGCAAG-30(forward),50-CA GAC-ACACTCCTGCGCATAGAC-30(reverse);LPL,50-GCCCAGCAACAT-TATCCAGT GTC-30(forward),50-AGCAGCATGGGCTCCAAGA-30 (reverse);CPT1A,50-CGCTCATG GTCAACAGCAACTAC-30(forward), 50-TCACGGTCTAATGTGCGACGA-30(reverse);PPAR c,50-TGTCGGT-TTCAGAAGTGCCTTG-30(forward),50-TTCAGCTGGTCGATAT CACTG-GAG-30(reverse);UCP2,50-GCTGGTGACCTATGACCTCATCAA-30 (forward),50-GTACTGGCCCAAGGCAGAGTTC-30(reverse).The quan-titative endpoint for real-time PCR is the threshold cycle(CT),which defined as the PCR cycle at which thefluorescent signal of the repor-ter dye crosses an arbitrarily placed threshold.The fold changes were calculated using the2À44Ct method with b-actin as the inter-nal control gene[23].Statistical analysisResults are expressed as means±standard error of the mean (SE).The data were analyzed with a one-way ANOVA,followed by LSD and Duncan’s test.Differences with P<0.01or P<0.05were considered significant.Results and discussionBody weight,food intake and adipose tissue weightCurrently,dietary fat is one of most commonly used environ-mental factors associated with the induction of obesity in rodents. In the present study,high-fat was used in the diet to induce obesity60X.Hu et al./Archives of Biochemistry and Biophysics519(2012)59–65of SD rats.Maeda et al.[24]reported that0.2%Fc significantly attenuated the body weight gain and WAT weight of diet-induced obese mice relative to the control mice.Recently,Woo et al. showed that0.05%and0.2%Fc both significantly suppressed body weight gain reaching15%and19%,respectively,in C57BL/6N mice fed20%high-fat[6].Likewise,the present study demonstrated that high-fat diet supplemented with FCL+CLA resulted in a significant reduction in body weight gain(P<0.05)and WAT weight gain (P<0.05)compared with control group without affecting food in-take(Fig.1,Table1and2).WAT weight of FCL and FCH rats was also inhibited compared to control group(P<0.05),though body weight was not affected by Fc supplement,which is possibly attrib-uted to the low levels of Fc(less than0.2%in diet)and/or short-term treatment.WAT is a primary site of energy storage in the form of triacylglycerol droplets,and it accumulates triacylglycerols during nutritional excess[25].Furthermore,according to the re-port of Hosokawa et al.[26],2.2%seaweed lipids containing Fc attenuated WAT weight gain of C57BL/6J mice by feeding of30% high-fat diet.In the present study,relative weights of adipose tis-sues were summarized in Table2.Mesenteric WAT weights were significantly attenuated in FCL,FCH and FCL+CLA groups in com-parison with control group whereas perirenal and inguinal WAT weights were markedly lowered only in FCL+CLA rats(P<0.05). Nevertheless,total WAT weight of rats in FCL,FCH and FCL+CLA groups was all significantly suppressed relative to control rats (P<0.05).Maeda et al.[5]reported that brown adipose tissue (BAT)weight was significantly greater in2.0%Undaria lipid-fed mice than in control mice.However,in the present study,there was no difference in BAT weight among all groups,which may be due to the little amount of BAT in rats and the lower dose of Fc sup-plemented in the diet.In addition,it was confirmed that a positive correlation between body weight and visceral fat weights exists (perirenal:r=0.840,P<0.01)[6].In the present study,a positive correlation also existed between body weight and perirenal WAT weight(r=0.704,P<0.01).As such,the body weight loss observed in FCL+CLA group was partly due to a decrease in WAT weight. Histology of liver and adipose tissue (Fig.2),moreover,liver weight of FCL+CLA rats was markedly lowered by17.3%relative to control rats(P<0.05)(Table2). Accordingly,Fc and FCL+CLA supplementation had a pleasant alle-viation on the accumulation of lipid droplets in liver cells.Obesity is characterized at the cell biological level by an in-crease in the number and size of adipocytes differentiated from fibroblastic pre-adipocytes in the adipose tissue[29].The present study revealed that perirenal adipocyte sizes of FCL,FCH and FCL+CLA groups were smaller than those of control group(Fig.3). Serum lipid profile,and levels of leptin,ghrelin and obestatinThe current study indicated that FCL,FCH and FCL+CLA supple-mentation was effective in improving serum lipid profile via signif-icantly affecting TG,TC and glucose levels,and maintaining a modest reduction in LDL-C level,though HDL-C concentrations were apparently reduced in rats fed FCH and FCL+CLA(Table3). Many studies have clarified that leptin secretions are elevated through the accumulation of fat in adipocytes and causes insulin resistance in obese animal models[30].Therefore,plasma/serum leptin level is used as an index of body fat accumulation.Here, two doses of Fc and FCL+CLA significantly decreased the serum leptin concentration compared with control group(P<0.01)(Table 4).Further,the leptin level in serum exhibited a positive correla-tion with WAT weight(r=0.571,P<0.01)(data not shown). Asakawa et al.reported that exogenous ghrelin stimulates food intake and promotes energy storage[31].Additionally,ghrelin treatment in rodents leads to sustained gain of fat mass induced via regulating food intake,decreased fat oxidation rates and ther-mogenesis,and increased lipogenesis in adipose tissue[32].In con-trast,obestatin,a peptide encoded by the ghrelin gene,opposes ghrelin’s effects on food intake,hence decreasing body-weight gain [33].In the present study,there were no differences in plasma lev-els of ghrelin and obestatin,however,a slight descending trend was observed in ghrelin levels in FCH and FCL+CLA groups and a mild increasing trend was revealed in obestatin levels in FCL and FCH groups(Table4).mRNA expression of lipid-regulating enzymes in adipose tissueCurrently,it is accepted that specific regulation of gene expres-sion in adipocytes is one of the most important targets for the intervention of obesity.In addition,WAT has been regarded as an active endocrine organ which secretes important molecules,like leptin and adiponectin,involved in the regulation of body weight [34].Adiponectin is a secreted protein which plays a major role in the regulation of glucose,insulin and fatty acids and which has an anti-obesity effect[35,36].In addition,adiponectin is very highly expressed in adipose tissues and it can increase b-oxidation1.Food intake of high-fat fed rats.Mean±SE,n=5for Control group,10for FCL,FCH and FCL+CLA groups,respectively.FCL,0.083mg/kg/bw fucoxanthin-supplemented group with a high-fat diet;FCH,0.167mg/kg/bw fucoxanthin-supplemented group with a high-fat diet;FCL+CLA,combination 0.083mg/kg/bw fucoxanthin and0.15g/kg/bw CLA-supplemented group with high-fat diet.Table1Effects of FCL,FCH and FCL+CLA supplementation on body weight in high-fat fed rats.Control FCL a FCH b FCL+CLA cInitial body weight(g)147±5.72148±4.32156±4.37142±5.55 Final body weight(g)522±11.37512±5.04531±7.34485±9.08** Body weight gain(g)375±10.15363±5.81375±8.80344±11.91* Mean±SE,n=5for Control group,and n=10for FCL,FCH and FCL+CLA groups, FCL+CLA vs.Control.*P<0.05.**P<0.01.a0.083mg/kg/bw fucoxanthin-supplemented group with a high-fat diet.b0.167mg/kg/bw fucoxanthin-supplemented group with a high-fat diet.c Combination of0.083mg/kg/bw fucoxanthin and0.15g/kg/bw CLA-supple-mented group with a high-fat diet.X.Hu et al./Archives of Biochemistry and Biophysics519(2012)59–6561in tissues and causes weight loss in mice[37].Moreover,adiponec-tin levels are inversely related with fat mass[38].In the present study,the mRNA expression of adiponectin was remarkably ele-vated in FCL,FCH and FCL+CLA groups in perirenal WAT compared to control group(P<0.05)(Fig.4).So far,a large body of work pro-posed that leptin exerts its actions on food intake and energy expenditure.However,in the present study,no significant differ-ence was observed in the mRNA expression of leptin in perirenal WAT among all groups.Deregulation of lipid metabolism has long been recognized as an essential factor in the development of obesity and WAT lipolysis plays a pivotal role in controlling the quantity of TG stored in fat depots[39].Reports showed that ATGL is the rate-limiting enzyme for thefirst step in TG hydrolysis,generating diacylglycerol(DG) and fatty acid(FA),whereas HSL is responsible for the subsequent degradation of DG,generating MG and FA[28].Apart from ATGL and HSL,LPL is another enzyme,rate-limiting for the hydrolysis of core TGs in chylomicrons and VLDLs[40,41].Furthermore,adi-pose LPL hydrolyzes the TG of lipoprotein particles in capillaries, thereby releasing FA and then transported into adipocytes where they are esterified to TG and stored for future energy use[42].Ele-vated LPL in adipocytes thus promotes the storage of excess FFA in adipose tissue[43].In the present study,the up-regulation of ATGL mRNA in perirenal WAT was observed in FCL,FCH and FCL+CLA groups(P<0.05).Further,FCL and FCH also enhanced mRNA expression of HSL(P<0.05)and LPL(P<0.01)respectively,which were predominantly involved in hydrolyzing triacylglycerols. These results suggest that the hydrolysis of excessive triacylglyce-rols stimulated by FCL,FCH and FCL+CLA contributed to diminish-ing the fat stores and combating obesity.The enzyme CPT1A regulates the entry of LCFAs into mitochondria,where they under-go b-oxidation[44,45].The present study showed that mRNA expression of CPT1A in perirenal WAT of rats fed FCL,FCH and FCL+CLA was all significantly increased compared with control rats(P<0.01,P<0.05),which supports the hypothesis that the enhancement of fatty acids mobilization and oxidation was poten-tially triggered by Fc and CLA.Moreover,PPAR c,a regulator of adi-pogenic gene expression,was significantly down-regulated by FCH and FCL+CLA(P<0.05).PPARs are nuclear hormone receptors that control lipid oxidation,adipocyte differentiation,glucose and lipidTable2Effects of FCL,FCH and FCL+CLA supplementation on WAT,BAT and liver weights in high-fat fed rats.liver in high-fat fed rats(100Â).Liver tissue of SD rats was stained with hematoxylin and eosin.FCL, high-fat diet;FCH,0.167mg/kg/bw fucoxanthin-supplemented group with a high-fat diet;FCL+CLA,CLA-supplemented group with a high-fat diet.62X.Hu et al./Archives of Biochemistry and Biophysics519(2012)59–65storage,and inflammation [46,47].Regulation of PPAR c would be one of the expected mechanisms underlying the anti-obesity effect of dietary Fc and CLA.Evidence to date indicates that the product of the UCP2gene is crucial for mammalian thermogenesis because of its high degree of sequence similarity (55–60%)to UCP1[48].UCP2is widely ex-pressed in human and rodent tissues [48–51],unlike UCP1,whichis expressed uniquely in BAT.The ubiquitous expression of UCP2suggests that the protein may be important for determining basal metabolic rate,and possibly regulating body weight in mammals including humans [52].In the present study,compared with con-trol group,there was a significant elevation of UCP2mRNA expres-sion in perirenal WAT in FCL group (P <0.05),which suggest a potential contribution to the underlyingthermogenesis.perirenal adipose tissue in high-fat fed rats (200Â).Perirenal adipose tissue of SD rats was stained with hematoxylin group with a high-fat diet;FCH,0.167mg/kg/bw fucoxanthin-supplemented group with a high-fat 0.15g/kg/bw CLA-supplemented group with a high-fat diet.Table 3Effects of FCL,FCH and FCL +CLA supplementation on TC,TG,HDL-C,LDL-C and glucose levels in high-fat fed rats.ControlFCL AFCH BFCL +CLA C TC (mmol/L) 2.15±0.081a 1.88±0.144ab 1.39±0.067b 1.61±0.107ab TG (mmol/L) 1.23±0.064a 0.76±0.046b 0.54±0.022b 0.48±0.036b HDL-C (mmol/L)0.71±0.035a 0.62±0.012a 0.45±0.049b 0.37±0.031b LDL-C (mmol/L)0.51±0.0580.37±0.0350.44±0.0360.28±0.027Glucose (mmol/L)4.56±0.242a3.94±0.202ab3.80±0.090b3.79±0.259bMean ±SE,n =5for Control group,and n =10for FCL,FCH and FCL +CLA groups,abcMeans in the row not sharing a common letter are significantly different between groups at P <0.05as determined by a one-way ANOVA test.A0.083mg/kg/bw fucoxanthin-supplemented group with a high-fat diet.B0.167mg/kg/bw fucoxanthin-supplemented group with a high-fat diet.CCombination of 0.083mg/kg/bw fucoxanthin and 0.15g/kg/bw CLA-supplemented group with a high-fat diet.Table 4Effects of FCL,FCH and FCL +CLA supplementation on serum insulin and leptin,plasma ghrelin and obestatin levels in high-fat fed rats.ControlFCL 1FCH 2FCL +CLA 3INS (mU/L) 2.35±0.08 2.40±0.13 2.19±0.13 2.11±0.06LEP (l g/L) 2.33±0.17a 1.60±0.06b 1.55±0.05bc 1.41±0.04c GHRE (ng/L)236.59±18.30234.71±21.13219.55±10.06201.36±11.24OBES (ng/L)70.09±4.0383.91±8.2179.90±4.0468.96±3.51Mean ±SE,n =5for Control group,and n =10for FCL,FCH and FCL +CLA groups,abc Means in the row not sharing a common letter are significantly different between groups at P <0.01as determined by a one-way ANOVA test.INS,insulin;LEP,leptin;GHRE,ghrelin;OBES,obestatin.10.083mg/kg/bw fucoxanthin-supplemented group with a high-fat diet.20.167mg/kg/bw fucoxanthin-supplemented group with a high-fat diet.3Combination of 0.083mg/kg/bw fucoxanthin and 0.15g/kg/bw CLA-supplemented group with a high-fat diet.ConclusionsThe present study showed that Fc reduced WAT weight while FCL +CLA decreased both body weight and WAT weight in rats.Both Fc and FCL +CLA could reduce serum concentration of TG,glucose and leptin.The mechanism underlying the anti-obesity ef-fect of FCL +CLA may be elucidated through up-regulating expres-sion of adiponectin,ATGL,CPT1A and down-regulating mRNA expression of PPAR c in WAT,which are involved in b -oxidation of fatty acids and triacylglycerol hydrolysis,however,the profound synergistic relationship between Fc and CLA need to be clarified by further study.AcknowledgmentsWe declare that there are not any potential conflicts of interest that are relevant to the manuscript.This work was supported by Grants from the National Natural Science Foundation of China (No.30972464)and the National Basic Research Program of China 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Astaxanthin and Peridinin Inhibit Oxidative Damage in Fe 2ϩ-Loaded Liposomes:Scavenging Oxyradicals or Changing Membrane Permeability?Marcelo P.Barros,*,1Ernani Pinto,*,†Pio Colepicolo,†and Marianne Pederse ´n**Department of Botany,Stockholm University,SE-10691Stockholm,Sweden;and †Departamento de Bioquimica,IQUSP,C.P.26077,05599-970,Sa ˜o Paulo,BrazilReceived August 30,2001Astaxanthin and peridinin,two typical carotenoids of marine microalgae,and lycopene were incorpo-rated in phosphatidylcholine multilamellar liposomes and tested as inhibitors of lipid oxidation.Contrarily to peridinin results,astaxanthin strongly reduced lipid damage when the lipoperoxidation promoters—H 2O 2,tert -butyl hydroperoxide (t -ButOOH)or ascor-bate—and Fe 2؉:EDTA were added simultaneously to the liposomes.In order to check if the antioxidant activity of carotenoids was also related to their effect on membrane permeability,the peroxidation pro-cesses were initiated by adding the promoters to Fe 2؉-loaded liposomes (encapsulated in the inner aqueous solution).Despite that the rigidifying effect of carote-noids in membranes was not directly measured here,peridinin probably has decreased membrane perme-ability to initiators (t -ButOOH >ascorbate >H 2O 2)since its incorporation limited oxidative damage on iron-liposomes.On the other hand,the antioxidant activity of astaxanthin in iron-containing vesicles might be derived from its known rigidifying effect and the inherent scavenging ability.©2001Academic PressKey Words:astaxanthin;peridinin;antioxidant;lipo-some;lipoperoxidation.Peridinin is an unusual C 37carbon skeleton carot-enoid with epoxy,hydroxy,and acetate groups on ␤-rings,an allene moiety and a lactone group conju-gated to the ␲-electron system (Fig.1)(1).In additionto the membrane-bound light harvesting complex of Photosystem II (PSII),dinoflagellates also contain a water-soluble external antenna complex,the peridinin-chlorophyll-protein (PCP).Peridinins in PCP and in model antenna systems effectively transfers electronic excitation to chlorophyll a (88to 95%)which is able to pass this excitation energy to membrane-bound light-harvesting complexes on PSII (1–4).Recently,Pinto et al.(5)have demonstrated that peridinin is the major singlet molecular oxygen [O 2(1⌬g )]quencher in Lingu-lodinium polyedra,despite being less efficient than ␤-carotene.However,it has not been clearly shown if dinoflagellates contain peridinin molecules on antenna complexes of the photosystems within thylakoid mem-branes (6).The ketocarotenoid astaxanthin (Fig.1)is a red pig-ment common to several aquatic organisms including algae,salmon,troute,and shrimp (7–9).Several re-ports indicate that astaxanthin is one of the most ef-fective antioxidant against lipid peroxidation and oxi-dative stress in many in vitro and in vivo systems (10–15).It has also been shown that simultaneous depletion of astaxanthin and ␣-tocopherol influences autoxidative defense,fatty acid metabolism and syn-thesis of coenzyme thiamine-pyrophosphate in Baltic Sea salmon affected by the M74syndrome (16–20).Another relevant property of carotenoids is how these compounds affect fluidity and permeability of natural and artificial membranes.Carotenoids with keto and hydroxy groups on both ends of the molecule (e.g.,zeaxanthin,astaxanthin,and canthaxanthin)strongly decrease water and small molecules perme-ability across the lipid bilayer (21).Thus,in addition to a direct scavenging ability against reactive oxygen spe-cies (ROS),some polar carotenoids also inhibit the penetration of oxidative substances and,consequently,the initiation of a lipid peroxidation process.The aim of this work is to study the antioxidant activity of astaxanthin and peridinin,two of the mostAbbreviations used:BHT,butylated hydroxytoluene;EDTA,eth-ylenediaminotetraacetic acid;Iron-PCL,Fe 2ϩ:EDTA-loaded egg-yolk phosphatidylcholine liposomes;MDA,malondialdehyde;PCL,egg-yolk phosphatidylcholine liposomes;PUFA,polyunsaturated fatty acids;ROS,reactive oxygen species;TBARS,thiobarbituric acid reactive substances;t -ButOOH,tert -butyl hydroperoxide;Trolox,(ϩ)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid.1To whom correspondence should be addressed at Departamento de Bioquı´mica,IQUSP,Bloco 9superior,C.P.26077,05599-970,Sa ˜o Paulo,Brazil.Fax:ϩ55-11-38182170.E-mail:mpbarros@botan.su.se.Biochemical and Biophysical Research Communications 288,225–232(2001)doi:10.1006/bbrc.2001.5765,available online at onabundant carotenoids among marine microalgal spe-cies.For that purpose,the carotenoids were incorpo-rated into egg-yolk phosphatidylcholine multilamellar liposomes(PCL)and challenged by different ROS which were generated by classical lipoperoxidation ini-tiators.In order to check if the carotenoid antioxidant activity is exclusively or partially derived from its ri-gidifying effect on membranes,the liposomes were pre-viously loaded with Fe2ϩ:EDTA complexes(Iron-PCL). Thus,to initiate ROS generation in Iron-PCL,the li-poperoxidation agents—H2O2,tert-butyl hydroperox-ide(t-ButOOH)and ascorbate—must cross the lipid bilayers and react with the metal ion present inside the vesicles.These experiments were also performed with lycopene and butylated hydroxytoluene(BHT),classi-cal antioxidants,as controls.MATERIALS AND METHODSMaterials.All chemicals were obtained from Sigma–Aldrich Swe-den AB,except FeSO4.7H2O and liquid chromatography grade sol-vents n-hexane,chloroform,methanol,and ethanol from Merck Co. (Darmstadt,Germany);ascorbic acid and Perdrogen(H2O230%) from Riedel-deHae¨n(Seelze,Germany);and(ϩ)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid(Trolox)from Fluka Chemika (Buchs,Switzerland).Peridinin was isolated from Lingulodinium polyedra as described by Pinto et al.(5).The dialysis membranes were Spectra/Por MWCO2000from Spectrum Medical Industries (Los Angeles,CA).Carotenoid stock solutions.All carotenoids were solubilized in organic solvents previously to their incorporation into egg-yolk phos-phatidylcholine liposomes(PCL)and the absorbances of these stock solutions were measured to evaluate their effective concentrations. Peridinin(␧469ϭ85.8ϫ103MϪ1cmϪ1)was solubilized in chromatog-raphy grade methanol while astaxanthin(␧468ϭ125ϫ103MϪ1cmϪ1)and lycopene(␧472ϭ186ϫ103MϪ1cmϪ1)were dissolved in purified n-hexane(22).The stock solutions were stored atϪ80°C freezer and protected from light to avoid oxidation.Preparation of multilamellar liposomes(PCL).In order to pre-vent aggregate formation and loss of material during the procedure, the carotenoids were isolated from stock solution byflushing the respective organic solvent with a N2stream until dryness.After that,500␮L of chloroform were added to eachflask and the egg-yolkphosphatidylcholine solution in CHCl3was mixed for afinal carote-noid:lecithin proportion of0.5%(25␮M and5mM,respectively).Eggyolk phosphatidylcholine was selected for its unsaturated fatty acid content which offers suitable oxidation targets for ROS(23,24).After brief mixing,chloroform was evaporated byflushing N2in a round-bottomflask adapted to a rotavapor apparatus working at a low speed to allow the formation of a homogeneous driedfilm.The lipid-carotenoidfilm was stored overnight in the dark under vacuum to eliminate traces of chloroform.The PCL vesicles were prepared by mixing100mM phosphate buffer(pH7.4)to the lipidfilm followed by strong vortexing for5min.The formation of carotenoid aggre-gates was avoided by preparing the PCL at40°C,which is high above the transition temperature of30°C for egg-yolk phosphatidylcholine (25).The suspension was centrifuged at15,000rpm for20min to eliminate eventual formed aggregates.Preparation of Fe2ϩ-incorporated multilamellar liposomes(Iron-PCL).Thefirst method tested for Iron-PCL preparation envolved sonication of the lipid-carotenoidfilm with100mM phosphate buffer (pH7.4)on ice until the dispersion becomes clean(26).However,this classic method of liposome preparation proved to be unefficient for our purposes since it caused a8.5-fold higher level of lipid oxidation (data not shown).Thus,the Iron-PCL was prepared as PCL:mixing the lipid-carotenoidfilm with5mL of100mM phosphate buffer(pH 7.4)plus5mM Fe2ϩ:EDTA solution(to afinal concentration of0.1 mM)and strong vortexation.A dialysis procedure was used to elim-inate external and loosely bound iron complexes from the liposomes. About5mL of uncleaned Iron-PCL were dialysed in Spectra/Por molecularporous membrane(MWCO2000)at room temperature against2L of destilled water for2h with smooth agitation by a magnetic stirrer.In the beginning,the liposome suspensions were dialysed against2L of100mM phosphate buffer(pH7.4)but this procedure did not efficiently remove the metal ions supposed to be placed outside the liposomes(data not shown).The iron content in the PCL was checked before and after every dialysis process to estimate loss of iron complexes during the procedure.Induction of lipid peroxidation.Either PCL or Iron-PCL,contain-ing significant concentrations of unsaturated lipids(27),were oxi-dized by incubation for45min at30°C with1mM solution of three different initiators:H2O2,t-ButOOH or ascorbic acid.To stimulate lipid oxidation in PCL,0.1mM Fe2ϩ:EDTA was simultaneously added.Trolox(0.5mM in0.1M phosphate buffer pH7.4)and5␮Mbutylated hydroxytoluene(BHT)were used as controls.Trolox,a water-soluble derivative of␣-tocopherol with similar scavenging ac-tivity(28),was used as a probe for checking the sites of ROS gener-ation in multilamellar vesicles since it is not supposed to permeate liposome lipid bilayers(Fig.2).Measurement of lipoperoxidation extent(TBARS test).After the incubation period,the oxidative reaction was stopped by adding20␮L of0.2M BHT(ethanol solution).To produce the coloured adduct, 350␮L of sample were incubated with700␮L of0.375%thiobarbi-turic acid(TBA)in0.25M HCl and1%Triton X-100at100°C for15 min.After reaching the room temperature,the absorbance of the solutions were measured at535nm using malondialdehyde(MDA) as standard(29).Controls for residual absorption of carotenoids at 535nm were made using0.25M HCl plus1%Triton X-100solution without TBA.Iron determination.The iron incorporation in the liposomes was checked before and after the dialysis procedure by a modification of the method described by Bralet et al.(30).Aliquotes of300␮L were taken from the liposome suspensions and3␮L of Triton X-100was added to disrupt the vesicles.The samples were added to50mM glycine hydrochloride buffer(pH2.5)with20mg/mL ascorbate,10 mg/mL pepsin and5mM2,2Ј-bipyridine.After incubation for2h at 37°C,the absorbance was measured at520nm and results compared to FeSO4.7H2O standardcurve. FIG.1.Chemical structures of peridinin and astaxanthin.Statistics.Data are presented as means ϮSD (standard devia-tion)and statistical analysis performed with the Student’s t test at significance level of 5%.RESULTS AND DISCUSSION Nonloaded Liposomes (PCL)The TBARS concentration after PCL preparations were (0.169Ϯ0.043nmol MDA/␮mol PC)and (0.209Ϯ0.031nmol MDA/␮mol PC),respectively for PCL and Iron-PCL.As expected,the encapsulation of Fe 2ϩ:EDTA complexes in PCL resulted in higher lipid oxi-dation level (c.a.25%).The coordination of Fe 2ϩwith EDTA does not prevent it to react with ROS and,hypothetically,it would be easier to eliminate (by di-alysis)a water-soluble Fe 2ϩ:EDTA complex than a membrane-associated Fe 2ϩ:phosphatidylcholine che-late (31,32).Probably,the osmotic pressure must have led to re-organization of PCL membranes and coalescence of lipid vesicles during the dialysis performed against distilled water (33).Even with distinguished polarity properties,lycopene and astaxanthin induced Fe 2ϩ:EDTA elimination from liposomes at the same extent (c.a.30%).When peridinin was associated,the effect was less intense (23%).On the other hand,a higher loss of iron chelate was measured in carotenoid-free liposomes (53%)(Fig.3).Ascorbic acid can behave as a prooxidant since it can reduce Fe 3ϩto Fe 2ϩ,a well-known strong promoter of lipoperoxidation (33).However,at millimolar concen-trations the ability of ascorbate to scavenge HO •be-comes more significant.Ascorbate is also able to reduce tocopheryl radicals,generated by hydrogen abstrac-tion from ␣-tocopherol,back to its active antioxidant form.Trolox,with similar scavenging mechanism as ␣-tocopherol,is supposed to be constantly regeneratedby ascorbate from the Trolox radical form in the aque-ous solution (28).Ascorbate is also supposed to be charged at pH 7.4(ascorbic acid pKa 1and pKa 2,4.17and 11.57,respectively)thus with low permeability throughout membranes.Butylated hydroxytoluene (BHT)was very efficient in scavenging free radicals generated by all lipoperoxi-dation agents in PCL even at micromolar range (Fig.4).This effect was probably due to its higher diffusibil-ity into membranes (34)which would allow this anti-oxidant to scavenge oxyradicals at several spots through out the lipid bilayer.Trolox,mostly present in the aqueous solution,required millimolar concentra-tions to inhibit lipid oxidation to the same extentionasFIG. 2.Trolox scavenging activity against ROS produced by H 2O 2,t -ButOOH and ascorbate in PCL andIron-PCL.FIG.3.Iron concentrations in Iron-PCL in the presence or ab-sence of lycopene (Iron-PCL/LYC),astaxanthin (Iron-PCL/AST)or peridinin (Iron-PCL/PER),during a dialysis process (nmols Fe 2ϩ/␮mol PC).Shown are the means ϮSD of 3experiments;*P Ͻ0.05.FIG.4.Effects of BHT and Trolox in lipoperoxidation of PCL in the absence of carotenoids induced by mixing lipoperoxidation promoters—H 2O 2,t -ButOOH,or ascorbate—to chelated Fe 2ϩions (nmols MDA/␮mol PC).Shown are the means ϮSD of 4experiments;*P Ͻ0.05.BHT in H 2O 2/Fe 2ϩsystem.Higher levels of TBARS were produced by addition of Fe 2ϩand t -ButOOH to PCL:1.9-fold higher compared to 94%obtained with H 2O 2.When lipoperoxidation process is initiated by t -ButOOH most of the free radicals detected in the lipid bilayer is peroxyl radical (ROO •)(35).A significant proportion of alkoxyl radical (RO •)and singlet oxygen [O 2(1⌬g )]have also to be considered (23,26,35–38).Trolox was not able to efficiently protect the PCL mem-branes in t -ButOOH-induced ually,Trolox is more reactive with ROS than BHT,especially concerning peroxyl radicals (39),but the higher perme-ability of the phenolic compound may have compen-sated for its lower reactivity.To evaluate the single effect of iron addition to PCL (with or without carotenoids),the TBARS measure-ments were also performed in the absence of peroxida-tion agents.As an extra control,PCL was also pre-pared containing 25␮M ␣-tocopherol as described by Palozza &Krinsky (40).As could be observed in Fig.5,astaxanthin and peridinin were able to inhibit lipoper-oxidation before the addition of iron complexes.The addition Fe 2ϩ:EDTA to PCL,in the absence of carote-noids,did not change TBARS production although an increase of 25%in MDA content was previously ob-served after Iron-PCL preparation.The lipid oxidation in astaxanthin-(PCL/AST)and peridinin-incorporated liposomes (PCL/PER)were both,approximately,25%lower than in PCL although only PCL/AST was insen-sitive to iron addition.Lycopene was the only carot-enoid which reduced (25%)the level of lipoperoxidation after Fe 2ϩ:EDTA addition (Fig.5).The carotenoid-incorporated liposomes were chal-lenged by ROS produced outside,when initiator and iron complexes were added simultaneously.These re-sults are presented in Fig.6.The simultaneous addi-tion of ferrous salt and ascorbate resulted in a more moderate increase of TBARS concentration (21%)thanthose observed for H 2O 2and t -ButOOH systems sug-gesting the previously described dual effect of ascor-bate concerning its action against free radicals.It is noteworthy that,usually,iron ions are contaminating ascorbic acid by 0.02%which would allow the initia-tion of lipid oxidation even without adding Fe 2ϩsolu-tion (31).Astaxanthin proved to be the best antioxidant in all experiments performed with both peroxidation initia-tor and iron chelate placed outside the PCL,as ex-pected from other authors (10,17).The ketocarotenoid was the only tested compound to avoid extreme high levels of lipid damage caused by concomitant addition of ferrous ions and H 2O 2,t -ButOOH or ascorbate:re-spectively,45,45,and 33%lower lipid oxidation than PCL added with iron (II).As observed with the exper-iments without peroxidation agents (Fig.5),astaxan-thin also induced the lowest enhancement of MDA production upon iron ions addition.No antioxidant activity was found for peridinin when incorporated into PCL and challenged by free radicals produced outside.Actually,peridinin led to intense augmentation of oxidated lipid levels after ferrous ions were added to H 2O 2-and t -ButOOH-treated liposomes:2.9-fold and 3.5-fold higher,respectively.The TBARS level obtained after incubation of PCL/PER with ascor-bate 1mM in the absence of Fe 2ϩ(0.65Ϯ0.14nmol MDA/␮mol PC)was one of the highest of all experi-ments performed.Lycopene,under the reaction conditions described here,could not inhibit the lipoperoxidation process in PCL.The effect of chelated iron (II)inclusion to ascorbate-treated PCL/LYC was lower than with other lipid peroxidation agents despite being the highest value measured (0.73Ϯ0.03nmol MDA/␮mol PC).Apolar carotenoids,e.g.,␤-carotene and lycopene,haveFIG.6.TBARS levels induced by H 2O 2,t -ButOOH or ascorbate and Fe 2ϩ:EDTA in carotenoid-associated PCL.Shown are the means ϮSD of 4experiments;*P Ͻ0.05.FIG.5.Levels of TBARS promoted by addition of Fe 2ϩ:EDTA in PCL containing ␣-tocopherol (PCL/TOC),lycopene (PCL/LYC),peri-dinin (PCL/PER)and astaxanthin (PCL/AST).Shown are the means ϮSD of 4experiments;*P Ͻ0.05.been reported to perturb the acyl chain packing and to increase bilayer permeability (41,42).In some circum-stances,efficient in vivo antioxidants like ␤-carotene and lycopene could also act,or partially offer,a prooxi-dative effect in lipid peroxidation process masking its antioxidant activity.Iron-Loaded Liposomes (Iron-PCL)After the dialysis,an insignificant concentration of Fe 2ϩ:EDTA was present outside the PCL.As it is shown in Fig.7,no significant variation was observed in H 2O 2-generating system when 25␮M,50␮M,0.25mM,or 0.5mM Trolox were added.This aspect sug-gests that these oxyradicals were generated in the internal aqueous solution,triggered by the permeation of the easily diffusible molecule,H 2O 2.When both iron (II)and H 2O 2were added to the external aqueous so-lution,0.5mM Trolox and 5␮M BHT inhibited lipoper-oxidation by 40and 55%,respectively (Fig.4).A constant (13%),but not significant,inhibition of MDA production in t -ButOOH-treated Iron-PCL was caused by Trolox in the concentration range from 25␮M to 0.25mM (Fig.7).However,0.5mM Trolox significantly suppressed lipid peroxidation:23.6%.Even also being a small and uncharged molecule,t -ButOOH was expected to permeate membranes in a less extention than H 2O 2.Paradoxically,higher lipid oxidation products were measured after incubation of Iron-PCL with t -ButOOH than with H 2O 2.BHT was not able to prevent lipid oxidation in this system al-though,when peroxyl and alkoxyl were generated out-side the liposomes (Fig.4)a 55%lowed MDA content was obtained.Ascorbate addition to Iron-PCL also resulted in a higher lipid oxidation despite being negatively charged at pH 7.4and not assumed to penetrate intensely the lipid bilayers.A possible explanation is the 0.02%usual iron contamination of commercial ascorbic acid (31).The effect of Trolox on lipoperoxidation is another indication that the oxidation process was initiated at the outer moiety.In fact,the addition of increasing concentrations of Trolox led to gradual higher protec-tion of the membranes against oxidative damage.An-other indication of external action of free radicals is the 53%inhibition of lipoperoxidation in Iron-PCL induced by 5␮M BHT.As shown in Fig.8,lipoperoxidation in Iron-PCL was intensely stimulated by the addition of peroxidation promoters—H 2O 2,t -ButOOH and ascorbate,respec-tively—2.4-,4-,and 3.8-fold higher than MDA concen-trations obtained without promoters (dotted line;0.12Ϯ0.02nmol MDA/␮mol PC).The MDA concentra-tions found when H 2O 2and t -ButOOH were added to Iron-PCL were significantly lower than those mea-sured when iron ions and promoter were added simul-taneously to the vesicles (Fig.4).When ascorbate/Fe 2ϩ:EDTA was used as lipoperoxidation initiator system,an equivalent MDA concentration was obtained for both types of vesicles:(0.45Ϯ0.06)and (0.45Ϯ0.07)nmol MDA/␮mol PC for,respectively,Iron-PCL and PCL.Astaxanthin was the more efficient antioxidant since it suppressed the H 2O 2-induced lipoperoxidation in Iron-PCL by 26%(Fig.8).Peridinin showed a more modest inhibition of lipid oxidation process (17.7%),suggesting that,due to its incorporation into lipid bi-layer,it could have limited the permeation of the per-oxidation agent,H 2O 2in these experiments.OnFIG.8.TBARS levels induced by addition of H 2O 2,t -ButOOH or ascorbate to Iron-PCL in the presence of lycopene (Iron-PCL/LYC),peridinin (Iron-PCL/PER)or astaxanthin (Iron-PCL/AST).Shown are the means ϮSD of 4experiments;*P Ͻ0.05.FIG.7.Effects of 5␮M BHT and 25␮M,50␮M,0.25mM and 0.5mM Trolox in lipoperoxidation of Iron-PCL (in the absence of caro-tenoids)induced by adding 1mM ROS promoters—H 2O 2,t -ButOOH,and ascorbate (nmols MDA/␮mol PC).Shown are the means ϮSD of 4experiments;*P Ͻ0.05.the other hand,lycopene showed evidences that ithas enhanced membrane permeability to H2O2andt-ButOOH,since increases of c.a.21%in MDA concen-trations(not significant)were observed in both sys-tems.When1mM ascorbate was added to Iron-PCL, peridinin significantly limited lipoperoxidation which was comparable to the values obtained with astaxan-thin:respectively,33.5%and46.4%.Lycopene was only able to protect liposome membranes when ascor-bate was used as a promoter of lipid oxidation(17.4% lower than control).CONCLUSIONSCarotenoids,especially astaxanthin and zeaxanthin, show high rate constants for reactions with peroxyl radicals(ROO•)and as a[O2(1⌬g)]quencher(24,37). Shimidzu et al.(43)developed in vitro assays to study the quenching efficiency of several carotenoids frommarine organisms against[O2(1⌬g)]and has evidenced astaxanthin as one of the most efficient.Recently, Pinto et al.(5)have demonstrated that peridinin,de-spite being less efficient than␤-carotene,is the major[O2(1⌬g)]quencher in Linulodinium polyedra,mainly due its elevated concentration in this organism.The antioxidant effect of carotenoids is probably also de-rived from its rigidifying effect on membranes which could lead to a limitation in metal ions or oxidative compound penetration into lipid bilayers(44,45).On the other hand,hydrophobic carotenoids(e.g.,lycopene and␤-carotene)make the membranes morefluid and, under some circumstances,morefluid and,under some circumstances,more susceptible to oxidative damage (41).The data reported here suggest that lycopene, under the described reaction conditions,was not able to protect membrane lipids against iron-induced oxida-tion process.This fact has also been recently pointed out as a possible explanation for the ambiguous action of␤-carotene challenged by oxyradicals in different lipid systems(42).Astaxanthin,as previously demonstrated in vitro and in vivo(10,17,46–50),was able to strongly inhibit the propagation step of lipoperoxidation in all tested systems.It is possible that two combined properties of astaxanthin were responsible for this fact:(i)the rigid-ifying effect on membrane,which could have limitedthe penetration of lipoperoxidation promoters—H2O2,t-ButOOH and ascorbate—into the liposome mem-branes(21,51,52);and(ii)the inherent antioxidant activity of this ketocarotenoid(53).However,the same explanation is not valid for antioxidant action of peri-dinin in Iron-PCL assays.Peridinin did not show any antioxidant property when the ROS were produced outside the liposomes.However,when the peridinin-associated vesicles were pre-loaded with Fe2ϩ:EDTA complexes,a significant inhibition of lipoperoxidation was observed(in all tested systems).Despite that no report about peridinin orientation on lipid bilayers was available in the literature,it is tempting to suggest,by a preliminary analysis of its chemical structure,that this carotenoid might have a vertical or more angular orientation in egg-yolk leci-thin liposomes.Thus,it is possible,despite being spec-ulative,that peridinin also shows polar-carotenoid ri-gidifying effect on membranes and,consequently,its detected inhibitory action on lipid oxidation might be related to a peridinin-induced decrease in the perme-ation of lipoperoxidation promoters in membranes. 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生物技术进展２０１５年㊀第５卷㊀第３期㊀１６４~１６９ＣｕｒｒｅｎｔＢｉｏｔｅｃｈｎｏｌｏｇｙ㊀ＩＳＳＮ２０９５￣２３４１进展评述Ｒｅｖｉｅｗｓ㊀收稿日期:２０１５￣０４￣０１ꎻ接受日期:２０１５￣０５￣０１㊀基金项目:哈尔滨工业大学优秀团队支持计划资助ꎮ㊀作者简介:李贵珍ꎬ博士研究生ꎬ研究方向为海洋微生物资源与利用ꎮＥ￣ｍａｉｌ:ｌｉｇｕｉｚｈｅｎ.ｏｋ＠１６３.ｃｏｍꎮ∗通信作者:闫培生ꎬ教授ꎬ博士生导师ꎬ研究方向为海洋微生物资源与利用㊁海洋生物质及其加工废物的高值资源化㊁有害微生物的生物防治与生物农药㊁微生物发酵工程与生物制药等ꎮＥ￣ｍａｉｌ:ｙｐｓ６＠１６３.ｃｏｍꎻ邵宗泽ꎬ研究员ꎬ博士生导师ꎬ研究方向为海洋微生物资源与海洋环境微生物ꎮ海洋石油污染及其微生物修复研究进展李贵珍１ꎬ２ꎬ㊀赖其良２ꎬ㊀闫培生１ꎬ３∗ꎬ㊀邵宗泽１ꎬ２∗１.哈尔滨工业大学市政环境工程学院ꎬ哈尔滨１５００９０ꎻ２.国家海洋局第三海洋研究所ꎬ海洋生物遗传资源国家重点实验室培育基地ꎬ福建厦门３６１００５ꎻ３.哈尔滨工业大学(威海)海洋科学与技术学院ꎬ山东威海２６４２０９摘㊀要:海洋石油污染严重影响了海洋生态系统平衡和人类健康ꎬ海洋石油污染的微生物修复技术因其自身的优势越来越受到人们的重视ꎮ介绍了海洋石油污染的现状和治理方法ꎬ并着重介绍了海洋中石油污染微生物修复中降解微生物的种类㊁降解机理和生物修复的研究进展ꎬ并指出了生物修复存在并需要克服的问题ꎬ以期为海洋石油污染环境修复研究提供参考ꎮ关键词:海洋ꎻ石油污染ꎻ海洋微生物ꎻ微生物修复ＤＯＩ:１０.３９６９/ｊ.ｉｓｓｎ.２０９５￣２３４１.２０１５.０３.０３ＡｄｖａｎｃｅｏｎＭａｒｉｎｅＰｅｔｒｏｌｅｕｍＰｏｌｌｕｔｉｏｎａｎｄＭｉｃｒｏｂｉａｌＲｅｍｅｄｉａｔｉｏｎＬＩＧｕｉ￣ｚｈｅｎ１ꎬ２ꎬＬＡＩＱｉ￣ｌｉａｎｇ２ꎬＹＡＮＰｅｉ￣ｓｈｅｎｇ１ꎬ３∗ꎬＳＨＡＯＺｏｎｇ￣ｚｅ１ꎬ２∗１.ＳｃｈｏｏｌｏｆＭｕｎｉｃｉｐａｌａｎｄＥｎｖｉｒｏｎｍｅｎｔａｌＥｎｇｉｎｅｅｒｉｎｇꎬＨａｒｂｉｎＩｎｓｔｉｔｕｔｅｏｆＴｅｃｈｎｏｌｏｇｙꎬＨａｒｂｉｎ１５００９０ꎬＣｈｉｎａꎻ２.ＢｒｅｅｄｉｎｇＢａｓｅｏｆＳｔａｔｅＫｅｙＬａｂｏｒａｔｏｒｙｏｆＭａｒｉｎｅＧｅｎｅｔｉｃＲｅｓｏｕｒｃｅｓꎬＴｈｉｒｄＩｎｓｔｉｔｕｔｅｏｆＯｃｅａｎｏｇｒａｐｈｙꎬＳａｔａｅＯｃｅａｎｉｃＡｄｍｉｎｉｓｔｒａｔｉｏｎꎬＦｕｊｉａｎＸｉａｍｅｎ３６１００５ꎬＣｈｉｎａꎻ３.ＳｃｈｏｏｌｏｆＭａｒｉｎｅＳｃｉｅｎｃｅａｎｄＴｅｃｈｎｏｌｏｇｙꎬＨａｒｂｉｎＩｎｓｔｉｔｕｔｅｏｆＴｅｃｈｎｏｌｏｇｙａｔＷｅｉｈａｉꎬＳｈａｎｄｏｎｇＷｅｉｈａｉ２６４２０９ꎬＣｈｉｎａＡｂｓｔｒａｃｔ:Ｍａｒｉｎｅｐｅｔｒｏｌｅｕｍｐｏｌｌｕｔｉｏｎｈａｖｅａｓｅｒｉｏｕｓｅｆｆｅｃｔｏｎｔｈｅｍａｒｉｎｅｅｃｏｓｙｓｔｅｍｓａｎｄｈｕｍａｎｈｅａｌｔｈ.Ｍｉｃｒｏｂｉａｌｒｅｍｅｄｉａｔｉｏｎｔｅｃｈｎｏｌｏｇｙｆｏｒｍａｒｉｎｅｐｅｔｒｏｌｅｕｍｐｏｌｌｕｔｉｏｎｉｓａｔｔｒａｃｔｉｎｇｅｘｔｅｎｓｉｖｅａｔｔｅｎｔｉｏｎｆｏｒｉｔｓａｄｖａｎｔａｇｅｓ.Ｔｈｉｓｐａｐｅｒｉｎｔｒｏｄｕｃｅｄｔｈｅｃｕｒｒｅｎｔｓｉｔｕａｔｉｏｎｏｆｍａｒｉｎｅｐｅｔｒｏｌｅｕｍｐｏｌｌｕｔｉｏｎꎬｔｈｅｔｒｅａｔｍｅｎｔｓｏｆｍａｒｉｎｅｐｅｔｒｏｌｅｕｍｐｏｌｌｕｔｉｏｎꎬａｎｄｍｉｃｒｏｂｉａｌｒｅｍｅｄｉａｔｉｏｎｏｆｍａｒｉｎｅｐｅｔｒｏｌｅｕｍｐｏｌｌｕｔｉｏｎ.Ｔｈｅｐａｐｅｒｍａｉｎｌｙｆｏｃｕｓｅｄｏｎｔｈｅｄｉｖｅｒｓｉｔｙｏｆｐｅｔｒｏｌｅｕｍｄｅｇｒａｄｉｎｇｍｉｃｒｏｏｒｇａｎｉｓｍｓꎬｍｅｃｈａｎｉｓｍｏｆｄｅｇｒａｄａｔｉｏｎａｎｄｔｈｅａｄｖａｎｃｅｏｆｂｏｉｒｅｍｅｄｉａｔｉｏｎ.Ｍｅａｎｗｈｉｌｅꎬｔｈｉｓｐａｐｅｒａｌｓｏｐｏｉｎｔｅｄｏｕｔｔｈｅｐｒｏｂｌｅｍｓｏｆｍｉｃｒｏｂｉａｌｒｅｍｅｄｉａｔｉｏｎｗｈｉｃｈｎｅｅｄｔｏｂｅｏｖｅｒｃｏｍｅｄꎬａｎｄｈｏｐｅｄｔｏｐｒｏｖｉｄｅｕｓｅｆｕｌｉｎｆｏｒｍａｔｉｏｎｓｆｏｒｔｈｅｓｔｕｄｙｏｎｍａｒｉｎｅｅｎｖｉｒｏｎｍｅｎｔａｌｍｉｃｒｏｂｉａｌｒｅｍｅｄｉａｔｉｏｎｏｆｐｅｔｒｏｌｅｕｍｐｏｌｌｕｔｉｏｎ.Ｋｅｙｗｏｒｄｓ:ｍａｒｉｎｅꎻｐｅｔｒｏｌｅｕｍｐｏｌｌｕｔｉｏｎꎻｍａｒｉｎｅｍｉｃｒｏｏｒｇａｎｉｓｍｓꎻｍｉｃｒｏｂｉａｌｒｅｍｅｄｉａｔｉｏｎ㊀㊀随着石油工业化进程的加快ꎬ环境污染问题变得越来越严重ꎮ近年来ꎬ由于海洋溢油事件不断发生ꎬ海洋石油污染受到越来越广泛的关注ꎮ据报道ꎬ全世界平均每年约有１.０ˑ１０１０ｋｇ石油流入海洋ꎬ我国每年有高达１.１５ˑ１０８ｋｇ的石油流入海洋[１]ꎬ石油已经成为海洋环境的主要污染物ꎮ海洋中石油污染的泛滥ꎬ不仅造成了巨大的直接经济损失ꎬ对海洋生态环境的破坏所造成的间接价值的损失更是无法估量ꎮ如何修复受污染的海洋ꎬ也引起人们越来越多的思考ꎮ生物修复(ｂｉｏｒｅｍｅｄｉａｔｉｏｎ)因其为自然降解过程ꎬ具有对人和环境的影响小㊁费用低㊁不易引起二次污染ꎬ并且可以定点修复[２]等优点而得到广泛研究和应用ꎮ本文介绍了石油污染的现状及主要的治理方法ꎬ并着重介绍了微生物修复的微生物种类㊁机理及相关研究进展ꎬ以期为石油污染环境修复提供参考ꎮ１㊀海洋石油污染现状１.１㊀海洋中石油污染的来源海洋中石油的来源主要有４个:①海上油运:主要通过压舱水㊁洗舱水㊁油轮事故和石油码头的泄漏等进入海洋ꎻ②海上油田:海底石油在开采过程中不可避免的油井的井喷㊁油管的破裂等事故会导致大量石油泄入海洋ꎻ③海岸排油:海岸上的各类石油废水直接排入海洋ꎻ④大气石油烃的沉降:由工厂㊁船坞和车辆等排出的石油烃挥发到大气后ꎬ有一部分最终落入海洋[３]ꎮ据统计ꎬ每年全世界石油总产量的０.５％最终会泄入海洋ꎬ每年井喷和运输事故造成的溢油就高达２.２ˑ１０７ｔꎬ我国各种溢油事故平均每年发生５００起ꎬ每年直接排入海洋的石油就有约１０万ｔꎬ大量的石油泄入海洋ꎬ无论是对整个海洋生态环境还是人类社会而言都是极为严重的破坏[４ꎬ５]ꎮ１.２㊀石油污染的危害石油进入海洋后ꎬ主要以水体表面形成的油膜㊁溶解分散㊁凝聚态３种形式存在[６]ꎮ石油污染对海洋造成的危害主要包括生态方面的危害和社会危害两大类[６]ꎮ生态方面危害表现在:①降低光合作用:海水表面的油膜ꎬ阻挡阳光射入海洋ꎬ破坏了海洋中的Ｏ２和ＣＯ２的平衡ꎬ从而影响光合作用ꎻ②影响海气交换:油膜覆盖于海水表面破坏海洋中溶解气体的循环平衡ꎻ③影响海水中的溶解氧ꎻ④毒化作用:石油中的有毒物质ꎬ如芳香烃等具有 三致 作用ꎬ对海洋生物和人类都有很大的危害ꎻ⑤引发赤潮:海洋中石油污染严重的区域ꎬ更容易引发赤潮ꎻ⑥全球效应:石油污染会加剧温室效应ꎬ从而间接引发全球问题ꎮ社会危害主要表现在:①对渔业造成的危害:石油进入海洋ꎬ在海水表面形成油膜ꎬ降低了光合作用效率ꎬ造成海水中的溶解氧含量降低ꎬ破坏海洋中的气体交换平衡ꎬ从而导致鱼类等大量死亡ꎬ严重影响渔业的发展ꎻ②对工农业的危害:石油污染增加了捕捞成本ꎬ许多海上作业企业受到严重影响ꎻ③对旅游业的危害:海洋中的石油会污染近海ꎬ从而影响海滨旅游业的发展ꎻ④对人类健康的危害:石油中含有大量有毒物质ꎬ这些有毒物质可以通过食物链和食物网进行生物累积ꎬ最终危害人类健康ꎮ２㊀海洋石油污染的治理方法海洋石油污染处理方法可以分为物理法㊁化学法和生物法３种ꎮ物理方法主要有:①围栏法:主要是阻止石油在海面上扩散ꎻ②撇油器:在不改变石油性质的基础上ꎬ对石油进行回收ꎻ③吸油材料:用亲油性的材料ꎬ将石油进行吸附回收ꎮ化学方法主要有:①分散剂:可以有效的减少石油与海水间的表面张力ꎬ从而使石油分散成小油株ꎬ有利于微生物对其进行降解ꎻ②凝油剂:可将石油凝成粘稠状或果冻状ꎬ从而有效的防止石油扩散ꎻ③其他化学品ꎮ生物方法主要是生物修复ꎮ生物修复的概念最早是１９９５年由Ｇｌａｚｅｒ和Ｎｉｋａｉｄｏ提出的[７]ꎬ描述微生物降解或清除环境中有害废物的过程ꎮ目前普遍认为ꎬ生物修复是指生物(尤其是微生物)催化降解环境有毒污染物ꎬ减少或最终消除环境污染的受控或自发过程[５]ꎮ生物修复一般可分为广义和狭义生物修复两方面[８]ꎮ广义生物修复指一切以生物技术为主的环境污染的治理技术ꎬ通常分为植物修复㊁动物修复和微生物修复３种类型ꎻ狭义生物修复指通过微生物的作用来清除土壤和水体环境中的污染物ꎬ或使污染物无毒化的过程ꎬ包括自然和人为控制条件下的降解或无毒化过程ꎮ与物理法和化学法相比ꎬ生物修复因其为自然降解过程ꎬ所以具有对人和环境的影响小㊁费用低㊁不易引起二次污染ꎬ并且可以定点修复[２]等优点ꎮ３㊀海洋石油污染的微生物修复３.１㊀可修复石油污染的微生物种类烃类降解菌早在２０世纪初就已发现[９]ꎬ据报道能够利用烃类作为唯一碳源和能源的有７９个细菌属㊁９个蓝藻属㊁１０３个真菌属和１４个藻属[１０]ꎮ据报道ꎬ从海洋环境分离到的可降解石油的微生物有７０个属ꎬ其中细菌就占了４０个属[１１]ꎮ就目前报道的石油降解菌来看ꎬ革兰氏阴性菌比革兰氏阳性菌要多ꎮ在长期的石油污染驯化过程中ꎬ海洋中出现了一类 噬石油烃 细菌ꎬ它们能以石油为唯一碳源生长繁殖ꎬ如利用多环芳香烃(ｐｏｌｙｃｙｃｌｉｃａｒｏｍａｔｉｃｈｙｄｒｏｃａｒｂｏｎｓ)为碳源的解环菌属(Ｃｙｃｌｏ￣５６１李贵珍ꎬ等:海洋石油污染及其微生物修复研究进展ｃｌａｓｔｉｃｕｓ)[１２~１５]㊁假单胞菌属(Ｐｓｅｕｄｏｍｏｎａｓ)[１６]㊁盐单胞菌属(Ｈａｌｏｍｏｎａｓ)[１６ꎬ１７]㊁海杆菌属(Ｍａｒｉ￣ｎｏｂａｃｔｅｒ)[１６ꎬ１７]㊁海旋菌(Ｔｈａｌａｓｓｏｓｐｉｒａ)[１６ꎬ１７]㊁海茎状菌(Ｍａｒｉｃａｕｌｉｓ)[１６]和假交替单胞菌属(Ｐｓｅｕｄ￣ｏａｌｔｅｒｏｍｏｎａｓ)[１７]ꎻ以饱和烷烃及支链烷烃为碳源生长的食烷菌属(Ａｌｃａｎｉｖｏｒａｘ)[１８~２１]ꎻ利用脂肪族烃㊁烷醇和链烷酸酯的嗜油菌属(Ｏｌｅｉｐｈｉｌｕｓ)和油螺旋菌属(Ｏｌｅｉｓｐｉｒａ)[２２ꎬ２３]ꎮ另外ꎬ还有降解荧蒽的速生杆菌属(Ｃｅｌｅｒｉｂａｃｔｅｒ)[２４]ꎮ除此之外ꎬ能够降解石油烃的细菌还有弧菌属(Ｖｉｂｒｉｏ)㊁诺卡氏菌属(Ｎｏｃａｒｄｉａ)㊁微球菌属(Ｍｉｃｒｏｃｏｃｃｕｓ)㊁乳杆菌属(Ｌａｃｔｏｂａｃｉｌｌｕｓ)㊁节杆菌属(Ａｒｔｈｒｏｂａｃｔｅｒ)㊁不动杆菌属(Ａｃｉｎｅｔｏｂａｃｔｅｒ)㊁葡萄球菌属(Ｓｔａｐｈｙ￣ｌｏｃｏｃｃｕｓ)㊁棒杆菌属(Ｃｏｒｙｈｅｂａｃｔｅｒｉｕｍ)㊁芽孢杆菌属(Ｂａｃｉｌｌｕｓ)㊁产碱杆菌属(Ａｌｃａｌｉｇｅｎｅｓ)㊁黄杆菌属(Ｆｌａｖｏｂａｃｔｅｒｉｕｍ)㊁气单胞菌属(Ａｅｒｏｍｏｎａｓ)㊁肠杆菌科(Ｅｎｔｅｒｏｂａｃｔｅｒｉａｃｅａｅ)和无色杆菌属(Ａｃｈｒｏ￣ｍｏｂａｃｔｅｒ)等[３]ꎮ海洋中能够降解石油烃的真菌主要是霉菌和酵母菌ꎬ霉菌如小克银汉霉菌(Ｃｕｎｎｉｎｇｈａｍｅｌｌａ)㊁曲霉属(Ａｐｅｒｇｉｌｌｕｓ)[２５]㊁头孢霉属(Ｃｅｐｈａｌｏｓｐｏｒｉｕｍ)㊁镰孢霉属(Ｆｕｓａｒｉｕｍ)和青霉属(Ｐｅｎｉｃｉｌｌｉｕｍ)等[２６]ꎬ但其数量远远少于细菌ꎮ能够降解石油烃的酵母菌主要有亚罗酵母属(Ｙａｒｒｏｗｉａ)[２７ꎬ２８]㊁假丝酵母属(Ｃａｎｄｉｄａ)[２５ꎬ２９ꎬ３０]㊁毕赤氏酵母菌属(Ｐｉｃｈｉａ)和红酵母菌属(Ｒｈｏｄｏｔｏｒｕｌａ)等[３ꎬ３０]ꎮ３.２㊀石油污染微生物修复机理石油是一种十分复杂的混合物ꎬ包括直链烷烃㊁环状烷烃㊁芳香烃和非烃类物质等ꎮ微生物对石油烃类的降解过程本质上为生物氧化过程ꎮ代谢用途主要分以下３大类:①石油烃被彻底氧化分解成二氧化碳和水ꎻ②石油烃被合成为微生物自身生命物质ꎬ如核酸㊁蛋白质和糖类等ꎻ③石油烃被转化为其他物质ꎬ例如脂肪酸㊁苯酚和醇等ꎮ石油烃类的降解主要分为以下几种:①烷烃的降解ꎮ烷烃的生物降解是一系列酶促反应过程[１１]ꎬ烷烃第一步氧化为相应的伯醇ꎬ伯醇再氧化成醛ꎬ醛再转化为相应的脂肪酸ꎬ脂肪酸再进行β￣氧化后转化为乙酰辅酶Ａꎬ乙酰辅酶Ａ再进行氧化分解或其他转化ꎮ链状烷烃可经脱氢步骤转变为烯ꎬ烯再氧化为醇ꎬ醇氧化成醛ꎬ然后醛可转化为脂肪酸ꎻ此外ꎬ链状烷烃还可以通过直接氧化成烷基过氧化氢ꎬ然后经脂肪酸途径进行降解ꎮ有些微生物可以通过亚末端氧化ꎬ形成仲醇ꎬ再转化成伯醇或脂肪酸进行氧化分解ꎮ也有些微生物将烯烃转化为不饱和脂肪酸ꎬ再通过双键位移或甲基化等ꎬ形成支链脂肪酸ꎬ进行氧化分解ꎮ②环烷烃的降解ꎮ环状烷烃的降解和链状烷烃亚末端氧化十分相似ꎬ首先氧化为环烷醇ꎬ再脱氢变为酮ꎬ而后氧化成内酯或直接开环变为脂肪酸[３]ꎮ③苯及其衍生物的降解ꎮ苯及短链烷基苯转化为二醇中间体ꎬ再进一步转化为邻苯二酚或取代基邻苯二酚ꎬ最后变为羧酸[３]ꎮ④多环芳烃的降解ꎮ多环芳烃具有 三致 作用ꎬ因此ꎬ人们对其降解十分重视ꎮ多环芳烃的降解ꎬ首先需要微生物产生加氧酶进行氧化定位[３]:细菌一般产生双加氧酶ꎬ两个氧原子加到苯环上ꎬ变成过氧化物ꎬ而后转化为顺式二醇ꎬ再脱掉氢变成酚ꎻ真菌一般能够产生单加氧酶ꎬ在单加氧酶的作用下ꎬ将一个氧原子直接加到苯环上ꎬ从而形成环氧化物ꎬ然后加水转化成反式二醇和酚ꎮ多环及杂环破裂是杂环化合物和多环芳烃降解的限速步骤[３１]ꎮ４㊀海洋石油污染微生物修复研究进展４.１㊀实验室模拟研究进展石油烃降解菌在海洋中广泛存在ꎮ早在２０世纪４０年代ꎬ各国就陆续开展了石油烃的生物降解及环境修复研究ꎮ我国在２０世纪７０年代开始研究石油烃的生物降解ꎬ也陆续出现了大量石油烃的相关报道ꎬ近年来ꎬ实验室研究主要集中于高效降解条件的优化㊁高效降解菌株的筛选及降解底物范围等方面ꎮ４.１.１㊀高效降解条件的优化㊀２０１１年ꎬ周瑜等[３２]使用寡营养培养基对威海金海湾油污进行富集培养ꎬ获得了６株石油降解菌ꎬ分属于假单胞菌属(Ｐｓｅｕｄｏｍｏｎａｓ)㊁芽孢杆菌属(Ｂａｃｉｌｌｕｓ)和无色杆菌属(Ａｃｈｒｏｍｏｂａｃｔｅｒ)ꎮ为了提高降解效率ꎬ他们将筛选到的细菌与分离到的微藻进行共培养ꎬ培养３ｄ后降解效率就可提高３.７９％~７ ９１％ꎮ数据表明ꎬ利用细菌与微藻的共生关系可以促进细菌对石油的降解ꎬ这在石油污染生物修复方面具有重要的实际应用价值ꎮ２０１３年ꎬＨｏｕ等[３３]筛选到一株不动杆菌Ａｃｉｎｅｔｏｂａｃｔｅｒｓｐ.Ｆ９ꎬ并将其固定化ꎬ研究发现ꎬ固定化后的菌剂在２ｄ后的降解率可以达到９０％ꎬ而游离状态下的６６１生物技术进展ＣｕｒｒｅｎｔＢｉｏｔｅｃｈｎｏｌｏｇｙ菌剂在７ｄ后的降解率还达不到９０％ꎮ２０１４年ꎬ李馨子等[３４]筛选到一株食烷菌Ａｌｃａｎｉｖｏｒａｘｓｐ.９７ＣＯ￣５ꎬ研究了其降解的石油效果ꎬ并进行了固定化ꎬ发现固定化后的菌剂对石油的降解率优于游离菌株ꎮ４.１.２㊀高效降解菌株的筛选及降解底物范围测定㊀２００８年ꎬ苏莹等[３５]从胜利油田污水中ꎬ以人工海水培养基进行富集培养得到一株适合海洋石油污染修复的菌株ＨＢ￣１ꎬ该菌株具有较强的原油降解能力ꎬ２００ｒ/ｍｉｎ振荡培养６ｄ后ꎬ原油的降解率可达５４.７４％ꎬ经１６ＳｒＤＮＡ序列分析ꎬ鉴定该菌为不动杆菌属(Ａｃｉｎｅｔｏｂａｃｔｅｒｓｐ.)ꎮ２０１０年ꎬ张月梅等[３６]从北极筛选到５０株以石油为唯一碳源的嗜冷降解菌ꎬ其中降解效率最高的３株ＢＪ１㊁ＢＪ９和ＢＪ１９都属于假交替单胞菌属(Ｐｓｅｕｄｏａｌｔｅｒ￣ｏｍｏｎａｓ)ꎮ这３株菌在１０~２０ħ的范围内均有生长ꎬ在温度为５ħ时的降解率均高于３０％ꎬ在最适温度下的降解率可达４５.７８％~６０.３２％ꎮ此外ꎬ这３株菌的碳源还具有广谱性ꎬ可分别以柴油㊁汽油㊁原油㊁海燃油㊁燃油㊁正十八烷㊁正二十四烷㊁萘和菲偶氮苯等为唯一碳源生长ꎮ２０１４年ꎬ同济大学的王鑫等[３７]从石油污染的海水中筛选到６株石油降解菌ꎬ并对其进行了菌群构建ꎬ结果表明ꎬ混合菌群对石油的降解率明显高于单菌ꎬ且菌株间具有明显的协同作用ꎮ２０１５年ꎬ张爱君等[３８]从渤海筛选到一株假交替单胞菌(Ｐｓｅｕｄｏａｌｔｅｒｏｍｏｎｓｐ.)ꎬ发现在最适条件下ꎬ其石油降解率可以达到７５.７１％ꎮＲａｇｈｕｋｕｍａｒ等[３９]的研究发现ꎬ海洋中的蓝细菌ＯｓｃｉｌｌａｔｏｒｉａｓａｌｉｎａＢｉｓ￣ｗａｓ㊁ＰｌｅｃｔｏｎｅｍａｔｅｒｅｂｒａｎｓＢｏｒｎｅｔｅｔＦｌａｈａｕｌｔ和Ａｐｈ￣ａｎｏｃａｐｓａｓｐ.在人工海水培养的条件下可以降解原油ꎬ通过重量法和气象色谱法测得１０ｄ内石油的去除率可以达到４５％~５５％(包括５０％的脂肪族化合物㊁３１％的石蜡和沥青㊁１４％的芳香烃和５％的极性化合物)ꎮ食烷菌(Ａｌｃａｎｉｖｏｒａｘ)是海洋中烷烃降解菌的重要组成部分ꎬＷａｎｇ等[４０]研究发现Ａ.ｄｉｅｓｅｌｏｌｅｉＢ５能够很好的降解链长为Ｃ６~Ｃ３６烷烃ꎬ包括支链烷烃ꎬ并深入研究了其降解长链烷烃的代谢网络调控机制ꎮ４.２㊀现场应用研究进展随着实验室对生物修复研究的不断成熟ꎬ生物修复技术从实验室开始ꎬ已经逐步进入了实际应用阶段ꎮ１９８９年美国ＡｌａｓｋａＥｘｘｏｎＶａｌｄｅｚ邮轮泄漏ꎬ约３５５００ｔ原油泄入海洋ꎬ泄漏发生后ꎬＢｒａｄｄｏｃｋ等[４１]连续３年对泄漏点威廉王子海湾的潮间带和潮下的沉积物中烃类降解微生物数量进行检测ꎬ数据显示ꎬ油膜路径地点的烃类降解微生物的数量远远超过对照组的数量ꎬ说明烃类降解菌有快速的环境适应性及修复污染环境的能力ꎮ１９９７年１月ꎬ约５０００ｔ石油从俄罗斯的纳霍德卡港泄漏ꎬ１２００ｋｍ的日本海岸受到严重污染ꎬ日本组织奥本海默生物科技公司(ＴｅｒｒａＺｙｍｅＴＭ)进行生物修复ꎬ３周后约３５％的石油得到降解[４２]ꎮ２０１２年ꎬ郑立等[４３]从海洋中筛选的石油降解菌剂在大连溢油污染岸滩修复实验中起到了良好的效果ꎬ在１２ｄ的潮间带油污生物修复中ꎬ喷洒菌剂处理区域的Ｃ１７/藿烷和Ｃ１８/藿烷降解率比对照组高４０％和３０％ꎬ总烷烃和总芳香烃降解率高８０％和７２％ꎬ说明此菌剂确实可以加快石油污染的生物修复过程ꎮ研究证明ꎬ海洋中存在着大量可降解石油的微生物ꎬ这为石油污染的生物修复治理提供了大量微生物资源ꎮ目前ꎬ微生物修复中的最大问题是生物降解能力不够理想ꎬ为了提高微生物降解石油的能力ꎬ目前采用的方法主要有接种高效石油降解菌㊁添加表面活性剂和添加营养盐等方法ꎮ①接种高效石油降解菌:通过接种高效石油降解菌改变污染区域的菌群结构ꎬ达到快速高效降解石油的目的ꎮ为了提高微生物降解石油的效率ꎬ许多学者还将菌剂进行固定化[３３ꎬ３４]ꎬ从而提高降解率ꎮ从目前的研究状况来看ꎬ通过接种高效石油降解菌的方法并不十分理想ꎬ因为海洋中存在的土著微生物会影响石油降解菌的活性ꎮ此外ꎬ也有学者对接种外来菌群是否会带来环境安全问题存有疑虑ꎮ②添加表面活性剂:表面活性剂可将石油疏散开ꎬ增大微生物与石油的接触面积ꎬ从而加速微生物对石油的降解ꎮ需要注意的是ꎬ不是所有的表面活性剂都可以加速石油的降解ꎬ许多表面活性剂由于自身具有很大毒性不仅不会加速石油的降解还会造成二次污染ꎮ例如ꎬ在１９６７年ＴｏｒｒｅｙＣａｎｙｏｎ油轮污染事件的修复中ꎬ约１００００ｔ的分散剂被投入使用ꎬ造成了严重的环境破坏[４４]ꎮ③添加营养盐:海洋受到石油污染ꎬ在碳源充足的条件下ꎬ环境中存在的石油降解菌群会大量７６１李贵珍ꎬ等:海洋石油污染及其微生物修复研究进展繁殖ꎬ但营养盐和氧气无法满足需求ꎬ因此通过投加营养盐的方法可以大大提高微生物降解石油的效率ꎬ降解效率甚至会提高几倍[４５]ꎮ营养盐类型一般分为缓释型㊁亲油型和水溶型３种[４６]ꎮ但由于海洋面积大ꎬ稀释能力强ꎬ所以要根据具体情况投加合适的营养盐ꎮ另外ꎬ海洋添加营养盐是否会引起环境某种程度的富营养化等问题也需要进一步探究ꎮ５　展望海洋石油污染呈现逐年加重的趋势ꎮ海洋中降解石油的微生物种类繁多ꎬ数量庞大ꎮ生物修复技术与化学修复㊁物理修复相比具有对人和环境影响小㊁费用低㊁不易引起二次污染等优势[２]ꎮ经过多年的研究ꎬ生物修复技术在石油污染修复中逐渐成为核心技术ꎮ但它也存在着一些不足ꎬ如见效慢㊁易受环境影响等ꎮ石油烃的生物降解过程十分复杂ꎬ降解效率主要受石油的理化性质㊁微生物的种类和环境参数的影响ꎮ环境参数主要是温度㊁盐度㊁营养浓度和ｐＨ等ꎬ这也是生物修复技术需要克服的问题ꎮ为解决这些问题ꎬ我们可以在以下方面进行改进:首先ꎬ在生物修复高效菌株的选择上ꎬ可以就地筛选出高效的石油降解菌ꎬ然后再投放回筛选地点进行生物修复ꎬ这样可以有效的避免外来微生物投加而引起的生态安全问题ꎮ其次ꎬ添加表面活性剂产生菌ꎬ许多微生物都可以产生表面活性剂ꎬ这些表面活性剂与化学表面活性剂相比较更安全可靠ꎬ我们可以将表面活性剂产生菌和高效降解菌株合理配比后投放到治理场地ꎬ这样表面活性剂产生菌株产生的表面活性剂可以有效提高石油降解菌株与石油的接触面积ꎬ从而在不添加化学分散剂的条件下ꎬ大大提高石油的降解效率ꎮ再次ꎬ营养盐的添加:大范围的营养盐开放式的添加不仅会造成营养盐的浪费而且还会造成水体富营养化ꎬ同时也大大增加了生物修复的成本ꎮ为了解决这个问题ꎬ可将营养盐与菌株进行漂浮固定ꎬ这样不仅大大降低了营养盐的添加量ꎬ而且也不会因大范围扩散而造成浪费ꎬ又可以在相当长的时间内满足降解菌株的需要ꎬ从而更经济㊁有效的提高生物修复的效率ꎮ总之ꎬ在经济快速发展的今天ꎬ海洋石油污染变得越来越严重ꎬ采用生物修复技术进行污染物降解清除ꎬ值得我们继续深入研究ꎮ参㊀考㊀文㊀献[１]㊀曲维政ꎬ邓声贵.灾难性的海洋石油污染[Ｊ].自然灾难学报ꎬ２００１ꎬ１０(１):６９－７４.[２]㊀ＶｉｄａｌｉＭ.Ｂｉｏｒｅｍｅｄｉａｔｉｏｎ.Ａｎｏｖｅｒｖｉｅｗ[Ｊ].ＰｕｒｅＡｐｐｌ.Ｃｈｅｍ.ꎬ２００１ꎬ７３(３):１１６３－１１７２.[３]㊀宋志文.海洋石油污染物的微生物降解与生物修复[Ｊ].生态学杂志ꎬ２００４ꎬ２３(３):９９－１０２.[４]㊀徐金兰ꎬ黄廷林ꎬ唐智新ꎬ等.高效石油降解菌的筛选及石油污染土壤生物修复特性的研究[Ｊ].环境科学学报ꎬ２００７ꎬ２７(４):６２２－６２８.[５]㊀ＡｔｌａｓＲＭ.Ｂｉｏｒｅｍｅｄｉａｔｉｏｎｏｆｐｅｔｒｏｌｅｕｍｐｏｌｌｕｔａｎｔｓ[Ｊ].Ｉｎｔｅｒｎａｔ.Ｂｉｏｄｅｔｅｒ.Ｂｉｏｄｅｇｒ.ꎬ１９９５ꎬ３５(１):３１７－３２７. [６]㊀赵庆良ꎬ李伟光.特种废水处理技术[Ｍ].哈尔滨:哈尔滨工业大学出版社ꎬ２００８.[７]㊀ＧｌａｚｅｒＡＮꎬＮｉｋａｉｄｏＨ.ＭｉｃｒｏｂｉａｌＢｉｏｔｅｃｈｎｏｌｏｇｙ:Ｆｕｎｄａ￣ｍｅｎ￣ｔａｌｓｏｆＡｐｐｌｉｅｄＭｉｃｒｏｂｉｏｌｏｇｙ[Ｍ]:Ｃａｍｂｒｉｄｇｅ:ＣａｍｂｒｉｄｇｅＵｎｉ￣ｖｅｒｓｉｔｙＰｒｅｓｓꎬ２００７.[８]㊀许晔ꎬ刘生瑶ꎬ曾铮.浅析生物修复技术在石油污染治理中的应用[Ｊ].油气田环境保护ꎬ２００８ꎬ１８(４):５０－５２. [９]㊀ＳöｈｎｇｅｎＮ.Ｂｅｎｚｉｎꎬｐｅｔｒｏｌｅｕｍꎬｐａｒａｆｆｉｎöｌｕｎｄｐａｒａｆｆｉｎａｌｓｋｏｈｌｅｎｓｔｏｆｆ￣ｕｎｄｅｎｅｒｇｉｅｑｕｅｌｌｅｆüｒｍｉｋｒｏｂｅｎ[Ｊ].ＺｅｎｔｒａｌｂｌａｔｔｆüｒＢａｋｔｅｒｉｏｌｏｇｉｅꎬＰａｒａｓｉｔｅｎｋｕｎｄｅｕｎｄＩｎｆｅｋｔｉｏｎｓｋｒａｎｋｈｅｉｔｅｎꎬ１９１３ꎬ３７:５９５－６０９.[１０]㊀ＰｒｉｎｃｅＲ.ＰｅｔｒｏｌｅｕｍＭｉｃｒｏｂｉｏｌｏｇｙ[Ｍ].ＷａｓｈｉｎｇｔｏｎＤＣ:Ａ￣ｍｅｒｉｃａｎＳｏｃｉｅｔｙｏｆＭｉｃｒｏｂｉｏｌｏｇｙＰｒｅｓｓꎬ２００５ꎬ３１７－３３６. [１１]㊀张士璀ꎬ晓范ꎬ马军英.海洋生物技术和应用(第二版) [Ｍ].北京:海洋生物技术出版社ꎬ１９９７.[１２]㊀ＳｔａｌｅｙＪ.Ｃｙｃｌｏｃｌａｓｔｉｃｕｓ:Ａｇｅｎｕｓｏｆｍａｒｉｎｅｐｏｌｙｃｙｃｌｉｃａｒｏｍａｔｉｃｈｙｄｒｏｃａｒｂｏｎｄｅｇｒａｄｉｎｇｂａｃｔｅｒｉａ[Ａ].Ｉｎ:Ｈａｎｄｂｏｏｋｏｆｈｙｄｒｏ￣ｃａｒｂｏｎａｎｄｌｉｐｉｄｍｉｃｒｏｂｉｏｌｏｇｙ[Ｍ].Ｓｐｒｉｎｇｅｒꎬ２０１０ꎬ１７８１－１７８６.[１３]㊀ＫａｓａｉＹꎬＫｉｓｈｉｒａＨꎬＨａｒａｙａｍａＳ.Ｂａｃｔｅｒｉａｂｅｌｏｎｇｉｎｇｔｏｔｈｅｇｅ￣ｎｕｓＣｙｃｌｏｃｌａｓｔｉｃｕｓｐｌａｙａｐｒｉｍａｒｙｒｏｌｅｉｎｔｈｅｄｅｇｒａｄａｔｉｏｎｏｆａｒｏ￣ｍａｔｉｃｈｙｄｒｏｃａｒｂｏｎｓｒｅｌｅａｓｅｄｉｎａｍａｒｉｎｅｅｎｖｉｒｏｎｍｅｎｔ[Ｊ].Ａｐｐｌ.Ｅｎｖｉｒｏｎ.Ｍｉｃｒｏｂｉｏｌ.ꎬ２００２ꎬ６８(１１):５６２５－５６３３. [１４]㊀ＷａｎｇＢꎬＬａｉＱꎬＣｕｉＺꎬｅｔａｌ..Ａｐｙｒｅｎｅ￣ｄｅｇｒａｄｉｎｇｃｏｎｓｏｒｔｉｕｍｆｒｏｍｄｅｅｐ￣ｓｅａｓｅｄｉｍｅｎｔｏｆｔｈｅＷｅｓｔＰａｃｉｆｉｃａｎｄｉｔｓｋｅｙｍｅｍｂｅｒＣｙｃｌｏｃｌａｓｔｉｃｕｓｓｐ.Ｐ１[Ｊ].Ｅｎｖｉｒｏｎ.Ｍｉｃｒｏｂｉｏｌ.ꎬ２００８ꎬ１０(８):１９４８－１９６３.[１５]㊀ＳｈａｏＺꎬＣｕｉＺꎬＤｏｎｇＣꎬｅｔａｌ..ＡｎａｌｙｓｉｓｏｆａＰＡＨ￣ｄｅｇｒａｄｉｎｇｂａｃｔｅｒｉａｌｐｏｐｕｌａｔｉｏｎｉｎｓｕｂｓｕｒｆａｃｅｓｅｄｉｍｅｎｔｓｏｎｔｈｅＭｉｄ￣ＡｔｌａｎｔｉｃＲｉｄｇｅ[Ｊ].ＤｅｅｐＳｅａＲｅｓ.ＰａｒｔＩ:Ｏｃｅａｎｏｇｒ.Ｒｅｓ.Ｐａ￣ｐｅｒｓꎬ２０１０ꎬ５７(５):７２４－７３０.[１６]㊀ＭａＹꎬＷａｎｇＬꎬＳｈａｏＺ.Ｐｓｅｕｄｏｍｏｎａｓꎬｔｈｅｄｏｍｉｎａｎｔｐｏｌｙｃｙｃｌｉｃａｒｏｍａｔｉｃｈｙｄｒｏｃａｒｂｏｎ￣ｄｅｇｒａｄｉｎｇｂａｃｔｅｒｉａｉｓｏｌａｔｅｄｆｒｏｍＡｎｔａｒｃｔｉｃｓｏｉｌｓａｎｄｔｈｅｒｏｌｅｏｆｌａｒｇｅｐｌａｓｍｉｄｓｉｎｈｏｒｉｚｏｎｔａｌｇｅｎｅｔｒａｎｓｆｅｒ[Ｊ].Ｅｎｖｉｒｏｎ.Ｍｉｃｒｏｂｉｏｌ.ꎬ２００６ꎬ８(３):４５５－４６５.[１７]㊀ＣｕｉＺꎬＬａｉＱꎬＤｏｎｇＣꎬｅｔａｌ..Ｂｉｏｄｉｖｅｒｓｉｔｙｏｆｐｏｌｙｃｙｃｌｉｃａｒｏ￣ｍａｔｉｃｈｙｄｒｏｃａｒｂｏｎ￣ｄｅｇｒａｄｉｎｇｂａｃｔｅｒｉａｆｒｏｍｄｅｅｐｓｅａｓｅｄｉｍｅｎｔｓｏｆｔｈｅＭｉｄｄｌｅＡｔｌａｎｔｉｃｒｉｄｇｅ[Ｊ].Ｅｎｖｉｒｏｎ.Ｍｉｃｒｏｂｉｏｌ.ꎬ２００８ꎬ１０８６１生物技术进展ＣｕｒｒｅｎｔＢｉｏｔｅｃｈｎｏｌｏｇｙ(８):２１３８－２１４９.[１８]㊀ＧｏｌｙｓｈｉｎＰＮꎬＤｏｓＳａｎｔｏｓＶＡＭꎬＫａｉｓｅｒＯꎬｅｔａｌ..ＧｅｎｏｍｅｓｅｑｕｅｎｃｅｃｏｍｐｌｅｔｅｄｏｆＡｌｃａｎｉｖｏｒａｘｂｏｒｋｕｍｅｎｓｉｓꎬａｈｙｄｒｏｃａｒｂｏｎ￣ｄｅｇｒａｄｉｎｇｂａｃｔｅｒｉｕｍｔｈａｔｐｌａｙｓａｇｌｏｂａｌｒｏｌｅｉｎｏｉｌｒｅｍｏｖａｌｆｒｏｍｍａｒｉｎｅｓｙｓｔｅｍｓ[Ｊ].Ｊ.Ｂｉｏｔｅｃｈｎｏｌ.ꎬ２００３ꎬ１０６(２):２１５－２２０. [１９]㊀ＬｉｕＣꎬＳｈａｏＺ.Ａｌｃａｎｉｖｏｒａｘｄｉｅｓｅｌｏｌｅｉｓｐ.ｎｏｖ.ꎬａｎｏｖｅｌａｌｋａｎｅ￣ｄｅｇｒａｄｉｎｇｂａｃｔｅｒｉｕｍｉｓｏｌａｔｅｄｆｒｏｍｓｅａｗａｔｅｒａｎｄｄｅｅｐ￣ｓｅａｓｅｄｉ￣ｍｅｎｔ[Ｊ].Ｉｎｔｅｒｎａｔ.Ｊ.Ｓｙｓｔ.Ｅｖｏｌ.Ｍｉｃｒｏｂｉｏｌ.ꎬ２００５ꎬ５５(３):１１８１－１１８６.[２０]㊀ＱｉａｏＮꎬＳｈａｏＺ.Ｉｓｏｌａｔｉｏｎａｎｄｃｈａｒａｃｔｅｒｉｚａｔｉｏｎｏｆａｎｏｖｅｌｂｉｏ￣ｓｕｒｆａｃｔａｎｔｐｒｏｄｕｃｅｄｂｙｈｙｄｒｏｃａｒｂｏｎ￣ｄｅｇｒａｄｉｎｇｂａｃｔｅｒｉｕｍＡｌｃａ￣ｎｉｖｏｒａｘｄｉｅｓｅｌｏｌｅｉＢ￣５[Ｊ].Ｊ.Ａｐｐｌ.Ｍｉｃｒｏｂｉｏｌ.ꎬ２０１０ꎬ１０８(４):１２０７－１２１６.[２１]㊀ＷｕＹꎬＬａｉＱꎬＺｈｏｕＺꎬｅｔａｌ..Ａｌｃａｎｉｖｏｒａｘｈｏｎｇｄｅｎｇｅｎｓｉｓｓｐ.ｎｏｖ.ꎬａｎａｌｋａｎｅ￣ｄｅｇｒａｄｉｎｇｂａｃｔｅｒｉｕｍｉｓｏｌａｔｅｄｆｒｏｍｓｕｒｆａｃｅｓｅａ￣ｗａｔｅｒｏｆｔｈｅｓｔｒａｉｔｓｏｆＭａｌａｃｃａａｎｄＳｉｎｇａｐｏｒｅꎬｐｒｏｄｕｃｉｎｇａｌｉ￣ｐｏｐｅｐｔｉｄｅａｓｉｔｓｂｉｏｓｕｒｆａｃｔａｎｔ[Ｊ].Ｉｎｔｅｒｎａｔ.Ｊ.Ｓｙｓｔ.Ｅｖｏｌ.Ｍｉ￣ｃｒｏｂｉｏｌ.ꎬ２００９ꎬ５９(６):１４７４－１４７９.[２２]㊀ＴｉｍｍｉｓＫＮꎬＭｃＧｅｎｉｔｙＴꎬＶａｎＤｅｒＭｅｅｒＪꎬｅｔａｌ..ＨａｎｄｂｏｏｋｏｆＨｙｄｒｏｃａｒｂｏｎａｎｄＬｉｐｉｄＭｉｃｒｏｂｉｏｌｏｇｙ[Ｍ].Ｈｅｉｄｅｌｂｅｒｇ:ＳｐｒｉｎｇｅｒＢｅｒｌｉｎꎬ２０１０ꎬ１７４９－１７５４.[２３]㊀ＨａｚｅｎＴＣꎬＤｕｂｉｎｓｋｙＥＡꎬＤｅＳａｎｔｉｓＴＺꎬｅｔａｌ..Ｄｅｅｐ￣ｓｅａｏｉｌｐｌｕｍｅｅｎｒｉｃｈｅｓｉｎｄｉｇｅｎｏｕｓｏｉｌ￣ｄｅｇｒａｄｉｎｇｂａｃｔｅｒｉａ[Ｊ].Ｓｃｉｅｎｃｅꎬ２０１０ꎬ３３０(６００１):２０４－２０８.[２４]㊀ＣａｏＪꎬＬａｉＱꎬＹｕａｎＪꎬｅｔａｌ..ＧｅｎｏｍｉｃａｎｄｍｅｔａｂｏｌｉｃａｎａｌｙｓｉｓｏｆｆｌｕｏｒａｎｔｈｅｎｅｄｅｇｒａｄａｔｉｏｎｐａｔｈｗａｙｉｎＣｅｌｅｒｉｂａｃｔｅｒｉｎｄｉｃｕｓＰ７３Ｔ[Ｊ].Ｓｃｉ.Ｒｅｐ.ꎬ２０１５ꎬｄｏｉ:１０.１０３８/ｓｒｅｐ０７７４１. [２５]㊀ＦｅｄｏｒａｋＰꎬＳｅｍｐｌｅＫꎬＷｅｓｔｌａｋｅＤ.Ｏｉｌ￣ｄｅｇｒａｄｉｎｇｃａｐａｂｉｌｉｔｉｅｓｏｆｙｅａｓｔｓａｎｄｆｕｎｇｉｉｓｏｌａｔｅｄｆｒｏｍｃｏａｓｔａｌｍａｒｉｎｅｅｎｖｉｒｏｎｍｅｎｔｓ[Ｊ].Ｃａｎ.Ｊ.Ｍｉｃｒｏｂｉｏｌ.ꎬ１９８４ꎬ３０(５):５６５－５７１. [２６]㊀ＣｅｒｎｉｇｌｉａＣꎬＰｅｒｒｙＪ.Ｃｒｕｄｅｏｉｌｄｅｇｒａｄａｔｉｏｎｂｙｍｉｃｒｏｏｒｇａｎｉｓｍｓｉｓｏｌａｔｅｄｆｒｏｍｔｈｅｍａｒｉｎｅｅｎｖｉｒｏｎｍｅｎｔ[Ｊ].ＺｅｉｔｓｃｈｒｉｆｔＦüｒＡｌｌｇｅ￣ｍｅｉｎｅＭｉｋｒｏｂｉｏｌ.ꎬ１９７３ꎬ１３(４):２９９－３０６.[２７]㊀ＯｓｗａｌＮꎬＳａｒｍａＰꎬＺｉｎｊａｒｄｅＳꎬｅｔａｌ..Ｐａｌｍｏｉｌｍｉｌｌｅｆｆｌｕｅｎｔｔｒｅａｔｍｅｎｔｂｙａｔｒｏｐｉｃａｌｍａｒｉｎｅｙｅａｓｔ[Ｊ].Ｂｉｏｒｅｓｏｕｒ.Ｔｅｃｈｎｏｌ.ꎬ２００２ꎬ８５(１):３５－３７.[２８]㊀ＡｈｅａｒｎＤＧꎬＭｅｙｅｒｓＳＰ.Ｆｕｎｇａｌｄｅｇｒａｄａｔｉｏｎｏｆｏｉｌｉｎｔｈｅｍａ￣ｒｉｎｅｅｎｖｉｒｏｎｍｅｎｔ[Ａ].Ｉｎ:Ｇａｒｅｔｈ￣ＪｏｎｅｓＥＢ.ＲｅｃｅｎｔＡｄｖａｎｃｅｓｉｎＡｑｕａｔｉｃＭｙｃｏｌｏｇｙ[Ｍ].ＮｅｗＹｏｒｋ:ＪｈｏｎＷｉｌｅｙ＆Ｓｏｎｓꎬ１９７６ꎬ１２５－１３３.[２９]㊀ＯｋｐｏｋｗａｓｉｌｉＧꎬＡｍａｎｃｈｕｋｗｕＳ.Ｐｅｔｒｏｌｅｕｍｈｙｄｒｏｃａｒｂｏｎｄｅｇｒａ￣ｄａｔｉｏｎｂｙＣａｎｄｉｄａｓｐｅｃｉｅｓ[Ｊ].Ｅｎｖｉｒｏｎ.Ｉｎｔｅｒｎａｔ.ꎬ１９８８ꎬ１４(３):２４３－２４７.[３０]㊀ＭａｒｇｅｓｉｎＲꎬＧａｎｄｅｒＳꎬＺａｃｋｅＧꎬｅｔａｌ..Ｈｙｄｒｏｃａｒｂｏｎｄｅｇｒａｄａ￣ｔｉｏｎａｎｄｅｎｚｙｍｅａｃｔｉｖｉｔｉｅｓｏｆｃｏｌｄ￣ａｄａｐｔｅｄｂａｃｔｅｒｉａａｎｄｙｅａｓｔｓ[Ｊ].Ｅｘｔｒｅｍｏｐｈｉｌｅｓꎬ２００３ꎬ７(６):４５１－４５８.[３１]㊀许俊强ꎬ郭芳ꎬ全学军ꎬ等.焦化废水中的杂环化合物及多环芳烃降解的研究进展[Ｊ].化工进展ꎬ２００８ꎬ２７(７):９７３－９７６.[３２]㊀周瑜ꎬ柴迎梅ꎬ杜宗军ꎬ等.海洋石油降解菌的分离㊁鉴定和与微藻共培养[Ｊ].海洋环境科学ꎬ２０１１ꎬ３０(２):２３０－２３３.[３３]㊀ＨｏｕＤꎬＳｈｅｎＸꎬＬｕｏＱꎬｅｔａｌ..Ｅｎｈａｎｃｅｍｅｎｔｏｆｔｈｅｄｉｅｓｅｌｏｉｌｄｅｇｒａｄａｔｉｏｎａｂｉｌｉｔｙｏｆａｍａｒｉｎｅｂａｃｔｅｒｉａｌｓｔｒａｉｎｂｙｉｍｍｏｂｉｌｉｚａｔｉｏｎｏｎａｎｏｖｅｌｃｏｍｐｏｕｎｄｃａｒｒｉｅｒｍａｔｅｒｉａｌ[Ｊ].ＭａｒｉｎｅＰｏｌｌ.Ｂｕｌｌ.ꎬ２０１３ꎬ６７(１):１４６－１５１.[３４]㊀李馨子ꎬ高伟ꎬ崔志松ꎬ等.海洋石油降解菌Ａｌｃａｎｉｖｏｒａｘｓｐ.９７ＣＯ￣５的固定化及其石油降解效果[Ｊ].海洋环境科学ꎬ２０１４ꎬ３３(３):３８３－３８８.[３５]㊀苏莹ꎬ陈莉ꎬ汪辉ꎬ等.海洋石油降解菌的筛选与降解特性[Ｊ].应用与环境生物学报ꎬ２００８ꎬ１４(４):５１８－５２２. [３６]㊀张月梅ꎬ祖国仁ꎬ那广水ꎬ等.北极耐冷石油降解菌的筛选㊁鉴定及其碳源利用广谱性[Ｊ].海洋环境科学ꎬ２０１０ꎬ２９(２):２１６－２２０.[３７]㊀王鑫ꎬ王学江ꎬ刘免ꎬ等.高效石油降解菌群构建及降解性能[Ｊ].海洋环境科学ꎬ２０１４ꎬ３３(４):５７６－５７９. [３８]㊀张爱君ꎬ郝建安ꎬ杨波ꎬ等.海洋石油降解菌的筛选㊁鉴定及降解活性[Ｊ].化学工业与工程ꎬ２０１５ꎬ３２(１):３１－３６. [３９]㊀ＲａｇｈｕｋｕｍａｒＣꎬＶｉｐｐａｒｔｙＶꎬＤａｖｉｄＪꎬｅｔａｌ..Ｄｅｇｒａｄａｔｉｏｎｏｆｃｒｕｄｅｏｉｌｂｙｍａｒｉｎｅｃｙａｎｏｂａｃｔｅｒｉａ[Ｊ].Ａｐｐｌ.Ｍｉｃｒｏｂｉｏｌ.Ｂｉｏ￣ｔｅｃｈｎｏｌ.ꎬ２００１ꎬ５７(３):４３３－４３６.[４０]㊀ＷａｎｇＷꎬＳｈａｏＺ.Ｔｈｅｌｏｎｇ￣ｃｈａｉｎａｌｋａｎｅｍｅｔａｂｏｌｉｓｍｎｅｔｗｏｒｋｏｆＡｌｃａｎｉｖｏｒａｘｄｉｅｓｅｌｏｌｅｉ[Ｊ].Ｎａｔ.Ｃｏｍｍｕｎ.ꎬ２０１４ꎬ５ꎬ５７５５. [４１]ＢｒａｄｄｏｃｋＪＦꎬＬｉｎｄｓｔｒｏｍＪＥꎬＢｒｏｗｎＥＪ.Ｄｉｓｔｒｉｂｕｔｉｏｎｏｆｈｙｄｒｏ￣ｃａｒｂｏｎ￣ｄｅｇｒａｄｉｎｇｍｉｃｒｏｏｒｇａｎｉｓｍｓｉｎｓｅｄｉｍｅｎｔｓｆｒｏｍＰｒｉｎｃｅＷｉｌ￣ｌｉａｍＳｏｕｎｄꎬＡｌａｓｋａꎬｆｏｌｌｏｗｉｎｇｔｈｅＥｘｘｏｎＶａｌｄｅｚｏｉｌｓｐｉｌｌ[Ｊ].ＭａｒｉｎｅＰｏｌｌ.Ｂｕｌｌ.ꎬ１９９５ꎬ３０(２):１２５－１３２.[４２]㊀ＭｉＴＨꎬＴｓｕｔｓｕｍｉＨＫꎬＫｏｎｏＭ.ＢｉｏｒｅｍｅｄｉａｔｉｏｎｏｎｔｈｅｓｈｏｒｅａｆｔｅｒａｎｏｉｌｓｐｉｌｌｆｒｏｍｔｈｅＮａｋｈｏｄｋａｉｎｔｈｅＳｅａｏｆＪａｐａｎ.Ｉ.ｃｈｅｍｉｓｔｒｙａｎｄｃｈａｒａｃｔｅｒｉｓｔｉｃｓｏｆｈｅａｖｙｏｉｌｌｏａｄｅｄｏｎｔｈｅＮａｋ￣ｈｏｄｋａａｎｄｂｉｏｄｅｇｒａｄａｔｉｏｎｔｅｓｔｓｂｙａｂｉｏｒｅｍｅｄｉａｔｉｏｎａｇｅｎｔｗｉｔｈｍｉｃｒｏｂｉｏｌｏｇｉｃａｌｃｕｌｔｕｒｅｓｉｎｔｈｅｌａｂｏｒａｔｏｒｙ[Ｊ].ＭａｒｉｎｅＰｏｌｌ.Ｂｕｌｌ.ꎬ２０００ꎬ４０(４):３０８－３１４.[４３]㊀郑立ꎬ崔志松ꎬ高伟ꎬ等.海洋石油降解菌剂在大连溢油污染岸滩修复中的应用研究[Ｊ].海洋学报ꎬ２０１２ꎬ３４(３):１６３－１７２.[４４]㊀刘金累ꎬ厦文香ꎬ赵亮ꎬ等.海洋石油污染及其生物修复[Ｊ].海洋湖沼通报ꎬ２００６ꎬ０３:４８－５３.[４５]㊀ＰｒｉｎｃｅＲＣꎬＢｒａｇｇＪＲ.ＳｈｏｒｅｌｉｎｅｂｉｏｒｅｍｅｄｉａｔｉｏｎｆｏｌｌｏｗｉｎｇｔｈｅｅｘｘｏｎｖａｌｄｅｚｏｉｌｓｐｉｌｌｉｎＡｌａｓｋａ[Ｊ].Ｂｉｏｒｅｍｅｄ.Ｊ.ꎬ１９９７ꎬ１(２):９７－１０４.[４６]㊀毛天宇ꎬ刘宪斌ꎬ李亚娟.海洋石油污染生物修复技术[Ｊ].海洋环境保护ꎬ２００８ꎬ３:１２－１３.９６１李贵珍ꎬ等:海洋石油污染及其微生物修复研究进展。