Optimization of the microwave-assisted extraction of phlorotannins from Saccharina japonica Aresch and evaluation of the inhibitory effects of phlorotannin-containing extracts on HepG2 cancer cells

The use of a microwave-assisted extraction(MAE) method for the extraction of phlorotannins from Saccharina japonica Aresch(S.japonica) has been evaluated with particular emphasis on the influential parameters,including the ethanol concentration,solid/liquid ratio,extraction time,extraction temperature,and microwave power.The MAE procedure was optimized using single-factor design and orthogonal array design(OAD).The content of total phlorotannins in S.japonica was determined using a Folin-Ciocalteu(FC) assay.A maximum total phlorotannin content of 0.644 mg of phloroglucinol equivalent per gram of dry weight plant(mg PGE/g DW) was obtained using the optimized model,which included an ethanol concentration of 55%,solid/liquid ratio of 1:8,extraction time of 25 min,irradiation power of 400 W,and temperature of 60°C.Under similar conditions,the application of a conventional extraction method led to a lower phlorotannin yield of 0.585 mg PGE/g WD.These results demonstrated that the MAE approach provided better results for the extraction of phlorotannins from S.japonica and was a promising technique for the extraction of phenolic compounds from S.japonica and other materials.In addition,screening tests for the inhibitory activity showed that the phlorotannin-containing extracts significantly inhibited the growth of human hepatocellular carcinoma cells(HepG2) by inducing their apoptosis.The morphological changes that occurred during cell apoptosis were characterized using Hoechst33258 staining.     

Comparison of lipids in organs of the starfish Asterias amurensis associated with different treatments

Lipids were extracted from organs of the starfish Asterias amurensis associated with different treatments(raw-control,boiling and heating),and then analyzed for lipid content,lipid oxidation index,lipid classes and fatty acid composition.Results showed that boiling softened the hard starfish shells,thus facilitating the collection of starfish organs.As compared with raw organs,the boiled organs had lower water content and higher lipid content,possibly due to the loss of water-holding capacity caused by protein denaturation.Both boiling and heating increased the peroxide value(PV),thiobarbituric acid(TBA) value and carbon value(CV) of lipids.Despite slight increases in the content of complex lipids,associated lipid composition had no substantial variations upon boiling and heating.For simple lipids,the content of 1,2-diglyceride decreased in boiled and heated organs,with free fatty acids observed on thin layer chromatography(TLC).However,neither boiling nor heating significantly changed the fatty acid compositions of simple or complex lipids in starfish organs,suggesting that these two treatments had no significant effects on complex lipids in starfish organs.Together,our results indicated that boiling of starfish soon after capture facilitated the handling and extraction of useful complex lipids consisting of abundant glucosylceramide and eicosapentaenoic acid(EPA)-bounded phospholipids.     

氯铝酸离子液体催化环己酮肟Beckmann重排制己内酰胺

以氯铝酸离子液体作为催化剂和反应介质,通过实验研制一种价格低廉且环保的环己酮肟Beckmann重排制己内酰胺的催化体系.结果表明:环己酮肟转化率和己内酰胺选择性随离子液体酸性的增强而逐渐增大,离子液体中溶解的HCl对环己酮肟Beckmann重排的影响不大;反应温度、反应时间和离子液体用量对环己酮肟Beckmann重排有着较大的影响;确定离子液体Et3NHCl-2AlCl3催化环己酮肟Beckmann重排反应的最佳条件,反应温度为100℃,反应时间为60min,离子液体与环己酮肟摩尔比为1∶1;在该反应条件下,环己酮肟转化率为99.7%,己内酰胺选择性为98.0%,己内酰胺收率为97.8%.     

盐度对威氏海链藻生长和生化指标的影响

【目的】研究威氏海链藻在不同盐度条件下的生长和生化组分变化。【方法】设置盐度梯度为5、15、25、35、45,采用血细胞计数板计数法、热乙醇提取法、尼罗红染色法、苯酚硫酸法和考马斯亮蓝法,分别测定威氏海链藻在不同盐度下的生物量、叶绿素a、类胡萝卜素、中性脂质、总糖和蛋白质含量。【结果】盐度对威氏海链藻细胞密度影响显著(P<0.05),当盐度25和盐度35时,培养8 d细胞密度最高,均为2.0×10⁶ mL^-1;盐度对威氏海链藻叶绿素a和类胡萝卜素含量有显著影响(P<0.05),当盐度15时,细胞叶绿素a和类胡萝卜素含量均最高,分别为3.4814μg/mL(4 d)、2.3549μg/mL(8 d)。盐度对威氏海链藻可溶性总糖含量有显著影响(P<0.05),当盐度15时,8 d时可溶性总糖积累含量最高,为123μg/mL。当盐度35时,6 d时可溶性蛋白积累含量最高,为142.9μg/mL,且显著高于其他实验组(P<0.05)。盐度45时,8 d时中性脂含量最高,显著高于其他实验组(P<0.05)。【结论】盐度为15~35时,威氏海链藻生长速度较快,生化组成较为稳定,盐度15时最有利于藻细胞叶绿素a和类胡萝卜素、总糖的合成;盐度25和盐度35时,藻细胞平均增长速度较快,盐度35时最有利于藻细胞可溶性蛋白积累。     

Nutrient effects of broodstocks on the larvae in Patinopecten yessoensis

Patinopecten yessoensis is a commercial valuable species. This study deals with the effect of nutrient effects of the broodstock(mainly ovaries) on the larvae. Concentrations of total carbohydrate, total protein and total lipid in the gonads of P. yessoensis from three Hatcheries(Hatchery 1, Hatchery 2, and Hatchery 3) were determined before and after spawning. The relationship between the nutrient concentration in ovaries before spawning(BC) and that of larvae(LC) was assessed as well as the change in nutrient levels in ovaries after spawning(DC). Results indicate that the BC of total carbohydrate(7.66%) and total lipid(14.48%) in ovaries were significantly higher than in testes(5.20%, 5.20% respectively), whereas the BC of total protein in the ovaries was lower(61.76%) than in the testes(81.67%). The different gonadal composition suggests the different nutrient demands between male and female broodstocks in breeding season. P atinopecten yessoensis gonads contained a higher proportion of lipids, in comparison to other bivalves, which might be a response to the low ambient water temperatures. Further analysis of fatty acids showed that the concentrations of n-3PUFA, EPA and DHA in larvae(LC) were positively correlated with BC and DC, indicating the significant nutrient influence of broodstocks on the larvae. As these fatty acids are important in metabolism, and have been demonstrated to be influential to the viability of the larvae, larval growth and the settlement, spat growth, and juvenile survival in many bivalves, they could possibly be used as indexes to evaluate, and predict condition of broodstocks and larvae.     

Lipid and fatty acid compositions of cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>), haddock (<Emphasis Type="Italic">Melanogrammus aeglefinus</Emphasis>) and halibut (<Emphasis Type="Italic">Hippoglossus hippoglossus</Emphasis>)

This study was conducted to compare lipid and fatty acid composition of cod, haddock and halibut. Three groups of cod (276 g ± 61 g), haddock (538 g ± 83 g) and halibut (3704 g ± 221 g) were maintained with commercial feeds mainly based on fish meal and marine fish oil for 12 weeks prior to sampling. The fatty acid compositions of muscle and liver were determined by GC/FID after derivatization of extracted lipids into fatty acid methyl esters (FAME). Lipids were also fractionated into neutral and polar lipids using Waters silica Sep-Pak?. The phospholipid fraction was further separated by high-performance thin-layer chromatography (HPTLC) and the FAME profile was obtained. Results of the present study showed that cod and haddock were lean fish and their total muscle lipid contents were 0.8% and 0.7%, respectively, with phospholipid constituting 83.6% and 87.5% of the total muscle lipid, respectively. Halibut was a medium-fat fish and its muscle lipid content was 8%, with 84% of the total muscle lipid being neutral lipid. Total liver lipid contents of cod, haddock and halibut were 36.9%, 67.2% and 30.7%, respectively, of which the neutral lipids accounted for the major fraction (88.1%–97.1%). Polyunsaturated fatty acids were the most abundant in cod and haddock muscle neutral lipid. Monounsaturated fatty acid level was the highest in halibut muscle neutral lipid. Fatty acid compositions of phospholipid were relatively constant. In summary, the liver of cod and haddock as lean fish was the main lipid reserve organ, and structural phospholipid is the major lipid form in flesh. However, as a medium-fat fish, halibut stored lipid in both their liver and muscle.     

Lipid and Fatty Acid Compositions of Cod(Gadus morhua),Haddock(Melanogrammus aeglefinus)and Halibut(Hippogiossus hippoglossus)

This study was conducted to compare lipid and fatty acid composition of cod,haddock and halibut.Three groups of cod(276 g±61 g),haddock(538 g±83 g)and halibut(3704 g±221 g)were maintained with commercial feeds mainly based on fish meal and marine fish oil for 12 weeks prior to sampling.The fatty acid compositions of muscle and liver were determined by GC/FID after derivatization of extracted lipids into fatty acid methyl esters(FAME).Lipids were also fractionated into neutral and polar lipids using Waters silica Sep-Pak(R).The phospholipid fraction was further separated by high-performance thin-layer chromatography(HPTLC)and the FAME profile was obtained.Results of the present study showed that cod and haddock were lean fish and their total muscle lipid contents were 0.8% and 0.7%,respectively,with phospholipid constituting 83.6% and 87.5% of the total muscle lipid,respectively.Halibut was a medium-fat fish and its muscle lipid content was 8%,with 84% of the total muscle lipid being neutral lipid.Total liver lipid contents of cod,haddock and halibut were 36.9%,67.2% and 30.7%,respectively,of which the neutral lipids accounted for the major fraction(88.1%-97.1%).Polyunsaturated fatty acids were the most abundant in cod and haddock muscle neutral lipid.Monounsaturated fatty acid level was the highest in halibut muscle neutral lipid.Fatty acid compositions of phospholipid were relatively constant.In summary,the liver of cod and haddock as lean fish was the main lipid reserve organ,and structural phospholipid is the major lipid form in flesh.However,as a medium-fat fish,halibut stored lipid in both their liver and muscle.     

Reproductive cycle and seasonal variations in lipid content and fatty acid composition in gonad of the cockle Fulvia mutica in relation to temperature and food

From March 2004 to February 2005,seasonal variations in lipid content and fatty acid composition of gonad of the cockle Fulvia mutica(Reeve) were studied on the eastern coast of China in relation to the reproductive cycle and environment conditions(e.g.,temperature and food availability).Histological analysis as well as lipid and fatty acid analyses were performed on neutral and polar lipids of the gonad.Results showed that gametogenesis occurred in winter and spring at the expense of lipids previously accumulated in summer and autumn,whereas spawning occurred in summer(20.4-24.6℃).The seasonal variation in lipid content was similar to that of the mean oocyte diameter.In both neutral and polar lipids,the 20:5n-3 and 22:6n-3 levels were relatively higher than saturated fatty acids,and polyunsaturated fatty acids were abundant,with series n-3 as the predominant component.Seasonal variations in the 20:5n-3 and 22:6n-3 levels and the principal n-3 fatty acids were clearly related to the reproductive cycle.The ∑(n-3) and ∑(n-6) values were relatively high during January-May,and the associated unsaturation index was significantly higher than that in other months.The results suggest that fatty acids play an important role in the gametogenesis of F.mutica.     

总状蕨藻多糖提取工艺的研究

采用酶法提取与热水抽提相结合的方法研究了总状蕨藻多糖的提取工艺,结果显示,采用固液质量比1∶10,蛋白酶用量136.5 U/g,于60℃下酶法提取2.5 h后,再将固液质量比调至1∶20,在温度95℃条件下继续进行热水提取2.5 h,多糖得率为6.7%,比单用热水提取法提高了131.0%。     

Analysis of phospholipids in microalga Nitzschia closterium by UPLC-Q-TOF-MS

Precise structural identification of phospholipids in the microalga Nitzschia closterium has been established using ultra performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (UPLC-ESI-Q-TOF-MS) for direct analysis of total lipid extracts. Mass spectrometry was performed in reflective time-of-flight using electron spraying ionization in negative mode. Phospholipid molecular species identification was based on the characteristic product ions and neutral loss yielded by different phospholipids under ESI-MS/MS mode. The molecular species were confirmed by the carboxylate anions produced by phospholipids in negative mode; the regiospecificity of the two acyl chains was determined from the ratio of sn-1 to sn-2 carboxylate anion abundances. As a result, 18 lipid molecular species were identified for the first time in this microalga, comprising seven phosphatidylcholines (PC), two phosphatidylethanolamines (PE), two phosphatidylinositols (PI), and seven phosphatidylglycerols (PG). Lipid standards of PC, PE, PI, and PG were added to the total lipids as internal standards for semiquantitative analysis, revealing concentrations of phospholipids in this species between 0.09 and 3.37 nmol/mg. This method can produce a full structural profile of intact phospholipid molecular species and can be used for study of the physiological and ecological functions of lipids by monitoring their individual changes over time.     

不同方式提取的近江牡蛎糖蛋白的体外抗氧化活性

比较研究了水浸提、盐液浸提及超声辅助盐液浸提的近江牡蛎糖蛋白粗提物对羟自由基、DPPH自由基、超氧阴离子自由基的清除效果。结果表明:水浸提、盐液浸提及超声辅助盐液浸提的糖蛋白中可溶性蛋白与总糖含量之比分别为1.83∶1、4.49∶1和2.51∶1;3种糖蛋白粗提物均具有一定的清除羟自由基、DPPH自由基和超氧阴离子自由基的能力;它们对羟自由基的清除能力高于对DPPH自由基和超氧阴离子自由基;盐液浸提物清除超氧阴离子的能力最弱;超声辅助盐液浸提的糖蛋白粗提物清除羟自由基、超氧阴离子自由基及DPPH自由基的能力比水浸提和盐液浸提的糖蛋白的能力强。     

Unraveling enhanced membrane lipid biosynthesis in Chlamydomonas reinhardtii starchless mutant sta6 by using an electrospray ionization mass spectrometry-based lipidomics method

The unicellular green alga Chlamydomonas reinhardtii, a well-established model organism, has been widely used in dissecting glycerolipid metabolism in oxygenating photosynthetic organisms. In previous studies, it has been found that shunting carbon precursors from the starch synthesis pathway can lead to a 10-fold increase in TAG content as compared to the wild type, but it is unknown whether inactivation of AGPase may affect membrane lipids biosynthesis. The study aims to investigate global changes in lipid metabolism and homeostasis in the starchless mutant C. reinhardtii sta6. By utilizing an electrospray ionization/mass spectrometry (ESI/MS)-based lipidomics approach, a total of 105 membrane lipid molecules of C. reinhardtii were resolved, including 16 monogalactosyldiacylglycerol (MGDG), 16 digalactosyldiacylglycerol (DGDG), 11 phosphatidylglycerol (PG), 6 sulfoquinovosyldiacylglycerol (SQDG), 49 diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), 2 phosphatidylethanolamine (PE), and 5 phosphatidylinositol (PI) molecules. The quantitative results indicated that the membrane lipid profiles were similar between the two C. reinhardtii strains grown under both low- and high-light conditions, but the cellular contents of a great number of lipids were altered in sta6 due to the defect in starch biosynthesis. Under low-light conditions, sta6 accumulated more PI, MGDG, DGDG but less amounts of DGTS as compared to WT. Under high light, sta6 cells contained higher content membrane lipids than cc-124, except for PG, which is more or less similar in both strains. Our results demonstrate that the cellular membrane lipid homeostasis underwent profound changes in the starchless mutant, and thereby its physiological impact remains to be explored.     

Polysaccharides in <Emphasis Type="Italic">Sipunculus nudus</Emphasis>: Extraction condition optimization and antioxidant activities

Marine organisms constitute unlimited resource of bioactive substances due to their high biodiversity and represent a valuable source of new compounds. This study optimized the alkali-extraction conditions and antioxidant activities of soluble polysaccharides from the body wall of Sipunculus nudus. The effects of solid-liquid ratio, extraction duration, extraction temperature, and alkali concentration on the yield of S. nudus polysaccharides (SNP) were examined, according to which the optimal combination of extraction parameters was obtained by an orthogonal test. The relative influencing importance of different extraction parameters on the yield of SNP followed the order as solid-liquid ratio > extraction temperature > alkali concentration > extraction duration. The highest extraction yield, 1.98%, was achieved under an optimal extraction condition: temperature 60°C, solid-liquid ratio 1:6 g mL^?1, duration 5 h, and alkali (NaOH) mass fraction 6%. The in vitro antioxidant activities examination showed that extracted SNP under this optimized condition had strong power in reducing certain hydroxyl and superoxide radical scavenging abilities. The promising results showed that extracted SNP could be a potent natural antioxidant.     

长松藻基本营养成分分析及其多糖的提取

对南海长松藻(Codium cylindricum Holmes)的主要营养成分进行了分析,结果显示:多糖、蛋白质和粗纤维是构成藻体的主要营养成分,占藻体干基的80%左右,其中多糖占55.57%、粗纤维占11.09%、蛋白质占12.57%,但脂肪含量仅为0.59%。水溶性营养成分较为丰富,水浸出物总量为34.55%,其中包括水溶性蛋白、水溶性多糖、游离氨基酸等。多糖是长松藻的主要有效成分。单因素及正交实验结果表明:长松藻多糖提取的最佳工艺参数为浸提温度95℃,浸提时间1.5 h,料水质量比1∶50,多糖得率为9.79%。     

Comparative study on the allergenicity of different Litopenaeus vannamei extract solutions

Allergen extracts are widely used for allergy diagnosis and treatment. The application of shrimp extract is hampered due to the low protein concentration and the inconsistent allergenicity. Extracting solutions are considered to be the primary limiting factor of protein extraction from crustaceans. This study aimed to select an optimal solution for shrimp protein extraction by comparing the allergenicity of different shrimp extracts. The effect of 7 existing or modified extracting solutions were evaluated, including the glycerol-NaCl solution, the glycerol Cocaine's solution, the buffered saline solution, the Cocaine's solution, the Glucose leaching solution, 1 mol L-1 KCl solution, and 0.01 mol L-1 phosphate buffered saline solution with and without dithiothreitolor(DTT). The quantitative(protein concentration) and qualitative parameters(SDS-PAGE protein patterns and immuno-reactivity) were determined using the sodium dodecyl sulfate polyacrylamide gel electrophoresis, enzyme linked immunosorbent assay and immunoblotting assay. Results showed that the 1 mol L-1 KCl solution with DTT was optimal for shrimp protein extraction, which yielded high concentration and allergenicity in the protein extract, including major and minor allergens. The 1 mol L-1 KCl solution with DDT is proposed for preparation of shrimp extract and associated allergy diagnosis, as well as potential applications for other crustaceans.     

超声波辅助提取河豚毒素的研究

以月腹刺鲀(Gastrophysus lunaris)卵巢为原料,利用超声波辅助提取河豚毒素(Tetrodotoxin,TTX)。通过单因素实验及二次回归旋转组合设计实验研究液料比、超声温度、超声时间等因素对提取率的影响,确定超声波辅助提取河豚毒素的最佳工艺条件。结果表明:在实验范围内影响河豚毒素得率的各因素大小依次为液料体积质量比、超声温度、超声时间;超声波辅助提取河豚毒素的最佳工艺条件为液料体积(mL)质量(g)比3.03∶1,超声温度39℃,超声时间22 min,该工艺条件下提取河豚毒素得率为63.57μg/g。     

Distribution characteristics of lipids in hadal sediment in the Yap Trench

In this study, cored sediment samples collected by the Jiaolong Submersible at 6 779 m depth from the hadal zone of the Yap Trench in May 2016 were sliced in 1-cm interval from top to bottom, and lipids in each sediment layer were quantitatively and qualitatively analyzed. The vertical distribution profiles of the lipids in the sediment sample, their main existing forms, and their possible sources were investigated. The results show that the concentration of lipid in the surface sediment was the highest with the carbon number from 12 to 27, dominated by medium and short-chain lipids. The total concentration of fatty acids in surface sediment was much higher than those in the offshore and deep-sea areas, being up to 325.77 μg/g due to the funnel effect caused by the “V” terrain of the trench. Fatty acid 18.0 was the most abundant lipids in the sediment sample. Abnormal high concentrations of fatty acid 18.ω7 and alkanes indicated the existence of hydrothermal fluids in the study area. In addition, saturated fatty acids and monounsaturated fatty acids existed mainly in free form, and polyunsaturated fatty acids existed mainly in bound form. Most of the alkanes were in bound form, and their major source was autochthounous input. The carbon number of alcohols in the sediment sample ranged from 12 to 20, mainly existed in bound form. The source of fatty acids was mainly autochthonous input, and the neutral lipids had both marine and terrestrial origin. This is the first study of lipids in hadal sediment of the Yap Trench. The results will promote deeper understanding of organic carbon cycle in marine environment.     

离子色谱法测定地下水中有机氮

本文探讨了K_2S_2O_8氧化离子色谱法测定有机氮的实验条件,考察了淋洗剂离子价态及淋洗剂总离子强度对低浓度NO_3~-与高浓度SO_4~2-分离的影响;测定了11种含氮有机化台物有机氮的回收率。本法测定地下水中有机氮的结果与经典蒸馏法结果较为一致,具有操作简便、快速、准确,效率高的特点。     

Isolation of protein from Chlorella sorokiniana CY1 using liquid biphasic flotation assisted with sonication through sugaring-out effect

Microalgae, a sustainable source of multi beneficial components has been discovered and could be utilised in pharmaceutical, bioenergy and food applications. This study aims to investigate the sugaring-out effect on the recovery of protein from wet green microalga, Chlorella sorokiniana CY1 which was assisted with sonication. A comparison of monosaccharides and disaccharides as one of the phaseforming constituents shows that the monosaccharides, glucose was the most suitable sugar in forming the phases with acetonitrile to enhance the production of protein(52% of protein). The protein productivity of microalgae was found to be significantly influenced by the volume ratio of both phases, as the yield of protein increased to 77%. The interval time between the sonication as well as the sonication modes were influencing the protein productivity as well. The optimum protein productivity was obtained with 10 s of resting time in between sonication. Pulse mode of sonication was suitable to break down the cell wall of microalgae compared to continuous mode as a lower protein yield was obtained with the application of continuous mode. The optimum condition for protein extraction were found as followed: 200 g/L glucose as bottom phase with volume ratio of I:1.25, 10 s of resting time for ultrasonication, 5 s of ultrasonication in pulse mode and 0.25 g of biomass weight. The high yield of protein about 81% could be obtained from microalgae which demonstrates the potential of this source and expected to play an important role in the future.     

Characterization of the growth,chlorophyll content and lipid accumulation in a marine microalgae Dunaliella tertiolecta under different nitrogen to phosphorus ratios

Microalgal lipids are regarded as main future feedstock of biofuels for its higher efficiency of accumulation and sustainable production.In order to investigate the effect of various nitrogen to phosphorus ratios on cells growth,chlorophyll content and accumulation of lipids in Dunaliella tertiolecta,experiments were carried out in modified microalgal medium with inorganic nitrogen(nitrate-nitrogen) or organic nitrogen(urea-nitrogen) as the sole nitrogen source at initial N:P ratios ranging from 1:1 to 32:1.The favorable N:P of 16:1 in the nitrate-N or urea-N medium yielded the maximum cell density and specific growth rate.Decrease in chlorophyll content were observed at the N:P of 4:1 in both nitrate-N and urea-N cultures.It was also observed that the maximum lipids concentration was obtained at the N:P of 4:1 in both nitrate and urea nutrient medium.The lipid productivity and lipid content of cultures in the urea-N medium at the N:P of 4:1were markedly higher than those from cultures with other N:P ratios(p 0.05).The results of this work illustrate the possibility that higher ratios of nitrogen to phosphorus have enhancing effect on cells growth of D.tertiolecta.Conversely,higher lipid accumulation is associated with a decrease in chlorophyll content under lower ratios of nitrogen to phosphorus.The results confirm the hypothesis of this study that a larger metabolic flux has been channeled to lipid accumulation in D.tertiolecta cells when the ratios of nitrogen to phosphorus drop below a critical level.