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.     

Detection of 36 antibiotics in coastal waters using high performance liquid chromatography-tandem mass spectrometry

Among pharmaceuticals and personal care products released into the aquatic environment, antibiotics are of particular concern, because of their ubiquity and health effects. Although scientists have recently paid more attention to the threat of antibiotics to coastal ecosystems, researchers have often focused on relatively few antibiotics, because of the absence of suitable analytical methods. We have therefore developed a method for the rapid detection of 36 antibiotic residues in coastal waters, including tetracyclines (TCs), sulfanilamides (SAs), and quinolones (QLs). The method consists of solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, using electrospray ionization (ESI) in positive mode. The SPE was performed with Oasis HLB and Oasis MCX cartridges. Chromatographic separation on a C18 column was achieved using a binary eluent containing methanol and water with 0.1% formic acid. Typical recoveries of the analytes ranged from 67.4% to 109.3% at a fortification level of 100 ng/L. The precision of the method, calculated as relative standard deviation (RSD), was below 14.6% for all the compounds. The limits of detection (LODs) varied from 0.45 pg to 7.97 pg. The method was applied to determine the target analytes in coastal waters of the Yellow Sea in Liaoning, China. Among the tested antibiotics, 31 were found in coastal waters, with their concentrations between the LOD and 212.5 ng/L. These data indicate that this method is valid for analysis of antibiotics in coastal waters. The study first reports such a large number of antibiotics along the Yellow Sea coast of Liaoning, and should facilitate future comprehensive evaluation of antibiotics in coastal ecosystems.     

Extraction of organochlorine pesticides in sediments using soxhlet,ultrasonic and accelerated solvent extraction techniques

The application of soxhlet, ultrasonic and accelerated solvent extraction techniques to the analysis of six organochlorine pesticides (α-HCH, β-HCH, γ-HCH, o, p'-DDT, p,p'-DDT and p, p'-DDE) in Taihu Lake sediment samples is described. It was found that the limits of quantification ranged from 0.002μgg-1 to 0.004μgg-1, and the recoveries of organochlorine pesticides with the three extraction techniques were acceptable (>80.7%). With a mass selective detector, better results were obtained by accelerated solvent extraction using hexane-acetone (1:1) as compared with soxhlet and ultrasonic extraction. It was shown that the accelerated solvent extraction was the optimum technique for the analysis of organochlorine pesticides in sediments. The general features of the three extraction techniques are also presented.     

Bioassay-Guided Extraction of Crude Fucose-Containing Sulphated Polysaccharides from Sargassum fusiforme with Response Surface Methodology

The response surface methodology(RSM) combined with bioassays was employed to optimize the extraction process of crude fucose-containing sulphated polysaccharides(c FCSP) from Sargassum fusiforme. The central composite design(CCD) was used with four variables, five levels, and four responses. The four variables were p H value of hydrochloric acid solution, extraction temperature(℃), ratio of liquid to raw material(m L g~(-1)), and extraction time(h), respectively. Chemical and bioassay indices were used in combination as the response parameters, which included the yield of c FCSP, fucose content, proliferation rate of spleen cells, and lipopolysaccharide-induced proliferation of splenocytes. The experimental data were fitted to a second-order polynomial equation using multiple regression analysis, and examined using the appropriate statistical methods. The best extraction conditions were as follows: the p H value of hydrochloric acid solution was 3.50; the extraction temperature was 100℃; the ratio of liquid to raw material was 15.00 m L g~(-1) and the extraction time was 2.50 h. The experimental yield was close to the predicted from the model. The extract could promote spleen lymphocyte proliferation, especially the lipopolysaccharide-induced lymphocyte proliferation in vitro, which suggested that its immunomodulatory effect on B lymphocytes. Therefore, c FCSP extracted from S. fusiforme could be utilized as an immunostimulant in functional foods and pharmaceutical industry in future.     

Optimization of ultrasonic extraction and clean-up protocol for the determination of polycyclic aromatic hydrocarbons in marine sediments by high-performance liquid chromatography coupled with fluorescence detection

The procedures of ultrasonic extraction and clean-up were optimized for the determination of polycyclic aromatic hydrocarbons (PAHs) in marine sediments. Samples were ultrasonically extracted, and the extracts were purified with a miniaturized silica gel chromatographic column and analyzed with high performance liquid chromatography (HPLC) with a fluorescence detector. Ultrasonication with methanol-dichloromethane (2:1, v/v) mixture gave higher extraction efficiency than that with dichloromethane. Among the three elution solvents used in clean-up step, dichloromethane-hexane (2:3, v/v) mixture was the most satisfactory. Under the optimized conditions, the recoveries in the range of 54.82% to 94.70% with RSDs of 3.02% to 23.22% for a spiked blank, and in the range of 61.20% to 127.08% with RSDs of 7.61% to 26.93% for a spiked matrix, were obtained for the 15 PAHs studied, while the recoveries for a NIST standard reference SRM 1941b were in the range of 50.79% to 83.78% with RSDs of 5.24% to 21.38%. The detection limits were between 0.75 ng L-1 and 10.99 ng L-1for different PAHs. A sample from the Jiaozhou Bay area was examined to test the established methods.     

Evaluating the performances of accelerated-solvent extraction, microwave-assisted extraction, and ultrasonic-assisted extraction for determining PCBs, HCHs and DDTs in sediments

Three extraction techniques have been recently used for the quantitative extraction of semi-volatile organic pollutants in sediments, i.e. accelerated solvent extraction (ASE), microwave assisted extraction (MAE) and ultrasonic assisted extraction (UAE). However, their extraction efficiencies have rarely been quantitatively compared using rigorous mathematical methods. In this paper, one-way analysis of variance (ANOVA) was employed to evaluate the performance of ASE, MAE and UAE in the overall extraction of PCBs, DDTs and HCHs by analysis of their recoveries from the Standard Reference Material IAEA-417, a sediment sample certified by many international laboratories. Conclusions were drawn at a significance level of P ≤ 0.05. No significant differences were found among the mean values for method recoveries using ASE, MAE and UAE. The mean values for real recoveries using ASE and MAE were nearly identical, but the real recovery using UAE was much lower. The concentrations of all PCBs, DDTs and HCHs except for CB52 and o,p′-DDT using UAE were the lowest. Comparing the results obtained using ASE with MAE, the concentrations of CB28, CB52, CB138, α-HCH, β-HCH, p,p′-DDE and p,p′-DDD were nearly identical, while the concentrations of other compounds were significantly different. Based on the low recoveries, it was concluded that UAE is a relatively inefficient extraction method, while ASE and MAE are equivalent methods. Taking into consideration the relative standard deviation (RSD) values, solvent volume, extraction time, and purchasing costs of the apparatus, MAE was considered superior to ASE for extraction of PCBs, DDTs and HCHs.     

Determination of lignin in marine sediment using alkaline cupric oxide oxidation-solid phase extraction-on-column derivatization-gas chromatography

Lignin serves as one of the most important molecular fossils for tracing Terrestrial Organic Matters (TOMs) in marine environment. Extraction and derivatization of lignin oxidation products (LOPs) are crucial for accurate quantification of lignin in marine sediment. Here we report a modification of the conventional alkaline cupric oxide (CuO) oxidation method, the modification consisting in a solid phase extraction (SPE) and a novel on-column derivatization being employed for better efficiency and reproducibility. In spiking blanks, recoveries with SPE for the LOPs are between 77.84% and 99.57% with relative standard deviations (RSDs) ranging from 0.57% to 8.04% (n=3), while those with traditional liquid-liquid extraction (LLE) are from 44.52% to 86.16% with RSDs being from 0.53% to 13.14% (n=3). Moreover, the reproducibility is greatly improved with SPE, with less solvent consumption and shorter processing time. The average efficiency of on-column derivatization for LOPs is 100.8% ± 0.68%, which is significantly higher than those of in-vial or in-syringe derivatization, thus resulting in still less consumption of derivatizing reagents. Lignin in the surface sediments sampled from the south of Yangtze River estuary, China, was determined with the established method. Recoveries of 72.66% to 85.99% with standard deviation less than 0.01mg/10g dry weight are obtained except for p-hydroxyben-zaldehyde. The lignin content Σ8 (produced from 10 g dry sediment) in the research area is between 0.231 and 0.587 mg. S/V and C/V ratios (1.028 ± 0.433 and 0.192 ± 0.066, respectively) indicate that the TOMs in this region are originated from a mixture of woody and nonwoody angiosperm plants; the high values of (Ad/Al) v suggest that the TOMs has been highly degraded.     

Determination of Steroid Hormone Residues in Fish Tissues Using Gel Permeation Chromatography and Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry

A highly sensitive method was developed for the simultaneous determination of 8 steroid hormones in high-fat fish tissues using ultra high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).The 8 steroid hormones were extracted from the tissues with diethyl ether.Differing from other common purification methods,the extract solutions were cleaned by gel permeation chromatography(GPC) using ethyl acetate-cyclohexane solution(1:1,v/v) as the mobile phase.The separation of target compounds was carried out by a BEH C18 column and a gradient elution consisting of acetonitrile and 0.2% aqueous formic acid(v/v).The compounds were detected under the multiple reaction monitoring(MRM) mode and quantified with external standard method.This method was validated with respect to linearity,specificity,accuracy and precision.A linearity with correlation coefficient larger than 0.995 was achieved in the range of 0.5 to 50 ng m L~(-1).The average recoveries at the spiked levels of 1.0,5.0,and 10.0 μg kg~(–1) varied between 81.7% and 90.8%,with the relative standard deviations(n=5) ranged from 3.50% to 10.0%.The limit of quantification(LOQ) for 8 steroid hormones ranged from 0.2 to 1.5 μg kg~(-1).It was concluded that this method can be successfully applied for the determination of 8 steroid hormones in complicated matrices including high-fat fish tissues.     

Estimates on age,growth,and mortality of Gymnocypris firmispinatus(Cyprinidae: Schizothoracinae) in the Anning River,China

The age, growth, and mortality of Gymnocypris firmispinatus were studied by 582 individuals that collected between July 2015 and June 2016 from the tributaries of the Anning River, China. The total length(TL) ranged from 27 mm to 242 mm, total weight(W) ranged from 0.2 g to 148.2 g. The relationship of total length and weight was expressed as W =7.754×10~(-6) TL~(3.304) for the total samples. The age classes of males were 1–9 a, and that of females were 1–13 a. The von Bertalanf fy functions based on otolith readings and observed length data were L_(t♂)=198.6(1–e~(-0.094(t +3.497))), and L_(t♀)=289.3(1–e~(-0.089(t +1.109))). The total mortality( Z) of male and female G. firmispinatus was estimated as 0.73/a and 0.40/a, respectively. The natural mortality( M) was evaluated as 0.25/a for males and 0.20/a for females, fishing mortality( F) was0.35/a for males and 0.16/a for females. The exploitation ratio of the population was 0.66/a for males and0.50/a for females. It was concluded that the growth of the fish is relatively slower than other congenericfishes. The population of G. firmispinatus in the tributaries of the Anning River might be in over-exploitation under the current fishing intensity.     

Isolation,Identification, and Quantitative Determination of Saponin in Apostichopus japonicus by HPLC-DAD

Apostichopus japonicus is an important invertebrate that is widely used as a tonic food in Asian countries.The purpose of this study is to purify and identify a class of compound,the saponins,from the body wall of A.japonicus,and to establish a new me-thod to determine the quantity of saponins in the sea cucumber.In this study,the saponins of A.japonicus,cladoloside A(CA),were ob-tained from 80%ethanol extract by column chromatography for the first time and were characterized using the spectral method.The resulting purified saponins were then profiled using ¹HNMR,¹³CNMR,and ESI-MS,which revealed the CA molecular formula of C₅₃H₈₂O₂ and contained a triterpenoid backbone,a methylglucopyranosyl moiety,a quinovopyranosyl,and two xylopyranosyls.A me-thod for the quantitative determination of CA,comprising microwave-assisted extraction,high-performance liquid chromatography,and diode array detector method,was established.Extraction efficiency was optimized by changing microwave power,extraction sol-vent,volume,time,and temperature.Results showed that under the optimum conditions(extraction time of 10 min,temperature of 45℃,and solvent of 25 mL 70%ethanol under 400 W),the detection limit of CA was 0.0015 mg mL^?1,and the recoveries of CA from samples at spiking levels of 10,20,and 50μg g^?1 ranged from 90.1%-104%.The proposed method was successfully applied to ana-lyze the saponins in different tissues of A.japonicus collected in different seasons.The method developed in this study can provide quantitative technical support for the quality control of A.japonicus.     

Screening of extraction methods for glycoproteins from jellyfish (Rhopilema esculentum) oral-arms by high performance liquid chromatography

In order to select an optimum extraction method for the target glycoprotein (TGP) from jellyfish (Rhopilema esculentum) oral-arms, a high performance liquid chromatography (HPLC)-assay for the determination of the TGP was developed. Purified target glycoprotein was taken as a standard glycoprotein. The results showed that the calibration curves for peak area plotted against concentration for TGP were linear (r = 0.9984, y = 4.5895x+47.601) over concentrations ranging from 50 to 400 mgL-1. The mean extraction recovery was 97.84% (CV2.60%). The fractions containing TGP were isolated from jellyfish (R. esculentum) oral-arms by four extraction methods: 1) water extraction (WE), 2) phosphate buffer solution (PBS) extraction (PE), 3) ultrasound-assisted water extraction (UA-WE), 4) ultrasound-assisted PBS extraction (UA-PE). The lyophilized extract was dissolved in Milli-Q water and analyzed directly on a short TSK-GEL G4000PWXL (7.8 mm×300 mm) column. Our results indicated that the UA-PE method was the optimum extraction method selected by HPLC.     

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。     

Comparative analysis of methods for concentrating venom from jellyfish Rhopilema esculentum Kishinouye

In this study, several methods were compared for the efficiency to concentrate venom from the tentacles of jellyfish Rhopilema esculentum Kishinouye. The results show that the methods using either freezing-dry or gel absorption to remove water to concentrate venom are not applicable due to the low concentration of the compounds dissolved. Although the recovery efficiency and the total venom obtained using the dialysis dehydration method are high, some proteins can be lost during the concentrating process. Comparing to the lyophilization method, ultrafiltration is a simple way to concentrate the compounds at high percentage but the hemolytic activities of the proteins obtained by ultrafiltration appear to be lower. Our results suggest that overall lyophilization is the best and recommended method to concentrate venom from the tentacles of jellyfish. It shows not only the high recovery efficiency for the venoms but high hemolytic activities as well.     

水产饲料中三聚氰胺检测方法的优化

通过对高效液相色谱法(HPLC)测定水产饲料中三聚氰胺含量的方法进行优化,得到最佳色谱条件和前处理方法,建立了水产饲料中三聚氰胺的检测方法。结果表明:以体积比8∶92的乙腈、庚烷磺酸钠离子对试剂为流动相,柱温40℃,流速1.2 mL/min,以波长236 nm检测;以提取液为18 mL50%(φ)乙腈水+1 mL乙酸锌+1 mL1mol/L盐酸,淋洗液3 mL水和3 mL甲醇,洗脱体积6 mL作样品前处理。三聚氰胺加标回收率为85%～100%,相对标准偏差(RSD)小于5%,线性范围为0.2 mg/L～5.0mg/L,相关系数0.999 8,方法检出限为0.2 mg/kg。     

平菇多糖的提取工艺

对平菇多糖提取进行优化研究 ,结果表明 ,平菇多糖提取最佳工艺条件为 :90℃浸提 8h ,m(原料 )∶m(水 ) =1∶30 ,醇析时乙醇体积分数为 80 % ,去除蛋白质时V(粗提液 )∶V(氯仿 +正丁醇 ) =1∶1,V(氯仿 )∶V(正丁醇 ) =10∶3,萃取 30min效果最佳 ,其得率为 1.5 4%。     

Effects of DNA extraction and universal primers on 16S rRNA gene-based DGGE analysis of a bacterial community from fish farming water

Among many reports investigating microbial diversity from environmental samples with denaturing gradient gel electrophoresis (DGGE), limited attention has been given to the effects of universal primers and DNA extraction on the outcome of DGGE analysis. In this study, these effects were tested with 16S rRNA gene-based DGGE on a bacterial community from farming water samples. The results indicate that the number of discernable bands in the DGGE fingerprint differed with the primer pairs used; the bands produced by 63f/518r, 341f/926r and 933f/1387r primer pairs were obviously fewer than those by 968f/1401r. Also, we found that each DNA extraction method resulted in different community profiles, reflected by the number and intensity of bands in the DGGE fingerprint. Furthermore, the main bands (theoretically representing dominant bacteria) differed with the extraction methods applied. It is therefore believed that the effects of universal primers and DNA extraction should be given more attention and carefully chosen before performing an investigation into a new environment with DGGE.     

Comparative analysis of methods for concentrating venom from jellyfish Rhopilema esculentum Kishinouye

In this study, several methods were compared for the efficiency to concentrate venom from the tentacles of jellyfish Rhopilema esculentum Kishinouye. The results show that the methods using either freezing-dry or gel absorption to remove water to concentrate venom are not applicable due to the low concentration of the compounds dissolved. Although the recovery efficiency and the total venom obtained using the dialysis dehydration method are high, some proteins can be lost during the concentrating process. Comparing to the lyophilization method, ultrafiltration is a simple way to concentrate the compounds at high percentage but the hemolytic activities of the proteins obtained by ultrafiltration appear to be lower. Our results suggest that overall lyophilization is the best and recommended method to concentrate venom from the tentacles of jellyfish. It shows not only the high recovery efficiency for the venoms but high hemolytic activities as well. Supported by the Award Foundation of Scientific Research for Excellent Young and Middle-age Scientist of Shandong Province (No. 2006BS07003) and the Knowledge Innovation Project of Chinese Academy of Sciences (KZCX2-YW-R-104)     

Spatiotemporal distribution of protozooplankton and copepod nauplii in relation to the occurrence of giant jellyfish in the Yellow Sea

The occurrence of the giant jellyfish, Nemopilema nomurai, has been a frequent phenomenon in the Yellow Sea. However, the relationship between the giant jellyfish and protozoa, in particular ciliates, remains largely unknown. We investigated the distribution of nanoflagellates, ciliates, Noctiluca scintillans, and copepod nauplii along the transect 33~N in the Yellow Sea in June and August, 2012, during an occurrence of the giant jellyfish, and in October of that year when the jellyfish was absent. The organisms studied were mainly concentrated in the surface waters in summer, while in autumn they were evenly distributed in the water column. Nanoflagellate, ciliate, and copepod nauplii biomasses increased from early June to August along with jellyfish growth, the first two decreased in October, while N. scintillans biomass peaked in early June to 3 571 pg C/L and decreased in August and October. In summer, ciliate biomass greatly exceeded that of copepod nauplii （4.61-15.04 ~tg C/L vs. 0.34-0.89 pg C/L）. Ciliate production was even more important than biomass, ranging from 6.59 to 34.19 ~tg C/（L.d） in summer. Our data suggest a tight and positive association among the nano-, micro-, and meso-zooplankton in the study area. Statistical analysis revealed that the abundance and total production of ciliate as well as loricate ciliate biomass were positively correlated with giant jellyfish biomass, indicating a possible predator-prey relationship between ciliates and giant jellyfish. This is in contrast to a previous study, which reported a significant reduction in ciliate standing crops due to the mass occurrence ofN. nomurai in summer. Our study indicates that, with its high biomass and, in particular, high production ciliates might support the mass occurrence of giant jellyfish.     

Inbreeding and genetic diversity analysis in a hatchery release population and clones of Rhopilema esculentum based on microsatellite markers

Ten microsatellite markers were used to analyze the levels of genetic diversity and inbreeding in a hatchery release population of Rhopilema esculentum Kishinouye(Scyphozoa: Rhizostomatidae). A total of 85 alleles were detected in 600 individuals. Within-population levels of observed( H o) and expected( H e) heterozygosity ranged from 0.152 to 0.839(mean=0.464) and from 0.235 to 0.821(mean=0.618), respectively. The polymorphism information content(PIC) of each marker ranged from 0.207 to 0.795 with an average of 0.580, indicating that the hatchery population maintained a high level of genetic diversity. Inbreeding levels were estimated in the hatchery population and the inbreeding coefficient was 0.203. This result revealed that a certain level of inbreeding occurred within the population. Meanwhile, we also determined genetic diversity at the clone level. Several polyps from the same scyphistomae were genotyped at the ten microsatellite loci and there was virtually no difference in their genotypes. Furthermore, we calculated the probabilities of exclusion. When both parents were known, the average exclusion probability of ten loci was 99.99%. Our data suggest that the ten microsatellite markers can not only be used to analyze the identity of individuals but they can also be applied to parentage identification. Our research provides a theoretical basis and technical support for genetic diversity detection and reasonable selection of R. esculentum hatchery populations. These findings support the use of releasing studies and conservation of R. esculentum germplasm resources.     

面向不同环境背景的Landsat影像水体提取方法适用性研究

快速、准确地从卫星影像中提取水体信息一直是遥感应用的热点问题,在水资源管理、水环境监测和灾害应急管理等领域极具应用价值。虽然目前已有多种针对Landsat系列影像的水体提取方法,但由于地理位置、地形和水体形态等环境背景因素的影响,导致同种方法在不同的环境背景中呈现出不同的提取效果。本文针对人为影响严重、影像明暗对比强烈的城区（北京怀柔县城周边）以及地形起伏明显、水体细小的非城区（北京密云水库周边） 2种典型背景环境,选择波段设置略有差异的Landsat 5（2009年）和Landsat 8（2019年）卫星影像,对比了常用的指数法（NDWI和MNDWI）和分类法（最大似然法和支持向量机）在水体信息提取方面的优势和不足。结果表明：在城区背景中,SVM的准确性最高（总体精度>97%）;在非城区背景中,MNDWI与SVM的精度相当（总体精度>95%）,前者更适用于水体的快速提取,而后者提取的山间细碎河流更完整,且在Landsat 8中应用的效果更好。该研究为不同环境背景下水体提取方法的选择提供了参考。