Ethyl acetate fraction of Sargassum pallidum extract attenuates particulate matter-induced oxidative stress and inflammation in keratinocytes and zebrafish

Objective:To evaluate the effect of the ethyl acetate fraction derived from Sargassum pallidum extract against particulate matter(PM)-induced oxidative stress and inflammation in HaCaT cells and zebrafish.Methods:HaCaT cells and zebrafish were used to evaluate the protective effects of the ethyl acetate fraction of Sargassum pallidum extract against PM-induced oxidative stress and inflammation.The production of nitric oxide(NO),intracellular ROS,prostaglandin E_(2)(PGE_(2)),and pro-inflammatory cytokines,and the expression levels of COX-2,iNOS,and NF-κB were evaluated in PM-induced HaCaT cells.Furthermore,the levels of ROS,NO,and lipid peroxidation were assessed in the PM-exposed zebrafish model.Results:The ethyl acetate fraction of Sargassum pallidum extract significantly decreased the production of NO,intracellular ROS,and PGE_(2) in PM-induced HaCaT cells.In addition,the fraction markedly suppressed the levels of pro-inflammatory cytokines and inhibited the expression levels of COX-2,iNOS,and NF-κB.Furthermore,it displayed remarkable protective effects against PM-induced inflammatory response and oxidative stress,represented by the reduction of NO,ROS,and lipid peroxidation in zebrafish.Conclusions:The ethyl acetate fraction of Sargassum pallidum extract exhibits a protective effect against PM-induced oxidative stress and inflammation both in vitro and in vivo and has the potential as a candidate for the development of pharmaceutical and cosmeceutical products.

Anti-obesity effects of fucoidan from Sargassum thunbergii in adipocytes and high fat diet induced obese mice through inhibiting adipogenic specific transcription factor

The prevalence of obesity has increased and is a health concern worldwide.Due to the concerns regarding synthetic anti-obesity treatments,nowadays natural products become a trend.Previous studies proved that there is a potential to use marine algae as anti-obesity agents.Therefore,in this study,the lipid inhibitory effect of crude polysaccharide of amyloglucosidase-assisted hydrolysate from Sargassum thunbergii(STAC)and its fucoidan fractions(STAFs)on 3T3-L1 cells and high-fat diet(HFD)-induced obese mice were investigated.According to the results,the STAF3,showed the highest xylose content and exhibited significant inhibitory effects on lipid accumulation by downregulating adipogenic and lipogenic proteins in 3T3-L1 cells.Furthermore,oral supplementation with STAC significantly declined gain in body weight and fat weight,and serum lipid contents in an HFD-induced obesity mouse model.Structural and chemical characterizations demonstrated that puritied STAF3 has consistent surface morphology and small particle size,with similar structural characteristics as commercial fucoidan.Together,these results indicate that STAC and purified STAF3 from Sargassum thunbergia is a potent source to develop as ananti-obesity agents or functional food products to counter obesity.

Effects of low molecular weight polysaccharide from Sargassum thunbergii against palmitic acid-induced intracellular lipid accumulation in 3T3-L1 adipocyte and HepG2 cells

Low molecular weight polysaccharides can be isolated from Sargassum thunbergii(LMPST)and in vitro experiments were conducted to evaluate the inhibitory effects on lipids.Two natures of LMPST were attained from S.thunbergii and appraised their LMPST on palmitic acid(PA)induced lipid accretion in Hep G2,and 3T3-L1 cells.LMPST treatment lessened lipid deposition and intracellular free fatty acid and triglyceride intensities in PA-treated above mentioned cells.The mechanistic study publicized that LMPST2 significantly suppressed adipogenesis and stimulated the PA-treated 3T3-L1 cells occupied in the lipolysis pathway.Furthermore,in PA-treated Hep G2 cells,the free fatty acid oxidation was significantly increased by LMPST2.Given these constructive properties of LMPST2 from S.thunbergii,is a potential candidate for diminishing the intracellular lipids,and for a therapeutic agent in those conditions.

Application of Sargassum fusiforme(Harv.)Setch.Extract in Cigarettes

[Objectives]This study was conducted to investigate the application of Sargassum fusiforme(Harv.)Setch.in cigarettes.[Methods]Tobacco-specific nitrosamines in the smoke of cigarettes added with ethanol extract of S.fusiforme were determined,and the compounds related to the aroma of S.fusiforme were identified by flavor-smelling experiment.[Results]With the addition of the ethanol extract of S.fusiforme,the decrease in the total amount of four tobacco-specific nitrosamines in mainstream cigarette smoke reached 16.42%.The results of the flavor-smelling experiment showed that the aroma of S.fusiforme might be related to(R)-5,6,7,7A-tetrahydro-4,4,7A-trimethyl-2(4H)-benzofuranone,glycerol,ethyl palmitate,methyl palmitate,ethyl linoleate,methyl(Z,Z,Z)-9,12,15-octadecatrienoate,ethyl(Z,Z,Z)-9,12,15-octadecatrienoate,phytol,and tetradecanoic acid.[Conclusions]The ethanol extract of S.fusiforme has the potential function of reducing the content of tobacco-specific nitrosamines in smoke and improving the taste of cigarettes.

In vitro antioxidant and wound healing activity of Sargassum polycystum hydroethanolic extract in fibroblasts and keratinocytes

Objective:To investigate the in vitro antioxidant and wound healing properties of the hydroethanolic extract of Sargassum polycystum,and elucidate the mechanism of its wound healing activity.Methods:Human dermal fibroblast and HaCaT cells were used to evaluate the proliferation by sulforhodamine B and dsDNA assay after treatment with Sargassum polycystum extracts.Scratch wound healing and phalloidin-rhodamine staining were employed to observe migratory activity and filopodia formation,respectively.Western blot and real-time RT-PCR assays were performed to determine the protein and gene expressions related to wound healing activities.Results:The phytochemical analysis found a higher level of flavonoid than phenolic compound in Sargassum polycystum extracts.In human dermal fibroblast cells,Sargassum polycystum extracts at 50 and 100μg/mL significantly increased fibroblast proliferation and the gene expressions of hyaluronic acid synthase 1(HAS1),HAS2,HAS3,collagen type 1 alpha 1 chain(COL1A1),collagen type 3 alpha 1 chain(COL3A1),and elastin.The phosphorylation of Akt,ERK1/2,and p38 MAPK was also significantly upregulated after treatment with Sargassum polycystum extracts.Additionally,50 and 100μg/mL of the extracts prominently enhanced the proliferation,migration,and filopodia formation of HaCaT cells,as well as the protein levels of pFAK/FAK,pSrc/Src,pAkt/Akt,pERK1/2/ERK1/2,Rac1 and Cdc42.Conclusions:Sargassum polycystum extracts show promising wound healing activities in human dermal fibroblasts and keratinocytes.

Diversity of culturable alginate lyase-excreting bacteria associated with Sargassum

Large numbers of bacteria live on the surface of various brown algae and can produce alginate lyases to consume alginate,an important component of the cell wall of brown algae.Sargassum is a genus of the largest canopyforming brown algae of more than 150 species,which are widely distributed in tropical and subtropical environments.However,our knowledge about the epiphytic bacteria and the alginate lyase-excreting bacteria from Sargassum is still primitive.Here,we investigated the diversity of the culturable epiphytic bacteria and alginate-degrading bacteria from Sargassum samples collected from the coastal seawaters of Shandong Province,China.In total,37 strains belonging to 21 genera in 3 phyla were isolated,including 15 previously unreported genera,of which Vibrio(6/37)and Pseudoalteromonas(5/37)are the dominant genera.Eight strains,mainly Vibrio and Pseudoalteromonas species,were further identified as alginate lyase-excreting strains that can utilize alginate for growth.The extracellular alginolytic activity of the 8 strains was determined,and strains Vibrio sp.C42and Pseudoalteromonas sp.M9 showed the highest activity.These results provide a better understanding of brown algae epiphytes and alginate-degrading bacteria,and are fundamental for further studies on the interactions between brown algae and their epiphytes.

南海马尾藻(Sargassum sp.)内生真菌No.ZZF36中甾醇化合物的研究

从南海马尾藻(Sargassumsp.)内生真菌No.ZZF36中分离得到4个甾醇化合物,利用波谱技术确定其结构分别为菜子甾醇(1),麦角甾醇(2),过氧化麦角甾醇(3),麦角甾-7,22(E)-二烯-3,β5,α6β-三醇(4)。

Comparative analysis on transcriptome sequencings of six Sargassum species in China

Species of Sargassum are distributed worldwide, and are of great ecological and economic importance in marine ecosystems and bioresources. In this study, transcriptome sequencings of six Sargassum species were performed for the first time using an Illumina platform. For each sample, a total of 2.1-2.5 Gb of nucle-otides are collected and assembled into 69 871-116 790 scaffolds, with an average length of 410-550 bp and N50 length of 756-1 462 bp. A total of 20 512-28 684 unigenes of each sample were annotated and compared well with known gene sequences from nr database. Clusters of Orthologous Groups （COG）, gene ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） analyses were also performed for further un-derstanding of gene functions and regulation pathways. Gene expression levels were calculated based on RPKM values and compared among these species, especially for those genes related to carbohydrate metab-olism. Cluster analyses indicated that the differences of global gene expression between S. fusiforme, which was nominated as Hizikia fusiformis before, and other five species were not significant. Further phylogenet-ic analysis of 108 orthologous genes confirmed that S. fusiforme had closer relationship with S. hemiphyllum rather than S. horneri. These transcriptome data provided valuable information for better understanding of genome and gene characteristics of Sargassum algae and benefiting comparative and phylogenetic studies of Phaeophyceae species in future studies.

基于聚类及简化基因组联合分析的浙江洞头栽培羊栖菜(Sargassum fusiforme)品系筛选研究

以浙江洞头栽培羊栖菜(Sargassum fusiforme)群体为研究对象,运用主成分分析法对其鲜重、藻体长度、茎宽、气囊形态和大小等表型特征进行分析,确定了羊栖菜簇生气囊中的“特征”大气囊为表型主成分;运用聚类分析法分析了21个羊栖菜差异表型样品的“特征”大气囊表型聚类特征,定量分析归纳了栽培羊栖菜表型品系类别;运用简化基因组分析法构建了34个羊栖菜样品的系统进化树,分析了栽培羊栖菜各样品及韩国丽水、辽宁大连、浙江舟山野生羊栖菜样品间的亲缘关系,定量分析归纳了栽培羊栖菜基因型品系类别。在栽培羊栖菜表型品系和基因型品系总重合率为86%的基础上,将浙江洞头栽培羊栖菜群体分为球囊、锥囊、宽棒囊、狭棒囊和梭镖囊等5个品系,并运用林奈的“双名法”加以学名命名。提供了一种科学的羊栖菜品系分类方法,以期为深度开展浙江洞头栽培羊栖菜的群体结构、亲缘关系、遗传稳定性和品质差异等基础研究,以及定向选育优质高产、逆境高耐受性和活性物质高富集品系等应用研究,提供方法和理论支撑。

我国沿海马尾藻属(Sargassum)的物种多样性及其区系分布特征

通过对我国沿海多次野外实地考查以及文献报道,采用统计学的分析方法,研究了马尾藻属海藻资源的物种构成和区系分布特征。结果表明,马尾藻属种数在我国海域的水平分布呈现出北少南多的趋势,黄、东海仅17种,南海达到124种、占世界马尾藻总数的36.47%。其中,黄海西区、东海西区、南海北区和南海南区种数及小区单一分布种类数分别为10、13、82和61种及2、3、52和42种。我国特有种类为64种、占世界尾藻总数的18.82%。在我国沿海马尾藻的垂直分布特征为:高潮带2种,低潮带91种,潮下带78种。

羊栖菜Sargassum fusiform化学成分的研究

从南澳岛海藻羊栖菜Sargassum fusiform(Harv.)Setch中分离到3个化合物,经化学方法和IR、NMR、MS等现代波谱分析,确定它们的结构分别为2,4-dihydroxy-2,6-trimethyl-△^(1,α)-cyclohexaneacetic-r-lactone(1)、saringosterol(2)、雪松醇(3)。

浙江沿海不同地理群体铜藻Sargassum horneri的AFLP分析

铜藻（Sargassum horneri）是浙江沿海重要的褐藻之一,具有较高的生态价值。对铜藻遗传多样性进行分析,可以为其资源保护、人工繁育以及海洋生态修复等提供理论参考。本研究利用AFLP分子标记技术,从64对选择性扩增引物中筛选出20对扩增效果好、带型清晰、条带分布均匀的引物,并用这20对引物对浙江4个铜藻群体进行了分析。结果显示,80个铜藻个体中共检测到有效结合位点407个,其中多态性结合位点270个;铜藻4个群体整体水平上的多态位点百分率（PPL）为66.34%,4个群体各自的多态位点百分率在30.49%～46.54%之间,均值为36.09%。4个铜藻群体的Nei基因多样性指数（H）为0.237 5,Shannon信息指数（I）为0.374 1,4个铜藻群体的总遗传变异（Ht）和群体内遗传变异（Hs）分别为0.314 2和0.256 9,群体间遗传分化系数（Gst）为0.182 3,即群体间遗传变异占总变异的18.23%,即群体内遗传变异占总变异的81.77%,表明群体间遗传分化程度较低。4个铜藻群体间的基因流动系数（Nm）为1.121 4,表明浙江沿海铜藻群体间有一定的基因交流。

鼠尾藻(Sargassum thunbergii)有性生殖过程与育苗

采用室内培养和野外实地观测的方法,研究了鼠尾藻的生长发育和有性生殖过程,并在此基础上进行了有性生殖人工育苗实验。结果表明,鼠尾藻在海水温度高于22℃时,生殖托开始发育,海水温度超过23℃以后,生殖托的生长速度加快,海水温度超过24℃时,生殖托生长速度最快并迅速成熟,在海水温度达到26℃时,观察到了卵的集中排放挂托现象。鼠尾藻卵的排放较为齐整且有一定的先后顺序,生殖托基部的卵最先排出,中部次之,最后是顶部,雄托排精时间要稍晚于雌托排卵时间。受精卵大约在受精1h后进行第一次细胞分裂,分裂面与长轴垂直,大约2h后进行第二次分裂,下端细胞的分裂面平行于第一次分裂方向,而上端细胞的分裂面则垂直,随后大约每2—4h细胞分裂一次。从卵挂托到幼胚脱落一般需要24—48h。观察发现,生殖窝不仅存在于生殖托上,在部分生殖枝上也有分布。在鼠尾藻的人工育苗的实验中,胚苗在苗帘上的生长状态良好。

我国沿海鼠尾藻(Sargassum thunbergii)遗传资源RAPD分析

采用RAPD分子标记技术分析了我国沿海五个海区(大连、威海、青岛、温州、福州)鼠尾藻种群的遗传多样性。从60条随机引物中筛选出21条引物,对五个鼠尾藻种群共150份DNA样本进行了扩增,共检测到207个有效位点,多态位点达89.5%,Nei基因多样性指数(H)为0.2966,Shannon信息指数(I)为0.4508;种群间的遗传分化系数(Gst)为0.6131,基因流(Nm)为0.3155。AMOVA分析表明Fst为0.6185。我国沿海鼠尾藻种群遗传多样性较低,遗传分化水平较高。

羊栖菜Sargassum fusiforme Setch繁殖与发育生物学的初步研究

对经济海藻羊栖菜的分类、形态结构、繁殖方式、发育方式。

桑沟湾筏式养殖鼠尾藻(Sargassum thunbergii)的生长特性

2014年4–6月在桑沟湾海区进行鼠尾藻(Sargassum thunbergii)海上筏式养殖实验,分析了鼠尾藻在桑沟湾的生长特性,调查了藻体上的附着生物。结果显示,(1)鼠尾藻在桑沟湾海域生长迅速,水温为10–17℃时特定生长率最高,可达6.10%/d;根据特定生长率与水温的关系,获得了鼠尾藻的最佳生长温度为14.9℃;(2)5月10日开始有生殖托形成,水温达到20.4℃时,鼠尾藻生殖托大量成熟,并有放散;(3)养殖期间,鼠尾藻最大长度达187.05 cm,均长可达112.31 cm;干湿比由0.147(4月)上升至0.189(6月);每公顷产量可达43.95 t(湿重),相当于干重为8.25 t;(4)藻体上有大型附着生物16种,主要优势种为尖嘴扁颌针鱼鱼卵、玻璃海鞘和海绵;附着生物的生物量随着水温升高而增加。研究表明,海区的附着生物对鼠尾藻的生长影响不大,在桑沟湾大规模养殖鼠尾藻是可行的。

不同氮、磷浓度及配比对铜藻(Sargassum horneri)幼苗生长的影响

采用铜藻(Sargassum horneri)幼苗为实验对象,分别以Na NO3和Na H2PO4为氮源和磷源,研究了不同氮磷质量浓度及氮磷配比对铜藻幼苗生长的影响。进行了氮、磷单因子实验、双因子实验以及不同氮磷配比实验,实验时间为8d。结果表明:氮和磷对铜藻幼苗生长影响极显著(P<0.01),且当氮、磷的质量浓度分别为8mg/L、0.4mg/L时,铜藻幼苗的特定生长率最大;交互作用影响不显著(P>0.05),不同质量浓度的氮为主效应;不同氮磷质量浓度比影响极显著(P<0.01),且当氮磷比为10:1时,铜藻幼苗的特定生长率最大。研究结果为铜藻幼苗培育过程中营养盐的合理调控提供了理论依据。

Growth and Resource Accumulation of Drifting Sargassum horneri(Fucales, Phaeophyta) in Response to Temperature and Nitrogen Supply

The abnormal increase of drifting brown alga Sargassum horneri was initially documented in 2007.It formed blooms along the coast of East China Sea and Yellow Sea in 2017.In this study,we investigated the changes of specific growth rate and resource accumulation of drifting S.horneri in response to temperature and nitrogen richness at different growth stages under laboratory condition.The investigation lasted from June 2015 to April 2016 with the observation made every two months.The results showed that the life cycle consists of a few growth stages dividable with morphological characteristics.The growth can be divided into shedding and withering(August),rapid growing(October to September),slow growing(February),rapid growing(April)and maturation(June)stages.Under the experimental condition,algal segments were found to grow at temperatures ranging from 5 to 25℃in 12 days even when nitrogen is deficient.A significant difference in the special growth rate(SGR)between nitrogen-enriched and nitrogen-removed treatments was found in most months(P<0.05).SGR was lower in August and February than that in other months.Nitrogen and chlorophyll contents in algal segments were different among different temperatures,nitrogen supply and seasons.Nitrogen content was higher in February and April than that in other months in both nitrogen-enriched and nitrogen-removed treatments.The results showed that the demand of S.horneri for nitrogen increased in spring when it grows fast.It is likely that the high temperature and nitrogen concentration in winter and spring lead to the high biomass accumulation of drifting S.horneri.

Decadal characteristics of the floating Ulva and Sargassum in the Subei Shoal,Yellow Sea

The data of field surveys during 2009 to 2018 was analyzed to understand the seasonality and inter-annual variability of the floating Ulva and Sargassum in the Subei Shoal,the southwestern Yellow Sea of China on decadal scale.The floating Ulva biomass was consistently originated from the central region of the Subei Shoal in middle to late April,increased rapidly,drifted and extended into the offshore water in May and June.The average floating Ulva biomass in the shoal generally increased over the years with evident inter-annual fluctuations.In contrast,pelagic Sargassum was accumulated in the Subei Shoal and formed the spring bloom only in 2013,2017 and 2018,and the biomass was higher than the co-occurring Ulva during the survey in these three years.Compared to the raft-origin floating Ulva,genesis and development of the pelagic Sargassum was distinct.Based on the current research,the Sargassum biomass was exotic and often initiated in the offshore water in March,and intruded into the shoal in April and May.The analysis on the environmental parameters was inconclusive since multiple anthropogenic and non-indigenous factors could influence the green tides in this region.Further research covering both the East China Sea and the Yellow Sea is needed to trace the origin of the floating Sargassum and to understand the interactions between these two co-occurring seaweeds.

Anti-oxidant and anti-inflammatory activities of ultrasonicassistant extracted polyphenol-rich compounds from Sargassum muticum

Polypehnol is an important,potentially bioactive component of Sargassum muticum.In this study,ultrasonic assisted extraction of polyphenol-rich substances was performed using a 38%ethanol solution at a solid:liquid ratio of 1:30 at 68℃ for 32min,determined by single-factor and response surface methodology(RSM)optimization.The content of polyphenol was 5.66mg/g in the crude extract.Further extraction showed that the polyphenol mainly distributed in ethyl acetate(SKEE)and water phases(SKEW).The anti-oxidation test by electron spin resonance(ESR)spectrum showed that the SKEE had the strongest scavenging activity on DPPH(1,1-diphenyl-2-picrylhydrazyl)and alkyl radicals.SKEE was shown noncytotoxic but could inhibit the generation of cellular ROS,showing protective effects in H2O2 and AAPHinduced Vero cells and UV-B irradiated HaCaT cells.SKEE also signifi cantly inhibited the release of NO of LPS-induced RAW 264.7 cells.Therefore,the polyphenol-rich extracts in ethanol and ethyl acetate showed excellent anti-oxidant and anti-infl ammatory activities,which is beneficial to the development of high-value bio-substances.