CN115490780A - 马尾藻岩藻多糖粗提物的提取方法及应用 - Google Patents
马尾藻岩藻多糖粗提物的提取方法及应用 Download PDFInfo
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- CN115490780A CN115490780A CN202211249016.7A CN202211249016A CN115490780A CN 115490780 A CN115490780 A CN 115490780A CN 202211249016 A CN202211249016 A CN 202211249016A CN 115490780 A CN115490780 A CN 115490780A
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Abstract
本发明公开了马尾藻岩藻多糖粗提物的提取方法及应用。所述方法包括如下步骤:将马尾藻经粉碎机粉碎后,再进行超微粉碎,得到超微粉碎马尾藻粉;向所述超微粉碎马尾藻粉中加水搅拌均匀后,置于热水中浸提;浸提完成后进行超声提取得到浸提液;将所述浸提液离心、去沉淀,将上清液浓缩后加入无水乙醇,离心,取沉淀;将所述沉淀用有机溶剂洗涤后加水溶解,再加入复合有机溶剂除蛋白,振荡后静置分层,取上清液于透析袋中进行透析,再经真空冷冻干燥后即得到岩藻多糖粗提物。本发明采用超声波辅助热水浸提法从超微粉碎马尾藻粉中提取岩藻多糖粗提物,效率更高,所得岩藻多糖粗提物具有良好的降血糖活性和免疫增强效果,可以得到广泛应用。
Description
技术领域
本发明涉及多糖提取技术领域,特别地涉及马尾藻岩藻多糖粗提物的提取方法及应用。
背景技术
近来,有关海洋藻类来源的生物活性物质研究被广泛报道,其中就有关于不同种类褐藻被证实含有许多生物活性丰富的蛋白和糖类的研究,而马尾藻作为褐藻的一种,是富硫多糖等多种营养成分的来源,因其具有许多生物活性功能的物质而广泛应用于食品保健和生物医药领域(周乾云,欧阳月红,李勇,许诺,吴明江,姜潮.马尾藻海藻多糖生物活性研究新进展[J].食品科学,2019,12:211-214.)。湛江马尾藻资源丰富,大多分布在硇洲岛等东南地区,其种类众多,现有研究的马尾藻种类包括亨氏马尾藻、张氏马尾藻、半叶马尾藻、围氏马尾藻等,由于马尾藻种类、生长季节、采集时间不同,所获得的生物活性物质化学组成也会不同。
岩藻多糖是从马尾藻中提取出的主要活性物质,因其主要单糖成分为岩藻糖而得名(Narayani S S,Saravanan S,Ravindran J,Ramasamy M S,Chitra J.In vitroanticancer activity of fucoidan extracted from Sargassum cinereum againstCaco-2cells[J].International Journal ofBiological Macromolecules,2019,138:618-628.)。同时,它也是一种水溶性的多聚阴离子同型杂多糖,富含硫酸基团,因此又被叫做岩藻聚糖硫酸酯(Wu L,Sun J,Su X,Yu Q,Yu Q,Zhang P.A review about thedevelopment of fucoidan in antitumor activity:Progress and challenges[J].Carbohydrate Polymers,2016,154:96-111.)。岩藻多糖的单糖组成除了主要的L-岩藻糖外,还包括少量的鼠李糖、阿拉伯糖、半乳糖等,各单糖间主要通过α-1,2、α-1,3或α-1,4糖苷键相连(Vo T-S,Kim S-K.Fucoidans as a natural bioactive ingredient forfunctional foods[J].Journal ofFunctional Foods,2013,5(1):16-27.)。大量研究表明,岩藻多糖有广泛的保健作用和治疗功效,具有降血脂(廖敏,谌素华,王维民,钟思燕,俞素素,廖森泰.张氏马尾藻多糖的体外降胆固醇活性[J].广东海洋大学学报,2017,37(3):80-85.)、降血糖(Shan X,Liu X,Hao J,Cai C,Fan F,DunY,Zhao X,Liu X,Li C,Yu G.Invitro and in vivo hypoglycemic effects of brown algal fucoidans[J].International Journal of Biological Macromolecules,2016,82:249-255.)、抗氧化(徐元庆,王哲奇,张静,范依霖.岩藻多糖的抗氧化功能研究进展[J].天然产物研究与开发,2020,32:1782-1793.)、抗癌(Narayani S S,Saravanan S,Ravindran J,Ramasamy MS,Chitra J.In vitro anticancer activity of fucoidan extracted from Sargassumcinereum against Caco-2 cells[J].International Journal ofBiologicalMacromolecules,2019,138:618-628.)、调节免疫功能(Amin M L,Mawad D,Dokos S,KoshyP,Martens P J,Sorrell C C.Immunomodulatory properties of photopolymerizablefucoidan and carrageenans[J].Carbohydrate Polymers,2020,230:115691.)等生物活性,其生物活性还与多糖的分子量大小、硫酸基含量以及化学结构有着密切的关系,深入了解岩藻多糖的构效关系也成为大量学者今后研究的重点。
岩藻多糖的组成和结构与其生物学活性和生物利用度有关,并且受提取方法的影响(Flórez-Fernández N,Torres M D,González- M J,Domínguez H.Potential ofintensification techniques for the extraction and depolymerization offucoidan[J].Algal Research,2018,30:128-148.)。常见的提取方法根据提取原理的不同可分为两大类,一类是物理化学提取,主要是利用物理化学手段如高温、加压、超声波、酸碱等使得多糖结构发生改变,进而通过多糖溶解和扩散等过程来得到多糖,包括热水浸提法、酸提和碱提法、加压液相提取等(He L,Yan X,Liang J,Li S,He H,Xiong Q,Lai X,Hou S,Huang S.Comparison of different extraction methods for polysaccharides fromDendrobium oficinale stem[J].Carbohydrate Polymers,2018,198:101-108.)。另一类是生物提取,以酶解法为主,主要利用生物活性酶来破坏细胞壁和细胞膜,促进多糖的分离,或是将多糖降解从而易于提取(Nadar S S,Rao P,Rathod V K.Enzyme assistedextraction of biomolecules as an approach to novel extraction technology:Areview[J].Food Research International,2018,108:309-330.)。然而,单一提取方法在时间成本和环保方面可能会存在一些不足,为了迫切解决这些问题,许多学者提出了将不同提取方法结合的观点。例如:热水浸提法是多糖提取的常用方法,但因其具有时间长、高温、低效等缺点,因此逐渐被更多的快速、便捷方法替代,Shi等(Shi F,Yan X,Cheong K-L,Liu Y.Extraction,purification,and characterization ofpolysaccharides frommarine algae Gracilaria lemaneiformis with anti-tumor activity[J].ProcessBiochemistry,2018,73:197-203.)发现,超声波-微波辅助热水浸提法的多糖提取率胜过传统的热水浸提法,超声波的振动空化和微波的辐射作用可以大大提高提取率,并且更经济环保和减少提取过程中副产物的产生。当然,其他不同提取方法的组合也被许多学者所发现,以便提高多糖的提取效率。
有关2型糖尿病的研究不断深入,但作为一种慢性的、不可阻挡的疾病,其发病机制尚不清楚。同时,目前用于治疗2型糖尿病的临床用药主要有双胍类、磺脲类以及α-葡萄糖苷酶抑制剂类如阿卡波糖等,但这类药物副作用较多,会造成肠胃功能紊乱、低血糖症等。因此,开发安全性高、副作用小的新型2型糖尿病治疗药物是新药研发的热点之一(陈雨豪,王周,牛宣,吴陈.螺旋藻肽对四氧嘧啶诱导的糖尿病小鼠作用研究[J].宁波大学学报(理工版),2020,33(3):13-18.)。已有研究表明,α-葡萄糖苷酶活性与餐后血糖水平的升高密切相关,因此对这种酶的控制对糖尿病的治疗尤其重要。Shan等(Shan X,Liu X,Hao J,Cai C,Fan F,Dun Y,Zhao X,Liu X,Li C,Yu G.In vitro and in vivo hypoglycemiceffects of brown algal fucoidans[J].International Journal of BiologicalMacromolecules,2016,82:249-255.)研究发现从褐藻中提取的岩藻多糖可以有效抑制α-葡萄糖苷酶的活性,从而降低体内血糖的水平以减少糖尿病的发生,且其IC50值显著低于广泛用于治疗糖尿病的阿卡波糖,因此其可以作为一种潜在的治疗高血糖的药物。
免疫系统与许多疾病的发生和发展密切相关。免疫是人体的一种生理功能,人体可以利用这种功能来识别“自己”和“非己”成分,从而清除进入人体的抗原物质或人体自身产生的损坏细胞或肿瘤细胞,进而维持身体健康。巨噬细胞是免疫细胞的一种,通过激活巨噬细胞,可以吞噬或杀死病原体,其分泌的NO、前列腺素、肿瘤坏死因子等也可以间接杀死病原体,因此活化巨噬细胞对免疫调节有很大的帮助(陈怡帆,沈成龙,曾英杰,阳辉蓉,陈炼红.甘孜松茸多糖的分离纯化及免疫调节活性研究[J].食品科技,2021,46(10):177-183.)。多糖的免疫调节活性机制正在不断地被人们发现。有研究表明,iNOS(诱导型一氧化氮合酶)是催化巨噬细胞分泌合成NO的关键酶,而COX-2(诱导型环氧化酶)是催化巨噬细胞合成前列腺素的关键酶,并且两种酶均主要在激活后的巨噬细胞中表达,因此,通过检测多糖样品干预后RAW264.7细胞NO的释放量以及利用Western Blot技术和荧光免疫技术研究多糖干预后RAW264.7细胞中iNOS和COX-2这两种酶在蛋白水平的表达情况都可以来反应多糖的免疫调节活性(任琪.银杏叶多糖的提取、分离纯化及生物活性研究[D].安徽农业大学,2018.)。越来越多不同来源的多糖被证明可以通过刺激细胞NO和前列腺素的分泌来改善免疫调节活性。任琪通过提取、分离纯化得到银杏叶多糖后,将其加入细胞培养基中发现银杏叶多糖可以显著促进细胞COX-2和iNOS在蛋白水平上的表达(任琪.银杏叶多糖的提取、分离纯化及生物活性研究[D].安徽农业大学,2018.)。Xu等(Xu Z,Lin R,Hou X,Wu J,Zhao W,Ma H,Fan Z,Li S,Zhu Y,Zhang D.Immunomodulatory mechanism of a purifiedpolysaccharide isolated from Isaria cicadae Miquel on RAW264.7 cells viaactivating TLR4-MAPK-NF-kappaB signaling pathway[J].International JournalofBiological Macromolecules,2020,164:4329-4338.)研究发现蝉花纯化多糖通过激活TLR4-MAPK-NF-κB信号通路激活RAW264.7细胞分泌NO,进而对RAW264.7细胞发挥免疫调节作用。陈怡帆等(陈怡帆,沈成龙,曾英杰,阳辉蓉,陈炼红.甘孜松茸多糖的分离纯化及免疫调节活性研究[J].食品科技,2021,46(10):177-183.)研究了甘孜松茸多糖的免疫调节活性,发现多糖可以提高RAW264.7细胞的增殖能力,且促进细胞释放NO和多种细胞因子,进而显著提升了RAW264.7的免疫调节能力。NO、COX-2以及iNOS作为免疫调节的显著标志,其变化可以很好地帮助我们进行免疫调节活性判断方面的研究。
发明内容
有鉴于此,本发明的主要目的是提供马尾藻岩藻多糖粗提物的提取方法。
本发明所提供的从马尾藻中提取岩藻多糖的方法,包括如下步骤:
将马尾藻粉碎后,再经超微粉碎,得到超微粉碎马尾藻粉;向所述超微粉碎马尾藻粉中加水搅拌均匀后,置于热水中浸提;浸提完成后进行超声提取得到浸提液;将所述浸提液离心、去沉淀,将上清液浓缩后加入无水乙醇,离心,取沉淀;将所述沉淀用有机溶剂洗涤后加水溶解,再加入复合有机溶剂除蛋白,振荡后静置分层,取上清液于透析袋中进行透析,再经真空冷冻干燥后,即得到岩藻多糖粗提物。
优选地,所述超微粉碎马尾藻粉的粒度大小为150-2000目,进一步优选为150-600目。
更优选地,所述超微粉碎马尾藻粉的粒度大小为300目。
优选地,所述超微粉碎马尾藻粉与水的料液比为1:10g/mL~1:50g/mL;所述水为蒸馏水。
更优选地,所述超微粉碎马尾藻粉与水的料液比为1:30g/mL;所述水为蒸馏水。
优选地,所述置于热水中浸提是指置于50℃~80℃热水浸提1h~8h。
更优选地,所述置于热水中浸提是指置于80℃热水浸提3.5h。
优选地,所述超声提取是在超声功率为50W~500W的条件下超声提取10min~60min;所述浸提液在3000r/min~8000r/min条件下离心5min~40min。
更优选地,所述超声提取是在超声功率为350W的条件下超声提取50min;所述浸提液在4000r/min条件下离心10min。
优选地,所述将所述上清液浓缩,加入无水乙醇,离心后取沉淀是指:将所述上清液用旋转蒸发仪浓缩至原体积的五分之一至二分之一后,加入无水乙醇至其体积百分比浓度为20%~40%,二次离心取上清液;上清液继续加入无水乙醇至其体积百分比浓度为50%~90%,离心后取沉淀。
更优选地,所述将所述上清液浓缩,加入无水乙醇,离心后取沉淀是指:将所述上清液用旋转蒸发仪浓缩至原体积的三分之一后,加入无水乙醇至其体积百分比浓度为30%,二次离心取上清液;上清液继续加入无水乙醇至其体积百分比浓度为80%,离心后取沉淀。
该步骤加入无水乙醇的目的是除去马尾藻中的海藻酸盐;30%为乙醇的最终浓度。按照溶液体积比公式,乙醇最终浓度=(无水乙醇浓度×所需无水乙醇体积)/总溶液体积;30%是根据文献以及海藻酸盐物质的特性决定的,可以使海藻酸盐沉淀除去;可行范围为20%~50%,浓度稍低时需静置一段时间待海藻酸盐都沉淀。此处沉淀即为提取的主要物质岩藻多糖;沉淀除包含岩藻多糖外,还含有蛋白质、多酚等物质。
优选地,将所述沉淀用有机溶剂进行洗涤是指:沉淀用无水乙醇和丙酮各洗2次。因多糖不溶于乙醇、丙酮,所以加入无水乙醇和丙酮可以除去一些可溶性杂质以及色素。可以用其他溶剂代替无水乙醇和丙酮,乙醇和丙酮为较为常用的有机试剂。
优选地,所述加入复合有机溶剂除蛋白是按照体积比为3:1~6:1的比例加入复合有机溶剂;所述透析在4℃~16℃冰箱中透析24h~48h。
更优选地,所述加入复合有机溶剂除蛋白是按照体积比为4:1的比例加入复合有机溶剂;所述透析在4℃冰箱中透析24h,透析期间更换3次水。
优选地,所述透析后的溶液在-50℃进行真空冷冻干燥。
除蛋白还可以用中性盐、金属化合物等物质,通过盐析以及金属沉淀法将蛋白除去,而复合有机溶剂法较适合本实验且效果好。透析的目的为除去多糖溶液中的有机试剂等。
所述马尾藻为半叶马尾藻或张氏马尾藻等。
所述方法提取得到的岩藻多糖提取物也属于本发明的保护范围。
所述的岩藻多糖提取物在制备降血糖和/或调节免疫活性的产品中的应用也属于本发明的保护范围。
本发明以超声波辅助热水浸提法提取出的马尾藻岩藻多糖粗提物为研究对象,对马尾藻岩藻多糖粗提物进行化学成分分析,明确其化学组成,进而探究其降血糖作用和免疫调节活性。通过研究马尾藻岩藻多糖粗提物对α-葡萄糖苷酶的抑制作用来初步判断其降血糖生物活性。通过RAW264.7细胞实验、荧光定量PCR、免疫荧光技术和Western Blot蛋白免疫印迹实验来判断马尾藻岩藻多糖粗提物的免疫活性作用,为进一步开发马尾藻岩藻多糖降血糖和调节免疫活性药物提供理论依据。
附图说明
为了说明而非限制的目的,现在将根据本发明的优选实施例、特别是参考附图来描述本发明,其中:
图1为岩藻糖标准曲线。
图2为牛血清蛋白标准曲线。
图3为没食子酸标准曲线。
图4为硫酸钾标准曲线。
图5为两种马尾藻岩藻多糖粗提物的高效凝胶色谱图。
图6为半叶马尾藻岩藻多糖粗提物的离子色谱图。
图7为张氏马尾藻岩藻多糖粗提物的离子色谱图。
图8为单糖混标的离子色谱图。
图9为两种马尾藻岩藻多糖粗提物的扫描电镜图(A:张氏马尾藻岩藻多糖粗提物;B:半叶马尾藻岩藻多糖粗提物)。
图10为两种马尾藻岩藻多糖提取物对α-淀粉酶(A)和α-葡萄糖苷酶(B)的抑制作用。
图11为两种马尾藻岩藻多糖提取物对α-葡萄糖苷酶的IC50值。
图12为不同浓度下多糖对RAW264.7细胞NO产气量的影响。
图13为采用荧光定量PCR分析两种不同浓度马尾藻岩藻多糖粗提物刺激下RAW264.7细胞中COX-2和iNOS的mRNA表达。
图14为两种马尾藻岩藻多糖粗提物干预下COX-2和iNOS蛋白表达的免疫荧光图,比例尺为50μm;其中,图14A为张氏马尾藻岩藻多糖粗提物干预下COX-2蛋白表达的免疫荧光图;图14B为张氏马尾藻岩藻多糖粗提物干预下iNOS蛋白表达的免疫荧光图;图14C为半叶马尾藻岩藻多糖粗提物干预下COX-2蛋白表达的免疫荧光图;图14D为半叶马尾藻岩藻多糖粗提物干预下iNOS蛋白表达的免疫荧光图。
图15为采用Western blot分析两种不同浓度马尾藻岩藻多糖粗提物刺激下RAW264.7细胞中COX-2和iNOS的蛋白表达(图A-C为张氏马尾岩藻多糖粗提物,图D-F为半叶马尾藻岩藻多糖粗提物)。
具体实施方式
实施例1、马尾藻岩藻多糖粗提物的提取方法
1材料与仪器
1.1原料
半叶马尾藻:2022年3月采摘于广东省湛江市硇洲岛地区附近海域。采摘后的马尾藻经清洗、晾晒、烘干、粉碎后过100目筛,放干燥器或冰箱里储藏备用。
张氏马尾藻:2021年9月采摘于广东省湛江市硇洲岛地区附近海域。采摘后的马尾藻经清洗、晾晒、烘干、粉碎后过100目筛,放干燥器或冰箱里储藏备用,提取多糖前进行超微粉碎。
1.2主要试剂和材料
表1主要试剂和材料
1.3主要仪器和设备
表2主要仪器和设备
2实验方法
2.1岩藻多糖粗提物的提取
称取超微粉碎马尾藻粉(300目)20.0g于1L烧杯,按1:30g/mL的料液比加入蒸馏水,玻璃棒搅拌均匀后,将转子轻轻放入,烧杯放入恒温磁力搅拌锅中,80℃热水浸提3.5h。浸提完成后,用350W超声辅助提取50min,浸提液在4000r/min条件下离心10min,弃去沉淀。上清液用旋转蒸发仪浓缩至原体积的三分之一后,加入无水乙醇至其体积百分比浓度为30%(v/v),第二次离心取上清液。上清液继续加入无水乙醇至其体积百分比浓度为80%,离心后取沉淀。沉淀用无水乙醇和丙酮各洗2次后,加入水溶解,再以4:1(v/v)的比例加入复合有机溶剂除蛋白,漩涡仪振荡20min后,静置分层,取上清液于透析袋中,在4℃冰箱中透析24h,期间更换2-3次蒸馏水,透析后,将多糖溶液进行真空冷冻干燥,即得岩藻多糖粗提物。岩藻多糖粗提物得率和提取率的计算公式如下:
2.2岩藻多糖粗提物的化学成分分析
2.2.1总糖含量的测定
总糖含量采用苯酚-硫酸法进行测定(刘海韵.马尾藻岩藻聚糖硫酸酯对血栓及HUVEC和HMVEC的作用研究[D].广东海洋大学,2019.)。
(1)试剂配制:0.1mg/mL岩藻糖标准溶液:0.005g岩藻糖加水定容至50mL。6%苯酚溶液:苯酚放在60℃水浴锅溶解后,吸取600μL苯酚,加水定容至10mL。
(2)标准曲线的制作:分别吸取0.2,0.4,0.6,0.8,1.0mL的岩藻糖标准液,加蒸馏水补至1.0mL,再在每管中加入1.0mL苯酚溶液,摇匀后迅速加入5.0mL浓硫酸,混匀后静置15min,直到冷却至室温,再用移液枪分别注入200μL/孔到96孔板中,以蒸馏水为空白,使用酶标仪在490nm的波长下测定吸光值,以标准岩藻糖浓度为横坐标,吸光值为纵坐标绘制标准曲线,得回归方程。
(3)样品总糖含量的测定;准确称取2.5mg干燥样品,加水溶解后定容至50mL,即样品溶液浓度为50μg/mL。取1.0mL样品溶液,按标准曲线的方法测定样品吸光值。代入制作好的标准曲线,根据以下公式即可计算总多糖含量。
式中:V-样品溶液的体积(mL);C-由标准曲线上计算得到的多糖浓度(mg/mL);W-样品的质量(mg)。
2.2.2蛋白质含量的测定
蛋白质含量采用考马斯亮蓝法进行测定(刘海韵.马尾藻岩藻聚糖硫酸酯对血栓及HUVEC和HMVEC的作用研究[D].广东海洋大学,2019.)。
(1)试剂配制:1.0mg/mL牛血清蛋白标准溶液:称取1.0mg牛血清蛋白于蒸馏水中溶解,后定容至10mL。考马斯亮蓝溶液:称取考马斯亮蓝10mg,溶解于5mL 95%的酒精中,再用10mL 85%磷酸进行酸化,再加蒸馏水定容至100mL。
(2)标准曲线的制作:分别吸取0、0.05、0.15、0.25、0.35、0.45mL牛血清蛋白溶液,加入蒸馏水至0.5mL,各管再加入考马斯亮蓝溶液2.5mL,振荡混匀后,避光静置反应10min,在595nm下测定吸光值。以牛血清蛋白溶液的浓度为横坐标,吸光值为纵坐标绘制标准曲线。
(3)样品蛋白质含量的测定:称取多糖样品20mg,加入蒸馏水定容至10mL。吸取0.5mL多糖溶液,按标准曲线的方法测定样品吸光值,用标准曲线计算多糖样品蛋白质含量。
2.2.3多酚含量的测定
多酚含量采用福林酚法进行测定(崔欣.石花菜多糖的提取纯化、理化性质和生物活性研究[D].南京农业大学,2019.)。
(1)试剂配制:10μg/mL没食子酸溶液:称取没食子酸1.0mg,用蒸馏水定容至100mL。福林酚溶液:买到的福林酚试剂稀释10倍后得到。10%碳酸钠溶液:称取10.0g碳酸钠固体,用蒸馏水溶解后定容至100mL。
(2)标准曲线的制作:分别吸取0、0.25、0.50、0.625、0.75、0.875mL没食子酸溶液,用蒸馏水补至1.0mL,再在各管中加入1.0mL福林酚溶液,于30℃温度下避光放置5min,再加入2.0mL碳酸钠溶液,继续在30℃温度下避光反应1h,在747nm下测定吸光值。以没食子酸溶液的浓度为横坐标,以吸光值为纵坐标绘制标准曲线。
(3)样品多酚含量的测定:称取多糖样品20mg,加入蒸馏水定容至10mL。吸取0.5mL多糖溶液,按上述方法测定样品吸光值,用标准曲线计算多糖样品中的多酚含量。
2.2.4硫酸基含量测定
硫酸基含量采用氯化钡-比浊法进行测定。
(1)试剂配制:0.5%明胶溶液:称取1.25g明胶于蒸馏水中,在60-70℃温度的水浴下溶解完全,再加蒸馏水定容至250mL,4℃下静置过夜,冷藏陈化。明胶-氯化钡溶液:称取1.0g氯化钡固体于5%明胶溶液中,定容至100mL,于4℃下静置2h,冷藏保存。0.6mg/mL硫酸钾标准溶液:称取硫酸钾固体3mg,用1mol/L的盐酸定容至5mL。3%TCA溶液:称取3g三氯乙酸固体,用蒸馏水溶解后定容至100mL。
(2)标准曲线的绘制:分别吸取0、0.04、0.08、0.12、0.16、0.20mL硫酸钾溶液,用1mol/L的盐酸补至0.20mL,再在各管中加入3%的TCA溶液3.8mL和明胶溶液1.0mL,摇匀,于30℃温度下避光放置15min,于360nm处测定吸光值1;用明胶溶液代替氯化钡-明胶溶液,于360nm处测定吸光值2。以硫酸钾溶液的浓度为横坐标,吸光值1-吸光值2为纵坐标绘制标准曲线。
(3)样品硫酸基含量的测定:称取多糖样品3mg,加入1mol/L的盐酸溶液2mL,摇匀至完全溶解,100℃温度下加热3h,冷却后过滤。吸取过滤后溶液0.2mL,按标准曲线的方法测定样品吸光值,用标准曲线计算多糖样品的硫酸基含量。
2.3岩藻多糖粗提物分子量测定
采用高效凝胶色谱法测定多糖的分子量。
(1)样品的准备:精密称取样品和标准品,样品溶液配制浓度为5mg/mL,12000rpm/10min离心后,吸取上清液于0.22μm的微孔滤膜中过滤,然后将过滤后溶液转置于1.8mL进样小瓶中。
(2)色谱条件:色谱柱:BRT105-104-102串联凝胶柱(8×300mm);流动相:0.05mol/LNaCl溶液;流速:0.6mL/min,柱温:40℃;进样量:20μL;检测器:示差检测器RI-10A。
2.4岩藻多糖粗提物单糖组成及含量的分析
采用离子色谱法进行单糖组成和含量的分析。
(1)样品的准备:精密称量5mg样品,加入2mL 3M TFA后,于120℃水浴中水解3h。将酸水解溶液吸取转移至离心管中,用氮吹仪吹干,加入5mL蒸馏水涡旋混匀后,用移液枪吸取50μL于950μL蒸馏水中,在12000rpm条件下离心5min。取上清进离子色谱仪中分析。
(2)色谱条件:色谱柱:Dionex Carbopac TMPA20(3mm×150mm);流动相:A:H2O;B:15mM NaOH;C:15mM NaOH&100mM NaOAC;流速:0.3mL/min;进样量:5μL;柱温:30℃;检测器:电化学检测器。
2.5岩藻多糖粗提物的微观形态分析
采用扫描电子显微镜(SEM)观察岩藻多糖粗提物的微观形态。取适量岩藻多糖样品置于样品台,用导电胶固定后进行镀金,在5kV电场下对多糖的表面形态进行观察并拍照。
2.6粒径和电位的测定
将两种岩藻多糖粗提物分别溶于蒸馏水中,配成5mg/mL的溶液,再采用马尔文纳米粒度电位仪进行测定,每个样品扫描3次。
2.7岩藻多糖粗提物的降血糖作用
2.7.1α-葡萄糖苷酶活性的抑制
(1)试剂配制:样品与阿卡波糖分别配置成0、0.01、0.1、0.5、0.75、1.0、1.5mg/mL浓度梯度的溶液。
(2)实验步骤:实验在96微孔板上进行,具体步骤参照表3。
表3α-葡萄糖苷酶活性测定实验步骤
(3)吸光值的测定:试验完成后,将96孔板于405nm处测量吸光值。按照下式计算酶活性抑制率。
酶活性抑制率=[(A对照-A对照空白)-(A样品-A样品空白)]/A对照-A对照空白
2.7.2岩藻多糖提取物对α-淀粉酶活性的抑制
样品配制成0、0.5、0.75、1.0mg/mL浓度梯度的溶液。实验在96孔板上进行,具体步骤参照表4。将96孔板于540nm处测量吸光值[20]。按照下式计算酶活性的抑制率:
酶活性抑制率=[(A对照-A对照空白)-(A样品-A样品空白)]/A对照-A对照空白
表4α-淀粉酶活性测定实验步骤
2.8岩藻多糖粗提物调节免疫活性研究
2.8.1 Griess试剂法检测RAW264.7细胞NO的释放量
(1)细胞培养:RAW264.7细胞在含有10%FBS、青霉素(100U/mL)和链霉素(100μg/mL)的DMEM培养基中,于含5%CO2的37℃恒温培养箱中培养。
(2)NO含量测定:将RAW264.7细胞悬液(1×104个/mL)接种至96孔板(100μL/孔),并将其放置含5%CO2培养箱中培养24h。培养过后,使用0.1mol/L的PBS缓冲液(pH=7.2)洗涤96孔板两次,以除去非贴壁细胞。向96孔板加入浓度为25、50和100μg/mL的两种多糖溶液(用新鲜的完全DMEM培养基溶解),孵育24h后收集96孔板上每个孔中的上清液进行NO含量的测定。NO含量使用一氧化氮检测试剂盒进行检测,在540nm处测定其吸光值。该试验用不含粗多糖的DMEM培养基作空白对照,根据NaNO2的校准曲线计算NO的含量。
2.8.2荧光定量PCR实验
RAW264.7细胞培养方法同2.8.1(1)。分别用两种马尾藻岩藻多糖粗提物(25,50和100μg/mL)处理细胞,处理时间24h。用Trizol试剂盒提取总RNA,加入SYBR green进行PCR扩增。采用荧光定量PCR仪测定COX-2和iNOS基因表达,以GAPDH为内参。COX-2的正向引物为:TGAGTACCGCAAACGC-TTCT,反向引物为:ACGAGGTTTTTCCACCAGCA;iNOS的正向引物为:CCTCCTCGTTCAGCTCACCT,反向引物为:CAATCCACAACTCGCTCCAA;GAPDH的正向引物为:GGTGAAGGTCGGTGTGAACG,反向引物为:CTCGC-TCCTGGAAGATGGTG。
2.8.3 Western blot和免疫荧光技术检测COX-2和iNOS蛋白的表达量
(1)细胞培养:RAW264.7细胞培养与2.8.1(1)相同。
(2)免疫荧光分析:参照张帅的方法略有改动(张帅.刺参岩藻聚糖硫酸酯对RAW264.7细胞炎症和自噬的影响[D].西北农林科技大学,2021.)。将RAW264.7细胞悬液(1×104个/mL)接种于铺有盖玻片的6孔板中(2mL/孔),设置空白对照组、张氏马尾藻岩藻多糖粗提物对照组(25、50和100μg/mL)和半叶马尾藻岩藻多糖粗提物对照组(25、50和100μg/mL),每个浓度做三个平行,培养24h至贴壁。弃去细胞培养液后,对照组加入2mL细胞培养液,实验组加入2mL对应浓度的多糖溶液,接着在细胞培养箱中培养1d。取出6孔板,再次弃去废液,用预冷的PBS缓冲液清洗3遍,每次5min。向孔内加入4%的多聚甲醛1mL保持20min以固定细胞,弃去固定液,用PBS缓冲液清洗3次,每次5min。加入0.5%TritonX-100溶液(PBS配制),15min细胞通透结束后,弃去破膜液,用PBS缓冲液清洗3遍,每次5min。加入山羊血清封闭30min,弃去封闭液,用PBS缓冲液清洗3遍,每次5min。每孔加入80-100μL iNOS或COX-2一抗,封上封口膜,4℃温度下孵育过夜,弃去一抗,用PBS缓冲液清洗3遍,每次5min。每孔加入150μLFITC-荧光二抗(1:800PBS),37℃温度下避光孵育1h,弃去二抗,用PBS缓冲液清洗3遍,每次5min。避光条件下,每孔加入150μLDAPI溶液(1:2000PBS)复染细胞核5-10min后,弃去染液,用PBS缓冲液清洗3遍,每次5min,以洗去多余的染液。取出盖玻片,将盖玻片附着有细胞的那一面沿着一个方向倒盖于滴有抗荧光猝灭剂的载玻片上,避免产生气泡。将固定好的载玻片置于荧光显微镜下观察并拍照分析结果。
(3)蛋白提取与分析:参照张帅的方法略有改动(张帅.刺参岩藻聚糖硫酸酯对RAW264.7细胞炎症和自噬的影响[D].西北农林科技大学,2021.)。将RAW264.7细胞悬液(1×104个/mL)接种于铺有盖玻片的6孔板中(2mL/孔),设置空白对照组、张氏马尾藻岩藻多糖粗提物对照组(25、50和100μg/mL)和半叶马尾藻岩藻多糖粗提物对照组(25、50和100μg/mL),每个浓度做三个平行,培养24h至贴壁。弃去细胞培养废液后,对照组加入2mL细胞培养液,实验组加入2mL对应浓度的多糖溶液,接着在细胞培养箱中培养24h。弃去培养液,用预冷的PBS洗3次,每孔加入细胞裂解液150μL,置于冰上裂解30min。接着在4℃、12000rpm离心15min,取上清液。然后将对照组和实验组依次加入倒好胶的电泳槽中。通过SDS-PAGE分离蛋白质样品(约1μg/μL,10-20μL)并转移到PVDF膜。然后将PVDF膜与5%BSA在室温下孵育2h,并用TBST洗涤3次,每次洗涤时间为10min,一抗加入后在4℃下孵育24h。最后,用HRP标记的二抗在室温下孵育2h后,用TBST洗去多余的二抗。加入显色液进行曝光拍照,利用Image J软件进行灰度分析。试验以GAPDH作为内参,每个样品最后测得的目的蛋白含量与本样品的GAPDH含量的比值即为每个样品中蛋白的相对含量,然后再进行样品与样品之间的比较。
2.9数据分析
每个实验做3次重复。采用GraphPad进行数据处理,数据间差异采用单因素方差分析中的Tukery’s多重比较分析数据间差异显著性(*代表测试组与对照组数据间的比较,*P<0.05,**P<0.01,***P<0.001,****P<0.0001;#代表测试组数据间的比较,#P<0.05,##P<0.01,###P<0.001,####P<0.0001)。数据结果采用“平均值±标准误差”表示。
3结果与分析
3.1岩藻多糖粗提物的得率、提取率及理化性质
采用超声波辅助热水浸提法提取马尾藻中的岩藻多糖,岩藻多糖提取物得率及提取率如表5所示。半叶和张氏马尾藻总糖含量分别为16.36%和27.88%,表5显示半叶马尾藻和张氏马尾藻岩藻多糖提取物的得率和提取率分别为13.60±0.15%、83.13±1.69%和19.95±0.37%、71.56±1.65%。与传统水浸提法相比,超声波辅助热水浸提法大大提高了多糖提取率。提取工艺为:料液比=1:30;水浴温度80℃;水浴时间3.5h;超声功率350W;超声时间50min。与传统水浸提法相比,超声波辅助热水浸提法大大提高了多糖提取率。在同一提取工艺下,谌素华等(谌素华,王维民,李春桃.不同提取方法对半叶马尾藻多糖提取率的影响研究[J].农产品加工·创新版,2009,4:27-29.)实验得到采用热水浸提法提取多糖,马尾藻多糖得率为4.50%;采用超声波辅助热水浸提法,马尾藻多糖得率为10.55%,我们以超微粉碎马尾藻粉为原料、采用超声波辅助热水浸提法所得岩藻多糖粗提物提取率远高于谌素华等的结果。因此在多糖提取的方法对比上,超微粉碎结合超声波辅助热水浸提法更有效率,可以得到广泛应用。此外,粗多糖得率与海藻原料总糖含量也有关系。
表5两种马尾藻岩藻多糖提取物的得率、提取率及理化性质比较
以L-岩藻糖为标准品,绘制岩藻糖标准曲线如图1所示,线性关系良好。采用苯酚-硫酸法对马尾藻岩藻多糖粗提物进行总糖含量的测定,测定结果半叶马尾藻和张氏马尾藻岩藻多糖粗提物总糖含量分别为75.35±1.46%和82.77±0.40%(表5)。
以牛血清蛋白为标准品,绘制牛血清蛋白标准曲线如图2所示,线性关系良好。采用考马斯亮蓝法对马尾藻岩藻多糖粗提物进行蛋白质含量的测定,测定结果半叶马尾藻和张氏马尾藻岩藻多糖粗提物蛋白质含量分别为2.66±0.67%和1.92±0.38%(表5)。
以没食子酸为标准品,绘制的没食子酸标准曲线如图3所示,线性关系良好。采用福林酚法对马尾藻岩藻多糖粗提物进行多酚含量的测定,测定结果半叶马尾藻和张氏马尾藻岩藻多糖粗提物多酚含量分别为0.49±0.01%和0.14±0.01%,即4.90mg/g和1.40mg/g(表5)。Banu等(BanuAT,Ramani P S,MuruganA.Effect of seaweed coating on qualitycharacteristics and shelf life of tomato(Lycopersicon esculentum mill)[J].Food Science and Human Wellness,2020,9(2):176-183.)研究隶属于红藻的麒麟菜和隶属于褐藻的软叶马尾藻中多酚含量,结果为0.45±0.001mg/g和0.31±0.001mg/g。相比较下,本实验马尾藻岩藻多糖粗提物中多酚含量相对较高。
以硫酸钾为标准品,绘制的硫酸钾标准曲线如图4所示,线性关系良好。采用氯化钡-明胶比浊法对马尾藻岩藻多糖粗提物物进行硫酸基含量的测定,测定结果半叶马尾藻和张氏马尾藻岩藻多糖粗提物硫酸基含量分别为44.11±0.01%和29.74±0.01%(表5)。
3.2岩藻多糖粗提物的分子量
如图5所示,4号为流动相的峰。半叶马尾藻岩藻多糖粗提物样品比张氏马尾藻岩藻多糖粗提物多一个峰,说明张氏马尾藻岩藻多糖粗提物的纯度更高。
有研究报道,岩藻多糖的分子量分布在400kDa至1400kDa之间,分子量的差异主要与提取原料的种类与提取的方法有关(Mohd Fauziee NA,Chang L S,Wan Mustapha W A,Md Nor A R,Lim S J.Functional polysaccharides of fucoidan,laminaran andalginate from Malaysian brown seaweeds(Sargassum polycystum,Turbinaria ornataand Padina boryana)[J].International Journal of Biological Macromolecules,2021,167:1135-1145.)。本实验采用重均分子量来进行表征,两种岩藻多糖提取物的重均分子量存在明显差别。表6中,半叶马尾藻岩藻多糖提取物中分子量在1166.48kDa附近的物质占比较高,分子量在100-200kDa附近的物质占比次之。分子量在3374.86kDa的物质占比为7.68%,这体现了提取物高分子的特性。剩余的两个峰,即分子量在20-50kDa附近的物质不在上述报道的分子量分布范围内,推测可能是提取物中的杂质。张氏马尾藻岩藻多糖提取物中分子量在111.278kDa附近的物质占比达到90.27%,说明张氏马尾藻岩藻多糖提取物纯度高,分子量较半叶马尾藻岩藻多糖提取物小。除此之外,张氏马尾藻岩藻多糖提取物中还含有分子量高达7121.794kDa以及分子量集中在739.711kDa附近的物质,但含量较低(分别为0.17%和9.01%)。
表6两种马尾藻岩藻多糖粗提物的相对分子量
3.3岩藻多糖粗提物的单糖组成
采用离子色谱法对两种马尾藻岩藻多糖粗提物进行单糖组成的分析,结果如表7所示。将两种岩藻多糖粗提物的离子色谱图(图6和图7)与混标色谱图(图8)进行对比,两种岩藻多糖粗提物所含单糖大体相同,包括岩藻糖、甘露糖、葡萄糖醛酸、葡萄糖、木糖、半乳糖及葡萄糖醛酸等酸性单糖,其中张氏马尾藻岩藻多糖粗提物还包括阿拉伯糖。结果表明:相同来源不同品种的岩藻多糖粗提物单糖组成相近,其中多个单糖含量存在差异,这可能与海藻品种不同有关。
表7两种马尾藻岩藻多糖粗提物的单糖摩尔比
3.4扫描电镜结果
两种马尾藻岩藻多糖提取物的扫描电镜图结果如图9所示。图9的A-1、B-1为低倍下观察到的两种马尾藻岩藻多糖提取物的表面微观形貌。图9的A-2、B-2为放大2000倍的条件下观察到的两种马尾藻岩藻多糖提取物的表面微观形貌。实验结果表明,张氏马尾藻岩藻多糖提取物表面较为粗糙,结构较松散,提高放大倍数后发现多糖表面含有较多孔洞且密集。而半叶马尾藻岩藻多糖提取物表面相对光滑,样品大致呈现片状结构,有屑状堆积的特征,放大倍数后,发现表面也相对光滑,表面附着有少许圆形颗粒。两种马尾藻岩藻多糖提取物微观结构存在明显差异,可能与其种类、化学组成以及分子量大小等因素不同有关。
3.5粒径和电位测定结果
多糖粒径的大小与其在生物体内的利用和代谢程度有很大的关系,而电位则反映多糖溶液的稳定性,研究多糖的粒径和电位有利于功能性食品或药物的开发。表8为两种马尾藻岩藻多糖提取物粒径和电位的测定结果。PDI值(polymer dispersity index,聚合物分散性指数)用来描述多糖分子量的分布情况。PDI值越小,多糖分子量分布越均匀,反之亦然。张氏马尾藻岩藻多糖提取物的PDI值小于半叶马尾藻岩藻多糖提取物,说明张氏马尾藻岩藻多糖提取物的分子量分布更均匀,与分子量分析结果一致。
Zeta电位反映溶液体系的稳定性,电位绝对值接近或者大于等于30mV时,表明溶液是较稳定的。如表8所示,半叶马尾藻岩藻多糖粗提物的平均zeta电位绝对值更接近于30mV,表明半叶马尾藻岩藻多糖提取物溶液体系较为稳定。理化性质分析结果表明半叶马尾藻岩藻多糖提取物的硫酸基含量较高,而张氏马尾藻岩藻多糖提取物硫酸基含量低,硫酸基含量的不同可能造成两种马尾藻岩藻多糖溶液zeta电位的差异。
表8粒径与电位结果
3.6岩藻多糖提取物对α-淀粉酶和α-葡萄糖苷酶的抑制作用
在不同的浓度下,张氏和半叶马尾藻岩藻多糖对α-淀粉酶(图10的A)和α-葡萄糖苷酶(图10的B)均有抑制作用。两种多糖对α-淀粉酶的抑制作用无剂量关系,由图10的A可以看出当多糖浓度在0.5mg/mL时抑制α-淀粉酶的效果最好。图10的B表明,两种多糖对α-葡萄糖苷酶的抑制作用呈现浓度依赖关系,随着多糖浓度的增加,对α-葡萄糖苷酶的抑制率逐渐升高,且张氏马尾藻岩藻多糖对α-葡萄糖苷酶的抑制率高于半叶马尾藻岩藻多糖。使用GraphPad Prism软件对α-葡萄糖苷酶抑制数据进行分析(图11),得到张氏马尾藻岩藻多糖和半叶马尾藻岩藻多糖的IC50值分别为0.033mg/mL和0.012mg/mL,说明半叶马尾藻岩藻多糖对α-葡萄糖苷酶的抑制效果优于张氏马尾藻岩藻多糖。Shan等(Shan X,Liu X,Hao J,Cai C,Fan F,DunY,Zhao X,Liu X,Li C,Yu G.In vitro and in vivo hypoglycemiceffects ofbrown algal fucoidans[J].International Journal ofBiologicalMacromolecules,2016,82:249-255.)通过研究11种不同来源的褐藻岩藻多糖对α-葡萄糖苷酶的影响,发现墨角藻来源的岩藻多糖对α-葡萄糖苷酶有明显的抑制作用,显著高于其他10种褐藻来源的岩藻多糖,其IC50值为0.068mg/mL。相比较下,本实验提取的马尾藻岩藻多糖抑制α-葡萄糖苷酶作用更佳。
3.7马尾藻岩藻多糖粗提物对RAW264.7细胞的免疫调节作用
3.7.1岩藻多糖粗提物对RAW264.7细胞NO释放量的影响
图12中各物质分别为A裙带菜岩藻多糖(高分子量)、B裙带菜岩藻多糖(低分子量)、C裙带菜岩藻多糖(中分子量)、D墨角藻岩藻多糖、E泡叶藻岩藻多糖、F海带岩藻多糖、G张氏马尾藻岩藻多糖粗提物和H半叶马尾藻岩藻多糖粗提物。由图12可知,8种不同多糖中除了F样品海带多糖刺激RAW264.7细胞释放NO的量与对照组相比没有明显差别外,其余7种多糖都有较明显的差别。A、B和C样品都是裙带菜来源的多糖,且在多糖浓度为25μg/mL时,三种多糖刺激下NO产生量与对照组相比没有明显的差异,但可发现NO释放量与多糖分子量大小有关,分子量大,NO释放量更多,这与Qi等(Qi J,Kim S M.Effects of the molecularweight and protein and sulfate content of Chlorella ellipsoideapolysaccharides on their immunomodulatory activity[J].International JournalofBiological Macromolecules,2018,107:70-77.)的研究一致。
G和H样品是本实验提取的两种不同品种的马尾藻岩藻多糖粗提物对RAW264.7细胞NO释放量的影响结果,可以看出岩藻多糖粗提物对细胞的免疫调节作用比纯化的多糖更好。两种马尾藻岩藻多糖粗提物均可以促进RAW264.7细胞释放NO,且呈现浓度依赖关系,其中G样品张氏马尾藻岩藻多糖粗提物对RAW264.7细胞释放NO水平的影响表现出较好的剂量关系。由图可知,当浓度达到25μg/mL时,两种岩藻多糖粗提物对RAW264.7细胞NO的产生量与对照组相比表现出无显著性差异(P>0.05)。当浓度在50-100μg/mL范围内时,H样品半叶马尾藻岩藻多糖粗提物促进RAW264.7细胞释NO的能力变化明显,对NO产生量的影响极显著(P<0.01和P<0.001),G样品张氏马尾藻岩藻多糖粗提物对RAW264.7细胞NO产生量的影响也极显著(P<0.0001)。相比较而言,张氏马尾藻岩藻多糖粗提物可更好地促进RAW264.7细胞释放NO,具有更好地激活巨噬细胞参与免疫反应、发挥免疫调节作用的潜力。
3.7.2两种岩藻多糖粗提物激活巨噬细胞COX-2和iNOS mRNA表达
免疫调节作用的共同机制与分泌一系列巨噬细胞来源的生物因子有关。据报道,iNOS是巨噬细胞中合成大量NO的关键控制者,COX-2和iNOS都是促炎症介质(Murakami A,Ohigashi H.Targeting NOX,INOS and COX-2 in inflammatory cells:chemopreventionusing food phytochemicals.International Journal of Cancer,2007,121(11),2357-2363.)。因此,为了证实岩藻糖对巨噬细胞的激活作用,我们选择通过荧光定量PCR分析两种马尾藻岩藻多糖粗提物对巨噬细胞中COX-2和iNOS mRNA表达的影响。结果显示,两种岩藻多糖粗提物在50μg/mL和100μg/mL浓度范围内显著促进COX-2mRNA表达(P<0.05)(图13);而在浓度为25μg/mL时,张氏马尾藻岩藻多糖粗提物对巨噬细胞COX-2mRNA表达显著增加(P<0.05),iNOS mRNA表达无显著变化(P>0.05);在25μg/mL时,半叶马尾藻岩藻多糖粗提物对巨噬细胞COX-2和iNOS mRNA的表达均无显著影响(P>0.05)。上述结果表明,在50~100μg/mL范围内,两种马尾藻岩藻多糖粗提物能提高靶基因的表达水平,促进NO的产生和分泌,进一步证实了二者可激活巨噬细胞的结论。此结果表明,两种马尾藻岩藻多糖粗提物通过上调COX-2和iNOS mRNA表达来促进NO的释放。
3.7.3岩藻多糖粗提物对RAW264.7细胞中COX-2和iNOS蛋白表达量的影响
iNOS是催化巨噬细胞分泌合成NO的关键酶,而COX-2是催化巨噬细胞合成前列腺素的关键酶,并且两种酶均主要在激活后的巨噬细胞中表达,因此,通过检测多糖样品干预后RAW264.7细胞NO的释放量以及采用Western Blot技术和免疫荧光技术研究多糖干预后RAW264.7细胞中iNOS和COX-2这两种酶在蛋白水平的表达情况都可以来反映多糖的免疫调节活性(任琪.银杏叶多糖的提取、分离纯化及生物活性研究[D].安徽农业大学,2018.)。iNOS和COX-2这两种酶在细胞免疫活性调节中发挥重要作用,是免疫调节的标志性蛋白,当发生免疫调节时,iNOS和COX-2蛋白表达量会增加。采用免疫荧光技术测定RAW264.7细胞内iNOS和COX-2蛋白的表达量,DAPI试剂染核呈现蓝色,荧光二抗标记所测的蛋白,合并后绿色荧光强度表示iNOS和COX-2蛋白的表达量。如图14所示,对照组在没有多糖的刺激下,绿色荧光十分微弱,随着多糖干预浓度的增加,绿色荧光点变多且亮度增强,这说明多糖促进RAW264.7细胞内iNOS和COX-2蛋白的表达,且具有剂量关系,与前面NO释放量和Westernblot结果一致。因此,在多糖的作用下,RAW264.7细胞中iNOS和COX-2蛋白表达增加,从而促进NO和前列腺素的分泌,发挥有效的免疫调节作用。
为了进一步了解RAW 264.7细胞COX-2和iNOS蛋白的激活情况,我们采用Westernblot方法检测COX-2和iNOS蛋白的表达。在两种岩藻多糖粗提物的干预后,RAW264.7细胞中COX-2、iNOS在蛋白水平的表达情况如图15所示。与内参相比,在两种多糖粗提物的刺激下,COX-2、iNOS蛋白的表达明显增加,并随着多糖浓度的增加而呈上升趋势。在多糖浓度为100μg/mL时,张氏马尾藻岩藻多糖粗提物刺激下COX-2和iNOS的表达量最高。
以上结果表明,两种多糖粗提物能显著促进RAW264.7巨噬细胞NO的分泌,具有良好的免疫促进作用。同时,免疫激活作用与两种多糖粗提物呈剂量依赖性,且通过上调COX-2和iNOS在基因和蛋白水平上增强RAW264.7巨噬细胞的免疫系统功能。
上述具体实施方式,并不构成对本发明保护范围的限制。本领域技术人员应该明白的是,取决于设计要求和其他因素,可以发生各种各样的修改、组合、子组合和替代。任何在本发明的精神和原则之内所作的修改、等同替换和改进等,均应包含在本发明保护范围之内。
Claims (4)
1.岩藻多糖提取物在制备降血糖和/或调节免疫活性的产品中的应用,所述岩藻多糖提取物的制备方法为:
将马尾藻经超微粉碎机,得到超微粉碎马尾藻粉;向所述超微粉碎马尾藻粉中加水搅拌均匀后,置于热水中浸提;浸提完成后,进行超声提取得到浸提液;将所述浸提液离心、去沉淀,将上清液浓缩后加入无水乙醇,离心,取沉淀;将所述沉淀用有机溶剂洗涤后加水溶解,再加入复合有机溶剂除蛋白,振荡后静置分层,取上清液于透析袋中进行透析,再经真空冷冻干燥后,即得到岩藻多糖粗提物;
所述马尾藻为半叶马尾藻或张氏马尾藻,超微粉碎马尾藻粉颗粒为150-2000目;
所述超微粉碎马尾藻粉与水的料液比为1:10g/mL~1:50g/mL;所述水为蒸馏水;
所述置于热水中浸提是指置于50℃~80℃热水浸提1h~8h;
所述超声提取是在超声功率为50W~500W的条件下超声提取10min~60min;所述浸提液在3000r/min~8000r/min条件下离心5min~40min;
所述除蛋白的复合有机溶剂是指二氯甲烷:戊醇=4:1(v/v);所述复合有机溶剂除蛋白是按照多糖溶液:复合有机溶剂体积比为3:1~6:1的比例加入有机溶剂试剂;
所述透析过程在4℃~16℃冰箱中透析24h~48h,透析期间更换2-6次蒸馏水。
2.根据权利要求1所述的应用,其特征在于:所述将所述上清液浓缩,加入无水乙醇,离心后取沉淀是指:将所述上清液用旋转蒸发仪浓缩至原体积的五分之一至二分之一后,加入无水乙醇至其体积百分比浓度为20%~40%,二次离心取上清液;上清液继续加入无水乙醇至其体积百分比浓度为50%~90%,离心后取沉淀。
3.根据权利要求1所述的应用,其特征在于:将所述沉淀用有机溶剂进行洗涤是指:沉淀用无水乙醇和丙酮各洗2次。
4.根据权利要求1所述的应用,其特征在于:透析后的溶液在-70℃~-35℃真空冷冻干燥。
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