CN110938152B - 一种纳豆菌发酵贝类制备的多糖rpp1及其纯化方法和应用 - Google Patents
一种纳豆菌发酵贝类制备的多糖rpp1及其纯化方法和应用 Download PDFInfo
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Abstract
本发明属于多糖的制备纯化及应用领域,具体涉及一种纳豆菌发酵贝类制备的多糖RPP1及其纯化方法和应用,多糖RPP1的重均分子量Mw为110.4KD,其单糖组成主要包括Glc、Ara、Gal、Xyl,其单糖摩尔比为Man:Rha:Glc:Gal:Xyl:Ara:Fuc=1:0.90:25.48:9.25:6.44:11.24:5.4。多糖RPP1对结肠癌细胞具有很好的抑制作用,本发明对多糖的结构进行初步解析和抗肿瘤活性机理进行了初步研究,为下一步揭示多糖RPP1在体内发挥抗肿瘤作用的机理提供一定的理论依据。
Description
技术领域
本发明属于多糖的制备纯化及应用领域,具体涉及一种纳豆菌发酵贝类制备的多糖RPP1及其纯化方法和应用。
背景技术
当前,多糖类化合物作为免疫调节剂具有抗肿瘤、抗突变、抗病毒、降血脂等作用,已应用于功能性食品或临床药物。其中,由于某些活性多糖的独特功能和无毒特性,其在肿瘤的治疗和预防上优于其他化合物。结肠癌的发病率和死亡率均位于所有肿瘤的第3位,在美国,结肠癌发病率占所有癌症的第四位,而死亡率为第二位,在我国结肠癌也是常见的消化道癌症,死亡率高居第5位。研究表明多糖类化合物具有提高机体免疫力,抑制肿瘤细胞的生长的作用,与化疗药物共同使用具有协同作用并可降低或免除化疗药物的毒副作用,因此活性多糖作为抗肿瘤药品的活性成分具有广阔的应用前景。
本申请人在对贝类利用的长期研究中,利用纳豆菌发酵菲律宾蛤仔得到了具有抗肿瘤活性的多糖粗提物,并申请了公布号为CN108410767A,名称为纳豆菌及其在发酵菲律宾蛤仔制备活性多糖的专利,但是在针对其粗提取到的多糖进行抗肿瘤研究时,效果并不是很突出,尤其是在结肠癌抗性方面没有得到很好的效果。
贝类多糖生物活性的实现依赖于其空间结构的完整性与高纯度,多糖的结构决定了多糖的功能活性,粗提得到的海洋贝类发酵多糖主要是性质、种类不同的大分子多糖混合物,其功能活性并没有得到很好的测定,因此需要对不同类型的多糖进行分离纯化,得到相对纯净的均一多糖,并针对纯化后的多糖研究其抗肿瘤活性。
发明内容
针对目前提取到的多糖粗提物的抗癌活性较低,本发明提出了一种纳豆菌发酵贝类制备的多糖RPP1及其纯化方法和应用,将多糖粗提物进一步纯化得到多糖RPP1,并进行了结构鉴定,多糖RPP1在体内外抗肿瘤活性研究中显示出很好的抑制结肠癌细胞的活性,为进一步揭示多糖RPP1的抗肿瘤作用提供了依据。
本发明的技术方案是:
一种纳豆菌发酵贝类制备的多糖RPP1,其特征在于,所述多糖RPP1的重均分子量为110.4KD,其单糖组成主要包括葡萄糖(Glc)、阿拉伯糖(Ara)、半乳糖(Gal)、木糖(Xyl),其单糖摩尔比为甘露糖(Man):鼠李糖(Rha):葡萄糖(Glc):半乳糖(Gal):木糖(Xyl):阿拉伯糖(Ara):岩藻糖(Fuc)=1:0.90:25.48:9.25:6.44:11.24:5.4。
进一步的,所述多糖RPP1的总糖含量为90.01%,蛋白质含量为0.24%,硫酸根含量为0.54%,糖醛酸含量为3.97%,不含有氨基己糖。
进一步的,核磁共振结果显示所述多糖RPP1含有α-1,4-糖苷键和β-1,4-糖苷键构型,并可能含有烷基或蛋白质中脂链氨基酸残基的存在。
进一步的,所述多糖RPP1的纯化方法为:将粗多糖经DEAE Sepharose Fast Flow阴离子交换柱和Sephacry-S-400HR凝胶柱分离纯化后,得到多糖RPP1。扫描电镜观察发现RPP1为不规则形状,表面呈花瓣状且发生结块,表明纯化后的多糖是无定形固体,分子聚集已经发生。
进一步的,所述多糖RPP1的纯度为90.01%;所述粗多糖为纳豆菌发酵贝类制备得到的活性多糖混合物。
一种纳豆菌发酵贝类制备的多糖RPP1在制备结肠癌治疗药物及功能食品中的应用。
本发明的有益效果:
(1)本发明提供的多糖RPP1为粗多糖经过离子交换和凝胶柱层析纯化后得到,经检测RPP1的蛋白质含量为0.24%、硫酸根含量为0.54%、糖醛酸含量为3.97%,未检测出氨基糖;采用GPC法测定多糖分子量,PMP衍生化法测定并得到单糖的主要组成,GPC色谱图的峰比较单一,分布较为均匀,RPP1的重均分子量Mw为110.4KD。紫外、红外光谱扫描显示RPP1含有D-吡喃葡萄糖,且含有以结合态形式存在的蛋白;核磁共振结果证明RPP1含有α-1,4-糖苷键和β-1,4-糖苷键构型,并可能含有烷基或蛋白质中脂链氨基酸残基的存在;经电子显微镜(SEM)扫描纯化的多糖,发现该多糖是无定形固体,表面呈花瓣状且发生结块。
(2)通过细胞凋亡实验以及动物实验得出,多糖RPP1对结肠癌具有很好的抑制作用,本发明对多糖的结构进行初步解析和抗肿瘤活性机理进行了初步研究,为下一步揭示多糖RPP1在体内发挥抗肿瘤作用的机理提供一定的理论依据。
附图说明
图1为多糖在DEAE Sepharose Fast Flow层析柱上的梯度洗脱图;
图2为RPP1、RPP2在Sephacryl S-400HR凝胶层析柱上的梯度洗脱图;
图3为蛋白质含量标准曲线;
图4为硫酸根含量标准曲线;
图5为糖醛酸含量标准曲线;
图6为单糖标准品的PMP柱前衍生液相色谱图;
图7为RPP1的PMP柱前衍生液相色谱图;
图8为RPP1紫外吸收图谱;
图9为RPP1的红外光谱图;
图10为RPP1的核磁共振图谱;
图11为RPP1的SEM图;
图12为流式细胞术测定不同浓度RPP1对HT-29细胞凋亡的作用;
图13为不同药物处理组HT29细胞凋亡率;
图14为裸鼠肿瘤组织图片;
图15为各组瘤体重量柱形图;
图16为不同处理对裸鼠抑瘤率的影响的柱形图。
具体实施方式
下面将结合具体实施例对本发明的技术方案进行清楚、完整的描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下,所获得的所有其他实施例,都属于本发明保护的范围。
为了进一步理解本发明,下面结合实施例对本发明进行详细说明。
实施例1
(1)粗多糖的提取
实施例中采用的粗多糖为发明人利用纳豆菌发酵菲律宾蛤仔得到的活性多糖混合物。具体步骤为:称取纳豆筛选并培养得到枯草芽孢杆菌纳豆亚种PS-1-HepG2种子菌悬液,取鲜活的菲律宾蛤仔取肉,匀浆,加入蒸馏水高温灭菌后接入上述种子菌悬液发酵,向发酵物中加入蒸馏水浸提、离心取上清液后加入乙醇进行醇沉,得到沉淀物后采用Sevag法除蛋白;超滤、冻干得到粗多糖粉末。
(2)多糖RPP1的纯化
步骤1:离子交换柱层析
将柱体积为20mL的DEAE Sepharose Fast Flow(16mm×100mm)弱阴离子交换柱连接到AKTA-FPLC快速蛋白质纯化系统上,用5~10柱体积缓冲液平衡体系,使系统的电导率≤0.02。取粗多糖0.05g溶于5mL蒸馏水中,配成10mg/ml的糖溶液,离心后取上清液过0.22μm滤膜,平稳上样。待样品充分吸附到DEAE Sepharose Fast Flow色谱柱上后进行梯度洗脱。分别用0、0.2、0.4、0.6、0.8、1.0mol/LNaCl溶液进行洗脱,每个梯度洗脱6个柱体积(即120mL),流速为1.5mL/min,每管收集6mL。
蛤仔发酵物中的粗多糖经离子交换柱洗脱后得到三个较对称尖峰,如图1所示,依次命名为RPP1、RPP2和RPP3。其中RPP1为以蒸馏水作为流动相的洗脱产物,说明RPP1不带电荷,收集主要洗脱峰RPP1、RPP2,冷冻干燥,采用苯酚-硫酸法检测RPP1多糖的得率为58.78%、RPP2得率为33.65%。
步骤2:凝胶渗透柱层析
将柱体积为20mL的HiPrep16/60Sephacryl S-300HR(17-1167-01)凝胶层析柱连接到AKTA-FPLC快速蛋白质纯化系统上,设置合适的报警压(<0.5MPa),1.5CV去离子水平衡(15cm/h,0.5mL/min),使系统的电导率≤0.02。取经过离子交换的多糖0.06g溶于2mL蒸馏水中,配成30mg/ml的糖溶液,过0.22μm滤膜过滤或10000g离心10min,平稳上样。待样品进入HiPrep 16/60Sephacryl S-300HR(17-1167-01)凝胶层析柱上后用水洗脱,流速为0.5mL/min,每管收集4mL。
RPP1、RPP2经凝胶层析柱分离纯化后,苯酚硫酸法测定收集多糖的吸光值,绘制洗脱曲线如图2所示。结果显示RPP1、RPP2的洗脱峰均为单一对称峰,收集RPP1峰尖部分,反复减压浓缩、冻干得纯品多糖RPP1。
实施例2
(1)多糖RPP1的理化性质分析
采用考马斯亮蓝法检测蛋白含量,标准曲线如图3所示,回归方程为y=0.0037x+0.0101,R2=0.99531,线性拟合在0~100μg/mL范围内较好。
采用明胶比浊法测定硫酸盐含量,图4为硫酸根含量标准曲线,回归方程为y=0.0037x+0.0101,R2=0.99531,线性拟合在0~600μg/mL范围内内良好。
采用间羟联苯法测定糖醛酸含量,糖醛酸含量的标准曲线如图5所示,回归方程为y=0.0219x-0.0046,R2=0.99558,线性关系在0~15μg/mL范围内良好。
上述检测分析可知,RPP1的总糖含量为90.01%,蛋白质含量为0.24%,硫酸根含量为0.54%,糖醛酸含量为3.97%,无氨基己糖。
采用凝胶渗透色谱(GPC)测定RPP1的分子量,GPC色谱图为单一对称洗脱峰,分布指数PD(Mw/Mn)仅为1.24,证明RPP1初度较高。RPP1的数均分子量(Mn)为88.9KD,重均分子量(Mw)为110.4KD,Z均分子量(Mz)为144.4KD。
(2)单糖组成分析
采用PMP衍生化法测定单糖组成,将单糖标准品及RPP1先进行PMP衍生化,然后采用高效液相仪测定,单糖标准品及RPP1的PMP柱前衍生液相色谱图如图6、7所示,主要单糖组成为葡萄糖(Glc)、阿拉伯糖(Ara)、半乳糖(Gal)、木糖(Xyl),单糖摩尔比为甘露糖(Man):鼠李糖(Rha):葡萄糖(Glc):半乳糖(Gal):木糖(Xyl):阿拉伯糖(Ara):岩藻糖(Fuc)=1:0.90:25.48:9.25:6.44:11.24:5.4。
(3)RPP1结构分析
紫外光谱分析
取一定量RPP1溶解在超纯水中,离心后取上清液,稀释成500μg/mL,用UV-2450分光光度计(Shimadzu Co.,Japan)在25℃下,记录200~500nm的范围内溶液的UV-Vis光谱。紫外吸收光谱如图8所示,RPP1在280nm处有微弱吸收,证明含少量蛋白质。
红外光谱分析
将RPP1冻干粉与KBr混匀(重量比为100:1),放于研磨帛中充分研磨、混匀后压片。使用Nicolet IN10型红外光谱仪(品牌Thermo Fisher)在波长4000~400cm-1处的进行红外光谱扫描,得到RPP1的IR吸收峰图谱。如图9所示,红外光谱显示RPP1含有D-吡喃葡萄糖,且含有以结合态形式存在的蛋白。
核磁共振分析
精确称取50mg分离纯化后的RPP1,用500μL D2O溶解。置于-80℃冰箱里预冷后冻干,重复该步骤3次。然后用500μL D2O完全溶解后移至核磁管中,在25℃环境下进行一维1H-NMR和13C-NMR分析,采用MestReNova软件进行数据辅助解析,标注峰的面积和大小。如图10所示,核磁共振结果显示RPP1含有α-1,4-糖苷键和β-1,4-糖苷键构型,并可能含有烷基或蛋白质中脂链氨基酸残基的存在。
扫描电镜观察
通过场发射扫描电子显微镜(SEM,JSM-7001F,JEOL,日本)获得多糖的扫描电子显微镜(SEM)图像。将干燥的样品借助双面胶带放置在样品架上,并使用溅射涂布机溅射金粉,在20kV的加速电位下观察样品,获得多糖的扫描电子显微镜(SEM)图像。如图11所示,扫描电镜观察发现RPP1为不规则形状,表面呈花瓣状且发生结块,表明纯化后的多糖是无定形固体,分子聚集已经发生。
实施例3
多糖RPP1的抗肿瘤活性研究
一、主要材料
人结肠癌细胞HT29(中国科学院上海细胞库)BALB/C,四周龄18-22g雄性裸鼠(上海斯莱克实验动物有限责任公司),菲律宾蛤仔发酵物中分离纯化获得的多糖RPP1。
二、多糖RPP1对HT-29细胞凋亡的作用
1、实验方法
从液氮罐中取出HT-29细胞,用含10%胎牛血清的McCoy's 5A培养基培养。将培养基重悬细胞混匀后接种到96孔培养板中,每个细胞可接种2个孔,培养板置于37℃、5%CO2的培养箱内培养。24h后,观察细胞状态,进行换液处理,继续培养。细胞生长至密度为90%左右,将细胞收集至离心管离心,弃上清。按1:2进行传代,将培养板置于37℃、5%CO2的培养箱内培养。培养细胞至密度70%时进行加药处理,分别用500μg/mL、1mg/mL、1.5mg/mL药物分别处理细胞72h。
将各组细胞离心5min后收集,小心吸除上清,用PBS洗涤细胞两次,离心5min收集细胞,小心吸除上清,残留约50μL左右的PBS,每管细胞样品中加入500μL Binding Buffer轻轻重悬细胞。加入5μLAnnexinV-FITC混匀后,加入10μLPropidium Iodide,混匀。室温避光孵育15min,随即进行流式细胞检测。
2、细胞凋亡结果分析
如图12和13所示,在药物作用72小时后,RPP1能明显提高HT29细胞中早期和晚期凋亡细胞比例,且凋亡比例呈浓度依赖性增加。
表1 RPP1对HT29细胞凋亡的作用
分组 | UL(%) | UR(%) | LL(%) | LR(%) |
对照组 | 0.40 | 0.94 | 95.97 | 2.69 |
500μg/mL药物处理组 | 1.78 | 3.30 | 90.11 | 4.81 |
1mg/mL药物处理组 | 3.55 | 4.72 | 84.30 | 7.43 |
1.5mg/mL药物处理组 | 8.60 | 13.04 | 72.59 | 5.76 |
用不同浓度的多糖RPP1体外作用于结肠癌HT29细胞72h后,用流式细胞仪检测发现随糖浓度增加,细胞凋亡率逐渐上升,与对照组相比,RPP1能明显提高HT29细胞中早期和晚期凋亡细胞比例,且凋亡比例呈浓度依赖性增加。
三、动物给药处理
1、结肠癌HT-29细胞裸鼠移植瘤模型的创建:
将将4-6周龄的雄性BALB/c裸鼠腹部皮下接种人结肠癌HT-29细胞(接种量:l×l06~2×107细胞/裸鼠),1周左右见裸鼠接种部位皮下成瘤直径约为0.3cm时视为建模成功。
2、裸鼠给药方案
以3只健康裸鼠为空白组,荷瘤裸鼠随机分成5组,每组5只,实验共需28只小鼠。给药方案如表2所示,整个实验周期为4周。末次给药后,小鼠禁食不禁水24h,放血后断髓处死小鼠,剥出瘤块称重,具体给药方案下表2所示。
表2裸鼠给药方案
3、结果分析
(1)实验结果发现,RPP1对HT29结肠癌细胞体内作用结果显示,与空白组相比,给药处理对裸鼠体重无显著性影响。模型组裸鼠的肿瘤体积增长迅速,各给药组裸鼠肿瘤体积增长速度均低于模型组,在造模成功后,各给药组裸鼠的肿瘤体积增长减缓,其中高剂量组在造模成功10天后肿瘤几乎不再生长。
(2)各组裸鼠肿瘤组织如图14所示,模型组与5-FU组、高中低剂量组存在极显著性差异(p<0.01),说明5-FU和不同剂量组的多糖均对肿瘤生长有一定的抑制作用。如图15所示,5-FU组与中低剂量组裸鼠的瘤体重量间无显著性差异(p>0.05),与高剂量组存在显著性差异(p<0.05)。
(3)取出各实验组裸鼠的全部实体瘤,用预冷的0.9%的生理盐水清洗称重后,按公式计算各组裸鼠的平均抑瘤率。各实验组抑瘤率如图16所示,结果显示抑瘤率:高剂量>中剂量组>低剂量组>5-FU组。
上述说明仅为本发明的优选实施例,并非是对本发明的限制,凡在本发明的内容范围内所做出的任何修改、等同替换、改型等,均应包含在本发明的专利保护范围之内。
Claims (4)
1.一种纳豆菌发酵贝类制备的多糖RPP1,其特征在于,所述多糖RPP1用于制备结肠癌治疗药物及功能食品;所述多糖RPP1的重均分子量Mw为110.4KD,其单糖组成主要包括Glc、Ara、Gal、Xyl,其单糖摩尔比为Man:Rha: Glc:Gal:Xyl:Ara:Fuc= 1:0.90:25.48:9.25:6.44:11.24:5.4;
所述多糖RPP1的纯化方法为:将粗多糖经DEAE Sepharose Fast Flow阴离子交换柱,以蒸馏水为流动相进行洗脱,经Sephacry-S-400 HR凝胶柱分离纯化后,得到多糖RPP1;所述粗多糖为纳豆菌发酵贝类制备得到的活性多糖混合物。
2.根据权利要求1所述的一种纳豆菌发酵贝类制备的多糖RPP1,其特征在于,所述多糖RPP1的总糖含量为90.01%,蛋白质含量为0.24%,硫酸根含量为0.54%,糖醛酸含量为3.97%,不含有氨基己糖。
3.根据权利要求1所述的一种纳豆菌发酵贝类制备的多糖RPP1,其特征在于,所述多糖RPP1含有α-1,4-糖苷键和β-1,4-糖苷键构型。
4.根据权利要求1所述的一种纳豆菌发酵贝类制备的多糖RPP1,其特征在于,所述多糖RPP1的纯度为90.01%。
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