CN112390898A - 一种魁蚶免疫调节和抗肿瘤多糖及其制备方法和应用 - Google Patents
一种魁蚶免疫调节和抗肿瘤多糖及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种魁蚶多糖及其制备方法和用途,本发明提供的一种魁蚶多糖JNY2PW,其特征在于,所述多糖含量为90‑95wt%,其单糖组成为葡萄糖,该多糖的重均分子量为7.0×106‑8.0×107Da,优选为5.25×107Da。本发明的多糖可以用于制备免疫增强剂和治疗肿瘤的药物和抗癌保健品。
Description
技术领域
本发明属于医药及保健食品技术领域,具体涉及一种魁蚶多糖及其制备方法和用途。
背景技术
生物体内活性物质的研究是研制功能药物、保健食品和生物制品的基本前提。多糖作为主要活性物质之一,是一种由10个以上的单糖通过脱水缩合形成糖苷键聚合而成的高分子糖链。多糖被发现参与许多生物学过程,如细胞之间通讯,胚胎发育,细菌或病毒感染以及体液免疫和细胞免疫。近年来,对多糖的研究涉及多个领域如临床医药、生物燃料、化妆品、营养保健品。多糖的多种生物学功能活性及多糖资源的开发与利用成为人们近代研究的热点。
我国是海洋贝类养殖和加工大国,海洋贝类中,扇贝、鲍鱼、牡蛎、魁蚶等是“药食同源”的海之珍品。海洋贝类多糖是广泛存在于海洋贝类体内的一种生物活性物质,海洋环境的特殊性赋予海洋多糖不同于陆地多糖的独特的合成途径,使其具有新颖的结构和功能。研究表明,海洋贝类多糖具有抗衰老、抗病毒、抗肿瘤、调节免疫、治疗糖尿病等多种生物活性。海洋贝类多糖成分复杂,其单糖组成有葡萄糖、氨基葡萄糖、半乳糖、葡萄糖醛酸、岩藻糖、甘露糖等,因其成分复杂,结构多变,导致其生物学活性存在差异。
魁蚶(Arca inflata Reeve)属于软体动物门,瓣鳃纲,蚶目,蚶科,是我国资源丰富的海产贝类之一。魁蚶个大体肥,肉质鲜美,古书中记载魁蚶有“令人能食、益血色、消血块和化痰积”之功效,营养价值和经济价值很高,是我国及亚洲沿海地区重要的经济贝类之一。通过查阅相关文献资料发现,国内外学者对魁蚶中的蛋白类成分进行了一些研究,从魁蚶中表征和表达了一种具有抗菌活性和免疫调节活性的多肽,从魁蚶中发现了两种具有抗肿瘤活性的多肽。而对魁蚶中多糖及其生物活性的研究较少,仅对魁蚶中存在葡糖氨基葡聚糖进行了报道,但对其连接方式,分子量范围和药理活性未作深入研究。故魁蚶多糖将高价值利用魁蚶这一海洋生物资源,扩大多糖结构资源的同时具有重大经济意义,为合理、高效利用海洋资源提供理论依据。
发明内容
本发明的目的在于提供魁蚶中多糖的提取方法。
本发明的另一目的在于提供新发现的魁蚶多糖。
本发明的再一目的在于提供上述魁蚶多糖在制备抗肿瘤、免疫调节药物或特医食品或功能食品中的应用。
本发明所采取的技术方案是:
一种魁蚶中多糖的提取方法,该方法包括以下操作步骤:
1)前处理:将新鲜魁蚶剥壳取斧足,用水清洗干净;
2)缓冲液提取:将清洗好的魁蚶斧足加入PBS缓冲液中,使用高速组织匀浆机匀浆处理,匀浆液置于冰上使用超声破碎仪破碎提取;离心后收集提取液。
3)盐析:将提取液浓缩后,加入硫酸铵使硫酸铵体积浓度为a%,静置,收集上清液;再向上清液中加入硫酸铵使硫酸铵体积浓度为b%,静置,收集沉淀,即得粗多糖;其中30<a≤70<b≤100。
4)纯化:对上述粗多糖进行纯化,即得魁蚶多糖。
进一步的,步骤2)中所述缓冲液提取的具体操作为用3~10倍体积的30-100mMPBS缓冲液对魁蚶斧足进行低温匀浆、低温超声破碎提取,匀浆时间为30~60s,提取时间20~60min,匀浆和超声破碎温度为4℃。
进一步的,步骤3)中所有所述浓缩为-40~-80℃减压冷冻浓缩,所有所述静置的时间为10~28h。
进一步的,步骤3)中30<a≤70,70<b≤100。
进一步的,将步骤4)中盐析后的粗多糖再进行阴离子交换柱层析,用含梯度浓度(0~1M)氯化钠的30mM PBS进行洗脱,采用苯酚-硫酸法跟踪洗脱曲线,根据洗脱曲线收集糖部分,浓缩,冷冻干燥;然后用双蒸水溶解,离心,取上清液进行分子筛凝胶柱层析,用双蒸水进行洗脱,采用苯酚-硫酸法跟踪洗脱曲线,根据洗脱曲线收集糖部分,浓缩,冷冻干燥后获得魁蚶多糖JNY2PW。
进一步的,上述离子交换柱为DEAE离子交换柱。
进一步的,上述分子筛凝胶层析选用SephadexG100分子筛色谱柱。
一种魁蚶多糖JNY2PW,该多糖由葡萄糖组成,重均分子量为5.25×107Da,由→4)-α-D-Glcp-(1→和→4,6)-α-D-Glcp-(1→构成主链,支链末端由-α-D-Glcp-(1→构成,其结构为:
魁蚶多糖JNY2PW,该多糖的提取方法为上述所述的提取方法。
魁蚶多糖JNY2PW在制备增强免疫力的药物或特医食品或功能食品中的应用。
魁蚶多糖JNY2PW在制备抗肿瘤药物或特医食品或功能食品中的应用。
本发明的有益效果是:
1.与传统水提醇沉法和Sevag除蛋白法提取多糖相比,本发明采用低温缓冲液提取,无需使用有毒的有机试剂,硫酸铵浓度由低到高进行分级盐析,对魁蚶多糖进行初步分离,同时高浓度硫酸铵能将极性大、水溶性好的多糖与极性小、水溶性差的脂质、疏水蛋白等低极性物质分离,使所提取的粗多糖的糖含量更高。且本制备工艺简单,无需使用有毒的有机试剂如正丁醇,氯仿等,仅使用价格低廉的无机盐溶液处理,操作方便,无后续有毒有机试剂残留风险,适合大规模生产。
2.本发明利用离子交换层析和分子筛凝胶柱层析方法纯化魁蚶多糖,首次制备出一种魁蚶精多糖JNY2PW,并且对其理化性质、分子量、单糖组成等进行了系统的分析鉴定,并成功得出了该魁蚶多糖的特征结构。本发明提供了魁蚶中粗多糖、精多糖的制备方法及魁蚶多糖在调节免疫和抗肿瘤方面的活性研究,为魁蚶多糖在医药、保健品等领域的应用提供依据。
3.本发明中魁蚶多糖JNY2PW具有显著的免疫调节和抗肿瘤活性。经体外免疫活性检测显示,魁蚶多糖JNY2PW具有增强免疫作用。其能刺激小鼠巨噬细胞RAW264.7的吞噬功能,还能刺激RAW264.7细胞分泌TNF-α、IL-6、NO等细胞因子。上述制备方法制得的魁蚶多糖JNY2PW体内抗肿瘤活性试验显示,魁蚶多糖JNY2PW可明显抑制小鼠乳腺癌细胞增殖,具有良好的抗癌活性。结果显示用100mg/kg浓度的魁蚶多糖JNY2PW口服给药乳腺癌细胞(4T1)移植瘤小鼠14天后,小鼠瘤体积明显减小,抗癌效果与阳性药多柔比星相似,但对肝肾无毒副作用。该多糖显示出良好的抗肿瘤作用且低毒性。以往研究中,β-葡聚糖的免疫增强和抗肿瘤活性研究较多,对α-葡聚糖的免疫增强和抗肿瘤活性研究较少,本发明公开了一种魁蚶来源的葡聚多糖JNY2PW,其为一种α-葡聚糖,具有良好的免疫增强和抗肿瘤生物活性。
该方法制备的多糖,因其来源于天然海洋贝类,加之纯化工艺简单,因此有较大的开发前景和应用价值。如:在医药方面,可作为其他药物的辅料或制备具有抗肿瘤活性的癌症辅助药物;在保健品方面,可制备具有抗肿瘤和抗氧化功能的保健口服液;另外,还可以作为天然食品添加剂应用在饮料和点心中,还可作为乳化剂、保湿剂添加在化妆品中。
四、附图说明
图1为JNY2PW的GPC图谱;
图2为JNY2PW的分子量图;
图3为JNY2PW的离子色谱图;
图4为JNY2PW的1H NMR图;
图5为JNY2PW的13C NMR图谱;
图6为JNY2PW的HSQC图谱;
图7为JNY2PW的HMBC图谱;
图8为JNY2PW对小鼠脾细胞的影响;
图9为JNY2PW对RAW264.7巨噬细胞的影响;
图10为JNY2PW对小鼠巨噬细胞RAW264.7吞噬荧光颗粒的影响;
图11为JNY2PW对小鼠巨噬细胞RAW264.7细胞分泌TNF-α的影响;
图12为JNY2PW对小鼠巨噬细胞RAW264.7细胞分泌IL-6的影响;
图13为JNY2PW对小鼠巨噬细胞RAW264.7细胞分泌NO的影响;
图14为给药14天内JNY2PW对乳腺癌荷瘤小鼠瘤体积的影响;
图15为给药14天时JNY2PW对乳腺癌荷瘤小鼠瘤重的影响;
图16为JNY2PW给药后对乳腺癌荷瘤小鼠肝、肾器官的影响。
五、具体实施方式
根据下述实施例可以更好的理解本发明。然而,本领域的技术人员容易理解,实施例所描述的内容仅用于说明本发明,而不应当也不会限制权利要求书所详细描述的本发明。
实施例1:魁蚶多糖JNY2PW的制备
一.实验方法
1)前处理:将新鲜魁蚶剥壳取斧足,用水清洗干净;
2)缓冲液提取:将清洗好的魁蚶斧足加入PBS缓冲液中,使用高速组织匀浆机匀浆处理,匀浆液置于冰上使用超声破碎仪破碎提取;离心后收集提取液。
3)盐析:将提取液浓缩后,加入硫酸铵使硫酸铵体积浓度为a%,静置,收集上清液;再向上清液中加入硫酸铵使硫酸铵体积浓度为b%,静置,收集沉淀,即得粗多糖;其中30<a≤70<b≤100。
4)纯化:对上述粗多糖进行纯化,即得魁蚶多糖。
优选的,步骤2)中所述缓冲液提取的具体操作为用3~10倍体积的30-100mM PBS缓冲液对魁蚶斧足进行低温匀浆、低温超声破碎提取,匀浆时间为30~60s,提取时间20~60min,匀浆和超声破碎温度为4℃。
优选的,步骤3)中所有所述浓缩为-40~-80℃减压冷冻浓缩,所有所述静置的时间为10~28h。
优选的,步骤3)中30<a≤70,70<b≤100。
优选的,将步骤4)中盐析后的粗多糖再进行阴离子交换柱层析,用含梯度浓度(0~1M)氯化钠的30mM PBS进行洗脱,采用苯酚-硫酸法跟踪洗脱曲线,根据洗脱曲线收集糖部分,浓缩,冷冻干燥;然后用双蒸水溶解,离心,取上清液进行分子筛凝胶柱层析,用双蒸水进行洗脱,采用苯酚-硫酸法跟踪洗脱曲线,根据洗脱曲线收集糖部分,浓缩,冷冻干燥后获得魁蚶多糖JNY2PW。
优选的,上述离子交换柱为DEAE离子交换柱。
优选的,上述分子筛凝胶层析选用SephadexG100分子筛色谱柱。
纯度检测:将上述获得的多糖JNY2PW配成2%浓度(W/V)的水溶液,HPGPC法测得保留时间。
二.实验结果
HPGPC结果显示经离子交换和凝胶过滤法分离纯化后得到的组分JNY2PW呈单一峰,说明JNY2PW为均一多糖(图1)。
实施例2:魁蚶多糖JNY2PW化学结构的研究
下面以上述实施例中提取的JNY2PW作进一步的结构分析。
一.实验方法
1、分子量测定
超高效液相-凝胶色谱-蒸发光散射检测器(UPLC-GPC-ELSD)仪器配置及色谱条件:美国Waters UPLC,TSK-3000GPC色谱柱,自动进样器,Millipore超纯水离子交换器制备高纯水(0.45μm醋酸纤维素膜过滤);流速0.3mL/min。
标准曲线的制备:分别称取适量的右旋糖苷标准品,加入去离子水,将其配置成浓度0.5mg/mL的对照品溶液,之后逐一进行UPLC检测。
样品溶液制备:分别称取一定量的实施例1制备的多糖蛋白JNY2PW,加入适量去离子水,将其配制成浓度为1mg/mL的溶液,Millipore 0.22μm水系滤膜过滤,进样检测。结果见图2。
2、单糖组成分析
完全酸水解:精密称取实施例1制备的多糖JNY2PW(10mg)放入厚壁耐压瓶中,加入4mL的2M三氟乙酸(TFA),充氮气,封管后,100℃水解2h,减压蒸干。
样品的离子色谱分析
仪器配置及色谱条件:DionexICS3000型离子色谱,CarboPac PA20分析柱,150×3mm,,CarboPac PA20保护柱,50*3mm,,淋洗液组成及流速1-25min,1mM KOH;25.1-32min,30mM KOH,;32.1-35min,1mM KOH;0.45mL/min,进样10μL。
对照品溶液的制备:取阿拉伯糖、半乳糖、葡萄糖、木糖、甘露糖对照品适量,用去离子水溶解成各含10.0mg/L的对照品溶液,摇匀,即得。
供试品溶液制备:将实施例1制备的多糖JNY2PW完全酸水解产物复溶于50mL去离子水,超声10分钟使溶解,取溶液适量,过孔径0.22μm水系滤膜及DIONEXRPⅡ固相萃取小柱。
分别精密吸取对照品溶液和供试品溶液各10μL,注入离子色谱仪,即得。结果见图3。
3、甲基化分析
多糖蛋白完全甲基化:称取适量实施例1制备的多糖蛋白JNY2PW于反应瓶中,放入真空干燥箱中干燥5h(50℃),加入经过分子筛处理过的DMSO 2mL,超声振荡5分钟,待样品完全溶解后,加入研磨成粉状NaOH 20mg,同时用氮气赶尽瓶中空气,室温下超声10分钟,静置90分钟,待反应瓶中的DMSO完全冰冻后,逐滴加入0.1mL碘甲烷(此过程大约需要15~20分钟),同时反应物会慢慢解冻,并逐渐澄清,直至成为亮黄色。然后超声10分钟,静置30分钟。室温减压蒸馏,去尽过量的碘甲烷,用水透析一天,减压蒸至2mL左右。冷冻干燥后再于干燥器中干燥5h,重复上述操作2次后,取少量样品进行红外检测,如果红外光谱中原3300cm-1处强而宽的羟基峰消失,而2900cm-1处甲基峰显著增强,表明样品的全甲基化反应已经完成。
部分甲基化阿尔迪醇乙酰酯制备:将已经完全甲基化的样品溶于3mL90%(v/v)的甲酸溶液中,封管,100℃下解聚6h,向反应瓶中加入2mL甲醇,40℃下减压浓缩蒸干,重复以上操作三次以除尽过量甲酸,然后向解聚后的样品中加入2M TFA溶液4mL,密封后于110℃下水解2h,反应瓶中的溶液于40℃下减压蒸干,再加入2mL甲醇,蒸干,重复以上操作多次以除尽过量的TFA。水解后的样品用3mL蒸馏水溶解后,加入约20mgNaBH4于室温下还原3h,然后用冰醋酸调pH值到5左右,加入2mL甲醇及一滴冰醋酸后,再减压蒸干,重复上述操作多次以除尽过量的乙酸。经上述处理的样品置于P2O5真空干燥器中减压干燥一天,再于110℃干燥10~15min后,加入3mL醋酐,110℃反应1h,向反应液中加入2mL甲苯,振荡后40℃下减压蒸去未反应的醋酐,如此重复多次以除尽醋酐。然后将乙酰化后的样品溶于氯仿中,再加入等体积的蒸馏水洗涤氯仿层3次,除尽水层,氯仿层加入无水硫酸钠干燥10min,过滤,将氯仿溶液室温减压浓缩至0.1mL左右后,进行气质联用分析(GC-MS)。气质的条件为:起始温度50℃,升温程序为40℃/min,至215℃,保持40min,检测器温度250℃,DB-5毛细管GC-MS色谱柱检测。结果见表1。
4、核磁共振分析
称取30mg实施例1制备的多糖蛋白JNY2PW,溶于D2O中,使用Bruker 600M核磁共振仪进行H谱,C13谱,HMBC谱,HSQC谱检测。结果见图4-7。
5、IR光谱分析:
称取1mg实施例1制备的多糖蛋白JNY2PW,真空干燥过夜,次日用溴化钾压片法进行IR检测。结果见图9。
二.实验结果
(1)分子量测定
根据标准曲线回归计算,如图1所示,JNY2PW重均分子量为5.25×107Da。
(2)单糖组成分析
由完全酸水解产物采用离子色谱法测谱,所得结果如图2所示。从图中可以看出JNY2PW仅有葡萄糖组成。
(3)甲基化分析
样品经甲基化,水解、还原,乙酰化后GC-MS分析,表1结果表明JNY2PW由-α-D-Glcp-(1→、→4)-α-D-Glcp-(1→和→4,6)-α-D-Glcp-(1→糖残基构成。
表1.JNY2PW甲基化分析
(4)多糖的核磁共振分析
将均一多糖JNY2PW样品置于核磁管中,用D2O溶解后测谱,所得结果如图4~7所示。
根据上述图4~7的核磁图谱可知各个碳和氢的归属,如下表2所示。
表2.JNY2PW核磁共振分析结果
经上述完全酸水解、甲基化分析、红外光谱检测及核磁分析,结果显示JNY2PW是一种由葡萄糖组成的葡聚多糖,从甲基化分析和二维核磁谱图分析可知JNY2PW由→4)-α-D-Glcp-(1→和→4,6)-α-D-Glcp-(1→构成主链,支链末端由-α-D-Glcp-(1→构成。JNY2PW的结构为:
实施例3:魁蚶多糖JNY2PW的免疫调节和抗肿瘤作用研究
一.实验方法
(1)魁蚶多糖JNY2PW对脾细胞的增殖作用
在无菌条件下将小鼠的脾脏制成均匀的细胞悬浮液。然后洗涤细胞悬浮液并离心(1500r/min,5min)以获得细胞沉淀。用Tris-NH4Cl溶液裂解红细胞并离心。将收集的淋巴细胞用PBS洗涤三次,制成细胞悬液。最后用RPMI-1640培养液调整细胞浓度为5×l06个/mL,接种到96孔板中(100μL/孔)。给药组分别加入100μL不同浓度的魁蚶多糖溶液(15.6~500μg/mL)。同时设阳性对照组(ConA,5μg/mL)和空白组(只加RPMI-1640培养基)。每组三个复孔。细胞放在培养箱中培养48h(37℃,5%CO2)。向每孔中加入20μL MTT(5mg/mL),并继续培养4h,离心,弃去上清液。最后向每个孔中加入150μL DMSO,震荡摇匀后,测定570nm处的吸光度并按下列公式计算细胞的增殖率。
细胞增殖率(%)=A给药组/A空白组×100%
(2)魁蚶多糖对RAW264.7巨噬细胞活性的影响
RAW264.7细胞于DMEM培养基中培养至对数生长期,每孔2300个细胞铺96孔板,将细胞培养24h使其贴壁。弃去孔板中上清液,在样品组中加入对应不同浓度的魁蚶多糖溶液(7.8~500μg/mL)。同时设阳性对照组(LPS,1μg/mL)和空白组(只加RPMI-1640培养基)。每组三个复孔。培养48h后,将20μL MTT溶液(5mg/mL)加入到每孔中,继续培养4h,弃去上清液。最后向每孔加入200μL DMSO,震荡摇匀后,在570nm下测定吸光度值并按下列公式计算细胞活力。
细胞活力(%)=A给药组/A空白组×100%
(3)FITC-dextran吞噬实验检测MEPB70-1对小鼠巨噬细胞RAW264.7吞噬荧光颗粒的影响
将RAW264.7细胞调整细胞悬液浓度为1.5×106cells/mL,接种于6孔板,每孔1mL细胞悬液,于37℃,5%CO2细胞培养箱中培养,使其贴壁。次日,弃上清,加入1mL不同浓度的羊肚菌多糖(终浓度分别为0、125、250及500μg/mL)或1μg/mL LPS溶液,于37℃,5%CO2培养箱中继续培养24h。作用24h后,加入终浓度为100μL 1mg/mL FITC-Dextran(40,000Da),于37℃,5%CO2细胞培养箱中继续培养1h。PBS洗2次后,加入500μL PBS重悬细胞,于流式细胞仪检测细胞吞噬荧光颗粒的情况。
(4)ELISA法检测魁蚶多糖对小鼠巨噬细胞RAW264.7中IL-6、TNF-α的分泌情况
将RAW264.7细胞调整细胞悬液浓度为1×105cells/mL,接种于96孔板,每孔100μL细胞悬液,于37℃,5%CO2细胞培养箱中培养,使其贴壁。次日,弃上清,加入200μL不同浓度的魁蚶多糖(终浓度分别为0、7.8125、16.25、31.25、62.5、125、250及500μg/mL)或1μg/mLLPS溶液,于37℃,5%CO2培养箱中继续培养24h。根据ELISA试剂盒内说明书介绍操作流程,检测细胞培养上清中IL-6、TNF-α的表达情况。
(5)Griess法检测魁蚶多糖对小鼠巨噬细胞RAW264.7中NO的分泌情况
将RAW264.7细胞调整细胞悬液浓度为1×105cells/mL,接种于96孔板,每孔100μL细胞悬液,于37℃,5%CO2细胞培养箱中培养,使其贴壁。次日,弃上清,加入200μL不同浓度的魁蚶多糖(终浓度分别为0、7.8125、16.25、31.25、62.5、125、250及500μg/mL)或1μg/mLLPS溶液,于37℃,5%CO2培养箱中继续培养24h。根据NO试剂盒内说明书介绍操作流程,测定细胞培养上清中NO的含量。
(6)魁蚶多糖JNY2PW对乳腺癌4T1荷瘤小鼠体内抗肿瘤试验
选取30只BALB/c雌性小鼠(6周龄,18-22g),通过在小鼠右肢的第二乳房垫下接种鼠源4T1乳腺癌细胞(每只小鼠1.5×106cells)构建小鼠乳腺癌原位荷瘤模型。当肿瘤体积大于80mm3时,将小鼠随机分为5组(每组6只):对照组(无肿瘤小鼠,自由饮食),模型组(生理盐水),多柔比星(DOX)组(2mg/kg),JNY2PW组(100mg/kg)。JNY2PW和DOX通过口服给药。每两天量取小鼠瘤体积。连续给药14天后,处死小鼠,称取瘤重,并通过苏木精和伊红(H&E)染色分析小鼠的肝,肾和肿瘤组织。
二.实验结果
(1)魁蚶多糖对脾细胞的增殖作用
由图8可知,魁蚶多糖样品JNY2PW具有促进小鼠脾细胞增殖的活性,且在一定浓度范围内呈现出良好的量效关系。
(2)魁蚶多糖对RAW264.7巨噬细胞的影响
由图9可知,魁蚶多糖样品JNY2PW在一定浓度范围内对RAW264.7巨噬细胞没有细胞毒性作用,并且对其增殖具有促进作用。
(3)魁蚶多糖对RAW264.7巨噬细胞吞噬功能的影响
由图10可知,在所测试浓度范围内,魁蚶多糖样品JNY2PW能明显促进RAW264.7细胞吞噬FITC-dextran的能力,且呈现明显的剂量依赖性。
(4)魁蚶多糖对小鼠巨噬细胞RAW264.7分泌IL-6、TNF-α的分泌情况
由图11和12可知,加药处理24h后,与对照组相比,魁蚶多糖样品JNY2PW能明显促进炎性细胞因子TNF-α和IL-6在巨噬细胞RAW264.7中的分泌。
(5)魁蚶多糖对小鼠巨噬细胞RAW264.7细胞分泌NO情况的影响
由图13可知,加药处理24h后,与对照组相比,魁蚶多糖样品JNY2PW能明显促进细胞毒性分子NO在巨噬细胞RAW264.7中的分泌。
(6)魁蚶多糖对乳腺癌4T1荷瘤小鼠体内抗肿瘤作用
使用具有高致瘤性的鼠源乳腺癌4T1细胞建立原位肿瘤模型,用于评估JNY2PW在体内的抗肿瘤作用。如图14和15所示,JNY2PW处理显著降低了BALB/c小鼠中4T1细胞的肿瘤发生能力。给药14天后,口服JNY2PW组的小鼠瘤重显著低于空白对照组,甚至略低于多柔比星组。此外,H&E染色测试暗示JNY2PW对荷瘤小鼠没有显著的肝和肾毒性(图16)。
本发明所提取的魁蚶多糖JNY2PW能显著促进脾细胞及RAW264.7巨噬细胞的增殖,增强巨噬细胞的吞噬功能,能明显促进RAW264.7分泌IL-6/TNF-α,具有显著的体外增强免疫作用。通过乳腺癌荷瘤小鼠体内试验,魁蚶多糖JNY2PW能够显著抑制肿瘤生长,且无肝肾毒性,具有显著的体内抗肿瘤作用,高效低毒,具有广阔的临床开发潜力。
Claims (4)
1.一种魁蚶多糖,所述多糖含量为90-95wt%。该多糖的单糖组成为葡萄糖。
2.根据权利要求1所述的多糖,其特征在于,所述多糖的重均分子量为7.0×106-8.0×107Da,优选为5.25×107Da。
3.根据权利要求1或2所述的多糖,其特征在于,该多糖的制备方法包括如下步骤:
(1)将魁蚶去壳洗净后用30mM PBS缓冲液匀浆、超声提取,采用100%硫酸铵盐析,收集沉淀糖类部分;
(2)将步骤(1)得到的糖类部分复溶于30mM PBS缓冲液,采用5000截留分子量的透析袋用水透析,收集袋内部分,冻干;
(3)使用DEAE-Sepharose Fast Flow阴离子交换柱色谱对步骤(2)收集到的袋内部分进行分离,其中使用的洗脱液为10-100mM PBS溶液、优选为30mM PBS溶液,收集洗脱峰,采用5000截留分子量的透析袋用水透析,收集袋内部分,冻干;
(4)使用Sephadex G-100分子排阻柱色谱对步骤(3)收集到的袋内部分进行分离,其中使用的洗脱液为双蒸水溶液,收集洗脱峰,采用5000截留分子量的透析袋用水透析,收集袋内部分,冻干。
4.权利要求1至3中任一项所述的多糖在制备免疫调节剂、治疗肿瘤的药物和抗癌保健品中的用途。
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