CN113817074A - 一种菱角中性多糖及其制备方法和用途 - Google Patents
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Abstract
本发明公开了一种菱角中性多糖及其制备方法和用途,属于农产品精深加工技术领域。该方法包括如下步骤:预处理、多糖提取、除杂、醇沉、分离纯化、透析、干燥等步骤。该方法操作简单、省时高效、实用性强,易于工业化放大生产;同时,采用本方法制备的菱角中性多糖具有免疫调节和抗肿瘤活性,为菱角精准开发免疫、抗肿瘤药物和营养保健产品提供新的资源,对推动健康产业发展具有重要的理论价值和经济效益。
Description
技术领域
本发明属于农产品精深加工技术领域,具体涉及一种菱角中性多糖及其制备方法和用途。
背景技术
植物多糖是一类存在于植物细胞中,具有广泛生物活性的天然大分子物质,由于其来源广泛、生物活性强、安全性高、毒副作用小等特点,近年来成为国内外研究的热点。随着多糖化学和生物学研究的不断深入,其增强免疫活性、抗肿瘤、抗炎、抗衰老、抗病毒、抗菌等生物活性已得到世界各领域学者的广泛共识。因此,优质多糖原料和高活性多糖资源的发掘业已成为国内外多糖研究的重要内容之一。
菱角(Trapa bispinosa L.)是一年生草本水生植物菱的果实,在我国已有2000多年的栽培历史,尤以长江中下游太湖地区和珠江三角洲栽种较多。菱角是我国传统的药食同源物,不仅含有丰富的淀粉、蛋白质、葡萄糖、不饱和脂肪酸及多种维生素和矿物元素等营养素,还含有多糖、多酚、甾醇和生物碱等多种生理活性物质,因此,具有极佳的食用和药用价值。我国菱角资源丰富,目前菱角仍以鲜售或蒸煮熟制的初加工销售为主,鲜见精深加工产品问世。多糖是菱角中主要的活性成分之一,但迄今关于菱角多糖结构和活性的研究鲜见报道。本课题组研究发现,菱角中富含中性多糖,并具有免疫调节和抗肿瘤活性,如能对其开发并加以利用,对于菱角资源化利用提高其附加值,丰富多糖资源,提高国民健康水平,推动健康产业发展将具有重要的理论价值和经济效益。基于此,本发明公开一种菱角中性多糖,并提供这种菱角中性多糖的制备方法和用途。
发明内容
针对菱角资源开发仍处于初期阶段的现状,本发明的目的在于提供一种菱角中性多糖及其制备方法和用途。本发明的方法操作简单、省时高效、实用性强,且得到的菱角中性多糖具有一定的免疫调节和抗肿瘤活性,可作为天然活性物质用于功能性食品和药品的开发,对提高菱角的资源利用率、深度开发和利用菱角资源具有现实意义。
为实现上述发明目的,本发明提供一种菱角中性多糖的制备方法,按照下述步骤进行:
(1)菱角预处理:菱角洗净、去壳、烘干、粉碎,过80目筛,得到菱角粉;取粉,按固料比1∶5(g/mL)加石油醚脱脂,脱脂后的残渣再用体积分数80%乙醇水溶液浸泡12h,除去游离糖和小分子物质,残渣烘干并粉碎过筛,得到脱脂预处理的菱角粉。
(2)多糖提取:将步骤(1)得到的预处理的菱角粉与蒸馏水按固料比1∶40-60(g/mL)混合,调节温度为90-100℃,在140-160W的超声功率下辅助提取10-20min,经过滤、浓缩,得到菱角粗多糖提取液。
(3)除杂:向步骤(2)得到的粗多糖提取液中按0.03%原料干重比加入α-淀粉酶除淀粉,过滤后再加入Sevag试剂(氯仿∶正丁醇=4∶1)除蛋白,浓缩上清液至原体积的1/10,按体积比1∶4加入无水乙醇进行醇沉过夜,收集沉淀物复溶于水中,得到脱淀粉和蛋白的菱角粗多糖水溶液。
(4)分离纯化:将步骤(3)得到的粗多糖水溶液上DEAE-52纤维素离子交换柱进行分离纯化,用去离子水洗脱,收集水洗组分,再用3500Da透析袋透析后,得到菱角中性多糖组分。
(5)干燥:将步骤(4)得到的中性多糖组分水溶液经喷雾或者冷冻干燥,得到菱角中性多糖,命名为TBPS-W。
本发明的菱角中性多糖的组成和结构特征:多糖含量大于95%,未检出蛋白质,是由鼠李糖(Rha)、阿拉伯糖(Ara)、木糖(Xyl)、甘露糖(Man)、葡萄糖(Glu)和半乳糖(Gal)组成的一种杂多糖,其摩尔比为3∶5∶9∶36∶937∶10;分子量分布在5000-8000Da之间,主要集中在6388Da,且不具有三股螺旋结构。其微米级表面形貌为有气孔、带褶皱、稍显卷曲、大小较均一的片状结构;纳米级结构为直径在89-403nm、高度在0.670-9.853nm的圆锥形颗粒状聚集体。经红外/紫外光谱分析、高碘酸氧化、Smith降解以及核磁共振测定,该菱角中性多糖为α型吡喃糖,由→6)-α-D-Glcp-(1→,α-Glcp-(1→,→4,6)-α-D-Glcp-(1→,→6)-α-D-Manp-(1→和→3)α-D-Manp-(1→类型的糖苷键组成。
本发明还提供上述菱角中性多糖的应用:
使用剂量在25-400μg/mL条件下,上述菱角中性多糖对RAW264.7细胞无毒性,并对RAW264.7细胞增殖活性具有显著促进作用(p<0.01,与空白对照组相比),且在25-100μg/mL浓度范围内增殖效应呈现剂量依赖性。
使用剂量在25-200μg/mL条件下,上述菱角中性多糖可有效激活巨噬细胞,显著促进RAW264.7细胞吞噬中性红能力(p<0.05,与空白对照组相比),且在25-100μg/mL浓度范围间呈现剂量依赖性。
使用剂量在25-400μg/mL条件下,上述菱角中性多糖可显著提高RAW264.7细胞的NO分泌量(p<0.05,与空白对照组相比),且在25-200μg/mL浓度范围内呈现剂量依赖性。
使用剂量在25-100μg/mL条件下,上述菱角中性多糖对HepG2肝癌细胞增殖活性有显著抑制性(p<0.05,与空白对照组相比),且在该浓度范围内呈现剂量依赖性。
本发明的优点和有益效果在于:提供一种菱角中性多糖及其制备方法和用途,可以高效低能耗地以菱角为原料,最大化地制备出高纯度的菱角中性多糖;且该多糖是一种从菱角中分离纯化出的新型植物活性多糖,具有低分子量,同时具有免疫增强和抗肿瘤的用途,不仅丰富了植物活性多糖的种类,同时也为延长菱角资源精深加工和提高菱角的经济效益提供了新的途径。
附图说明
图1为TBPS-W的高效排阻色谱图;
图2为TBPS-W的气相色谱图(1.鼠李糖;2.阿拉伯糖;3.木糖;4.甘露糖;5.葡萄糖;6.半乳糖;7.内标);(A)标品;(B)TBPS-W的单糖组成分析;
图3为TBPS-W的红外光谱图;
图4为TBPS-W的史密斯降解产物气相色谱图(1.甘油;2.赤藓醇;3.鼠李糖;
4.阿拉伯糖;5.木糖;6.甘露糖;7.葡萄糖;8.半乳糖;9.内标):(A)标品;(B)TBPS-W的史密斯降解产物
图5和图6为TBPS-W的一维核磁共振波谱;(图5)1H NMR;(图6)13C NMR;
图7为TBPS-W的三股螺旋结构测定图;
图8为TBPS-W的原子力显微镜图;(A)二维和三维图;(B)高度分析切面图;
图9为TBPS-W的扫描电子显微镜图。
具体实施方法
下面结合具体实施例,对本发明的具体实施方式作进一步详细的描述,但本发明的实施方法不限于此。下述实施例中所使用的试验方法若无特殊说明均为常规方法,所涉及的物料若无特殊说明均可通过商业途径获取。
实施例1
取1.0kg脱脂菱角粉,按固料比1∶40(g/mL)加水混合,调节温度为90℃,在140W超声辅助下提取10min;过滤并浓缩提取液,按原料干重0.03%加入α-淀粉酶除淀粉,过滤后再加入Sevag试剂除蛋白。随后,将除杂后提取液浓缩至原体积1/10,以终体积分数80%乙醇浓度醇沉过夜,收集沉淀物加水复溶,浓缩后过DEAE-52离子交换柱,收集去离子水洗脱组分,浓缩、透析、冻干,所得菱角中性多糖为11.82g,得率为1.182%。由苯酚硫酸法测得,糖含量为95.53%,
实施例2
取1.0kg脱脂菱角粉,按固料比1∶60(g/mL)加水混合,调节温度为100℃,在160W超声辅助下提取20min;过滤并浓缩提取液,按原料干重0.03%加入α-淀粉酶除淀粉,过滤后再加入Sevag试剂除蛋白。随后,将除杂后提取液浓缩至原体积1/10,以终体积分数80%乙醇溶液醇沉过夜,收集沉淀物加水复溶,浓缩后过DEAE-52离子交换柱,收集去离子水洗脱组分,浓缩、透析、冻干,所得菱角中性多糖为13.22g,得率为1.322%。由苯酚硫酸法测得,糖含量为96.45%,
实施例3
实施例1中所制菱角中性多糖,高效排阻色谱法(HPSEC)测定其分子量,结果如附图1所示,结果表明,TBPS-W是分子量分布均一的纯化多糖,分子量集中在6388Da。气相色谱法(GC)测定其单糖组成,结果如附图2所示,结果表明,该多糖由鼠李糖、阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖组成,各单糖摩尔比为3∶5∶9∶36∶937∶10。经红外光谱、高碘酸氧化和史密斯降解产物GC分析、核磁共振波谱对其结构进行分析和表征,如附图3、图4、图5、图6所示,结果显示,该菱角中性多糖为α型吡喃糖,由→4,6)-α-D-Glcp-(1→,→6)-α-D-Manp-(1→,→3)α-D-Manp-(1→,→6)-α-D-Glcp-(1→和α-Glcp-(1→类型的糖苷键组成。刚果红实验测定结果如附图7所示,表明TBPS-W不含有三股螺旋结构。扫描电子显微镜和原子力显微镜对其表观形态和尺寸进行测定,结果如附图8和附图9所示,结果表明,该多糖微米级结构表面有气孔、带褶皱、稍显卷曲、大小较均一的片状结构,纳米级结构为直径在89-403nm、高度在0.670-9.853nm的圆锥形颗粒状聚集体。
实施例3
实施例2中所制菱角中性多糖,MTT法测定该菱角中性多糖使用剂量在25~400μg/mL条件下对RAW264.7细胞增殖活力的影响,结果表明该浓度范围内这种菱角中性多糖对RAW264.7细胞无毒性,且与空白对照对比,其对RAW264.7细胞的增殖活性具有显著促进作用(143%~158%)。在25~100μg/mL浓度范围间发现增殖效应呈现剂量依赖性。
实施例4
实施例2中所制菱角中性多糖,中性红吞噬实验测得该菱角中性多糖使用剂量在25~200μg/mL条件下可显著促进RAW264.7细胞吞噬中性红的能力(19.0%~34.5%),且在25~100μg/mL浓度范围间呈现剂量依赖性;NO检测试剂盒测得该菱角中性多糖使用剂量在25~400μg/mL条件下可显著促进RAW264.7细胞分泌细胞因子NO的分泌量(27.50~37.79μM),且在25~200μg/mL浓度范围间呈现剂量依赖性。
实施例5
实施例2中所制菱角中性多糖,MTT法测得该菱角中性多糖使用剂量在25~400μg/mL条件下可显著抑制HepG2肝癌细胞的增殖活性(14.8%~47.9%),且在25~100μg/mL浓度范围间呈现剂量依赖性。
综合以上结果,本发明中的新型菱角中性多糖具有一定的免疫调节活性,可通过促进RAW264.7细胞的增殖,有效激活巨噬细胞,增强其吞噬中性红的能力,并且显著刺激RAW.264.7小鼠巨噬细胞分泌细胞因子NO,从而达到免疫增强的效果。此外,该菱角中性多糖还可显著抑制HepG2肝癌细胞的增殖,具有一定的抗肿瘤能力。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的原理和精神实质下所做的改变、修饰、替代、组合、简化,均视为等效的置换方法,都包含在本发明的保护范围内。
Claims (3)
1.一种菱角中性多糖及其制备方法和用途,其特征在于按照以下步骤进行:
(1)将菱角烘干粉碎过80目筛,按固料比1∶5(g/mL)加石油醚脱脂,脱脂后残渣再用体积分数80%乙醇水溶液浸泡12h,经烘干、过筛,得到脱脂菱角粉;
(2)将步骤(1)得到的脱脂菱角粉与蒸馏水按固料比为1∶40-60(g/mL)混合,调节温度为90-100℃,在140-160W的超声功率下辅助提取10-20min,经过滤、浓缩,得到菱角粗多糖提取液;
(3)向步骤(2)得到的菱角粗多糖提取液中按原料干重0.03%加入α-淀粉酶除淀粉,过滤后再加入Sevag试剂除蛋白;离心取上清,浓缩至原体积1/10,按体积比1∶4加入无水乙醇醇沉过夜,收集沉淀物复溶于水中,得到脱淀粉和蛋白的菱角粗多糖水溶液;
(4)将步骤(3)得到的粗多糖水溶液经DEAE-52离子交换柱进行分离纯化,用去离子水洗脱,收集水洗组分,经3500Da透析袋透析后,得到菱角中性多糖组分;
(5)将步骤(4)得到的中性多糖组分水溶液经喷雾或冷冻干燥,得到菱角中性多糖。
2.一种菱角中性多糖,其特征在于:其多糖含量大于95%,是由鼠李糖、阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖组成的一种杂多糖,摩尔比为3∶5∶9∶36∶937∶10;该菱角中性多糖分子量分布在5000-8000Da之间,主要集中在6388Da,且不具有三股螺旋结构;其微米级表面形貌为有气孔、带褶皱、稍显卷曲、大小较均一的片状结构,纳米级结构为直径在89-403nm、高度在0.670-9.853nm的圆锥形颗粒状聚集体;该中性多糖为α型吡喃糖,由→4,6)-α-D-Glcp-(1→,→6)-α-D-Manp-(1→,→3)α-D-Manp-(1→,→6)-α-D-Glcp-(1→和α-Glcp-(1→类型的糖苷键组成。
3.一种菱角中性多糖及其制备方法和用途,其特征在于,该中性多糖具有一定的免疫增强和抗肿瘤活性,可用于大健康食品、功能性食品或医药品等制造领域中,发挥其功能活性的用途。
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