CN113621085A - 一种蒲公英根多糖硫酸化修饰产物的制备方法及蒲公英根多糖硫酸化修饰产物的应用 - Google Patents
一种蒲公英根多糖硫酸化修饰产物的制备方法及蒲公英根多糖硫酸化修饰产物的应用 Download PDFInfo
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Abstract
本发明公开了一种蒲公英根多糖硫酸化修饰产物的制备方法及蒲公英根多糖硫酸化修饰产物的应用,多糖硫酸化修饰产物中多糖含量较未修饰前含量提高60%。本发明通过多组试验选出最优提取工艺,采用水提醇沉法得到粗多糖,Sevag法脱蛋白,然后采用DEAE Sepharose Fast Flow进行分离,葡聚糖凝胶纯化,制得纯度高的蒲公英根多糖,再采用氯磺酸‑二甲基甲酰胺法修饰得到蒲公英根多糖硫酸化修饰产物。本发明充分利用废弃的蒲公英根部资源,变废为宝,得到具有免疫调节活性的蒲公英根多糖及其硫酸化修饰产物,实现蒲公英药用资源的可持续化应用。
Description
技术领域
本发明涉及一种植物多糖,具体涉及一种具有免疫调节活性的蒲公英根多糖及其硫酸化修饰产物的制备方法及蒲公英根多糖硫酸化修饰产物的应用。
背景技术
蒲公英是菊科属多年生的草本植物,广泛分布于我国各个地域,且种类繁多,产量大。其药用价值早就载入各种医学古典中,关于蒲公英的记载最早见于《唐本草》:“味甘,平,无毒。主妇人乳痈肿。”,《本草纲目》中记载蒲公英主治妇人乳痈肿,解食毒,散滞气,化热毒,消恶肿、结核、丁肿。蒲公英根、茎、叶均具有较高的药用价值,并且蒲公英的化学成分复杂,其中多糖类占干重达到30%~50%,而多糖含量最高的部分则为蒲公英的根部,但蒲公英在采收过程中其根部多被丢弃,造成资源的极大浪费。
多糖是由单糖分子通过糖苷键结合而成的,广泛存在于植物动物和微生物中,具有提高机体免疫力,降血糖、抗菌、增强免疫力、抗肿瘤、护肝、延缓衰老等药理活性,但由于多糖的活性直接受结构影响,水溶性差、活性低等,因此,对天然多糖进行结构修饰,可显著提高其生物活性。硫酸化修饰是通过引入聚阴离子硫酸根使得多糖类分子空间构象发生改变,进而导致活性产生较大变化,硫酸化多糖不仅硫酸化多糖不仅具有抗凝血及免疫增强的作用,还具有降血脂,降血糖等作用,延长药物作用时间,增强药效降低药物毒副作用等。
目前,国内外对蒲公英根多糖研究主要集中在其化学成分分析上,对其活性研究较少。因此,利用硫酸化修饰技术改变蒲公英根多糖结构,增强其免疫活性,对蒲公英根部资源高效利用,变废为宝具有重大意义。
发明内容
为解决上述背景技术中提出的问题。本发明提供了一种具有提高免疫活性的蒲公英根多糖及其硫酸化修饰产物的制备方法,通过优选方法制备得到蒲公英根粗多糖,然后利用硫酸化修饰技术改变蒲公英根多糖结构,得到具有免疫调节活性的蒲公英根多糖硫酸化修饰产物,实现蒲公英根部资源高效利用,变废为宝,实现蒲公英药用资源的可持续化应用。
为了实现上述目的,本发明采用了如下技术方案:
一种蒲公英根多糖硫酸化修饰产物,蒲公英根多糖硫酸化修饰产物中多糖含量较未修饰前含量提高60%。
作为优选方案,以上所述的蒲公英根多糖硫酸化修饰产物的制备方法,包括以下步骤:
(1)将原料蒲公英根进行粉碎,后置于提取罐中,加入其10-30倍体积的水,沸水提取3-5小时,重复2-3次。将上清液通过滤网除杂,适当浓缩,采用Sevag试剂离心除蛋白,离心,取上清液,加入2-4倍体积的无水乙醇,搅拌均匀,沉淀过夜。用布氏漏斗和滤纸进行抽滤,得到沉淀物。将沉淀物重新用蒸馏水溶解,50-60℃水浴挥去乙醇,用冷冻干燥机进行冻干,得蒲公英根粗多糖;
(2)蒲公英根粗多糖的分离
称取步骤(1)制备得到的蒲公英根粗多糖,加蒸馏水溶解,8000-12000rpm转速离心,取上清液上样,用流速为10-20ml/min的蒸馏水进行洗脱。再用0.0、0.2、0.5、1.0mol/LNaCl溶液梯度洗脱。并采用苯酚硫酸法进行追踪检测多糖含量,分别收集不同的洗脱峰,减压浓缩,2000-4000Da透析袋透析,冷冻干燥,获得蒲公英根多糖;
(3)蒲公英根多糖的硫酸化修饰
取步骤(2)制备得到的蒲公英根多糖,采用氯磺酸-二甲基甲酰胺法进行修饰:准确称取蒲公英根多糖,加入吡啶,在冰浴条件下加入2mL氯磺酸溶液,室温下搅拌10-20min,待混合溶剂逐渐融化变成淡黄色澄清液体,再加入3mL甲酰胺,加热至全部溶解。冰浴至0℃,加入到硫酸衍生化试剂中搅拌溶解,于冰浴中反应1-2h至常温,用NaOH,调节pH=7~8,透析2天,取透析液,浓缩,冻干,得到蒲公英根多糖硫酸化修饰产物。
作为优选方案,以上所述的蒲公英根多糖硫酸化修饰产物的制备方法,步骤(3)准确称取蒲公英根多糖100mg,加入1mL吡啶,在冰浴条件下加入2mL氯磺酸溶液,室温下搅拌10-20min,待混合溶剂逐渐融化变成淡黄色澄清液体,再加入3mL甲酰胺,加热至全部溶解。冰浴至0℃,加入到硫酸衍生化试剂中搅拌溶解,于冰浴中反应1-2h至常温,用NaOH,调节pH=7~8,透析2天,取透析液,浓缩,冻干,得到蒲公英根多糖硫酸化修饰产物。
本发明通过实验研究表明,经过硫酸化修饰得到的蒲公英根多糖产物具有显著的降低小鼠溃疡性结肠炎炎症反应的作用,可用于制备免疫调节的保健食品或药品中。
本发明所述的蒲公英根多糖硫酸化修饰产物在制备具有免疫调节的保健食品或药品中的应用,可将蒲公英根多糖硫酸化修饰产物与配料制成胶囊、颗粒、片剂及包埋剂等。
有效效果:本发明提供的具有免疫调节活性的蒲公英根多糖及其硫酸化修饰产物和现有技术相比具有优点:
本发明通过多组试验选出最优提取工艺,采用水提醇沉法得到粗多糖,Sevag法脱蛋白,然后采用DEAE Sepharose Fast Flow进行分离,葡聚糖凝胶纯化,制得纯度高的蒲公英根多糖,再采用氯磺酸-二甲基甲酰胺法修饰得到蒲公英根多糖硫酸化修饰产物。本发明充分利用废弃的蒲公英根部资源,变废为宝,得到具有免疫调节活性的蒲公英根多糖及其硫酸化修饰产物,实现蒲公英药用资源的可持续化应用。
附图说明
图1为蒲公英根粗多糖的红外光谱图;
图2为蒲公英根多糖硫酸化修饰产物的红外光谱图;
图3为蒲公英根多糖硫酸化修饰前后红外光谱对比图。
图中,将附图向左旋转90°后,纵轴为透过率(%),横轴为波数(cm-1)。
具体实施方式
根据下述实施例,可以更好地理解本发明。然而,本领域的技术人员容易理解实施例所描述的具体的物料配比、工艺条件及其结果仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1
蒲公英根多糖硫酸化修饰产物的制备方法,包括以下步骤:
(1)将原料蒲公英根进行粉碎,后置于提取罐中,加入其10倍体积的水,沸水提取3小时,重复2次。将上清液通过滤网除杂,适当浓缩,采用Sevag试剂离心除蛋白,离心,取上清液,加入2倍体积的无水乙醇,搅拌均匀,沉淀过夜。用布氏漏斗和滤纸进行抽滤,得到沉淀物。将沉淀物重新用蒸馏水溶解,50℃水浴挥去乙醇,用冷冻干燥机进行冻干,得蒲公英根粗多糖;
(2)蒲公英根粗多糖的分离
称取步骤(1)制备得到的蒲公英根粗多糖,加蒸馏水溶解,8000rpm转速离心,取上清液上样,用流速为10ml/min的蒸馏水进行洗脱。再用0.0、0.2、0.5、1.0mol/LNaCl溶液梯度洗脱。并采用苯酚硫酸法进行追踪检测多糖含量,分别收集不同的洗脱峰,减压浓缩,2000Da透析袋透析,冷冻干燥,获得蒲公英根多糖;
(3)蒲公英根多糖的硫酸化修饰
取步骤(2)制备得到的蒲公英根多糖,采用氯磺酸-二甲基甲酰胺法进行修饰:准确称取蒲公英根多糖100mg,加入1mL吡啶,在冰浴条件下加入2mL氯磺酸溶液,室温下搅拌10min,待混合溶剂逐渐融化变成淡黄色澄清液体,再加入3mL甲酰胺,加热至全部溶解。冰浴至0℃,加入到硫酸衍生化试剂中搅拌溶解,于冰浴中反应1h至常温,用NaOH,调节pH=7,透析2天,取透析液,浓缩,冻干,得到蒲公英根多糖硫酸化修饰产物。
实施例2
(1)将原料蒲公英根进行粉碎,后置于提取罐中,加入其10-30倍体积的水,沸水提取5小时,重复3次。将上清液通过滤网除杂,适当浓缩,采用Sevag试剂离心除蛋白,离心,取上清液,加入4倍体积的无水乙醇,搅拌均匀,沉淀过夜。用布氏漏斗和滤纸进行抽滤,得到沉淀物。将沉淀物重新用蒸馏水溶解,60℃水浴挥去乙醇,用冷冻干燥机进行冻干,得蒲公英根粗多糖;
(2)蒲公英根粗多糖的分离
称取步骤(1)制备得到的蒲公英根粗多糖,加蒸馏水溶解,12000rpm转速离心,取上清液上样,用流速为20ml/min的蒸馏水进行洗脱。再用0.0、0.2、0.5、1.0mol/LNaCl溶液梯度洗脱。并采用苯酚硫酸法进行追踪检测多糖含量,分别收集不同的洗脱峰,减压浓缩,4000Da透析袋透析,冷冻干燥,获得蒲公英根多糖;
(3)蒲公英根多糖的硫酸化修饰
取步骤(2)制备得到的蒲公英根多糖,采用氯磺酸-二甲基甲酰胺法进行修饰:准确称取蒲公英根多糖100mg,加入1mL吡啶,在冰浴条件下加入2mL氯磺酸溶液,室温下搅拌20min,待混合溶剂逐渐融化变成淡黄色澄清液体,再加入3mL甲酰胺,加热至全部溶解。冰浴至0℃,加入到硫酸衍生化试剂中搅拌溶解,于冰浴中反应2h至常温,用NaOH,调节pH=8,透析2天,取透析液,浓缩,冻干,得到蒲公英根多糖硫酸化修饰产物。
(4)红外光谱分析
取经过干燥的蒲公英根粗多糖和蒲公英根多糖硫酸化修饰产物样品各1mg,与100mg经干燥的溴化钾粉末于玛瑙研钵研磨均匀;分别压成薄片,分别在红外光谱仪上测定4000-400cm-1的红外光谱。如图1、图2和图3所示,蒲公英根粗多糖在1240cm-1左右的吸收峰是S=O伸缩振动,硫酸化之后明显增强,蒲公英根粗多糖在808cm-1左右的吸收峰是C-O-S的拉伸振动,硫酸化后明显增强,说明硫酸化成功。
Claims (4)
1.一种蒲公英根多糖硫酸化修饰产物,其特征在于,蒲公英根多糖硫酸化修饰产物中多糖含量较未修饰前含量提高60%;
蒲公英根多糖硫酸化修饰产物的制备方法包括以下步骤:
(1)将原料蒲公英根进行粉碎,后置于提取罐中,加入水进行沸水提取,将上清液经除杂浓缩后采用Sevag试剂离心除蛋白,离心,取上清液,加入无水乙醇混合均匀后沉淀过夜,然后进行抽滤后得到沉淀物,将沉淀物重新用蒸馏水溶解,水浴挥去乙醇,冷冻干燥后得蒲公英根粗多糖;
(2)蒲公英根粗多糖的分离
取步骤(1)制备得到的蒲公英根粗多糖,加蒸馏水溶解,离心后取上清液上样,用蒸馏水进行洗脱,再用不同浓度NaCl溶液梯度洗脱,并采用苯酚硫酸法进行追踪检测多糖含量,分别收集不同的洗脱峰,减压浓缩,透析袋透析,冷冻干燥,获得蒲公英根多糖;
(3)蒲公英根多糖的硫酸化修饰
取步骤(2)制备得到的蒲公英根多糖,采用氯磺酸-二甲基甲酰胺法进行修饰:准确称取蒲公英根多糖,加入吡啶,在冰浴条件下加入氯磺酸溶液,室温下搅拌,待混合溶剂逐渐融化变成淡黄色澄清液体,再加入甲酰胺,加热至全部溶解,冰浴至0℃,加入到硫酸衍生化试剂中搅拌溶解,于冰浴中反应至常温,用NaOH调节pH=7~8,经透析后取透析液,浓缩,冻干,得到蒲公英根多糖硫酸化修饰产物。
2.权利要求1所述的蒲公英根多糖硫酸化修饰产物的制备方法,其特征在于,蒲公英根多糖硫酸化修饰产物的制备方法包括以下步骤:
(1)将原料蒲公英根进行粉碎,后置于提取罐中,加入其10-30倍体积的水,沸水提取3-5小时,重复2-3次,将上清液通过滤网除杂,适当浓缩,采用Sevag试剂离心除蛋白,离心,取上清液,加入2-4倍体积的无水乙醇,搅拌均匀,沉淀过夜,用布氏漏斗和滤纸进行抽滤,得到沉淀物,将沉淀物重新用蒸馏水溶解,50-60℃水浴挥去乙醇,用冷冻干燥机进行冻干,得蒲公英根粗多糖;
(2)蒲公英根粗多糖的分离
称取步骤(1)制备得到的蒲公英根粗多糖,加蒸馏水溶解,8000-12000rpm转速离心,取上清液上样,用流速为10-20ml/min的蒸馏水进行洗脱,再用0.0、0.2、0.5、1.0mol/LNaCl溶液梯度洗脱,并采用苯酚硫酸法进行追踪检测多糖含量,分别收集不同的洗脱峰,减压浓缩,2000-4000Da透析袋透析,冷冻干燥,获得蒲公英根多糖;
(3)蒲公英根多糖的硫酸化修饰
取步骤(2)制备得到的蒲公英根多糖,采用氯磺酸-二甲基甲酰胺法进行修饰:准确称取蒲公英根多糖,加入吡啶,在冰浴条件下加入氯磺酸溶液,室温下搅拌10-20min,待混合溶剂逐渐融化变成淡黄色澄清液体,再加入甲酰胺,加热至全部溶解,冰浴至0℃,加入到硫酸衍生化试剂中搅拌溶解,于冰浴中反应1-2h至常温,用NaOH,调节pH=7~8,透析2天,取透析液,浓缩,冻干,得到蒲公英根多糖硫酸化修饰产物。
3.根据权利要求2所述的蒲公英根多糖硫酸化修饰产物的制备方法,其特征在于,步骤(3)准确称取蒲公英根多糖100mg,加入1mL吡啶,在冰浴条件下加入2mL氯磺酸溶液,室温下搅拌10-20min,待混合溶剂逐渐融化变成淡黄色澄清液体,再加入3mL甲酰胺,加热至全部溶解,冰浴至0℃,加入到硫酸衍生化试剂中搅拌溶解,于冰浴中反应1-2h至常温,用NaOH,调节pH=7~8,透析2天,取透析液,浓缩,冻干,得到蒲公英根多糖硫酸化修饰产物。
4.权利要求1-3任一项所述的蒲公英根多糖硫酸化修饰产物应用于调节免疫力的保健食品或药品中。
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