CN112125983B - 一种水溶性玉竹多糖及其硫酸化玉竹多糖和制备方法与应用 - Google Patents
一种水溶性玉竹多糖及其硫酸化玉竹多糖和制备方法与应用 Download PDFInfo
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- CN112125983B CN112125983B CN202011054472.7A CN202011054472A CN112125983B CN 112125983 B CN112125983 B CN 112125983B CN 202011054472 A CN202011054472 A CN 202011054472A CN 112125983 B CN112125983 B CN 112125983B
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- polysaccharide
- polygonatum odoratum
- soluble
- sulfated
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
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- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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Abstract
本发明涉及一种水溶性玉竹多糖及其硫酸化玉竹多糖和制备方法与应用。水溶性玉竹多糖糖苷键组成如下:→6)‑α‑D‑Glcp‑(1→,α‑Glcp‑(1→,→4,6)‑α‑D‑Glcp(1→,→4)‑β‑D‑Man(1→,→3)α‑D‑Manp‑(1→,其分子量分布范围为2000‑5000Da,分子量集中在4025Da。对其进行硫酸化修饰,通过控制不同的硫酸化时间2h和1h得取代度分别为0.54和0.41的硫酸化水溶性玉竹多糖。水溶性玉竹多糖及硫酸化水溶性玉竹多糖具有一定的体外抗氧化活性。本发明对PORP‑1的一级结构、高级结构和宏观结构进行了研究,为进一步研究玉竹多糖结构和活性的构效关系建立基础。
Description
技术领域
本发明属于天然高分子技术领域,特别涉及一种水溶性玉竹多糖及其硫酸化玉竹多糖和制备方法与应用。
背景技术
玉竹,为百合科植物玉竹Polygonatum ordoratum(Mill.)Druce的干燥根茎,广泛分布于我国东北、华北及西南地区,是一种具有食药两用价值的药食同源食品。玉竹中含有多糖、甾体皂苷、黄酮、挥发油等多种活性成分,表现出了降血糖、抗肿瘤、抗氧化、免疫调节、调节糖脂代谢等多种活性,是一种极具潜在应用价值的中国传统药材。
其中,玉竹多糖作为玉竹主要的活性成分之一,是影响其生物活性的关键组分。目前,关于玉竹多糖的结构研究仍然存在一级结构表征不完善、高级与宏观结构研究缺乏等不足。同时,关于玉竹多糖的活性研究集中于降血糖、抗氧化和抗肿瘤活性,已有研究表明相较于合成药物的药理活性,玉竹多糖表现出的生物活性与合成药物仍然存在着一定差距,而玉竹多糖的改性研究是一种改善玉竹多糖已有的生物活性与探索新的生物活性的有效手段。
硫酸化改性是改善天然多糖活性的有效手段,因具有低成本、制备简单、生成物低毒或无毒性等优点,硫酸化修饰已成为多糖改性的重要手段之一。此外,通过硫酸化修饰改性的多糖比原天然多糖具有更好的抗肿瘤、抗氧化、抗凝血等功效。已有研究证实,通过硫酸化改性的多糖由于羟基中被硫酸基团取代,多糖的供氢能力得到了显著的提高从而具有更好的抗氧化功效。
发明内容
为了克服上述现有技术的缺点与不足,本发明的首要目的在于提供一种水溶性玉竹多糖,初步预测其结构为:
本发明以玉竹为原料,通过分离纯化得到了一种水溶性玉竹多糖,明确了了这种水溶性玉竹多糖的一级结构、高级结构与宏观结构,为近一步研究玉竹多糖结构和活性的构效关系建立基础,同时通过硫酸化修饰的手段对这种水溶性玉竹多糖进行改性研究,制备了硫酸化基团修饰的玉竹多糖,为玉竹多糖改善活性提供新的思路。本发明旨在为开发玉竹多糖奠定理论基础,对于深度开发和利用玉竹资源具有现实意义。
本发明另一目的在于提供一种上述水溶性玉竹多糖的制备方法。
本发明再一目的在于提供一种基于上述水溶性玉竹多糖的硫酸化玉竹多糖。本发明的硫酸化玉竹多糖通过对水溶性玉竹多糖进行硫酸化改性,可获得显著改善的活性。
本发明再一目的在于提供上述水溶性玉竹多糖及硫酸化玉竹多糖作为天然抗氧化剂在食品及化妆品等领域中的应用。
本发明的目的通过下述方案实现:
一种水溶性玉竹多糖,命名为PORP-1,其糖苷键组成如下所示:→6)-α-D-Glcp-(1→,α-Glcp-(1→,→4,6)-α-D-Glcp-(1→,→4)-β-D-Man-(1→,→3)-α-D-Manp-(1→。
本发明的水溶性玉竹多糖,其分子量分布范围为2000-5000Da,分子量集中在4025Da。
本发明的水溶性玉竹多糖,是由甘露糖(Man)、葡萄糖醛酸(GlcA)、葡萄糖(Glc)、阿拉伯糖(Ara)、岩藻糖(Fuc)组成的杂多糖,摩尔比为 19.28:0.41:39.76:1.54:1.08。
本发明的水溶性玉竹多糖,是一种不具有三股螺旋结构的多糖,其微米级结构为直径范围在123-151nm的圆顶形颗粒状聚集体,其纳米级表面形貌为致密、较光滑、带褶皱的网络状结构。
本发明还提供一种上述水溶性玉竹多糖的制备方法,包括以下具体步骤:将玉竹醇提后,固形物加水热提,过滤,浓缩所得的水溶液得到玉竹粗多糖溶液;加入Sevag试剂除蛋白和AB-8大孔树脂摇床脱色素后,配成除蛋白除色素的玉竹粗多糖水溶液,醇沉,沉淀物复溶于水中,透析,冻干,得到玉竹粗多糖;采用DEAE-Sepharose离子交换柱层析法进行分离纯化,将玉竹粗多糖复溶于水中制成水溶液上样后,采用梯度洗脱法,依次用去离子水和0.1-0.4mol/L的NaCl盐溶液进行洗脱,收集去离子水组分,冷冻干燥后得到分离纯化的水溶性玉竹多糖PORP-1。
优选地,所述玉竹使用前先清洗干净、烘干并进行粉碎,粉碎后过100 目筛。
优选地,所述醇提条件为在70-80℃醇提2-4h;所述醇提后,过滤去掉滤液,固形物烘干备用。
优选地,所述加水热提的时间为0.5-3h,更优选为1.14h;所述加水热提优选重复多次,更优选为3次,并将多次提取的水溶液合并再进行浓缩。
优选地,所述加水热提的液固比为每1质量份(g)固形物添加10-50体积份(mL)水,更优选为40.23体积份水。
优选地,所述加水热提的温度为50-99℃,更优选为70.5℃。
优选地,所述加水热提中固形物重复提取次数为3次。
优选地,所述加入Sevag试剂除蛋白和AB-8大孔树脂摇床脱色素,具体可为将粗多糖溶于水中,采用Sevage法除蛋白,配制Sevage试剂(氯仿:正丁醇的体积比=4:1),多糖水提液:Sevage试剂为4:1的体积比进行脱蛋白处理。采用AB-8大孔树脂脱色,向脱蛋白多糖水提液中加入AB-8大孔树脂,摇床(150rpm)室温脱色2h,得到除蛋白除色素的多糖水提液。
优选地,所述醇沉在4℃静置过夜;所述醇沉的乙醇终浓度优选为80% (V/V)。
优选地,所述透析的透析袋截留分子量为3500-8000Da,透析时间为48h,期间换水4-8次。透析袋在使用前应使用沸水煮沸10min。
优选地,上述冻干步骤中,压强为0.01MPa,温度为-80℃。
优选地,上述DEAE-Sepharose离子交换柱层析法纯化玉竹粗多糖的准备步骤包括装柱和平衡。
更优选地,准备步骤包括:DEAE-52填料在去离子水中溶胀并超声30min,轻轻搅动下用玻璃棒引流装入层析柱中,避免填料和去离子水的对流翻动,去离子水从柱中排出。泵入去离子水冲柱,直到柱床高度恒定,柱床体积约为 600mL。
本发明还提供一种基于上述玉竹多糖的硫酸化玉竹多糖,具体可通过包括以下步骤获得:向置于冰水浴中正丁醇溶液中缓慢加入浓硫酸,并加入硫酸铵后搅拌均匀,缓慢加入PORP-1,搅拌反应一定时间;反应后的溶液用NaOH 溶液中和,透析,离心,冷冻干燥后得到硫酸化修饰的水溶性玉竹多糖。
优选地,上述水溶性玉竹多糖的硫酸化步骤中,PORP-1的用量为1g,正丁醇的用量为10mL,浓硫酸的用量为30mL,硫酸铵的用量为0.5g。
优选地,上述水溶性玉竹多糖的硫酸化步骤中,反应后的溶液用NaOH 溶液中和后,pH值的范围为6.5-7.5。
优选地,上述水溶性玉竹多糖的硫酸化步骤中,透析袋的截留分子量为 3000Da,透析时间为48h,期间换水4-8次。透析袋在使用前应使用沸水煮沸10min。
优选地,上述水溶性玉竹多糖的硫酸化步骤中,离心条件为8000r/min。
优选地,通过控制硫酸化反应时间分别为2h和1h得到两种取代度分别为0.54和0.41的硫酸化水溶性玉竹多糖SPORP-1和SPORP-2。
优选地,上述水溶性玉竹多糖的硫酸化步骤中,冷冻干燥的条件为压强为0.01MPa,温度为-80℃。
本发明的硫酸化玉竹多糖通过对水溶性玉竹多糖进行硫酸化改性,可获得显著改善的活性。本发明的水溶性玉竹多糖及硫酸化玉竹多糖可作为天然抗氧化剂应用于食品及化妆品等领域中。
附图说明
图1为玉竹粗多糖的洗脱曲线;
图2为PORP-1的高效渗透色谱图;
图3为PORP-1的单糖组成分析(Man:甘露糖,Rha:鼠李糖,GalA:半乳糖醛酸:Glc葡萄糖,GlcA:葡萄糖醛酸,Gal:半乳糖,Ara:阿拉伯糖, Fuc:岩藻糖);
图4为PORP-1的红外光谱图;
图5为PORP-1的史密斯降解产物的气相色谱图(A)标品(B)PORP-1 的史密斯降解产物;
图6和图7为PORP-1的一维核磁图谱;(图6)1H NMR;(图7)13C NMR;
图8为PORP-1的三股螺旋结构测定图;
图9为PORP-1的原子力显微镜图;
图10为PORP-1的扫描电镜图;
图11为SPORP-1和SPORP-2的红外光谱图;
图12为PORP-1、SPORP-1和SPORP-2的DPPH自由基清除能力图;
图13为PORP-1、SPORP-1和SPORP-2的ABTS自由基清除能力图;
图14为PORP-1、SPORP-1和SPORP-2的超氧自由基清除能力图;
图15为PORP-1、SPORP-1和SPORP-2的羟基自由基清除能力图。
具体实施方式
下面结合实施例对本发明作进一步详细的描述,但本发明的实施方式不限于此。下列实施例中涉及的物料若无特殊说明均可从商业渠道获得。
实施例1:水溶性玉竹多糖PORP-1的制备
(1)多糖提取:取1000g洗净烘干的玉竹,经粉碎机粉碎后过100目筛,添加无水乙醇在70℃水浴条件下回流两小时,过滤,滤渣在60℃的鼓风烘箱中烘干,加入10L蒸馏水,在95℃水浴条件下浸提2小时,离心,过滤,滤渣重复上述加水热提步骤3次,合并滤液,45℃减压浓缩至1L,得到玉竹粗多糖溶液。
(2)除蛋白除色素:配制Sevage试剂(氯仿:正丁醇=4:1),多糖水提液:Sevage试剂为4:1的体积比对步骤(1)得到的玉竹粗多糖溶液进行脱蛋白处理,振荡,静置,离心分离,取上清液,重复2-4次,于45℃下减压蒸发除去残留的Sevage试剂。加入AB-8大孔树脂,室温下摇床(150rpm) 脱色2h,抽滤,滤液减压浓缩,得到除蛋白除色素的多糖水提液。
(3)粗多糖的制备:多糖水提液:无水乙醇为1:4的体积比进行醇沉处理,于4℃冰箱静置12h,离心(8000r/min,10min)分离,收集沉淀。沉淀复溶于去离子水,使用3500Da透析袋去离子水透析48h,透析液在0.01MPa 和-80℃条件下冷冻干燥,得到玉竹粗多糖样品30.2g。
(4)多糖纯化:取2g步骤(3)获得的玉竹粗多糖样品,溶于100mL 蒸馏水,经纤维素DEAE-52阴离子交换树脂进行分离,依次以蒸馏水、0.1M、 0.2M、0.3M及0.4M的NaCl溶液洗脱,流速控制为1mL/min,每10分钟收集一管洗脱液。用苯酚-硫酸法测定糖含量,绘制洗脱曲线,如图1所示,根据洗脱曲线的吸收峰,收集蒸馏水洗脱组分,浓缩,冷冻干燥得到水溶性玉竹多糖PORP-1。经高效凝胶排阻色谱检测,PORP-1在凝胶色谱图上仅有一个均一对称的吸收峰,如图2所示,表明其是分子量分布均一的多糖。
实施例2:硫酸化玉竹多糖SPORP-1和SPORP-2的制备
采用浓硫酸法进行PORP-1的硫酸化修饰。取10mL正丁醇于烧杯中,将其置于冰水浴中,缓慢加入30mL的浓硫酸,加入0.5g的硫酸铵后搅拌均匀,缓慢加入PORP-1,磁力搅拌反应一定时间。反应后的溶液用2.5M的NaOH 溶液中和至pH=7,使用3000Da透析袋去离子水透析48h,透析液在0.01MPa 和-80℃条件下冷冻干燥。通过控制硫酸化反应时间分别为2h和1h并采用氯化钡—明胶法测定,得到两种取代度分别为0.54和0.41的硫酸化玉竹多糖SPORP-1和SPORP-2。
实施例3:PORP-1、SPORP-1和SPORP-2的结构鉴定
(1)分子量的测定:称取2-4mg的PORP-1溶解在2mL的磷酸二氢钾缓冲液(20mM)中,过0.22μm滤膜,4℃低温保存于色谱瓶中进行色谱分析。以普鲁兰系列葡聚糖为标品。采用配备GPC色谱柱(TSK G-5000PWXL 和TSK G-3000PWXL凝胶色谱柱串联)的Waters HPLC系统。流动相为磷酸二氢钾缓冲液(20mM),流速为0.6mL/min,柱温为35℃,进样量20uL,检测器为Waters 2414示差检测器。结果表明,PORP-1是分子量分布均一的纯化多糖,分子量集中在4025Da。
(2)单糖组成的测定:取5mg的PORP-1用三氟乙酸水解、经PMP衍生化、氯仿萃取后吸取上层水相过0.22μm滤膜后进行液相分析。色谱条件: Symmetry C18色谱柱(5μm,4.6×250mm);柱温:30℃;流动相:A液(乙腈)和C液(乙腈+蒸馏水+三乙胺+磷酸二氢钾);流速:1.0mL/min;检测器波长:245nm。同样地,单糖标品用相同方法衍生化后进行液相色谱分析。单糖组成结果如图3所示,结果表明,PORP-1为主要由甘露糖(Man)、葡萄糖醛酸(GlcA)、葡萄糖(Glc)、阿拉伯糖(Ara)、岩藻糖(Fru)组成的杂多糖,摩尔比含量分别为19.28%、0.41%、39.76%、1.54%、1.08%。
(3)红外光谱的测定:称取5mg的PORP-1,采用KBr压片法,进行傅里叶红外光谱测试,扫描波长为4000-400cm-1。红外光谱结果如图4所示,结果表明,PORP-1是一种含有羧基、β型甘露糖残基、可能含有α型糖苷键的吡喃环多糖。
(4)高碘酸氧化和史密斯降解:用容量瓶配制PORP-1溶液(5mg/mL),取5mL于25mL的棕色容量瓶中,加入12.5mL NaIO4(30mM)溶液,去离子水定容至25mL,制得样品反应溶液。将容量瓶放置于黑暗处进行反应,每隔12h避光取样0.1mL,用去离子水定容至25mL,在波长223nm处测定其吸光度值,至吸光度值稳定为止。最后向样品反应溶液中加入2mL的乙二醇终止氧化反应。将剩余终止反应后的样品反应溶液透析48h,透析液减压旋蒸至10mL左右,与70mg的NaBH4过夜反应后,用50%的醋酸溶液(w/w) 调节溶液pH至6.5到7,透析48h,减压蒸干透析液,得到氧化还原后的样品。取氧化还原后的样品用三氟乙酸完全水解、乙酰化衍生、二氯甲烷萃取、取有机相过0.22μm滤膜后用气相色谱进行分析。色谱条件为色谱柱:HP-5MS 色谱柱(30m×0.32mm×0.25μm);进样口温度:250℃;升温程序:140℃,保持1min;2℃/min,上升至220℃,保持1min,10℃/min,上升至250℃,保持2min。进样量:1μL。流动相:氦气。流速:1mL/min。结果如图5所示,结果表明PORP-1中的(1→6)或(1→)糖苷键摩尔百分比为17.50%; (1→2)、(1→2,6)、(1→4)或(1→4,6)糖苷键摩尔百分比为74.30%;(1 →3)、(1→3,6)、(1→2,4)、(1→3,4)或(1→2,3,4)糖苷键摩尔百分比为8.2%。
(5)核磁共振波谱的测定:称取20mg的PORP-1溶于1.1mL的D2O中,于核磁共振波谱仪中进行分析。结果如图6和图7所示,结果表明PORP-1是由→6)-α-D-Glcp-(1→,α-Glcp-(1→,→4,6)-α-D-Glcp(1→,→4)-β-D-Man(1→,→3)α-D-Manp-(1→类型的糖苷键所组成。表1为PORP-1的1H和13C化学位移分类表。
表1
(6)采用刚果红试剂法测定POPR-1是否具有三股螺旋结构。配制1 mg/mL的PORP-1样品溶液,以昆布多糖为阳性对照,与等体积的与刚果红试剂(100μmol/L)进行反应,滴加NaOH水溶液(1mol/L)调节混合液中NaOH 的浓度为0-0.5mol/L,于200-600nm波长数下进行光谱扫描,确定不同浓度 NaOH样品溶液的最大吸收波长。结果如图8所示,结果表明PORP-1是一种不具有三股螺旋结构的多糖。
(7)原子力显微镜的测定:配制1mg/mL的PORP-1样品溶液,过0.22μm 的滤膜,取10μL滤液滴加在剥片后的云母片上,室温风干,制得测试样品。测试样品黏附在贴有双面胶的样品台上,放入原子力显微镜中,调节探针高度和观察区域,运行扫描软件,改变扫描速度和观察区域进行纳米结构形态的观察。结果如图9所示,结果表明PORP-1的微米级结构为直径范围在123-151nm 的圆顶形颗粒状聚集体。
(8)扫描电镜的测定:取少量干燥的PORP-1样品均匀撒在贴有双面胶的样品台表面,洗耳球吹去未粘牢粉末,于真空镀膜仪中喷镀一层导电金,制得测试样品。测试样品放入扫描电镜中,抽真空,电子枪加压至10kV,改变放大倍数后聚焦进行表面形貌的观察。结果如图10所示,结果表明PORP-1 的纳米级表面形貌为致密、较光滑、带褶皱的网络状结构。
(9)红外光谱的测定:称取5mg的SPORP-1和SPORP-2,采用KBr压片法,进行傅里叶红外光谱测试,扫描波长为4000-400cm-1。红外光谱结果如图11所示,结果表明硫酸根基团被成功地结合在PORP-1上。
实施例4:PORP-1、SPORP-1和SPORP-2的抗氧化活性研究
分别利用PORP-1、SPORP-1和SPORP-2配制不同浓度的多糖水溶液 (0.2mg/mL、0.4mg/mL、0.6mg/mL、0.8mg/mL、1.6mg/mL、2mg/mL、4mg/mL 和8mg/mL)。
(1)用无水乙醇配制DPPH工作液(0.1mmol/L)。用150μL不同浓度的多糖水溶液与等体积的DPPH工作液在30℃下避光反应30min,于517nm 处测定吸光度。以去离子水为样品空白对照,VC为样品阳性对照,以无水乙醇为DPPH溶液背景对照。结果如图12所示。由图12可见,本发明水溶性玉竹多糖具有一定的清除DPPH活性,对其进行硫酸化改性后,活性得到显著的增强;当SPORP-1的浓度为8mg/mL时,其清除率可高达近80%,与 VC相接近。
(2)配制7mmol/L的ABTS溶液和2.45mmol/L的过硫酸钾溶液,等体积混合上述两种溶液,25℃避光保存12h后,用无水乙醇稀释至在波长732nm 处吸光度值为0.70±0.02,制得ABTS+·工作液。用20μL不同浓度的多糖溶液与150μL的ABTS+·工作液试剂在25℃下避光静置反应30min,于732nm 处测定吸光度,以去离子水为样品空白对照,VC为样品阳性对照,以无水乙醇为ABTS+·溶液背景对照。结果如图13所示。由图13可见,本发明水溶性玉竹多糖具有一定的清除ABTS活性,对其进行硫酸化改性后,活性得到显著的增强,SPORP-1和SPORP-1均可达到50%以上的清除率,且当SPORP-1 的浓度为8mg/mL时,其清除率可高达70%以上。
(3)配制pH=8.2的Tris-HCl缓冲液(0.1mol/L)和1.6mol/L的HCl溶液,配制24mmol/L的邻苯三酚溶液。在20μL多糖样品中添加100μL的 Tris-HCl缓冲液,25℃下静置反应10min后添加20μL邻苯三酚溶液,在25℃下反应5min,最后加入10μL的HCl终止反应,于320nm处测定吸光度,以去离子水为样品空白对照,VC为样品阳性对照,以去离子水为邻苯三酚溶液背景对照。结果如图14所示。由图14可见,本发明水溶性玉竹多糖具有一定的清除超氧自由基活性,对其进行硫酸化改性后,活性得到显著的增强, SPORP-1和SPORP-1均可达到60%以上的清除率。
(4)配制1.5mmol/L的硫酸亚铁溶液和6mmol/L的双氧水溶液,配制 20mmol/L的水杨酸溶液。1mL的多糖样品溶液中分别加入1mL硫酸亚铁溶液试剂、0.7mL的双氧水和0.3mL的水杨酸,37℃孵育1h,于562nm处测定吸光度,以去离子水为样品空白对照,VC为样品阳性对照,以去离子水为水杨酸溶液背景对照。结果如图15所示。由图15可见,本发明水溶性玉竹多糖具有一定的清除羟基自由基活性,对其进行硫酸化改性后,活性得到显著的增强,当SPORP-1的浓度为8mg/mL时,其清除率可高达近70%。
结果表明,本发明的水溶性玉竹多糖具有一定的抗氧化活性,对其进行硫酸化改性后,获得的硫酸化玉竹多糖具有显著增强的抗氧化活性,可作为天然抗氧化剂应用于食品及化妆品等领域中。
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精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (6)
1.一种硫酸化玉竹多糖,其特征在于,具体可通过包括以下步骤获得:向置于冰水浴中正丁醇溶液中加入浓硫酸,并加入硫酸铵后搅拌均匀,加入水溶性玉竹多糖,搅拌反应1-2h;反应后的溶液用NaOH溶液中和,透析,离心,冷冻干燥后得到硫酸化修饰的水溶性玉竹多糖;水溶性玉竹多糖的用量为1g,正丁醇的用量为10mL,浓硫酸的用量为30mL,硫酸铵的用量为0.5g;
所述水溶性玉竹多糖通过以下步骤制得:将玉竹醇提后,固形物加水热提,过滤,浓缩所得的水溶液得到玉竹粗多糖溶液;加入Sevag试剂除蛋白和AB-8大孔树脂摇床脱色素后,配成多糖水溶液,醇沉,沉淀物复溶于水中,透析,冻干,得到粗多糖;采用DEAE-Sepharose离子交换柱层析法进行分离纯化,将玉竹粗多糖复溶于水中制成水溶液上样后,采用梯度洗脱法,依次用去离子水和0.1-0.4mol/L的NaCl盐溶液进行洗脱,收集去离子水组分,冷冻干燥后得到分离纯化的水溶性玉竹多糖;所述水溶性玉竹多糖是由甘露糖、葡萄糖醛酸、葡萄糖、阿拉伯糖、岩藻糖组成的杂多糖,摩尔比为19.28:0.41:39.76:1.54:1.08;水溶性玉竹多糖糖苷键组成如下所示:→6)-α-D-Glcp-(1→,α-Glcp-(1→,→4,6)-α-D-Glcp-(1→,→4)-β-D-Man-(1→,→3)-α-D-Manp-(1→,其分子量分布范围为2000-5000Da,分子量集中在4025Da。
2.根据权利要求1所述的一种硫酸化玉竹多糖,其特征在于,反应后的溶液用NaOH溶液中和后,pH值的范围为6.5-7.5;
透析袋的截留分子量为3500Da,透析时间为48h,期间换水4-8次;离心条件为8000r/min。
3.根据权利要求1所述的一种硫酸化玉竹多糖,其特征在于,通过控制硫酸化反应时间分别为2h和1h得到两种取代度分别为0.54和0.41的硫酸化玉竹多糖SPORP-1和SPORP-2。
4.根据权利要求1所述的一种硫酸化玉竹多糖,其特征在于,冷冻干燥的条件为压强为0.01MPa,温度为-80℃。
5.根据权利要求1所述的一种硫酸化玉竹多糖,其特征在于:
所述玉竹使用前先清洗干净、烘干并进行粉碎,粉碎后过100目筛;
所述醇提条件为在70-80℃醇提2-4h;所述醇提后,过滤去掉滤液,固形物烘干备用;
所述加水热提的时间为0.5-3h;所述加水热提重复多次,并将多次提取的水溶液合并再进行浓缩;
所述加水热提的液固比为每1质量份固形物添加10-50体积份水;
所述加水热提的温度为50-99℃;
所述加入Sevag试剂除蛋白和AB-8大孔树脂摇床脱色素,具体可为将粗多糖溶于水中,采用Sevage法除蛋白,配制Sevage试剂,多糖水提液:Sevage试剂为4:1的体积比进行脱蛋白处理;采用AB-8大孔树脂脱色为,向脱蛋白多糖水提液中加入AB-8大孔树脂,150rpm摇床室温脱色2h,得到除蛋白除色素的多糖水提液;
所述醇沉在4℃静置过夜;所述醇沉的乙醇终浓度为80%(V/V);
所述透析的透析袋截留分子量为3500-8000Da,透析时间为48h,期间换水4-8次;
冻干的条件为压强为0.01MPa,温度为-80℃。
6.如权利要求1~5任一项所述硫酸化玉竹多糖在食品和化妆品领域中的应用。
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