CN113698502A - 白及须根低聚糖及其制备方法和应用 - Google Patents
白及须根低聚糖及其制备方法和应用 Download PDFInfo
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- CN113698502A CN113698502A CN202110835038.0A CN202110835038A CN113698502A CN 113698502 A CN113698502 A CN 113698502A CN 202110835038 A CN202110835038 A CN 202110835038A CN 113698502 A CN113698502 A CN 113698502A
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- bletilla striata
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Abstract
本发明提供了一种白及须根低聚糖及其制备方法和应用,制备方法包括:将白及须根烘干后,粉碎,并溶解在热水中得到白及须根溶液;将白及须根溶液通过提取,脱色,浓缩,醇沉,得到白及须根粗提物;将白及须根粗粗提物离心,洗涤,除蛋白,冻干即可,白及须根低聚糖的分子量为1000‑1500Da,其主要由鼠李糖、阿拉伯糖、岩藻糖、木糖、甘露糖、葡萄糖和半乳糖组成;本发明的白及须根多糖具有分子量较小、无毒、体系稳定、良好的抗氧化活性及酪氨酸酶抑制活性等特点,原料廉价易得,实现了白及属植物资源的充分利用,安全无毒,具有开发美白和化妆品的潜力;本发明的制备工艺简单,耗能少,经济无毒,方便易行,适用于工业化生产。
Description
技术领域
本发明属于药食两用多糖领域,具体涉及一种白及须根低聚糖及其制备方法和应用。
背景技术
白及是一种广泛分布在东亚国家的传统药食两用的中药,其干燥的块茎被广泛用作一种止血药,据报道,从白及块茎中能够分离得到多种多糖,这些多糖是文状芽孢杆菌最重要的活性成分,表现出各种生物活性。在临床上主要被用来治疗咯血、吐血、创伤出血、消化道黏膜损伤、溃疡、瘀伤和烧伤、肛肠疾病、妇科纤维瘤、肿瘤栓塞和前列腺手术等等。
已有文献研究表明,白及须根也含有相似功能的多糖。已报道的白及须根多糖由阿拉伯糖、半乳糖、葡萄糖和甘露糖四种单糖组成,分子量约为9.1x104Da,且有多种生物活性,如抗菌、抑菌、抗肿瘤等。
目前对白及块茎中的活性成分研究较多,相较而言对白及须根里面的活性成分研究较少。现有研究发现白及块茎和白及须根中的糖主要为高分子量多糖,而对于低分子量的糖研究较少。
专利CN111004335A公开了一种从白及块茎中分离得到低聚糖的提取方法,但是尚未对白及须根中组分及提取方法进行研究,对所得的低聚糖也未进行相关生物药物活性的研究。
发明内容
针对现有技术中的不足,本发明的目的之一是提供一种白及须根低聚糖的制备方法。本发明以药食两用的中药材白及为原料,对白及须根进行提取得到一种低分子量的新型多糖,该多糖表现出良好的抗氧化活性及酪氨酸酶抑制活性。
本发明的目的之二是提供上述白及须根低聚糖。
本发明的目的之三是提供上述白及须根低聚糖的用途。
为达到上述目的之一,本发明的解决方案是:
一种白及须根低聚糖的制备方法,其包括如下步骤:
(1)、将白及须根烘干后,粉碎,并溶解在热水中得到白及须根溶液;
(2)、将白及须根溶液通过提取,脱色,浓缩,醇沉,得到白及须根粗提物;
(3)、将白及须根粗提物离心,洗涤,得到白及须根多糖;
(4)、将白及须根多糖除蛋白,离心,冻干,得到白及须根低聚糖。
其中,白及须根低聚糖的分子量为1000-1500Da;
白及须根低聚糖主要由鼠李糖、阿拉伯糖、岩藻糖、木糖、甘露糖、葡萄糖和半乳糖组成。
作为本发明的一种优选实施例,步骤(1)中,烘干的温度为40-70℃。
作为本发明的一种优选实施例,步骤(1)中,白及须根和热水的质量体积比为1g:10mL。
作为本发明的一种优选实施例,步骤(2)中,提取的温度为80-90℃,时间为3±0.1h。
作为本发明的一种优选实施例,步骤(3)中,离心的转速为3000-5000r/min,时间为5±0.1min。
作为本发明的一种优选实施例,步骤(3)中,洗涤的过程为:先将得到的固体用乙醇洗涤,然后用丙酮溶液洗涤,最后用等体积的乙醇溶液漂洗。
作为本发明的一种优选实施例,步骤(4)中,除蛋白的过程为:将白及须根多糖加少量蒸馏水溶解,然后加入三氯乙酸调pH至3.0。
为达到上述目的之二,本发明的解决方案是:
由上述的制备方法得到一种白及须根低聚糖。
为达到上述目的之三,本发明的解决方案是:
一种上述的白及须根低聚糖在化妆品中的应用。
一种上述的白及须根低聚糖作为抗氧化剂在医药和食品等领域中的应用。
由于采用上述方案,本发明的有益效果是:
第一、本发明的低分子量的白及须根多糖可显著抑制酪氨酸酶活性,是一种天然的美白物质;同时具有很好的抗氧化活性,是一种天然的抗氧化剂;另外,本发明的白及须根多糖分子量较小、无毒、体系稳定;故具有活性高、无污染的优点。
第二、本发明的传统药食两用白及须根原料廉价易得,验证了白及须根的经济价值,实现了白及属植物资源的充分利用,为其进一步的科学研究提供了参考价值。另外,本发明的多糖来源于白及,安全无毒,具有开发美白和化妆品的潜力。
第三、本发明的制备工艺简单,耗能少,经济无毒,方便易行,适用于工业化生产。
附图说明
图1为本发明的实施例中白及须根低聚糖的高效凝胶渗透色谱图(横坐标Retention time为保留时间,纵坐标Electrical signal response value为电信号响应值)。
图2a为本发明的实施例中白及须根低聚糖为放大倍数为x70k的扫描电镜示意图。
图2b为本发明的实施例中白及须根低聚糖为放大倍数为x400k的扫描电镜示意图。
图2c为本发明的实施例中白及须根低聚糖为放大倍数为x2.00k的扫描电镜示意图。
图3为本发明的实施例中白及须根低聚糖对羟基自由基的清除作用示意图(横坐标Concentration为浓度)。
图4为本发明的实施例中白及须根低聚糖对酪氨酸酶的抑制作用示意图(横坐标Concentration为浓度,纵坐标Inhibition ratio为抑制率)。
具体实施方式
本发明提供了一种白及须根低聚糖及其制备方法和应用。首先以新鲜的白及须根为原料,通过水浴,醇沉,脱色,除蛋白,冻干等工艺得到白及须根低聚糖,其次通过柱分离的方法将白及须根粗提物进行纯化。
<白及须根低聚糖的制备方法>
本发明的白及须根低聚糖的制备方法包括如下步骤:
(1)、将白及须根在烘箱中烘干后,粉碎称重,并溶解在热水中配制得到白及须根溶液;
(2)、将白及须根溶液通过提取,抽滤,剩余残渣进行复提,合并两次滤液,然后用1.5wt%的活性炭脱色50min,将脱色后的滤液减压浓缩至大约0.3倍体积,取浓缩液3倍体积的无水乙醇溶液进行醇沉得到明显分层的固体和液态物质,即白及须根粗提物;
(3)、将白及须根粗提物离心,弃上清液,先将得到的固体用酒精洗涤,然后用丙酮溶液洗涤,最后用等体积的乙醇溶液漂洗,挥干酒精,得到白及须根多糖;
(4)、将白及须根多糖加少量蒸馏水溶解,然后加入三氯乙酸调pH至3.0,搅拌均匀后放置过夜,次日离心,取上清液冻干,得到白及须根低聚糖。
其中,白及须根低聚糖的分子量为1000-1500Da;
白及须根低聚糖主要由鼠李糖、阿拉伯糖、岩藻糖、木糖、甘露糖、葡萄糖和半乳糖组成。
具体地,在步骤(1)中,烘干的温度可以为40-70℃,优选为40℃。
在步骤(1)中,白及须根和热水的质量体积比为1g:10mL。
在步骤(2)中,提取的温度可以为80-90℃,优选为80℃;时间为3±0.1h。
在步骤(3)中,离心的转速可以为3000-5000r/min,优选为3000r/min;时间为5±0.1min。
本发明除了热水浸提的方法提取白及须根低聚糖外,还可以通过超临界CO2萃取法、超声提取法、回流提取法提取多糖。
<白及须根低聚糖>
本发明的白及须根低聚糖由上述的制备方法得到。
<白及须根低聚糖的应用>
本发明的白及须根低聚糖可以在化妆品中得以应用。
另外,本发明的白及须根低聚糖还可以作为抗氧化剂在医药和食品等领域中得以应用。
以下结合实施例对本发明作进一步的说明。
实施例:
本实施例的白及须根低聚糖的制备方法包括如下步骤:
(1)、以新鲜的白及须根为原料,在40℃的烘箱中烘干后,粉碎称重,并溶解在热水中配制得到白及须根溶液;其中,白及须根和热水的质量体积比为1g:10mL。
(2)、将白及须根溶液在80℃下水提取3h,抽滤,剩余残渣进行复提,合并两次滤液,然后用1.5wt%的活性炭脱色50min,将脱色后的滤液减压浓缩至大约0.3倍体积,取浓缩液3倍体积的无水乙醇溶液进行醇沉得到明显分层的固体和液态物质,即白及须根粗提物。
(3)、将白及须根粗提物在3000r/min下离心5min,弃上清液,先将得到的固体用酒精洗涤,然后用丙酮溶液洗涤,最后用等体积的乙醇溶液漂洗,挥干酒精,得到白及须根多糖。
(4)、将白及须根多糖加少量蒸馏水溶解,然后加入三氯乙酸调pH至3.0,搅拌均匀后放置过夜,次日离心,取上清液冻干,得到白及须根低聚糖。
<实验>
将上述实施例得到的白及须根低聚糖进行如下实验。通过HPGPC法对制备的白及须根低聚糖进行分子量测定;通过GC-MS法对该产物的单糖组成进行分析;以白及须根低聚糖为原料,探讨采用酶标法测定不同浓度的多糖对羟基自由基清除率的影响,来评价其体外抗氧化活性;以白及须根低聚糖为原料探讨不同浓度的多糖对酪氨酸酶的抑制作用。
<实验1>
分子量测定:
采用高效凝胶渗透色谱(HPGPC)对纯化出来的白及须根低聚糖进行分子量测定。将白及须根低聚糖溶解在0.02mol/L磷酸盐缓冲液(pH=6.8)中,制备浓度为2mg/mL的样品溶液。将制备好的样品在10000r/min离心10min,吸取20μL样品溶液通过0.22μm过滤器注入HPGPC系统。其中柱(Shodex SB-804,300×8mm)用磷酸盐缓冲液以0.3mL/min的流速洗脱,柱温保持在25℃。使用不同分子量(T3、T6、T10、T40、T100、T500和T1000)的葡聚糖标准品建立校准标准曲线。所得结果如图1所示。
从图1中可以看出白及须根低聚糖(pFSP)主峰的保留时间为36.117min,带入线性回归方程估算得到pFSP的分子量在1000-1500Da范围内。
<实验2>
扫描电镜(SEM)观察:
取约1mg干燥的白及须根低聚糖(pFSP)置于离子喷溅仪中镀一层导电金后,扫描电镜观察其在放大倍数为x70k(图2a)、x400k(图2b)、x2.00k(图2c)下的多糖形貌。
通过电镜扫描对所纯化的白及须根低聚糖进行了微观结构的观察。图2显示,白及须根低聚糖(pFSP)为不规则的片状结构。这表明官能团之间的强烈吸引作用产生了多糖链的聚集。另外,白及须根低聚糖(pFSP)表面较粗糙,多糖光滑的表面可能对富水性能产生负面影响,从而降低多糖本身的溶解度,这是pFSP溶解度较高的原因。纯化后的多糖分子内氢键发生变化导致白及须根低聚糖的形状和形态发生变化。可以预测,pFSP中多糖链的相互作用减弱,多糖片段更容易被撕碎。
<实验3>
单糖组成测定:
精确称取10mg的白及须根低聚糖在圆底烧瓶中,加2mL、2mol/L的三氟乙酸溶液,封口,100℃水浴下水解6h,40℃减压旋转蒸发除尽水解液。在水解产物中加入10mg盐酸羟胺和0.5mL吡啶,封口,在90℃恒温下水浴30min,并时常振荡,取出冷却至室温;加入0.6mL乙酸酐,封口,在90℃恒温下水浴30min,并时常振荡,冷却至室温;50℃减压旋转蒸干;加入4mL三氯甲烷溶解,4000r/min离心5min,取上清液,用GC-MS分析产物,GC-MS测单糖组成的柱长为150mm×3mm,柱温为30℃。以岩藻糖、鼠李糖、阿拉伯糖、氨基葡萄糖、半乳糖、葡萄糖、木糖、甘露糖、果糖、GalA、GlcA11种单糖标准品为参照物,测量结果如表1所示。
表1 pFSP各单糖摩尔比结果
表1测量结果即白及须根低聚糖(pFSP)各单糖摩尔比结果,从表1中可以看到pFSP主要由以下几种单糖组成,分别为:鼠李糖、阿拉伯糖、岩藻糖、木糖、甘露糖、葡萄糖、半乳糖。它们的摩尔比为1∶7∶7∶4∶20∶23∶59。
<实验4>
羟基自由基清除率测定:
分别配制浓度为1.0mg/mL、2.0mg/mL、3.0mg/mL、4.0mg/mL、5.0mg/mL的多糖溶液2mL在试管中,分别等体积加入FeSO4溶液和H2O2溶液,摇匀并静置5-10min后加入等体积的水杨酸溶液,混合均匀后静置半个小时。同样方法分别用蒸馏水代替水杨酸和多糖溶液配制对照溶液和空白对照溶液。以蒸馏水为空白在510nm波长处测定样品溶液、对照溶液和空白对照溶液吸光度,并根据式(1)计算多糖羟基清除率,测量结果如图3所示。
羟基清除率=[1-(A样品-A对照)/A空白对照]x100% (1)
式中:A样品:样品溶液吸光度;A对照:对照溶液吸光度;A空白对照:空白对照溶液吸光度。
由图3可知,在浓度为1-5mg/mL的浓度范围内,白及须根低聚糖(pFSP)对羟基自由基具有较好的清除作用,且随着浓度的增大而增大。当白及须根低聚糖的浓度为5mg/mL时,清除率达到81.06%,对实验结果进行线性拟合得到的方程为y=15.999x+10.851,R2=0.8582,根据方程计算出白及须根低聚糖对羟基自由基的半清除率IC50为2.45mg/mL。
<实验5>
酪氨酸酶抑制率测定:
对提取出来的白及须根低聚糖通过测定酪氨酸酶活性抑制率,来评价白及须根低聚糖的抑酶作用,采用酶标法测定所得多糖的酪氨酸酶抑制率。
(1)酪氨酸酶溶液的配制:称取一定质量的去皮切碎马铃薯,按1∶1(m/v)比例加入磷酸盐缓冲液(pH=6.8),然后离心。用移液管移取上清液并按一定比例加入PBS(pH=6.8)溶液定容在25mL棕色瓶中,冰水浴备用,1-2h内用完。
(2)磷酸缓冲液(PBS,pH=6.8)的配制:各取0.0717g/mL的Na2HPO4.12H2O溶液和0.0179g/mL的NaH2PO4·2H2O溶液100mL在500mL容量瓶中,用去离子水定容。
(3)0.05wt%的L-酪氨酸溶液的配制:称取0.05wt%的L-酪氨酸溶于一定量的盐酸,再加入PBS缓冲液。
(4)样品溶液的配置:分别配制浓度为1.0mg/mL、2.0mg/mL、3.0mg/mL、4.0mg/mL、5.0mg/mL的多糖溶液于试管中。
(5)实验用水为去离子水。
表2 pFSP溶液配置方案
(1)空白对照吸光度测定:先按表1要求,准确称取各溶液摇匀,配置C0溶液;然后将C0溶液在37℃条件下水浴10min,最后在475nm处测定吸光度。再按表2要求,准确量取各溶液,均匀配置C1溶液;然后37℃条件下水浴5-10min,然后加入酪氨酸酶溶液,仍在37℃加热5-10min;最后在475nm光谱条件下测定C1吸光度。
(2)、样品吸光度测定:同样,按表2具体要求,准确量取各溶液,均匀配制T1溶液、T0溶液;然后依据上述测定C1的步骤,常温475nm光谱条件下测定T1吸光度,最后须根多糖对酪氨酸酶的抑制结果如图4所示。
(3)、根据式2计算抑制率
抑制率T=(C1-T1)/C1 (2)
由图4可知,在浓度为1-5mg/mL的浓度范围内,白及须根低聚糖(pFSP)对酪氨酸酶具有较好的抑制作用,且随着浓度的增大而增大。当白及须根低聚糖的浓度为5mg/mL时,清除率达到79.33%,对实验结果进行线性拟合得到的方程为y=9.089x+36.881,R2=0.9724,根据方程计算出白及须根低聚糖对酪氨酸酶的半抑制率IC50为1.44mg/mL。
上述对实施例的描述是为了便于该技术领域的普通技术人员能理解和使用本发明。熟悉本领域技术人员显然可以容易的对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中,而不必经过创造性的劳动。因此,本发明不限于上述实施例。本领域技术人员根据本发明的原理,不脱离本发明的范畴所做出的改进和修改都应该在本发明的保护范围之内。
Claims (10)
1.一种白及须根低聚糖的制备方法,其特征在于:其包括如下步骤:
(1)、将白及须根烘干后,粉碎,并溶解在热水中得到白及须根溶液;
(2)、将所述白及须根溶液通过提取,脱色,浓缩,醇沉,得到白及须根粗提物;
(3)、将所述白及须根粗提物离心,洗涤,得到白及须根多糖;
(4)、将所述白及须根多糖除蛋白,离心,冻干,得到白及须根低聚糖;
所述白及须根低聚糖的分子量为1000-1500Da;
所述白及须根低聚糖主要由鼠李糖、阿拉伯糖、岩藻糖、木糖、甘露糖、葡萄糖和半乳糖组成。
2.根据权利要求1所述的白及须根低聚糖的制备方法,其特征在于:步骤(1)中,所述烘干的温度为40-70℃。
3.根据权利要求1所述的白及须根低聚糖的制备方法,其特征在于:步骤(1)中,所述白及须根和热水的质量体积比为1g:10mL。
4.根据权利要求1所述的白及须根低聚糖的制备方法,其特征在于:步骤(2)中,所述提取的温度为80-90℃,时间为3±0.1h。
5.根据权利要求1所述的白及须根低聚糖的制备方法,其特征在于:步骤(3)中,所述离心的转速为3000-5000r/min,时间为5±0.1min。
6.根据权利要求1所述的白及须根低聚糖的制备方法,其特征在于:步骤(3)中,所述洗涤的过程为:先将得到的固体用乙醇洗涤,然后用丙酮溶液洗涤,最后用等体积的乙醇溶液漂洗。
7.根据权利要求1所述的白及须根低聚糖的制备方法,其特征在于:步骤(4)中,所述除蛋白的过程为:将所述白及须根多糖加蒸馏水溶解,然后加入三氯乙酸调pH至3.0。
8.一种白及须根低聚糖,其特征在于:其由权利要求1-7任一项所述的制备方法得到。
9.一种如权利要求8所述的白及须根低聚糖在化妆品中的应用。
10.一种如权利要求8所述的白及须根低聚糖作为抗氧化剂在医药和食品领域中的应用。
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