CN109010456A - 一种从覆盆子中提取α-葡萄糖苷酶抑制剂的方法 - Google Patents
一种从覆盆子中提取α-葡萄糖苷酶抑制剂的方法 Download PDFInfo
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Abstract
本发明涉及一种从中药覆盆子中提取α‑葡萄糖苷酶抑制剂的方法,属于中药、天然药物化学技术领域。其提取方法包括如下步骤:(1)将覆盆子粉碎,得到覆盆子颗粒;(2)以亲水性有机溶剂为提取剂,采用溶剂提取法处理覆盆子颗粒得到提取液;再将提取液浓缩干燥,得到覆盆子粗提取物;(3)用水溶解覆盆子粗提取物配制为覆盆子粗提取物水溶液,采用柱层析法进一步纯化,得到α‑葡萄糖苷酶抑制剂。本发明工艺简单,操作方便,产物不含有害物质,对环境无污染,安全性高,对从覆盆子中开发中药降血糖新药具有重要意义。
Description
技术领域
本发明属于中药、天然药物化学技术领域,涉及一种从中药覆盆子中提取α-葡萄糖苷酶抑制剂的方法。
背景技术
糖尿病(Diabetes Mellitus,DM)是由于人体中胰岛素含量绝对或相对不足而引发的内分泌代谢性紊乱的疾病。目前,糖尿病已成为全球性的重大公共健康问题,2015年,全球大约有4.15亿人患有糖尿病,因糖尿病导致的死亡人数约占全球总死亡人数的9%。
治疗糖尿病的有效方法之一是通过α-葡萄糖苷酶抑制剂(AGIs)降低机体餐后和空腹血糖水平。α-葡萄糖苷酶位于小肠刷状缘膜上皮细胞,它能将双糖,如蔗糖、麦芽糖等水解成可被小肠吸收的单糖,是碳水化合物水解的关键酶。当α-葡萄糖苷酶的活性被抑制时,碳水化合物的总体消化时间被延长,从而降低餐后血糖水平,达到治疗糖尿病并减少糖尿病并发症的效果。但合成的AGIs,如阿卡波糖和米格列醇可能有肠胃气胀、腹泻和急性肝炎等副作用。另外,此类药物多为国外进口,对于多数长期服用的糖尿病患者来说,经济压力较大。
覆盆子为蔷薇科悬钩子属植物华东覆盆子Rubus chingii Hu的干燥未成熟果实,夏初由绿变为黄绿时采收因叶裂如掌也称掌叶覆盆子,为中国药典收载的常用中药,具有益肾、固精、缩尿和养肝明目之功效,用于肾虚尿频、阳痿早泄、遗精滑精等。主要产于江苏、安徽、浙江、江西、福建、广西等地区。目前国内外学者对覆盆子的化学成分及其应用研究取得了一定进展,在抗肿瘤、抗氧自由基、抗衰老等方面有明确活性。
现有技术中还没有文献报道从覆盆子中制备α-葡萄糖苷酶抑制剂的相关提取富集工艺。
发明内容
针对上述现有技术不足,本发明的目的在于提供一种从覆盆子中提取α-葡萄糖苷酶抑制剂的方法,和由上述方法得到的α-葡萄糖苷酶抑制剂,以及α-葡萄糖苷酶抑制剂的应用。
本发明的目的由以下技术方案实现:
一种从覆盆子中提取α-葡萄糖苷酶抑制剂的方法,其包括以下步骤:
(1)将覆盆子粉碎,得到覆盆子颗粒;
(2)以亲水性有机溶剂为提取剂,采用溶剂提取法处理覆盆子颗粒得到提取液;再将提取液浓缩干燥,得到覆盆子粗提取物;
(3)用水溶解覆盆子粗提取物配制为覆盆子粗提取物水溶液,采用柱层析法进一步纯化,得到α-葡萄糖苷酶抑制剂。
步骤(1)中的覆盆子为干燥的覆盆子果实,粉碎至粒度为180~4000μm。
步骤(2)中的亲水性有机溶剂可以是浓度为100%及以下的甲醇或乙醇的水溶液,优选浓度为40%的乙醇的水溶液。亲水性有机溶剂的加入量为覆盆子颗粒5~20倍,即覆盆子颗粒与亲水性有机溶剂的比例为1g:5mL~1g:20mL。
步骤(2)中的溶剂提取法可以为震荡法浸提,浸提时间为12~24小时;也可以为超声波提取,超声提取时间为30min~60min,超声功率为250~500W。
步骤(3)中柱层析法的条件可以为:覆盆子粗提取物水溶液的浓度为20~100mg/mL,以经过预处理的大孔吸附树脂为固定相,上样量为1BV,上样流速为1~5BV/h,吸附方式为静态吸附,吸附时间为2~8小时;用0~95%乙醇或甲醇为洗脱剂进行梯度洗脱,洗脱体积为3BV,洗脱流速为1~5BV/h。
其中,大孔吸附树脂为XAD-16、AB-8或D101等大孔吸附树脂。大孔吸附树脂进行预处理的方法可以为:将大孔吸附树脂用乙醇或甲醇浸泡,再用乙醇或甲醇冲洗,洗至流出的醇无浑浊,然后用水洗至无醇味,后用2%~4%HCl溶液浸泡,用水洗至中性,再用2~4%NaOH溶液浸泡,用去水洗至中性,得到处理后的大孔吸附树脂。大孔吸附树脂进行预处理所用乙醇或甲醇、HCl溶液、NaOH溶液浸泡时间皆为2~8小时,所用水皆为蒸馏水或去离子水。
所述从覆盆子中提取的α-葡萄糖苷酶抑制剂可以用于制备治疗餐后高血糖的药物或保健食品。后续可利用所述从覆盆子中提取的α-葡萄糖苷酶抑制剂配合药物载体,更好地将覆盆子应用于临床。
与现有技术相比,本发明具有如下优点:本发明的覆盆子提取物制备方法简单、得率高、成本低;此方法操作简单,容易实现。制得的α-葡萄糖苷酶抑制剂组分含量较大,且抑制效率高,抑制效果良好,具有很高的实用价值;其原料为天然产物,毒副作用小。
附图说明
图1为对覆盆子果实使用不同方法提取粗提取物得率的示意图。
具体实施方式
称取10g覆盆子粉末(颗粒),分别以20%、40%、60%、80%和100%的乙醇和甲醇按1:10(g/mL)的比例超声或浸提。超声条件为400W、室温超声提取1h,浸提时间为24h,然后抽滤,分别收集上清液,残渣再按1:10(g/mL)的比例分别添加20%、40%、60%、80%和100%的乙醇和甲醇,分别在相同条件下重复提取、离心2次,合并上清液,浓缩,用各自的提取溶剂定容到100mL,最终得到20个样品,4度冰箱保存。每个样品分别取2个2mL用20mL的样品瓶浓缩、冻干,称量样品的质量m,计算每个样品提取物的得率,计算公式为:
样品提取物得率见图1。
下面结合几个实施例对本发明做进一步详细的描述,但本发明的实施方式不限于此。
实施例1
一种从覆盆子中提取α-葡萄糖苷酶抑制剂的方法,包括以下步骤:将新鲜采摘的覆盆子果实洗净后晒干,用粉碎机粉碎后过20目筛,取1000g覆盆子粉末加入料液比(质量比)1:10的40%乙醇-水混合溶剂,于30℃下超声提取30min(超声功率为300w),冷却过滤后,滤渣按照上述提取方式重复提取2次,合并3次分别所得的滤液,于-0.1MPa真空浓缩后得到粗提物浸膏,该粗提物浸膏为原覆盆子原料的22%。采用湿法上样,按料液比为500mg/mL将粗提物浸膏分散于蒸馏水中,离心后取上清液得上样溶液。取内径为5.5cm的大孔吸附树脂层析柱并向其内部加入XAD-16填料至填料高度为25cm。将上样溶液加入大孔吸附树脂层析柱中吸附8小时后,用乙醇-水溶液作为洗脱液按照乙醇在洗脱液中的体积含量依次为0%、10%、40%、60%、80%、95%对上样溶液进行梯度洗脱。其中,梯度洗脱的洗脱体积分别为2000mL、1500mL、1500mL、1500mL、1500mL、1500mL,洗脱流速为30mL/min。收集洗脱组分,得到α-葡萄糖苷酶抑制剂。
由上述实施例中所得的α-葡萄糖苷酶抑制剂(样品)在体外对α-葡萄糖苷酶的抑制实验叙述如下:
以α-葡萄糖苷酶(西格玛公司生产)和对硝基苯基-α-D-葡萄糖苷(西格玛公司生产)为原料。具体实验方法如下:50μL适宜浓度的样品溶液与50μL0.1U/mL的α-葡萄糖苷酶混匀,室温反应6min后加入50μL5.0mM的对硝基苯基-D-吡喃葡萄糖苷(0.1M PBS,pH 6.8),再于37℃反应10min后,向反应体系中加100μL,0.2M Na2CO3终止反应。以不加酶和样品的反应体系为空白,采用酶标仪于405nm测吸光值(Ai),实验重复测三次。酶抑制作用计算公式为:
AC为样品溶液被50μL 90%甲醇替代的反应体系的吸光值(控制组);Aj为α-葡萄糖苷酶溶液被100μL0.1M,pH 6.9PB替代的反应体系的吸光值(样品空白组);Ab为样品和α-葡萄糖苷酶溶液分别被50μL 90%甲醇和100μL 0.1M,pH 6.9PBS替代的反应体系的吸光值。IC50值为α-葡萄糖苷酶的活性被抑制50%时所需要的样品浓度。以阿卡波糖为阳性对照,按规定方法进行实验。最终结果如下表1所示。
表1覆盆子提取物对α-葡萄糖苷酶抑制活性
从上表可以看出,40%乙醇-水溶液洗脱组分对α-葡萄糖苷酶的抑制效果最佳,该活性组分的重量占覆盆子原料重量的2.2%。
实施例2
一种从覆盆子中提取α-葡萄糖苷酶抑制剂的方法,包括以下步骤:将新鲜采摘的覆盆子果实洗净后晒干,用粉碎机粉碎后过40目筛,取1000g覆盆子粉末加入料液比(质量比)1:10的40%乙醇-水混合溶剂,于24℃、常压条件下,用磁力搅拌器对待提物进行搅拌提取24h,过滤后,滤渣按照上述提取方式重复提取2次,合并3次分别所得的滤液,于-0.1MPa真空浓缩后得到粗提物浸膏,该粗提物浸膏为原覆盆子原料的19.75%。采用湿法上样,按料液比为1g/mL将粗提物浸膏分散于蒸馏水中,离心后取上清液得上样溶液。取内径为6cm的大孔吸附树脂层析柱并向其内部加入XAD-16填料至填料高度为30cm。将上样溶液加入大孔吸附树脂层析柱中吸附8小时后,用乙醇-水溶液作为洗脱液按照乙醇在洗脱液中的体积含量依次为0%、10%、40%、60%、80%、100%对上样溶液进行梯度洗脱。其中,梯度洗脱的洗脱体积分别为2500mL、1800mL、1800mL、1800mL、1800mL、1800mL,洗脱流速为40mL/min。收集各洗脱流分,并对其进行α-葡萄糖苷酶抑制活性测试,测试方法同实例1所述,结果显示40%乙醇-水溶液洗脱组分抑制效果最佳(IC50=1.26μg/mL),该活性组分的重量占覆盆子原料重量的2%。
实施例3
一种从覆盆子中提取的α-葡萄糖苷酶抑制剂的制备方法,包括以下步骤:将新鲜采摘的覆盆子果实洗净后晒干,用粉碎机粉碎后过80目筛,取500g覆盆子粉末加入料液比(质量比)1:10的80%乙醇-水混合溶剂,于24℃、常压条件下,用磁力搅拌器对待提物进行搅拌提取24h,过滤后,滤渣按照上述提取方式重复提取2次,合并3次分别所得的滤液,于-0.1MPa真空浓缩后得到粗提物浸膏,该粗提物浸膏为原覆盆子原料的24.15%。采用湿法上样,按料液比为0.5g/mL将粗提物浸膏分散于蒸馏水中,离心后取上清液得上样溶液。取内径为4cm的大孔吸附树脂层析柱并向其内部加入XAD-16填料至填料高度为20cm。将上样溶液加入大孔吸附树脂层析柱中吸附8小时后,用乙醇-水溶液作为洗脱液按照乙醇在洗脱液中的体积含量依次为0%、10%、40%、60%、80%、100%对上样溶液进行梯度洗脱。其中,梯度洗脱的洗脱体积分别为700mL、500mL、500mL、500mL、500mL、500mL,洗脱流速为15mL/min。收集各洗脱流分,并对其进行α-葡萄糖苷酶抑制活性测试,测试方法同实例1所述,结果显示,40%乙醇-水溶液洗脱组分抑制效果最佳(IC50=1.58μg/mL),该活性组分的重量占覆盆子原料重量的2.1%。
实施例1为本发明较佳的实施方式,以上所描述的实施例是本发明一部分实施例,而不是全部的实施例。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,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
Claims (10)
1.一种从覆盆子中提取α-葡萄糖苷酶抑制剂的方法,包括以下步骤:
(1)将覆盆子粉碎,得到覆盆子颗粒;
(2)以亲水性有机溶剂为提取剂,采用溶剂提取法处理覆盆子颗粒得到提取液;再将提取液浓缩干燥,得到覆盆子粗提取物;
(3)用水溶解覆盆子粗提取物配制为覆盆子粗提取物水溶液,采用柱层析法进一步纯化,得到α-葡萄糖苷酶抑制剂。
2.根据权利要求1所述的方法,其特征在于,步骤(1)中的覆盆子为干燥的覆盆子果实,粉碎至粒度为180~4000μm。
3.根据权利要求1所述的方法,其特征在于,步骤(2)中的亲水性有机溶剂是甲醇或乙醇,或者,亲水性有机溶剂是甲醇或乙醇的水溶液。
4.根据权利要求1所述的方法,其特征在于,步骤(2)中的溶剂提取法为震荡浸提,浸提时间为12~24小时;或者为超声波提取,超声提取时间为30~60min,超声功率为250~500W。
5.根据权利要求1所述的方法,其特征在于,步骤(3)中柱层析法的条件为:覆盆子粗提取物水溶液的浓度为20~100mg/mL,以经过预处理的大孔吸附树脂为固定相,上样量为1BV,上样流速为1~5BV/h,吸附方式为静态吸附,吸附时间为2~8小时;用0~95%乙醇或甲醇为洗脱剂进行梯度洗脱,洗脱体积为3BV,洗脱流速为1~5BV/h。
6.根据权利要求5所述的方法,其特征在于,大孔吸附树脂为XAD-16、AB-8或D101大孔吸附树脂。
7.根据权利要求5所述的方法,其特征在于,大孔吸附树脂进行预处理的方法为:将大孔吸附树脂用乙醇或甲醇浸泡,再用乙醇或甲醇冲洗,洗至流出的醇无浑浊,然后用水洗至无醇味,后用2%~4%HCl溶液浸泡,用水洗至中性,再用2~4%NaOH溶液浸泡,用去水洗至中性,得到处理后的大孔吸附树脂。
8.根据权利要求1~7所述的方法得到的α-葡萄糖苷酶抑制剂。
9.根据权利要求8所述的α-葡萄糖苷酶抑制剂的应用。
10.根据权利要求9所述的应用,其特征在于,所述的α-葡萄糖苷酶抑制剂用于制备治疗餐后高血糖的药物或保健食品。
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Application publication date: 20181218 |