CN111773257B - 沙棘提取物及其用途 - Google Patents
沙棘提取物及其用途 Download PDFInfo
- Publication number
- CN111773257B CN111773257B CN202010774383.3A CN202010774383A CN111773257B CN 111773257 B CN111773257 B CN 111773257B CN 202010774383 A CN202010774383 A CN 202010774383A CN 111773257 B CN111773257 B CN 111773257B
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- Prior art keywords
- extract
- methanol
- hippophae
- solution
- hippophae rhamnoides
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Classifications
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Abstract
本发明公开了一种沙棘提取物及其用途,通过热水溶解、过滤的前处理步骤就可得到粗提取物;再经C18填料富集后,可得到多种不同的沙棘提取物。另外,本发明不同洗脱剂得到的多种沙棘提取物均能有效抑制α‑葡萄糖苷酶的活性,抑制能力均优于现有的α‑葡萄糖苷酶抑制剂,可用于制备调节糖化学代谢紊乱、降低餐后血糖、预防和/或治疗糖尿病及其并发症的产品,还可用于制备预防和/或治疗肥胖症、高血压、慢性乙肝、艾滋病、肿瘤等以α‑葡萄糖苷酶为靶点的疾病的产品,安全高效。
Description
技术领域
本发明涉及天然植物中有效物质的提取和应用领域,特别是涉及沙棘提取物及其用途。
背景技术
沙棘(拉丁学名:Hippophae rhamnoides Linn.),是一种胡颓子科、沙棘属落叶性灌木,其特性是耐旱、抗风沙,可以在盐碱化土地上生存,因此被广泛用于水土保持。中国西北部大量种植沙棘,用于沙漠绿化。
沙棘果实营养丰富,据测定其果实中含有多种维生素、黄酮类化合物、三萜类化合物、油和脂肪酸、酚类、挥发油类、微量元素、磷脂类、5-羟色胺等活性物质和人体所需的各种氨基酸和蛋白质。
目前沙棘广泛应用于沙棘果汁、沙棘原浆液的生产,在生产过程中,将沙棘榨汁后会产生大量的沙棘果渣,往往会被当作废料丢弃,或生产成干粉当作饲料,价格非常低廉。倘若能更多的开发出沙棘果渣的利用价值,将具有极大的经济意义。
发明内容
本发明主要解决的技术问题是提供一种沙棘提取物及其用途,可有效抑制α-糖苷酶的活性。
为解决上述技术问题,本发明采用的一个技术方案是:
提供一种沙棘提取物,采用如下方法制备得到:将沙棘浸膏与水混合,加热至浸膏完全溶解,静置沉淀,固液分离得固形物;进一步地,加热至浸膏完全溶解的温度为50~80℃,优选60~70℃,更优选65℃。
进一步地,沙棘浸膏:水的料液比为1g:(1~6)mL;进一步为1g:(2~5)mL;更进一步为1g:(3~4)mL。
为了进一步对提取物中的活性物质进行富集,将固形物进行柱层析,以体积分数为10~90%的甲醇水溶液、甲醇为流动相,以流动相中甲醇浓度依次增大的顺序进行洗脱。
进一步地,所述柱层析依次采用如下洗脱剂:10~30%甲醇水溶液,50~70%甲醇水溶液,70~90%甲醇水溶液,甲醇;其中,甲醇洗脱部分呈现2个以上的色带,至少单独收集甲醇洗脱出的第一个色带的洗脱液;
所述“至少单独收集甲醇洗脱出的第一个色带的洗脱液”,是指,可以是,只单独收集甲醇洗脱出的第一个色带的洗脱液,得到α-糖苷酶抑制活性最高的一部分提取物;也可以是,除了单独收集甲醇洗脱出的第一个色带的洗脱液以外,对10~30%甲醇水溶液、50~70%甲醇水溶液、70~90%甲醇水溶液、甲醇洗脱的第二个色带中一种或几种等得到的洗脱液各自单独收集;最后,将收集到的各个洗脱液移除溶剂后,可得到不同的沙棘提取物。
总之,甲醇洗脱出的第一个色带的洗脱液是肯定收集的,才能得α-糖苷酶抑制活性最高的沙棘提取物,从本发明的具体实施例中可以看出,各个洗脱剂得到的沙棘提取物中α-糖苷酶抑制活性不同,其中由甲醇洗脱出的第一个色带得到的提取物α-糖苷酶抑制活性最高,IC50仅为0.040mg/mL。
更进一步地,所述柱层析依次采用如下洗脱剂:20%甲醇水溶液,60%甲醇水溶液,80%甲醇水溶液,甲醇。
进一步地,每个浓度的甲醇水溶液为洗脱剂的用量为2~3个柱体积,甲醇为洗脱剂的用量为6~8个柱体积。
或者,为了进一步对提取物中的活性物质进行富集,将固形物进行柱层析,以体积分数为10~90%的甲醇水溶液为流动相,以流动相中甲醇浓度依次增大的顺序进行洗脱。
进一步地,所述柱层析依次采用如下洗脱剂:10~30%甲醇水溶液,50~70%甲醇水溶液,70~90%甲醇水溶液;其中,至少单独收集70~90%甲醇水溶液的洗脱液;
进一步地,所述柱层析依次采用如下洗脱剂:20%甲醇水溶液,60%甲醇水溶液,80%甲醇水溶液。
更进一步地,每个浓度的甲醇水溶液的用量为2~3个柱体积。
所述“至少单独收集70~90%甲醇水溶液的洗脱液”,是指,可以是,只单独收集70~90%甲醇水溶液的洗脱液,得到对应部位的提取物;也可以是,除了单独收集70~90%甲醇水溶液的洗脱液以外,对10~30%甲醇水溶液、50~70%甲醇水溶液洗脱中一种或两种得到的洗脱液各自单独收集;最后,将收集到的各个洗脱液移除溶剂后,可得到不同的沙棘提取物。
总之,70~90%甲醇水溶液洗脱出的洗脱液是肯定收集的,才能得具有显著的降血糖活性的沙棘提取物,从本发明的具体实施例中可以看出,各个洗脱剂得到的沙棘提取物中α-糖苷酶抑制活性不同,其中由70~90%甲醇水溶液洗脱出的提取物α-糖苷酶抑制活性高,与甲醇洗脱得到第一个色带的提取物相当。
进一步地,所述柱层析的柱填料为C18填料。
进一步地,所述柱层析采用干法上样;
进一步地,所述干法上样是将样品或样品溶液与柱填料混合后上样,采用样品溶液与柱填料混合时,将溶剂移除后再上样,干法上样中柱填料的质量用量为样品质量的1~1.5倍。
进一步地,所述沙棘浸膏是通过乙醇提取沙棘得到。
进一步地,所述沙棘为沙棘果渣。
本发明还提供了上述沙棘提取物在制备预防和/或治疗糖耐量受损、糖尿病、肥胖症、慢性乙肝、艾滋病、肿瘤中的一种或几种病症的产品中的用途。
糖耐量受损即糖尿病前期,主要包括空腹血糖受损(impaired fasting glucose,IFG)和糖耐量减低(impaired glucose tolerance,IGT)。可有效改善糖调节受损,预防和/或治疗糖耐量受损和/或糖尿病,同时也可预防或改善糖尿病并发症,可用于制备相关产品。
本发明还提供了上述沙棘提取物在制备α-糖苷酶抑制中的用途。
α-葡萄糖苷酶在食物吸收过程中起着重要的作用,必须与之结合后,食物才能消化和吸收。α-葡萄糖苷酶抑制剂的降糖机制是,通过抑制肠黏膜上的α-葡萄糖苷酶,使淀粉分解为葡萄糖的速度减缓,减少和延缓小肠对葡萄糖的吸收,以降低血糖,对餐后高血糖的作用比较明显。
本发明通过实验表明,本发明沙棘提取物对α-葡萄糖苷酶的活性具有很好的抑制作用,抑制能力远优于现有的α-葡萄糖苷酶抑制剂阿卡波糖,可用于制备α-葡萄糖苷酶抑制剂。
α-葡萄糖苷酶抑制剂不刺激胰岛素的分泌,单独使用此类药物通常不会引发低血糖,因此可帮助减少血糖的波动,可以明显降低糖尿病患者发生心血管病变的概率,对心肌梗死的改善作用最为显著。是少数可干预糖耐量受损的口服降糖药之一。据报道,α-葡萄糖苷酶抑制剂不仅对糖尿病有确切的疗效,而且对于肥胖症,慢性乙肝,艾滋病及肿瘤都有一定的治疗作用。
本发明还提供一种产品,所述产品包含上述的沙棘提取物。
在本发明中,所述产品包括但不限于药品、保健品、食品等。
本发明的有益效果是:
(1)本发明的沙棘粗提取物和经C18填料富集后的不同沙棘提取物,通过试验验证了其都可抑制α-葡萄糖苷酶的活性,效果优于阿卡波糖,可用于制备α-葡萄糖苷酶抑制剂类产品,也可用于制备预防和/或治疗以α-葡萄糖苷酶为靶点的疾病的产品,如治疗糖耐量受损、糖尿病、高血压、肥胖症、慢性乙肝、艾滋病、肿瘤等疾病的产品,原料为沙棘果渣,实现了废物利用,变废为宝。
(2)本发明提取方法步骤简单,简便快捷,原料利用率高,可有效降低沙棘有关产品的成本,值得推广。
具体实施方式
下面对本发明的技术方案进行清楚、完整地描述,显然,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。基于本发明中的实施例,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。
实施例1
1、浸膏的提取:
将干燥的沙棘果渣粉碎后用95%乙醇溶液热回流提取,温度70℃、提取时间1.5h/次,提取三次、料液比1:12g:mL,移除溶剂,冷冻保存。
2、浸膏的初处理:
(1)称取含有部分水分的沙棘浸膏30.9g,加水100mL,加热至浸膏完全溶解,约65℃左右。
(2)静置沉淀,过滤分离得到滤渣。
(3)将滤渣在自然条件下进行干燥后得到沙棘粗提物,粗提物质量为6.2g。
3、C18填料富集:
(1)取一定量甲醇将沙棘粗提物溶解并过滤。
(2)将步骤(1)过滤后的溶液用少量C18填料进行拌样,填料:样品质量比为1:1~1:1.5,并旋干溶剂。
(3)取一定量的C18填料,YMC C18规格:ODS-A S-50um 12nm,装柱,上样体积:未上样体积≥1:3,上样前用20%的甲醇冲柱。
(4)上样后在填料顶部加塞棉花,防止填料飞溅。
(5)依次用20%甲醇水溶液v/v、60%甲醇水溶液v/v、80%甲醇水溶液v/v、纯甲醇冲柱,每个浓度的甲醇水溶液的冲柱体积为2~3个柱体积,纯甲醇部分共冲洗8个柱体积。
(6)纯甲醇冲柱时,可看出柱中存在三个颜色不同的条带,分别按照被洗脱出的先后顺序收集前两个色带为100%-1、100%-2,100%-1得到的提取物质量为1.848g。
(7)最后用10%的甲醇-氯仿溶液v/v进行洗脱,将吸附物质冲干净即可。
(8)再用纯甲醇替换柱中的氯仿,最后将填料保存在甲醇溶液中即可。
试验例1
1、试剂的配制
(1)甲液的配置:NaH2PO4·2H2O称取15.603g,定容至500mL,4℃,棕色瓶保存,备用。
(2)乙液的配置:Na2HPO4·12H2O称取35.822g,定容至500mL,4℃,棕色瓶保存,备用。
(3)0.1M磷酸缓冲液的配置:量取甲液51mL,乙液49mL,加入100mL水,混匀,得pH值6.8的磷酸缓冲液,4℃,棕色瓶保存,备用。
(4)酵母α-葡萄糖苷酶:将100U/ml酶原液用磷酸缓冲液(pH6.8)稀释为20U/mL的酶溶液,冷冻备用,使用前用磷酸缓冲液(pH6.8)稀释为1U/mL,备用。
(5)底物pNPG配制:精密称取0.3766g pNPG,加入适量的磷酸钠缓冲液中溶解,再定容至50mL,配制成25mmol/L母液,使用前用磷酸钠缓冲液配制成0.5mmol/L,备用。
(6)阿卡波糖抑制剂配制:精密称取13.9mg阿卡波糖,用DMSO定容至1mL,配制成13.9mg/mL,备用。
(7)0.1mol/L的Na2CO3配制:称取1.06g Na2CO3于烧杯中,加入适量蒸馏水溶解,并定容到100mL,4℃下保存,备用。
2、抑制剂的制备
精密称取10mg沙棘提取物至1mL容量瓶中,用少量DMSO溶解后定容至刻度,并用DMSO稀释4倍浓度为2.5mg/mL作为母液,需要使用时再稀释至适当的浓度,即得系列不同浓度沙棘提取物的抑制剂。
3、沙棘提取物对α-葡萄糖苷酶的活性抑制效果实验
分别测定实施例1中提取到的不同提取物对α-糖苷酶活性的抑制作用,测定方法为本领域内常规的测定对α-糖苷酶活性的抑制效果的方法,如下:
试验过程:在96孔板上按表1所示步骤进行试验,每组3个平行,将待测试剂、磷酸缓冲液和酶溶液混合均匀,在恒温振荡器中37℃保温10min,结束后,取出,加入50μL0.5mmol/L pNPG溶液,充分混匀,于37℃水浴反应20min,结束后加入50μL 0.1mol/L的Na2CO3溶液中止反应,在405nm波长下测定吸光值,根据公式便可计算出各样品α-葡萄糖苷酶的抑制率及IC50值。
计算IC50值,测定结果见表1。
表1试验步骤及方法(单位:μL)
(酶溶液按原方法稀释完再稀释一半,浓度为0.5U/mL)
表2
阿卡波糖为一种α-葡萄糖苷酶抑制剂,可抑制各种α-葡萄糖苷酶如麦芽糖酶、异麦芽糖酶、葡萄糖淀粉酶及蔗糖酶的活性,使淀粉分解成寡糖如麦芽糖(双糖)、麦芽三糖及糊精(低聚糖)进而分解成葡萄糖的速度减慢,使蔗糖分解成葡萄糖和果糖的速度减慢,因此造成肠道葡萄糖的吸收减缓,从而缓解餐后高血糖,达到降低血糖的作用。长期服用,可降低空腹血糖和糖化血红蛋白的浓度。
从上述实验结果可以看出,通过本发明提取方法的不同洗脱剂得到的不同洗脱部位的沙棘提取物,均能有效抑制α-葡萄糖苷酶的活性,且效果均优于阿卡波糖。其中,甲醇洗脱得到的第一个色带100%-1的提取物抑制作用最强,对α-葡萄糖苷酶的半抑制浓度远低于阿卡波糖对α-葡萄糖苷酶的半抑制浓度,效果显著,可用于制备α-葡萄糖苷酶抑制剂,也可用于制备治疗以α-葡萄糖苷酶为靶点的疾病的药物,如治疗糖耐量受损、糖尿病、高血压、肥胖症、慢性乙肝、艾滋病、肿瘤等疾病的药物。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (14)
1.一种抑制α-葡萄糖苷酶活性的沙棘提取物,其特征在于,采用如下方法制备得到:
将沙棘浸膏与水混合,加热至浸膏完全溶解,静置沉淀,固液分离得固形物;所述沙棘浸膏是通过95%乙醇提取沙棘果渣得到;
将固形物通过柱层析进一步纯化,柱层析的柱填料为 C18 填料,柱层析选自如下 A、B方案之一:
A、以体积分数为20%甲醇水溶液、60%甲醇水溶液、80%甲醇水溶液、纯甲醇为流动相,以流动相中甲醇浓度依次增大的顺序进行洗脱;其中,纯甲醇洗脱部分呈现 2 个以上的色带,单 独收集纯甲醇洗脱出的第一个色带的洗脱液;
B、所述柱层析依次采用如下洗脱剂:20%甲醇水溶液、60%甲醇水溶液、80%甲醇水溶液;其中,单独收集 80%甲醇水溶液的洗脱液。
2.根据权利要求 1 所述的沙棘提取物,其特征在于,加热至浸膏完全溶解的温度为50~80℃。
3.根据权利要求 2 所述的沙棘提取物,其特征在于,温度选60~70℃。
4.根据权利要求3所述的沙棘提取物,其特征在于,温度选自65℃。
5.根据权利要求 1 所述的沙棘提取物,其特征在于,沙棘浸膏:水的料液比为 1g:1~6mL。
6.根据权利要求 5 所述的沙棘提取物,其特征在于,沙棘浸膏:水的料液比为 1g:2~5mL。
7.根据权利要求 6 所述的沙棘提取物,其特征在于,沙棘浸膏:水的料液比为 1g:3~4mL。
8.根据权利要求 1 所述的沙棘提取物,其特征在于,方案 A 中,每个浓度的甲醇水溶液的用量为 2~3 个柱体积,纯甲醇的用量为 6~8 个柱体积。
9.根据权利要求 1 所述的沙棘提取物,其特征在于,方案 B 中,每个浓度的甲醇水溶液的用量为 2~3 个柱体积。
10.根据权利要求 1 所述的沙棘提取物,其特征在于,所述柱层析采用干法上样。
11.根据权利要求 10 所述的沙棘提取物,其特征在于,所述干法上样是将样品或样品溶液与柱填料混合后上样,采用样品溶液与柱填料混合时,将溶剂移除后再上样,干法上样中柱填料的质量用量为样品质量的 1~1.5 倍。
12.权利要求 1~11任一项所述的沙棘提取物在制备预防和/或治疗糖耐量受损、糖尿病中的一种或几种病症的产品中的用途。
13.权利要求 1~11 任一项所述的沙棘提取物在制备α-葡萄糖苷酶抑制剂中的用途。
14.一种产品,其特征在于,包含权利要求 1~11 任一项所述的沙棘提取物。
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