CN110123878A - 一种油橄榄果渣提取物的制备及其在制备α-葡萄糖苷酶抑制剂中的应用 - Google Patents
一种油橄榄果渣提取物的制备及其在制备α-葡萄糖苷酶抑制剂中的应用 Download PDFInfo
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Classifications
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- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
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Abstract
本发明公开了一种油橄榄果渣提取物的制备及其在制备α‑葡萄糖苷酶抑制剂中的应用,将油橄榄果渣二次利用提取油橄榄果渣提取物,并公开具体制备方法为以油橄榄果渣为原料,用乙醇水溶液提取溶解,再加入水,离心过滤,得到油橄榄果渣提取物沉淀物;将沉淀物用80~95%的乙醇水溶液复溶,充分混匀后,用少量大孔吸附树脂拌样,挥干溶剂;加入解吸液进行解吸附,合并解吸液,减压浓缩,喷雾干燥后,得到粉末状的油橄榄果渣提取物;首次将制得的油橄榄果渣提取物用于制备α‑葡萄糖苷酶抑制剂的药物上,起到抑制α‑葡萄糖苷酶的作用。
Description
技术领域
本发明涉及植物制药领域,尤其涉及一种油橄榄果渣提取物的制备及其在制备α-葡萄糖苷酶抑制剂中的应用。
背景技术
糖尿病是一组以高血糖为特征的代谢性疾病。高血糖则是由于胰岛素分泌缺陷或其生物作用受损,或两者兼有引起;糖尿病时长期存在的高血糖,导致各种组织,特别是眼、肾、心脏、血管、神经的慢性损害、功能障碍。
糖尿病又可以分为I型糖尿病和II型糖尿病。II型糖尿病原名叫成人发病型糖尿病,多在35~40岁之后发病,占糖尿病患者90%以上;对II型糖尿病进行治疗的一种思路是使用α-糖苷酶抑制剂;α-葡萄糖苷酶抑制剂通过抑制小肠黏膜刷状缘的α-葡萄糖苷酶以延缓碳水化合物的吸收,降低餐后高血糖。主要特点包括平稳降糖、安全性高,以及可降低心血管并发症的发生率,是少数可干预糖耐量受损的口服降糖药之一。
油橄榄果渣由油橄榄果皮、果核及残余果肉组成,其中主要含有三萜类、黄酮类、挥发油及维生素等有效成分。研究结果表明三萜酸是油橄榄果渣的主要有效成分,其中以山楂酸和齐墩果酸含量最高。山楂酸和齐墩果酸均为五环三萜类有机弱酸,可溶于甲醇、丙酮、氯仿,难溶于水。山楂酸和齐墩果酸具有降血糖血脂,抗HIV,抗肿瘤,清除自由基等多种活性作用,是重要的药品、保健品与化妆品的原料和添加剂。采用油橄榄果渣提取物制备糖苷酶抑制剂,不仅可以有效抑制α-糖苷酶活性,治疗II型糖尿病,而且还可以避免由于使用Acarbose(阿卡波糖)而产生的胃肠道功能紊乱等症状。
发明内容
针对上述技术中存在的问题,本发明提供一种油橄榄果渣提取物的制备及其在制备α-葡萄糖苷酶抑制剂中的应用,能够将油橄榄果渣综合利用,通过采用了操作简单,实现性强的提取工艺对油橄榄果渣进行提取,所获提取物中功效性成分的活性更强;进一步被运用到预防和治疗II型糖尿病药物以及其它具备预防和治疗II型糖尿病的功能性食品的制备中。
为实现上述目的,本发明提供一种油橄榄果渣提取物的制备方法,包括以下步骤:
1)取油橄榄果渣,用80~95%的乙醇水溶液提取,回收溶剂得油橄榄果渣浸膏,制得油橄榄果渣乙醇水溶液提取物;
2)将步骤1)中的浸膏加入95%乙醇水使油橄榄果渣提取物浸膏充分溶解,再加入水使油橄榄果渣中溶液乙醇浓度为30~60%,静置6~8h,离心过滤,得到油橄榄果渣提取物沉淀物;
3)将步骤2)中所得油橄榄果渣提取物沉淀物用80~95%的乙醇水溶液复溶,充分混匀后,用少量大孔吸附树脂拌样,挥干溶剂;
4)将步骤3)中的拌样树脂装入层析柱上层中,树脂柱先用水洗涤以除去水溶性杂质,弃去洗涤液,加入解吸液进行解吸附,合并解吸液,减压浓缩,干燥后,得到粉末状的油橄榄果渣提取物。
其中,所述油橄榄果渣提取物主要成份为山楂酸和齐墩果酸。
其中,步骤1)中所述的油橄榄果渣重量与提取溶剂体积比优选为1:3~1:15。
其中,步骤3)中所述的大孔吸附树脂的柱径与柱高比为1:10~1:20。
其中,步骤4)中所述的大孔吸附树脂柱吸附后解吸液为乙醇甲醇或水中的一种。
其中,其特征在于,步骤4)中所述对的吸附流速为2~15BV/h。
本发明还提供一种以上述基础的一种油橄榄果渣提取物在制备预防和/或治疗糖尿病药物中的应用。
其中,所述油橄榄果渣提取物用于制备葡萄糖苷酶活性抑制剂。
其中,所述葡萄糖苷酶活性抑制剂为α-葡萄糖苷酶活性抑制剂。
本发明的有益效果是:与现有技术相比,本发明提供的一种油橄榄果渣提取物对的制备及其在制备α-葡萄糖苷酶活性抑制剂中的应用,通过从油橄榄果渣中提取有效提取物,使油橄榄果渣得到综合利用;采用了操作简单,实现性强的提取工艺对油橄榄果渣进行提取,所获提取物中功效性成分的活性更强;另一方面,首次将油橄榄果渣提取物的葡萄糖苷酶抑制活性应用在药品及保健食品领域中。
具体实施方式
为了更清楚地表述本发明,下面结合具体实施例对本发明作进一步地描述。
本发明在测试油橄榄果渣提取物对α-葡萄糖苷酶活性的抑制作用时,用Acarbose作为对照组,Acarbose为阿卡波糖,即是一种新型口服降糖药。在肠道内竞争性抑制葡萄糖甙水解酶。降低多糖及蔗糖分解成葡萄糖,使糖的吸收相应减缓,因此可具有使饭后血糖降低的作用。一般单用,或与其它口服降血糖药,或胰岛素合用。配合餐饮,治疗胰岛素依赖型或非依赖型糖尿病。
使用计算抑制率(%)的公式为:
抑制率(%)=(ODcontrol-ODneg)-(ODtest-ODtest control)/(ODcontrol-ODneg)*100%;
其中ODcontrol为对照组吸光度;ODneg为空白组吸光度;ODtest为样品吸光度;ODtest control为样品本底吸光度。
本发明的优选实施例中公开了一种油橄榄果渣提取物的制备方法,包括以下步骤:
1)取油橄榄果渣,用80~95%的乙醇水溶液提取,回收溶剂得油橄榄果渣浸膏,制得油橄榄果渣乙醇水溶液提取物;
2)将步骤1)中的浸膏加入95%乙醇水使油橄榄果渣提取物浸膏充分溶解,再加入水使油橄榄果渣中溶液乙醇浓度为30~60%,静置6~8h,离心过滤,得到油橄榄果渣提取物沉淀物;
3)将步骤2)中所得油橄榄果渣沉淀物用80~95%的乙醇水溶液复溶,充分混匀后,用少量大孔吸附树脂拌样,挥干溶剂;
4)将步骤3)中的拌样树脂装入层析柱上层中,树脂柱先用水洗涤以除去水溶性杂质,弃去洗涤液,加入解吸液进行解吸附,合并解吸液,减压浓缩,喷雾干燥后,得到粉末状的油橄榄果渣提取物。
在进一步的实施例中,油橄榄果渣提取物为山楂酸和齐墩果酸;步骤1)中的油橄榄果渣重量与提取溶剂体积比优选为1:3~1:15;步骤3)中的大孔吸附树脂的柱径与柱高比为1:10~1:20;步骤4)中的大孔吸附树。
通过采用醇提、醇提水沉分离、大孔树脂纯化等操作,得到了功效性成分活性较强的油橄榄提取物,且将油橄榄果渣得到二次利用。
本发明还提供一种以上述基础的一种油橄榄果渣提取物在制备预防和/或治疗糖尿病药物中的应用。
在进一步的实施例中,油橄榄果渣提取物用于制备葡萄糖苷酶活性抑制剂;葡萄糖苷酶活性抑制剂为α-葡萄糖苷酶活性抑制剂。
该油橄榄果渣提取物可以被用于预防和治疗II型糖尿病的药物或药物组合物,该药物或药物组合物可以采用片剂、颗粒剂、胶囊、口服液、滴丸等;也可以被用于制备具备预防和治疗II型糖尿病的功能性食品和饮料,比如泡腾片、保健茶等。
如口服剂:口服液:将油橄榄果渣提取物与其他功能性成分搅拌溶于水或其它可食用溶剂中,充分混匀后进行巴氏消毒,接着用高压均质机进行均质,最后进行罐装、封口得到产品。
滴丸:将滴丸壁材制剂(如聚乙二醇)油浴加热下完全溶解,加入油橄榄果渣提取物进行充分混匀后,用滴丸机将混合物滴入冷却剂中制成滴丸,接着再用离心机将冷却剂去除,得到滴丸产品。
保健茶:将油橄榄果渣提取物溶解于水中,充分溶解之后用喷雾装置将药液均匀喷洒至茶叶干品上,让茶叶吸收药液中的油橄榄果渣提取物,接着采用低温烘干的方法将茶叶干品的含水量减少至6—10%后,经过检验、包装之后得到保健茶产品。
下述结合具体实施例对本发明优选实施例中的油橄榄果渣提取物的制备方法作进一步说明,并结合采用Acarbose作参考比较。
将Acarbose由二甲基亚砜(DMSO)配制10mg/ml的溶液,并配制67mmol的磷酸缓冲液,4-硝基酚-α-D-吡喃葡萄糖苷(PNPG)底物溶液(5Mm,磷酸缓冲液配制),和0.2M的NaCO3溶液(磷酸缓冲液配制)。
实施例1
称取阴干的油橄榄果渣2000g,加入10000ml 95%乙醇水溶液冷浸提取2次,每次3天,过滤;合并滤液,浓缩得浸膏(醇提物)40.7g。将浸膏用300ml 90%乙醇水复溶,溶解液中加水使乙醇浓度调制40%乙醇,静置沉淀6h,5000rpm,离心10min,得到油橄榄果渣提取物沉淀物25.5g。将该沉淀物用100ml 90%乙醇溶解后,缓慢加样在200ml的干燥的大孔吸附树脂上面,自然挥干溶剂,即得拌样树脂;量取大孔吸附树脂800ml,湿法装入内径5.0cm、柱高60cm的层析柱中;将拌样树脂加到大孔吸附树脂层析柱上层,先用水进行洗脱,弃去水洗脱液,然后用洗后的树脂加入12BV的70%乙醇进行解吸附,收集解吸液,进行减压浓缩和冷冻干燥,制得粉末状油橄榄果渣提取物I15.5g。
实施例2
称取阴干的油橄榄果渣2000g,加入10000ml 80%乙醇水溶液冷浸提取2次,每次3天,过滤;合并滤液,浓缩得浸膏(醇提物)38.6g。将浸膏用300ml 90%乙醇水复溶,溶解液中加水使乙醇浓度调制50%乙醇,静置沉淀6h,5000rpm,离心10min,得到油橄榄果渣提取物沉淀物25.5g;将该沉淀物用100ml 90%乙醇溶解后,缓慢加样在200ml的干燥的大孔吸附树脂上面,自然挥干溶剂,即得拌样树脂。量取大孔吸附树脂800ml,湿法装入内径5.0cm、柱高60cm的层析柱中;将拌样树脂加到大孔吸附树脂层析柱上层,先用水进行洗脱,弃去水洗脱液,然后用洗后的树脂加入12BV的50%乙醇进行解吸附,收集解吸液,进行减压浓缩和喷雾干燥,制得粉末状油橄榄果渣提取物II12.2g。
实施例3
称取阴干的油橄榄果渣2000g,加入10000ml 85%乙醇水溶液冷浸提取2次,每次3天,过滤;合并滤液,浓缩得浸膏(醇提物)37.1g;将浸膏用300ml 90%乙醇水复溶,溶解液中加水使乙醇浓度调制60%乙醇,静置沉淀6h,5000rpm,离心10min,得到油橄榄果渣提取物沉淀物25.5g;将该沉淀物用100ml 90%乙醇溶解后,缓慢加样在200ml的干燥的大孔吸附树脂上面,自然挥干溶剂,即得拌样树脂;量取大孔吸附树脂800ml,湿法装入内径5.0cm、柱高60cm的层析柱中;将拌样树脂加到大孔吸附树脂层析柱上层,先用水进行洗脱,弃去水洗脱液,然后用洗后的树脂加入12BV的90%乙醇进行解吸附,收集解吸液,进行减压浓缩和喷雾干燥,制得粉末状油橄榄果渣提取物III13.1g。
下面对实施例1-3制得的粉末状油橄榄提取物I-III和Acarbose分别由二甲基亚砜(DMSO)配制10mg/ml的溶液,并配制67mmol的磷酸缓冲液,4-硝基酚-α-D-吡喃葡萄糖苷(PNPG)底物溶液(5Mm,磷酸缓冲液配制),和0.2M的NaCO3溶液(磷酸缓冲液配制)。
采用比色法对油橄榄果渣提取物和Acarbose对α-葡萄糖苷酶的半数抑制浓度进行测定;将20μL的α-葡萄糖苷酶(0.8U)加入样品孔中,然后将测试样品溶液用磷酸缓冲液按一定比例稀释,每孔加入样品溶液120μL,使测试样品的最终浓度为500μg/mL,250μg/mL,125μg/mL,62.5μg/mL,31.25μg/mL,15.625μg/mL,最后再加入反应底物PNPG20μL(5mM);37℃水浴反应15min后,每隔样品孔中加入80μLNaCO3(0.2M)终止反应,在405nm波长处比色测定;相同体积的磷酸缓冲液代替酶溶液;抑制率由样品OD值对于空白和对照OD值计算。
表1实施例1-3制得的油橄榄果渣提取物I-III和Acarbose的IC50的结果
样品 | IC50 |
I | 108.65+1.34 |
II | 286.12+5.26 |
III | 312.47+3.81 |
Acarbose | 408.78+0.15 |
从表中的IC50的大小可以看出,对照中Acarbose对α-葡萄糖苷酶活性的半数抑制浓度(IC50)为408.78+0.15,实施例制得的油橄榄果渣提取物I-III对α-葡萄糖苷酶活性的半数抑制浓度(IC50)最大的为286.12+5.26,小于Acarbose的IC50的值,看出本发明的油橄榄果渣提取物对α-葡萄糖苷酶活性的抑制作用比Acarbose的更好;此外,从表中看出,在实施例I的IC50的值最小,也就是其对α-葡萄糖苷酶活性的抑制作用最好,即在使用70%乙醇进行解吸附时,本发明所获提取物中功效性成分的活性最强。
本发明的优势在于:
(1)、从油橄榄果渣中提取有效提取物,使油橄榄果渣得到综合利用;
(2)、采用了操作简单,实现性强的提取工艺对油橄榄果渣进行提取,所获提取物中功效性成分的活性更强;
(3)、首次将油橄榄果渣提取物的α-葡萄糖苷酶抑制活性应用在药品及保健食品领域中。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的技术人员来说,在不脱离本发明构思的前提下,还可以做出若干等同替代或明显变型,而且性能或用途相同,都应当视为属于本发明的保护范围。
Claims (9)
1.一种油橄榄果渣提取物的制备方法,其特征在于,包括以下步骤:
1)取油橄榄果渣,用80~95%的乙醇水溶液提取,回收溶剂得油橄榄果渣浸膏,制得油橄榄果渣乙醇水溶液提取物;
2)将步骤1)中的浸膏加入95%乙醇水使油橄榄果渣浸膏充分溶解,再加入水使油橄榄果渣中溶液乙醇浓度为30~60%,静置6~8h,离心过滤,得到油橄榄果渣提取物沉淀物;
3)将步骤2)中所得油橄榄果渣提取物沉淀物用80~95%的乙醇水溶液复溶,充分混匀后,用少量大孔吸附树脂拌样,挥干溶剂;
4)将步骤3)中的拌样树脂装入层析柱上层中,树脂柱先用水洗涤以除去水溶性杂质,弃去洗涤液,加入解吸液进行解吸附,合并解吸液,减压浓缩,干燥后,得到粉末状的油橄榄果渣提取物。
2.根据权利要求1所述的一种油橄榄果渣提取物的制备方法,其特征在于,所述油橄榄果渣提取物的主要成份为山楂酸和齐墩果酸。
3.根据权利要求1所述的一种油橄榄果渣提取物的制备方法,其特征在于,步骤1)中所述的油橄榄果渣重量与提取溶剂体积比优选为1:3~1:15。
4.根据权利要求1所述的一种油橄榄果渣提取物的制备方法,其特征在于,步骤3)中所述的大孔吸附树脂的柱径与柱高比为1:10~1:20。
5.根据权利要求1所述的一种油橄榄果渣提取物的制备方法,其特征在于,步骤4)中所述的大孔吸附树脂柱吸附后解吸液为乙醇甲醇或水中的一种或两种。
6.根据权利要求1所述的一种油橄榄果渣提取物的制备方法,其特征在于,步骤4)中所述对的吸附流速为2~15BV/h。
7.根据权利要求1所述的一种油橄榄果渣提取物在制备预防和/或治疗糖尿病药物中的应用。
8.根据权利要求7所述的应用,其特征在于,所述油橄榄果渣提取物用于制备葡萄糖苷酶活性抑制剂。
9.根据权利要求8所述的应用,其特征在于,所述葡萄糖苷酶活性抑制剂为α-葡萄糖苷酶活性抑制剂。
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CN102060893A (zh) * | 2009-11-13 | 2011-05-18 | 中国科学院兰州化学物理研究所 | 从油橄榄叶中同步制备橄榄苦苷和橄榄总黄酮的工艺方法 |
CN103169771A (zh) * | 2011-12-23 | 2013-06-26 | 中国科学院兰州化学物理研究所 | 从油橄榄果渣中提取含有山楂酸和齐墩果酸混合物的方法 |
CN103830333A (zh) * | 2012-11-20 | 2014-06-04 | 中国科学院兰州化学物理研究所 | 采用大孔树脂吸附提取油橄榄叶抗糖尿病有效部位的方法 |
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Publication number | Priority date | Publication date | Assignee | Title |
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CN102060893A (zh) * | 2009-11-13 | 2011-05-18 | 中国科学院兰州化学物理研究所 | 从油橄榄叶中同步制备橄榄苦苷和橄榄总黄酮的工艺方法 |
CN103169771A (zh) * | 2011-12-23 | 2013-06-26 | 中国科学院兰州化学物理研究所 | 从油橄榄果渣中提取含有山楂酸和齐墩果酸混合物的方法 |
CN103830333A (zh) * | 2012-11-20 | 2014-06-04 | 中国科学院兰州化学物理研究所 | 采用大孔树脂吸附提取油橄榄叶抗糖尿病有效部位的方法 |
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