CN108096299B - 猕猴桃籽活性部位提取分离方法及其应用 - Google Patents
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Abstract
本发明涉及具降血糖活性,特别是指猕猴桃籽活性部位提取分离方法及其应用,本发明利用新的提取分离工艺,首次对猕猴桃籽的药用及营养价值进行了系统研究,发现并得到两个具有降血糖的活性部位(KWO和KWS‑B),单独或者合用均可作为膳食添加或制成胶囊、软胶囊、片剂、粉剂等作为保健品、食品或药品应用,解决了现有技术中猕猴桃籽的浪费及常用降血糖药对人体副作用大且无安全有效的替代药的技术问题。
Description
技术领域
本发明涉及降血糖药物的,特别是指猕猴桃籽活性部位提取分离方法及其应用。
背景技术
近些年来,随着对野生水果猕猴桃研究的深入,“水果之王” 的美誉便应运而生。猕猴桃作为药食两用植物,在我国资源丰富,分布广泛。猕猴桃可以预防老年骨质疏松;抑制胆固醇在动脉内壁的沉积,从而防治动脉硬化;可改善心肌功能,防治心脏病等。在抗癌方面,具有抑制肠道内亚硝胺对组织的诱变作用。一些癌病人食用猕猴桃后,可以减轻厌食和恶病质,还可以减轻病人作X线照射和化疗中产生的副作用或毒性反应。多食用猕猴桃,还具有阻止体内产生过多的过氧化物,防止老年斑的形成,延缓人体衰老。据报道,单个猕猴桃果实中的籽粒平均可达250-800余粒,1 kg果实约含干燥种子33-46 g,而且营养丰富,猕猴桃籽中含有丰富的蛋白质、脂肪和矿物质。此外,有测定显示,从猕猴桃籽提取到的猕猴桃籽油中富含黄酮类、多酚类、微量元素硒及其他生物活性物质,其中亚油酸、α-亚麻酸等不饱和脂肪酸占75%以上。随着食品加工技术的发展,猕猴桃被加工成果干、罐头、果汁等半成品,然而加工副产物猕猴桃籽,除少量用于动物饲料和油脂加工外,绝大多数被丢弃,造成了资源的浪费。
姚茂君等在《猕猴桃籽油的开发利用探讨》中进一步公开了猕猴桃籽油的理化特性及其脂肪酸组成和VE含量,并指出猕猴桃籽油是一种极具开发潜力的营养保健油资源。但关于猕猴桃籽的药用价值的研究很少,本申请的发明人尝试多种工艺,并对每个提取物进行研究,发现猕猴桃籽的提取物部位中具有多种新的药用价值,特别对高血糖的治疗效果显著。
糖尿病是常见的慢性疾病,常用的降血糖药种类多样,其中胰岛素受体增敏剂类降血糖药如罗格列酮、吡格列酮均为西药,每日初始剂量为4mg,该类药不良反应严重,如可能会导致停经,停止排卵妇女的再次排卵,故服药期间应注意避孕,且用药禁忌多,如过敏者、肝肾功能不全者、妊娠等不能服用;且市场上并无较佳的替换产品。
发明内容
本发明提出猕猴桃籽活性部位提取分离方法及其应用,解决了现有技术中猕猴桃籽的浪费及常用降血糖药对人体副作用大且无安全有效的替代药的技术问题。
本发明的技术方案是这样实现的:
猕猴桃籽活性部位提取分离方法,步骤为:
(1)称取一定质量的猕猴桃籽,加入8-12倍猕猴桃籽质量的无水乙醇,利用“闪式提取器”提取,共重复提取3-5遍,得提取液Ⅰ;
(2)提取液Ⅰ经过滤后合并得滤液Ⅰ,收集残渣备用,滤液Ⅰ经减压回收至无醇味后移至烧杯中,水浴加热挥发去残留乙醇,得滤液Ⅱ;
(3)滤液Ⅱ离心后取上层油状物,即为猕猴桃籽降血糖活性部位Ⅰ(KWO);
(4)向残渣中加入7-10倍残渣量的乙醇,利用“闪式提取器”提取,共重复提取3-5遍,得提取液Ⅱ;
(5)提取液Ⅱ经过滤后合并滤液,减压回收乙醇,并适当浓缩制成混悬液;(6)混悬液经石油醚-乙酸乙酯超声萃取后,减压回收萃取液并浓缩干燥,得到猕猴桃籽降血糖活性部位Ⅱ(KWS-B)。
所述步骤(1)、(4)中利用“闪式提取器”提取的时间为3min。
所述步骤(3)中离心的条件为2000 r/min-3500 r/min转速下离心3-5min。
所述步骤(4)中乙醇的质量分数为65-85%。
所述步骤(6)中石油醚:乙酸乙酯的体积比为(1-5):(2-10)。
所述步骤(3)中的猕猴桃籽降血糖活性部位Ⅰ为油状物,猕猴桃籽降血糖活性部位Ⅰ的得率为10-30%,其中α-亚麻酸含量≥50%。
所述步骤(6)中猕猴桃籽降血糖活性部位Ⅱ为棕色固体,得率为5-20%,其中总酚酸≥50%。
所述的猕猴桃籽活性部位作为制备降血糖药物、保健品或食品的应用。
所述的猕猴桃籽活性部位的应用,1-8ng/mL的猕猴桃籽降血糖活性部位Ⅰ和1-4ng/mL的猕猴桃籽降血糖活性部位Ⅱ作为罗格列酮替代药的应用。
本发明的有益效果在于:
(1)现有的猕猴桃籽作为副产物绝大多数被丢弃,造成资源的浪费,本发明利用新的提取分离工艺,使副产物猕猴桃籽资源得到充分利用,首次对猕猴桃籽的药用及营养价值进行了系统研究,发现并得到两个具有降血糖的活性部位(KWO和KWS-B),单独或者合用均可作为膳食添加或制成胶囊、软胶囊、片剂、粉剂等作为保健品或药品应用。
(2)罗格列酮作为Ⅱ型糖尿病的一线治疗药物,可有效提高肝脏、脂肪和肌肉组织对胰岛素的敏感性。但是,长期使用可能会引起水肿、贫血和血脂增高等不良反应,与其他降糖药物合用会有低血糖的风险,并会损伤肝功能;本发明是从食用猕猴桃籽中提取纯天然活性成分,对细胞无毒副作用,在较低的实验浓度(1ng/mL)下,就能取得罗格列酮在4mg/mL浓度下取得的效果,对细胞葡萄糖消耗有明显改善,纯植物萃取的KWO和KWS-B较罗格列酮更加安全有效。
(3)本发明不经过柱层析,方法简便、环保,生产周期短,便于产业化;制备的活性成分具有生产工艺简单、成本低等特点,本发明以天然产物猕猴桃籽为原料,无需昂贵设备,也不会引入新的杂质,仅用萃取的方法就能得到,在降血糖方面具有广阔的应用前景。
附图说明
图1为24h和48 h猕猴桃籽KWS-B部位对HepG2细胞活力的影响(n=4)。
图2为24h和48 h猕猴桃籽KWS-B部位对HepG2细胞绝对葡萄糖消耗量的影响(n=4),#:与正常对照组比较,P<0.05。
图3为24h和48 h猕猴桃籽KWS-B部位对HepG2细胞相对葡萄糖消耗量的影响(n=4),#:与正常对照组比较,P<0.05。
图4为24h和48 h猕猴桃籽KWO部位对HepG2细胞活力的影响(n=4)。
图5为24h和48 h猕猴桃籽KWO部位对HepG2细胞绝对葡萄糖消耗量的影响(n=4),#:与正常对照组比较,P<0.05;*:与罗格列酮组比较,P<0.05。
图6为24h和48 h猕猴桃籽KWO部位对HepG2细胞相对葡萄糖消耗量的影响(n=4),#:与正常对照组比较,P<0.05;*:与罗格列酮组比较,P<0.05。
具体实施方式
下面将结合本发明实施例,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅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付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
称取1Kg的猕猴桃籽,加入8倍量的无水乙醇,利用“闪式提取器”提取,共重复提取3遍,每次提取时间为3min。过滤,残渣备用。合并滤液,减压回收至无醇味,移至烧杯中,水浴加热挥去残留乙醇,离心机离心3 min(2000 r/min),取上层油状物,得到猕猴桃籽降血糖活性部位Ⅰ(KWO)。
KWO性状:油状物;得率:平均25%;检验指标:α-亚麻酸含量≥50%。
将残渣中加入7倍65%乙醇,利用“闪式提取器”提取,共重复提取3遍,每次提取时间为3min。过滤,合并滤液,减压回收乙醇,并适当浓缩后制成混悬液(比重约1.1左右),利用石油醚-乙酸乙酯(1: 10)超声萃取,减压回收萃取液并浓缩干燥,得到猕猴桃籽降血糖活性部位Ⅱ(KWS-B)。
KWS-B性状:棕色固体;得率:平均10%;检验指标:总酚酸≥50%。
将实施例1制备的猕猴桃籽降血糖活性部位Ⅰ作为胶囊填充剂,制成胶囊供病人服用。
实施例2
称取1Kg的猕猴桃籽,加入12倍量的无水乙醇,利用“闪式提取器”提取,共重复提取5遍,每次提取时间为5min。过滤,残渣备用。合并滤液,减压回收至无醇味,移至烧杯中,水浴加热挥去残留乙醇,离心机离心5 min(3500 r/min),取上层油状物,得到猕猴桃籽降血糖活性部位Ⅰ(KWO)。
KWO性状:油状物;得率:平均25%;检验指标:α-亚麻酸含量≥50%。
将残渣中加入8倍85%乙醇,利用“闪式提取器”提取,共重复提取5遍,每次提取时间为5min。过滤,合并滤液,减压回收乙醇,并适当浓缩后制成混悬液(比重约1.1左右),利用石油醚-乙酸乙酯(5:2)超声萃取,减压回收萃取液并浓缩干燥,得到猕猴桃籽降血糖活性部位Ⅱ(KWS-B)。
KWS-B性状:棕色固体;得率:平均10%;检验指标:总酚酸≥50%。
将实施例2制备的猕猴桃籽降血糖活性部位Ⅱ制成片剂供病人服用。
实施例3
称取1Kg的猕猴桃籽,加入10倍量的无水乙醇,利用“闪式提取器”提取,共重复提取4遍,每次提取时间为4min。过滤,残渣备用。合并滤液,减压回收至无醇味,移至烧杯中,水浴加热挥去残留乙醇,离心机离心4 min(2800r/min),取上层油状物,得到猕猴桃籽降血糖活性部位Ⅰ(KWO)。
KWO性状:油状物;得率:平均25%;检验指标:α-亚麻酸含量≥50%。
将残渣中加入10倍70 %乙醇,利用“闪式提取器”提取,共重复提取4遍,每次提取时间为5min。过滤,合并滤液,减压回收乙醇,并适当浓缩后制成混悬液(比重约1.1左右),利用石油醚-乙酸乙酯(1:2)超声萃取,减压回收萃取液并浓缩干燥,得到猕猴桃籽降血糖活性部位Ⅱ(KWS-B)。
KWS-B性状:棕色固体;得率:平均10%;检验指标:总酚酸≥50%。
实施例4
称取1Kg的猕猴桃籽,加入9倍量的无水乙醇,利用“闪式提取器”提取,共重复提取3遍,每次提取时间为4min。过滤,残渣备用。合并滤液,减压回收至无醇味,移至烧杯中,水浴加热挥去残留乙醇,离心机离心4 min(3000 r/min),取上层油状物,得到猕猴桃籽降血糖活性部位Ⅰ(KWO)。
KWO性状:油状物;得率:平均25%;检验指标:α-亚麻酸含量≥40%。
将残渣中加入9倍80 %乙醇,利用“闪式提取器”提取,共重复提取3遍,每次提取时间为3min。过滤,合并滤液,减压回收乙醇,并适当浓缩后制成混悬液(比重约1.1左右),利用石油醚-乙酸乙酯(5:2)超声萃取,减压回收萃取液并浓缩干燥,得到猕猴桃籽降血糖活性部位Ⅱ(KWS-B)。
KWS-B性状:棕色固体;得率:平均10%;检验指标:总酚酸≥50%。
应用例
对实施例1的产品进行疗效验证:
1、实验方法:
将HepG2细胞接种于96孔板中,培养24 h之后,分别用阳性对照药罗格列酮(10μM),KWS-B部位(1、4和8ng/mL)及KWO部位(1、2和3 ng/mL)作用于细胞,加上阴性对照与空白对照组。共设置9个组别,每个组别设置4个副孔。
各组药物作用于细胞24和48h后,用葡萄糖氧化酶(GOD)试剂盒及CCK-8试剂盒对细胞糖消耗及细胞活力进行测定结果如表1、表4、图1、图4。
采用葡萄糖氧化酶(GOD)法进行葡萄糖含量测定,具体操作为:各活性部位处理细胞24和48 h后,每孔取10μL上清液,加入到96孔板中,再加入150μL的工作液,放入37°C恒温震荡器中,震摇40 min,使用酶标仪在505 nm处测定其吸光度。根据吸光度值求各个孔的葡萄糖含量与绝对葡萄糖消耗量,结果如表2、表5、图2、图5。相对葡萄糖消耗量=绝对葡萄糖消耗量/细胞活力值。
采用CCK-8试剂进行细胞活力测定,具体操作为:各活性部位作用于细胞24和48 h后(细胞葡萄糖消耗量检测完毕后),向96孔板中每孔加入5μL CCK-8试剂,37℃培养4 h,使用酶标仪在450 nm处测定其吸光度,即为细胞活力OD值,结果如表3、表6、图3、图6。
2、KWS-B部位对细胞活力及细胞糖消耗的测量结果
表1 24h和48 h猕猴桃籽KWS-B部位对HepG2细胞活力的影响(n=4)
表2 24h和48 h猕猴桃籽KWS-B部位对HepG2细胞葡萄糖消耗量的影响(n=4)
#:与正常对照组比较,P<0.05。
3、KWO部位对细胞活力及细胞糖消耗的影响。
表3 24h和48 h猕猴桃籽KWO部位对HepG2细胞活力的影响(n=4)
表4 24h和48 h猕猴桃籽KWO部位对HepG2细胞葡萄糖消耗量的影响(n=4)
#:与正常对照组比较,P<0.05;*:与罗格列酮组比较,P<0.05。
4、结果分析
由表1、2和图1、2可知各个浓度的猕猴桃籽KWS-B部位和KWO部位对细胞活力没有明显影响,没有细胞毒性。由表2和图2、3可知猕猴桃籽KWS-B部位在较低的浓度1、4和8 ng/mL时对HepG2细胞的葡萄糖消耗量有明显的改善效果且可以达到阳性药罗格列酮的效果。由表4和图5、6可知猕猴桃籽KWO部位在较低的浓度1、2和3 ng/mL时对HepG2细胞的葡萄糖消耗量有明显的改善效果,且在1 ng/mL可以达到阳性药罗格列酮的效果。实验结果表明:猕猴桃籽KWS-B和KWO部位在体外具有良好的促进糖消耗作用。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (5)
1.猕猴桃籽降血糖活性部位在制备降血糖药物中的应用,其特征在于,所述猕猴桃籽降血糖活性部位为猕猴桃籽降血糖活性部位Ⅰ和/或猕猴桃籽降血糖活性部位Ⅱ;
猕猴桃籽降血糖活性部位的制备步骤为:
(1)称取一定质量的猕猴桃籽,加入8-12倍猕猴桃籽质量的无水乙醇,利用“闪式提取器”提取,共重复提取3-5遍,得提取液Ⅰ;
(2)提取液Ⅰ经过滤后合并得滤液Ⅰ,收集残渣备用,滤液Ⅰ经减压回收至无醇味后移至烧杯中,水浴加热挥发去残留乙醇,得滤液Ⅱ;
(3)滤液Ⅱ离心后取上层油状物,即为猕猴桃籽降血糖活性部位Ⅰ;
(4)向残渣中加入7-10倍残渣量的乙醇,利用“闪式提取器”提取,共重复提取3-5遍,得提取液Ⅱ;
(5)提取液Ⅱ经过滤后合并滤液,减压回收乙醇,并适当浓缩制成混悬液;
(6)混悬液经石油醚-乙酸乙酯超声萃取后,减压回收萃取液并浓缩干燥,得到猕猴桃籽降血糖活性部位Ⅱ;
所述步骤(3)中的猕猴桃籽降血糖活性部位Ⅰ为油状物,猕猴桃籽降血糖活性部位Ⅰ的得率为10-30%,其中α-亚麻酸含量≥40%;
所述步骤(6)中猕猴桃籽降血糖活性部位Ⅱ为棕色固体,得率为5-20%,其中总酚酸≥50%;
所述猕猴桃籽降血糖活性部位Ⅰ的浓度为1-8ng/mL,猕猴桃籽降血糖活性部位Ⅱ的浓度为1-4ng/mL。
2.如权利要求1所述的猕猴桃籽降血糖活性部位在制备降血糖药物中的应用,其特征在于:所述步骤(1)、(4)中利用“闪式提取器”提取的时间为3min。
3.如权利要求1所述的猕猴桃籽降血糖活性部位在制备降血糖药物中的应用,其特征在于:所述步骤(3)中离心的条件为2000 r/min-3500 r/min转速下离心3-5min。
4.如权利要求1所述的猕猴桃籽降血糖活性部位在制备降血糖药物中的应用,其特征在于:所述步骤(4)中乙醇的质量分数为65-85%。
5.如权利要求1所述的猕猴桃籽降血糖活性部位在制备降血糖药物中的应用,其特征在于:所述步骤(6)中石油醚:乙酸乙酯的体积比为(1-5):(2-10)。
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