CN105395927B - 青稞麸皮提取物在制备α-葡萄糖苷酶活性抑制剂中的应用 - Google Patents
青稞麸皮提取物在制备α-葡萄糖苷酶活性抑制剂中的应用 Download PDFInfo
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- CN105395927B CN105395927B CN201510850185.XA CN201510850185A CN105395927B CN 105395927 B CN105395927 B CN 105395927B CN 201510850185 A CN201510850185 A CN 201510850185A CN 105395927 B CN105395927 B CN 105395927B
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Abstract
本发明公开了一种青稞麸皮提取物在制备α‑葡萄糖苷酶活性抑制剂中的应用,特别是用于制备预防和/或治疗II型糖尿病的药物、保健品或食品。青稞麸皮提取物中含有质量百分比为10%‑60%的总甾醇。该总甾醇包括β‑谷甾醇、菜油甾醇、岩藻甾醇、豆甾醇和链甾醇。
Description
技术领域
本发明涉及农副产品深加工及综合利用领域;更具体地涉及青稞麸皮提取物在制备α-葡萄糖苷酶活性抑制剂中的应用。
背景技术
糖尿病(Diabetes Mellitus,DM)及其并发症严重威胁着人类的健康,第17届国际糖尿病大会报道,全球已确诊1.5亿人,预测2025年将增至3亿。在发达国家DM患病率已达3%~7%,成为仅次于癌症、艾滋病、心血管病之后第4位需要优先考虑的疾病,已成为世界第5位死亡主因。我国的情况也不容乐观,2015年国家卫计委发布的《中国居民营养与慢性病状况报告》中指出,当前我国18岁及以上成人糖尿病患病率为9.7%,约已经达到了1.06亿,还有上亿糖尿病高危人群。因而研究开发预防糖尿病发生的功能食品和高效、无毒副作用的降糖药物,是刻不容缓的事情。
α-葡萄糖苷酶(α-Glucosidase)位于小肠刷状细胞表面。多糖只有水解成单糖后才能被人体吸收,而α-葡萄糖苷酶是多糖水解中的关键酶类[Kim SD,Nho HJ.Isolationand characterization ofα-glucosidase inhibitor from the fungusganodermalucidm.J.Microbiology,2004,42,223-227],是人体糖代谢中最重要的酶之一。研究表明,α-葡萄糖苷酶抑制剂类药物能够通过降低α-葡萄糖苷酶的活性、调节餐后胃肠激素的方式而有效控制餐后血糖的升高,产生较为理想的降糖效果[Requejo F,UttenthalLO,Bloom SR.Effects ofα-glucosidase inhibition and viscous fibre on diabeticcontrol and postprandial gut hormone response.Diabet Med,1990,7,515-520][Moritoh Y,Takeuchi K,Hazama M.Voglibose,and alpha-glucosidase inhibitor,toincrease active glucagon-like peptide-1levels.Mol Cell Pharmacol,2009,1,188-192]。α-葡萄糖苷酶抑制剂,依其来源可划分为微生物代谢物、天然动植物或微生物提取物和化学合成物三大类。目前已被批准用于临床糖尿病治疗的α-葡萄糖苷酶抑制剂主要为化学合成药物,包括:阿卡波糖(Acarbose)、伏格列波糖(Voglibose)和米格列醇(Miglitol)。因长期服用化学合成药物会产生副作用,因此从天然植物中获得更安全、高效的抑制剂的寻求仍然是Ⅱ型糖尿病治疗药物和功能性食品开发的研究热点。
目前,应用到临床的α-葡萄糖苷酶抑制剂多为糖及其衍生物类的竞争性抑制剂,而α-葡萄糖苷酶非竞争性抑制剂通过与酶的非活性部位结合导致其酶活性降低,基本上属于非糖基化合物。通常,非竞争性抑制剂大都是通过筛选随机得到的。已报道的从天然产物中得到的α-葡萄糖苷酶非糖基化合物抑制剂并没有统一的结构模型,种类繁多,包括:姜黄素类、黄酮类、呫吨酮类、喹唑啉类、Schulzeine、芪类和萜类等[Melo EB,Gomes AS andCarvalho I.α-andβ-glucosidase inhibitors:chemical structure and biologicalactivity.Tetrahedron,2006,62:10277-10302]。因此,从天然产物中筛选α-葡萄糖苷酶非竞争性抑制剂是寻找高效治疗Ⅱ型糖尿病药物的有效途径。CN101849973.A公开了具有降血糖作用的桦褐孔菌总三萜的制备方法,方法中依次采用乙醇水溶液提取、石油醚萃取、正丁醇萃取、干燥等工艺,获得三萜含量70-300mg/g的桦褐孔菌总三萜提取物。CN101953867.A公开了具有α-葡萄糖苷酶抑制作用的天山雪莲石油醚提取物的制备方法。CN101810625A公开一种含量为5%~100%的蒲公英甾醇对α-葡萄糖苷酶和α-淀粉酶的抑制作用。CN103705593A公开了从矮生二裂叶委陵菜中提取α-葡萄糖苷酶抑制剂的方法,具体步骤:原料经粉碎后用6倍体积的70%乙醇回流提取,提取液浓缩后依次进行石油醚、乙酸乙酯萃取,萃取液真空干燥得α-葡萄糖苷酶抑制剂。已报道的技术通常含有多步提取、萃取过程,步骤均较为繁琐。
青稞(Hordeumvulgare L.var.nudum hook.f.)是我国青藏高原地区特有的一种高蛋白、高纤维、高维生素、低脂肪、低糖的多棱裸粒大麦。由于青稞生长在高海拔地区,高寒、缺氧、强光照的极端环境,青稞相比其他谷物含有更丰富的次生代谢产物和更明显的保健特性。人群调查试验已对青稞降血脂、降体重、改善胰岛素敏感性等保健作用进行了验证,同时,青稞中的β-葡聚糖降血脂、降胆固醇、改善胰岛素敏感性等药理活性也已被证实。此外,朱颖秋等研究结果显示超临界CO2萃取的青稞麸皮油对实验性高脂血症大鼠的血脂有很明显的调节作用,可预防动脉粥样硬化的发生[朱颖秋,蒋思萍,包善飞,等.超临界CO2萃取青稞麸皮油对高脂血症大鼠降脂作用研究[J].四川动物,2013,32(2):272-275]。报道并未涉及超临界CO2萃取的工艺参数,且该研究认为青稞麸皮油中的亚油酸、十八碳烯酸是降低血清TC、LDL-C的有效成分。
总体而言,对青稞麸皮提取物的制备工艺及其功效作用的研究报道仍较少,且以对β-葡聚糖、花色苷、黄酮等为主。中国专利文献CN101555294A公开了利用二次纤维素酶酶解青稞麸皮,后续经过碱提离心、酸提离心除蛋白、高温α-淀粉酶酶解、离心醇沉、硫酸铵沉淀等系列工艺后分别制得β-葡聚糖和青稞膳食纤维;CN104725526A公开了利用碱提酸沉、过滤脱色、浓缩等步骤制备β-葡聚糖的方法;CN201410350638.8公开了一次性连续分离青稞蛋白、淀粉、纤维素和β-葡聚糖的方法,具体包括以下步骤:将青稞粉碎,粉碎产物中加水并调节pH提取,过筛,筛上物烘干研磨后得青稞纤维粉;筛下物离心得第一沉淀物为青稞淀粉;第一上清液调节pH离心后得第二沉淀物为青稞蛋白;第二上清液超滤得透过液浓缩离心得第三沉淀物为青稞β-葡聚糖;CN200710039964.7公开了以β-葡聚糖为主成分的青稞提取物在治疗神经干细胞缺血药物中的应用;CN200910168946.8公开了含青稞β-葡聚糖的降血脂药物的配方及其制剂;CN201310196869.3公开从紫色青稞中分离花色苷的方法;CN201310400124.4公开了黑青稞籽皮类黄酮及其制备方法与应用,该黑青稞籽皮类黄酮具有预防和治疗缺氧损伤的作用;CN101696381A公开了用常规含水醇溶媒回流提取青稞幼苗中黄酮类化合物的制备新工艺,方法中采用管式离心机除去杂质、陶瓷膜过滤澄清、超滤膜截留分离和纳滤膜脱盐浓缩等工序,最后经喷雾干燥或微波干燥制得含总黄酮80.1%~14.9%的青稞黄酮提取物;CN201410604115.1公开了青稞膳食纤维-多酚复合物的制备方法;CN201410603523.5公开了青稞膳食纤维中多酚化合物的分离方法;CN201410151798.X公开了青稞胚芽油闪式提取的制备方法,获得的胚芽油富含维生素E、维生素D和谷维素;CN201410851568.4公开了一种对糖尿病患者有保健功能的蚕豆青稞饼干及其生产方法;CN201310506264.X公开了一种具有抗疲劳功能的含青稞苗提取物的青稞全粉饼干,该饼干能显著提高小鼠的爬杆和负重游泳时间;另有多项专利涉及了降低血脂、提高人体免疫力、提高记忆力、预防心血管疾病等功能的含有青稞或青稞提取物的保健食品、饮品。此外,贺敏等利用50%乙醇浸泡提取制备黑青稞籽皮提取物,提取物有效成分总花色苷的含量为7.6%,并通过动物实验证明能够提高小鼠耐缺氧及抗疲劳能力。(贺敏,王庆军,丁雪洁,等.黑青稞籽皮提取物提高小鼠耐缺氧及抗疲劳能力的初步研究[J].中国医药导报,2014,11(28):7-10)。
青稞是西藏农区的主要粮食作物,占到西藏粮食总产量的55%左右,占农牧民粮食消耗量的60%,在藏区农业生产和社会经济发展中同样起着非常重要的作用。随着对青稞产业的重视和青稞保健作用的认识,青稞的商业产品产量逐步提高,青稞生产加工中的副产品的量也将快速提高,青稞麸皮是青稞主要的加工副产物,含有大量活性物质,但是目前青稞麸皮的综合利用率低,大部分作为饲料。如果能够综合利用青稞麸皮,则会大大推动青稞深加工产业的发展,从而提高农牧民的生产积极性,拉动西藏经济的发展。
发明内容
本发明要解决的技术问题是提供青稞麸皮提取物在制备α-葡萄糖苷酶活性抑制剂中的应用。
为了解决上述技术问题,本发明提供一种青稞麸皮提取物在制备α-葡萄糖苷酶活性抑制剂中的应用。
作为本发明的青稞麸皮提取物在制备α-葡萄糖苷酶活性抑制剂中的应用的改进:用于制备预防和/或治疗II型糖尿病的药物、保健品或食品。
作为本发明的青稞麸皮提取物在制备α-葡萄糖苷酶活性抑制剂中的应用的进一步改进:青稞麸皮提取物含有质量百分比为10%-60%的总甾醇。所述总甾醇包括β-谷甾醇、菜油甾醇、岩藻甾醇、豆甾醇和链甾醇。
作为本发明的青稞麸皮提取物在制备α-葡萄糖苷酶活性抑制剂中的应用的进一步改进:
青稞麸皮提取物的获取方法为:将青稞麸皮粉碎后过筛(50目),按照1g/4~8ml(较佳为1g/4~6ml)的料液比,加入有机溶剂后进行回流提取,提取次数为至少一次,提取总时间为0.5~6h;然后过滤,滤液减压浓缩去除有机溶剂,得青稞麸皮提取物(收率约为2%~6.5%);
所述有机溶剂为石油醚、正己烷或环己烷。
该青稞麸皮提取物为青稞麸皮中的非极性部位。备注说明:本发明中所用的提取溶剂为低极性溶剂,得到的提取物为非极性成分;区别于水、乙醇等溶剂的提取物。通常,非极性成分为油脂、甾醇、三萜、烷烃等成分。
作为本发明的青稞麸皮提取物在制备α-葡萄糖苷酶活性抑制剂中的应用的进一步改进:所述有机溶剂为正己烷,共回流提取3次;每次提取时的料液比为1g/6ml、时间为1.5h。
作为本发明的青稞麸皮提取物在制备α-葡萄糖苷酶活性抑制剂中的应用的进一步改进:青稞麸皮提取物的获取方法为:将青稞麸皮粉碎后过筛,进行超临界CO2流体萃取,萃取条件为:萃取压力为10~40MPa,萃取温度25~60℃,CO2流速10~25L/h,萃取时间1~5h,分离温度28~50℃(例如为28~35℃),得青稞麸皮提取物。
作为本发明的青稞麸皮提取物在制备α-葡萄糖苷酶活性抑制剂中的应用的进一步改进:所述超临界CO2流体的萃取条件为:萃取压力20~25MPa、萃取温度40~45℃、萃取时间2.0~2.5h,CO2流量15~20L/h(收率为4%~8%)。
本发明的总甾醇含量用Liebermann Burchard分光光度法检测,以β-谷甾醇为标准品,测得提取物中总甾醇含量为10~60%。
本发明的青稞麸皮提取物非极性部位具有α-葡萄糖苷酶抑制活性,经体外实验观察青稞麸皮提取物对α-葡萄糖苷酶的抑制作用,实验结果表明青稞麸皮提取物的抑制活性(IC50=0.021~2.17mg/mL)明显强于市售α-葡萄糖苷酶抑制剂——阿卡波糖(IC50=7.87mg/mL)。
可用于本发明的青稞品种没有特别限制,可以是不同种的青稞。
本发明中所涉及的各项测定方法具体如下:
1)青稞提取物总甾醇含量测定——Liebermann Burchard比色法
精密称取人参皂苷标准品10.0mg于100mL量瓶中,加无水乙醇溶解并稀释至刻度。从中吸取标准品溶液0、0.2、0.4、0.8、1.2、1.6、2.0mL,于加热状态下氮气吹干溶剂,分别加入5%香草醛-冰醋酸0.5mL,然后加入高氯酸0.8mL,混匀后于65℃水浴反应15min,取出放置冰水中迅速冷却至室温后,加冰醋酸5mL,振荡混匀;以零管为空白,用1cm的比色杯在560nm处测定吸光度,以人参皂苷的质量为横坐标,吸光度为纵坐标绘制标准曲线。
取青稞麸皮提取物0.1g,氯仿溶解并定容至50mL,取0.2mL,按标准曲线绘制制备中的操作步骤,测定总甾醇含量。
2)青稞麸皮提取物的游离甾醇组成分析——气相色谱-质谱法(GC-MS)
确称取50mg青稞麸皮提取物,加入20μg胆固醇作为内标,以5mL色谱纯正己烷溶解,取0.5g/6cc silica填料SPE柱,加入1g无水硫酸钠(600℃加热4h以除水),以10ml正己烷活化,常压下上样,以10ml正己烷:乙醚(90:10)溶液淋洗,10mL正己烷:乙醚(70:30)洗脱吸附的游离甾醇,氮气吹干有机试剂,加入5μL MHFBA:1-MIM(95:5;v/v)105℃衍生15min;待冷却后以9.950ml正己烷定容,取1mL进行GC-MS检测。
GC-MS条件:DB-5MS(30m×0.25mm×0.25μm,Agilent Technologies,USA)。氦气载气,流速1.2mL/min。程序升温:100℃保持1min,然后50℃/min上升到200℃,再20℃/min上升到250℃,最后1.5℃/min上升到300℃并保持10min。不分流进样1μL,进样口温度300℃,离子源温度250℃,传输线温度300℃。扫描范围核质比50-600m/z。
本发明相对于现有技术具有如下的优点及效果:
1、从农产品加工副产物青稞麸皮中提取出非极性部位作为α-葡萄糖苷酶抑制剂,具有作为预防和治疗II型糖尿病的药物、保健品或食品开发的前景;
2、通过有机溶剂萃取或超临界CO2萃取技术加工青稞麸皮提取物,工艺步骤少、操作简便、条件温和,获得的产品质量好、活性高。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细说明。
图1为青稞麸皮提取物的游离甾醇组成气相色谱-质谱图。
具体实施方式
下面结合具体实施例对本发明作进一步的说明,但本发明并不限于实施例。
实施例1、一种青稞麸皮提取物的制备:
以石油醚为有机溶剂;
将1kg青稞麸皮粉碎成50目,过筛,加5倍量(即,5L)的石油醚回流提取0.5h,过滤,滤液减压浓缩去除有机溶剂,得青稞麸皮提取物(记为E-1)20.3g。
实施例2、一种青稞麸皮提取物的制备:
将1kg青稞麸皮粉碎成50目,过筛,加6倍量的环己烷回流提取3次,每次1h,过滤,滤液合并后减压浓缩去除有机溶剂(环己烷),得青稞麸皮提取物(E-2)35.6g。
实施例3、一种青稞麸皮提取物的制备:
将1kg青稞麸皮粉碎成50目,过筛,加4倍量的正己烷回流提取1次,每次1.5h,过滤,滤液减压浓缩去除有机溶剂(正己烷),得青稞麸皮提取物(E-3)29.2g。
实施例4、一种青稞麸皮提取物的制备:
将1kg青稞麸皮粉碎成50目,过筛,加6倍量的正己烷回流提取3次,每次1.5h,过滤,滤液合并后减压浓缩去除有机溶剂(正己烷),得青稞麸皮提取物(E-4)59.2g。
该提取物中,β-谷甾醇:菜油甾醇:岩藻甾醇:豆甾醇:链甾醇的比例为34.1:11.4:4.8:3.6:1。
实施例5、一种青稞麸皮提取物的制备:
将0.5kg青稞麸皮粉碎成50目,将其放入1L的超临界萃取釜中,先对萃取釜和分离釜进行预热,贮罐进行冷却,达到45℃时,打开CO2气瓶送气,并打开高压泵升压,达到10MPa时,开始循环萃取,调节CO2流量至15L/h,恒温恒压萃取1h后多次从分离釜1和分离釜2收集萃取物,至无萃取物为止,分离釜1的压力8MPa,温度35℃,分离釜2的压力5MPa,温度28℃。从分离釜中收集萃取物,经低温干燥(于60℃干燥2h)后得青稞麸皮提取物(E-5)11g。
实施例6、一种青稞麸皮提取物的制备:
将0.5kg青稞麸皮粉碎成50目,将其放入1L的超临界萃取釜中,先对萃取釜和分离釜进行预热,贮罐进行冷却,达到35℃时,打开CO2气瓶送气,并打开高压泵升压,达到20MPa时,开始循环萃取,调节CO2流量至15L/h,恒温恒压萃取3h后,从分离釜中收集萃取物,经低温干燥后得青稞麸皮提取物(E-6)19.5g。
实施例7、一种青稞麸皮提取物的制备:
将0.5kg青稞麸皮粉碎成50目,将其放入1L的超临界萃取釜中,先对萃取釜和分离釜进行预热,贮罐进行冷却,达到45℃时,打开CO2气瓶送气,并打开高压泵升压,达到15MPa时,开始循环萃取,调节CO2流量至15L/h,恒温恒压萃取2.0h后从分离釜中收集萃取物,经低温干燥后得青稞麸皮提取物(E-7)23.8g。
实施例8、一种青稞麸皮提取物的制备:
将0.5kg青稞麸皮粉碎成50目,将其放入1L的超临界萃取釜中,先对萃取釜和分离釜进行预热,贮罐进行冷却,达到45℃时,打开CO2气瓶送气,并打开高压泵升压,达到20MPa时,开始循环萃取,调节CO2流量至20L/h,恒温恒压萃取2.5h后从分离釜中收集萃取物,经低温干燥后得青稞麸皮提取物(E-8)31.3g。
实验1:青稞麸皮提取物的α-葡萄糖苷酶抑制活性的测定
10μL适量浓度的供测样品溶液与45μL 0.5U/mLα-葡萄糖苷酶磷酸盐溶液(0.1mol/L,pH=6.8),振荡混匀后于37℃水浴中孵育10min。加入45μL 5mmol/L的pNPG溶液开始启动反应,振荡混匀,37℃水浴中反应20min后,加入100μL 0.2mol/L Na2CO3水溶液终止反应,振荡混匀后,于405nm测定吸光值。以磷酸盐缓冲液(0.1mol/L,pH=6.8)替代α-葡萄糖苷酶溶液测定样品空白;以磷酸盐缓冲液替代供测样品测定酶液空白。按照下式计算抑制率,并用Origin软件求出相应的IC50值。
其中,Ac为未添加样品反应液的吸光值,A0为酶液空白,As为添加样品的反应液的吸光值,Ab为样品空白。
实施例1-8所制备的青稞麸皮提取物的α-葡萄糖苷酶抑制活性如表1所示。
青稞麸皮提取物 | 得率 | 总甾醇含量(g/kg) | IC50(mg/mL) |
E-1 | 2.03% | 13.4 | 2.17 |
E-2 | 3.56% | 21.7 | 1.15 |
E-3 | 2.92% | 31.2 | 0.16 |
E-4 | 5.92% | 39.8 | 0.046 |
E-5 | 2.20% | 19.2 | 1.52 |
E-6 | 3.90% | 29.5 | 0.58 |
E-7 | 4.76% | 40.6 | 0.077 |
E-8 | 6.26% | 54.3 | 0.021 |
阿卡波糖 | 7.87 |
对比例1-1、将实施例4中的萃取剂由正己烷改成50%乙醇,其余等同于实施例4,得青稞麸皮提取物200.3g。
对比例1-2、将实施例4中的萃取剂由正己烷改成乙酸乙酯,其余等同于实施例4,得青稞麸皮提取物55.7g。
对比例1-3、将实施例4中的“青稞麸皮”改成“青稞”,其余等同于实施例4,得青稞提取物15.7g。
对比例1-4、将青稞麸皮利用如下常规技术进行提取:
1kg青稞麸皮用10倍量50%乙醇(用0.2%柠檬酸调pH至2)53℃水浴浸提3h,过滤,滤液减压浓缩去除有机溶剂,得青稞麸皮提取物235.1g。
对比例2-1、将实施例8的超临界萃取的工艺参数改成如下:萃取压力10MPa、温度35℃、萃取时间3h、CO2流量为10L/h,其余等同于实施例8。
对比试验,将上述所有对比例所得的青稞麸皮提取物(除对比例1-3为青稞提取物)如同上述实验1所述方法进行检测,所得结果如下表2所述。
表2
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
Claims (6)
1.青稞麸皮提取物在制备α-葡萄糖苷酶活性抑制剂中的应用,其特征是:用于制备预防和/或治疗II型糖尿病的药物、保健品或食品;
青稞麸皮提取物中含有质量百分比为10%-60%的总甾醇。
2.根据权利要求1所述的青稞麸皮提取物在制备α-葡萄糖苷酶活性抑制剂中的应用,其特征是:所述总甾醇包括β-谷甾醇、菜油甾醇、岩藻甾醇、豆甾醇和链甾醇。
3.根据权利要求1所述的青稞麸皮提取物在制备α-葡萄糖苷酶活性抑制剂中的应用,其特征是:
青稞麸皮提取物的获取方法为:将青稞麸皮粉碎后过筛,按照1g/4~8ml的料液比,加入有机溶剂后进行回流提取,提取次数为至少一次,提取总时间为0.5~6h;然后过滤,滤液减压浓缩去除有机溶剂,得青稞麸皮提取物;
所述有机溶剂为石油醚、正己烷或环己烷。
4.根据权利要求3所述的青稞麸皮提取物在制备α-葡萄糖苷酶活性抑制剂中的应用,其特征是:所述有机溶剂为正己烷,共回流提取3次;每次提取时的料液比为1g/6ml、时间为1.5h。
5.根据权利要求1所述的青稞麸皮提取物在制备α-葡萄糖苷酶活性抑制剂中的应用,其特征是:
青稞麸皮提取物的获取方法为:将青稞麸皮粉碎后过筛,进行超临界CO2流体萃取,萃取条件为:萃取压力为10~40MPa,萃取温度25~60℃,CO2流速10~25L/h,萃取时间1~5h,分离温度28~50℃,得青稞麸皮提取物。
6.根据权利要求5所述的青稞麸皮提取物在制备α-葡萄糖苷酶活性抑制剂中的应用,其特征是:所述超临界CO2流体的萃取条件为:萃取压力20~25MPa、萃取温度40~45℃、萃取时间2.0~2.5h,CO2流量15~20L/h。
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