CN110663788A - 一种茶叶提取物及其工业化制备方法和用途 - Google Patents
一种茶叶提取物及其工业化制备方法和用途 Download PDFInfo
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- CN110663788A CN110663788A CN201910946691.7A CN201910946691A CN110663788A CN 110663788 A CN110663788 A CN 110663788A CN 201910946691 A CN201910946691 A CN 201910946691A CN 110663788 A CN110663788 A CN 110663788A
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- tea
- alcohol
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- tea extract
- drying
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Classifications
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Abstract
本发明涉及一种茶叶提取物及其工业化制备方法和用途,所述茶叶提取物中总儿茶素含量不低于85wt%,表没食子儿茶素没食子酸酯含量在65wt%以上,咖啡因含量不高于0.3%。本发明将茶叶提取、纯化、浓缩、干燥后,得到茶叶提取物,收率比现有工艺提高近3倍。未使用任何有毒有机溶剂,较为适合大规模生产。所制备的茶叶提取物具有较好的抗雾霾、保护肺功能,并且对神经细胞具有保护作用。
Description
技术领域
本发明涉及一种茶叶提取物的产品,具有较高含量的表没食子儿茶素没食子酸酯和总儿茶素类的化学成分。本发明还涉及到茶叶提取物的制备工艺,通过提取、纯化、浓缩干燥而成,该提取物可用作食品、保健食品或者药品。
背景技术
茶在我国有着数千年的悠久历史,自古就有“神农尝百草,日遇七十二毒,得茶而解之”的传说,现代饮茶有益健康已成为普遍的常识。我国茶叶品种很多,其中绿茶独占大头,覆盖面最广,也最受大众喜爱。茶多酚约占绿茶干重的20-30%,其中主要活性成分为儿茶素类化合物。儿茶素类主要有以下几种组成:儿茶素(C)、表儿茶素(EC)、没食子儿茶素(GC)、表没食子儿茶素(EGC)、表儿茶素没食子酸酯(ECG)、表没食子儿茶素没食子酸酯(EGCG)、儿茶素没食子酸酯(CG)、没食子儿茶素没食子酸酯(GCG)等。国内外大量的科学研究表明,儿茶素类化合物具有抑菌消炎、抗肿瘤、抗病毒、抗氧化、降血脂、降血糖、抑制肥胖、抗老年痴呆、调节免疫等多种药理功效。其中EGCG是绿茶茶多酚的主成分,也是绿茶儿茶素类的主成分。许多研究表明EGCG具有抗自由基DNA损害,抗辐射和紫外线,阻止油脂过氧化,减少血清中低密度胆固醇、超低密度胆固醇和甘油三酯的含量,干扰癌细胞生存所需的信号传递,抑制饮食中的致癌物质,与肠、肝、和肺中的其他酶和抗氧化剂作用共同阻止某些致癌物质的活力,清除自由基,抵御污染、日晒和吸烟的影响,防治皮肤老化和起皱。
由于绿茶成分复杂,从中制取高纯度的儿茶素类且含有较高含量EGCG化学成分的茶叶提取物具有很大的技术难度。美国专利[US7763291]使用热水对经干燥后的绿茶进行提取,再使用乙酸乙酯对水提物进行萃取,将乙酸乙酯浓缩后上大孔树脂,收集体积浓度为40%甲醇溶液的洗脱液,浓缩干燥后得到总儿茶素类成分和EGCG含量分别占85%和55%以上的茶叶提取物,收率为2.2wt%。虽然制备了较高含量的儿茶素类和EGCG成分,但工序中使用了大量的乙酸乙酯和甲醇,此有机溶剂有毒有害,易燃易爆,给人体健康造成危害,给生产环境带来风险,既不安全也环保,更不是绿色生产,此外,此工序获得的茶叶提取物收率较低,造成资源利用率低,从而使成本上升,也无法满足绿色生产。
乙醇是一种常见的有机试剂,并且具备无毒性、价格低、易回收等优势,和使用较昂贵的仪器和有毒的化学试剂相比,既节约了成本,又减少了废液排放对环境的污染。本申请针对这一问题,使用低度乙醇水溶液对茶叶进行提取,再使用柱层析纯化后,获得了较高含量的儿茶素类和EGCG成分的茶叶提取物,收率提高了近3倍。本工艺资源利用率高、操作简便、能源消耗低,降低了生产成本,绿色环保,适合大规模生产。
发明内容
本发明的目的是提供了一种具有较高含量的没食子儿茶素没食子酸酯和总儿茶素类的茶叶提取物。
本发明的另一目的是提供了一种具有较高含量的没食子儿茶素没食子酸酯和总儿茶素类的茶叶提取物的制备方法。
本发明的另一目的是提供了本发明茶叶提取物用于制备预防、改善或者治疗神经相关疾病的食品、保健食品或者药品的应用。
本发明的另一目的是提供了本发明茶叶提取物用于制备改善或治疗记忆力衰退的食品、保健食品或者药品的应用。
本发明的另一目的是提供了本发明茶叶提取物用于制备治疗或预防帕金森症和阿尔茨海默症的食品、保健食品或者药品的应用。
本发明的另一目的是提供了本发明茶叶提取物用于制备治疗或预防PM 2.5颗粒物引起的症状的食品、保健食品或者药品的应用。
本发明的另一目的是提供了本发明茶叶提取物用于制备保护或提高肺部功能的食品、保健食品或者药品的应用。
本发明的另一目的是提供了本发明茶叶提取物用于制备治疗或预防肺部疾病的食品、保健食品或者药品的应用。
本发明的目的是通过下述技术方案实现的:
一种茶叶提取物,其特征在于通过下述方法制备得到:将茶叶鲜品或干品与醇水溶液混合,依次进行提取、纯化、干燥得到茶叶提取物。
优选地,所述的茶叶提取物,其特征在于茶叶选用地上或地下部分,其中地上部分优选自叶、茎或种子。
优选地,所述的茶叶提取物,其特征在于通过下述方法制备得到:
(1)将茶叶洗净、晾干、或者粉碎获得茶叶提取原料。
(2)将一定量的茶叶提取原料与醇水溶液(醇的体积浓度为20%~50%)按质量比至少为1:5混合,恒温70~90℃提取至少30min,提取次数至少为2次;
(3)提取完成后,将步骤(2)中的药材提取液冷却至室温,过滤,滤液浓缩至无醇味;
(4)将步骤(3)中的滤液先进行1号柱层析分离,所得洗脱液浓缩至无醇味后,再进行2号柱层析分离,将所得的洗脱液浓缩至50℃时的相对比重为1.05~1.20时,经干燥得到茶叶提取粉。
优选地,所述1号柱层析的填料为聚酰胺树脂以及LXD-200、HP-20、LX-8、D101、AB-8等大孔树脂,更优选地选用聚酰胺树脂。
优选地,所述2号柱层析的填料为LXD-200、HP-20、LX-8、D101、AB-8等大孔树脂,更优选地选用LXD-200或HP-20大孔树脂。
优选地,所述1号柱层析分离方法为:将提取液加至装有聚酰胺树脂层析柱中,先使用至少2BV(柱体积)水洗脱,再使用至少2BV体积浓度至少为60%醇溶液洗脱,收集醇洗脱液。
优选地,所述2号柱层析分离方法为:将1号柱层析醇水洗脱液浓缩至无醇味后,进入2号柱层析分离,先使用至少2BV水洗脱,再使用至少2BV体积浓度至少为20%醇溶液洗脱,收集醇洗脱液。
优选地,所涉及到的醇可以为甲醇、乙醇、正丁醇、异丙醇等,更优选地选用乙醇。
优选地,所述茶叶提取物的收率不低于6wt%。
优选地,所述的茶叶提取物其特征在于:总儿茶素含量不低于85wt%,没食子儿茶素没食子酸酯含量在65wt%以上,咖啡因含量不高于0.3%。
优选地,所述的茶叶提取物,其特征在于其制备方法中的浓缩方法可以为真空浓缩、常压浓缩、膜浓缩法,优选地使用真空浓缩法。
优选地,所述的茶叶提取物,其特征在于其制备方法中的干燥方法可以为真空干燥、加热干燥、晾干、风干、冷冻干燥和喷雾干燥法。
一种组合物,其含有上述茶叶提取物,和药物或食品上可接受的助剂。
优选地,所述组合物的剂型选自素片、薄膜包衣片、糖衣片、肠衣片、分散片、胶囊、颗粒剂、口服溶液或口服混悬液,以及液体、乳液、膏霜、粉、块状等化妆品剂型。
本发明所述的茶叶提取物及其组合物用于制备预防、改善或者治疗神经相关疾病的食品、保健食品或者药品的应用;用于制备治疗或预防PM 2.5颗粒物引起的症状的的食品、保健食品或者药品的应用。
与现有技术相比,本发明具有如下优点:
(1)本发明涉及到的茶叶提取物制备工艺中,仅使用乙醇和水作为溶剂,没有使用其它任何有毒有害,易燃易爆的化学物质或者溶剂,对人体无害,工艺安全系数高,绿色环保,无污染,无残留。
(2)本发明涉及到的茶叶提取物的制备工艺的收率是现有工艺的3倍,资源利用率高,节约生产成本。
(3)本发明涉及到的茶叶提取物制备工艺,既保证了有效成分的含量,也降低了咖啡因的含量,低至0.3%以下。
实施例
下面通过实施例对本发明作进一步说明。应该理解的是,本发明实施例所述方法仅仅是用于说明本发明,而不是对本发明的限制,在本发明的构思前提下对本发明制备方法的简单改进都属于本发明要求保护的范围。如无特别说明,实施例中用到的所有原料和溶剂均为市售产品。
制备实施例1
取干绿茶(都匀毛尖)100kg,加入1000L体积浓度为20%的乙醇水溶液,升温至80℃时提取30min,提取2次后,合并两次提取液,冷却至室温,过60目筛网,浓缩至无醇味。
提取浓缩液按1BV/h的流速上样至装有聚酰胺树脂的1号柱层析,上样完成后,使用2BV纯化水洗脱后,再使用2BV体积浓度为80%的乙醇水溶液洗脱,收集洗脱液并浓缩至为无醇味。
将1号柱层析洗脱液的浓缩液上样至装有LXD-200树脂的2号柱层析,使用2BV纯化水洗脱后,再使用2BV体积浓度为25%的乙醇水溶液洗脱,收集洗脱液并浓缩50℃时的相对比重为1.20时,喷雾干燥后,得到6.1kg茶叶提取物(C-1),总儿茶素类含量为90.3%,EGCG含量为68.2%,咖啡因含量为0.14%,收率为6.1wt%。
制备实施例2
取干绿茶(信阳毛尖)100kg,加入1000L体积浓度为20%的乙醇水溶液,升温至70℃时提取30min,提取3次后,合并三次提取液,冷却至室温,过80目振荡筛,浓缩至无醇味。
提取浓缩液按1BV/h的流速上样至装有聚酰胺树脂的1号柱层析,上样完成后,使用2BV纯化水洗脱后,再使用2BV体积浓度为75%的乙醇水溶液洗脱,收集洗脱液并浓缩至为无醇味。
将1号柱层析洗脱液的浓缩液上样至装有LXD-200树脂的2号柱层析,使用2BV纯化水洗脱后,再使用2BV体积浓度为40%的乙醇水溶液洗脱,收集洗脱液并浓缩50℃时的相对比重为1.10时,真空干燥后,得到6.4kg茶叶提取物(C-2),总儿茶素类含量为86.9%,EGCG含量为67.1%,咖啡因含量为0.16%,收率为6.4wt%。
制备实施例3
取干绿茶(碧螺春)100kg,加入1000L体积浓度为20%的乙醇水溶液,升温至90℃时提取30min,提取2次后,合并两次提取液,冷却至室温,板框过滤,浓缩至无醇味。
提取浓缩液按1BV/h的流速上样至装有聚酰胺树脂的1号柱层析,上样完成后,使用2BV纯化水洗脱后,再使用2BV体积浓度为70%的乙醇水溶液洗脱,收集洗脱液并浓缩至为无醇味。
将1号柱层析洗脱液的浓缩液上样至装有LXD-200树脂的2号柱层析,使用2BV纯化水洗脱后,再使用2BV体积浓度为20%的乙醇水溶液洗脱,收集洗脱液并浓缩50℃时的相对比重为1.20时,冷冻干燥后,得到6.4kg茶叶提取物(C-3),总儿茶素类含量为89.0%,EGCG含量为66.4%,咖啡因含量为0.26%,收率为6.4wt%。
制备实施例4
取干绿茶(都匀毛尖)100kg,加入1000L体积浓度为50%的乙醇水溶液,升温至80℃时提取30min,提取2次后,合并两次提取液,冷却至室温,板框过滤,浓缩至无醇味。
提取浓缩液按1BV/h的流速上样至装有聚酰胺树脂的1号柱层析,上样完成后,使用2BV纯化水洗脱后,再使用2BV体积浓度为70%的乙醇水溶液洗脱,收集洗脱液并浓缩至为无醇味。
将1号柱层析洗脱液的浓缩液上样至装有HP-20树脂的2号柱层析,使用2BV纯化水洗脱后,再使用2BV体积浓度为30%的乙醇水溶液洗脱,收集洗脱液并浓缩50℃时的相对比重为1.20时,冷冻干燥后,得到6.5kg茶叶提取物(C-4),总儿茶素类含量为87.1.0%,EGCG含量为66.7%,咖啡因含量为0.23%,收率为6.5wt%。
制备实施例5
取干绿茶(都匀毛尖)100kg,加入1000L体积浓度为20%的乙醇水溶液,升温至70℃时提取30min,提取2次后,合并两次提取液,冷却至室温,板框过滤,浓缩至无醇味。
提取浓缩液按1BV/h的流速上样至装有聚酰胺树脂的1号柱层析,上样完成后,使用2BV纯化水洗脱后,再使用2BV体积浓度为85%的乙醇水溶液洗脱,收集洗脱液并浓缩至为无醇味。
将1号柱层析洗脱液的浓缩液上样至装有HP-20树脂的2号柱层析,使用2BV纯化水洗脱后,再使用2BV体积浓度为30%的乙醇水溶液洗脱,收集洗脱液并浓缩50℃时的相对比重为1.20时,冷冻干燥后,得到6.4kg茶叶提取物(C-5),总儿茶素类含量为86.6%,EGCG含量为68.4%,咖啡因含量为0.19%,收率为6.4wt%。
制备实施例6
取干绿茶(都匀毛尖)100kg,加入1000L体积浓度为30%的乙醇水溶液,升温至85℃时提取30min,提取2次后,合并两次提取液,冷却至室温,板框过滤,浓缩至无醇味。
提取浓缩液按1BV/h的流速上样至装有聚酰胺树脂的1号柱层析,上样完成后,使用2BV纯化水洗脱后,再使用2BV体积浓度为65%的乙醇水溶液洗脱,收集洗脱液并浓缩至为无醇味。
将1号柱层析洗脱液的浓缩液上样至装有HP-20树脂的2号柱层析,使用2BV纯化水洗脱后,再使用2BV体积浓度为20%的乙醇水溶液洗脱,收集洗脱液并浓缩50℃时的相对比重为1.20时,喷雾干燥后,得到6.1kg茶叶提取物(C-6),总儿茶素类含量为86.6%,EGCG含量为65.9%,咖啡因含量为0.25%,收率为6.1wt%。
生物活性实施例1(神经细胞的保护作用评价)
使用含10%胎牛血清的高糖DMEM培养基培养PC12细胞,细胞传代时用0.125%的胰酶消化约50s,用含10%血清的DMEM培养基终止消化,加入新鲜的培养基将细胞吹打均匀。以105个/mL的细胞密度传代。每瓶细胞加入4mL含细胞的培养液。在37℃,5%CO2条件下培养。PC12细胞在培养瓶中生长至融合状态,采用0.125%胰蛋白酶溶液消化,并反复吹打至细胞悬液,以含10%FBS的高糖DMEM培养基稀释成1.0×105个/mL,每孔100μL接种于96孔培养板中,每组5-6个复孔,在37℃,5%CO2条件下培养24h,成融合状态。
96孔板于各孔中以一定浓度梯度分别给予药物100μL,培养24h后,MTT法检测细胞活力。50mg MTT溶解于10mL PBS,0.22μm微孔滤膜过滤。临用前稀释到0.5mg/mL。各组细胞弃去培养基,PBS洗两次,每加入0.5mg/mL MTT,37℃,5%CO2条件下孵育3h,除去MTT工作液,每孔加入150μL DMSO溶解结晶,振摇10min,测定每孔的OD值(测定波长570nm,参比波长650nm)。以对照组OD值平均值为100%细胞活力,计算模型组和给药组的细胞活力。
实验分组:1)空白组(高糖DMEM);2)模型组(高糖DMEM培养3h,再加入H2O2使其终浓度为100μM,刺激1h);3)阳性药(NAC)组:先加入含一定浓度(500μM)阳性药NAC的高糖DMEM培养3h,再加100μM H2O2刺激1h;4)给药组:先加入含各浓度梯度茶叶提取物(C-1)的高糖DMEM培养3h,再加100μM H2O2刺激1h。上述各组于相同条件下培养,随后进行实验,并用MTT法检测细胞活力,测定结果如表1和表2所示。
表1茶叶提取物对正常PC12细胞活力的影响
与对照组比较,***p<0.001
表2茶叶提取物对H2O2诱导的PC12细胞氧化损伤的影响
与对照组比较,###p<0.001;与模型组比较,***p<0.001
如表1所示,茶叶提取物在浓度为1~3μg/mL时,对正常PC12细胞活力没有显示抑制作用,在浓度为10~500μg/mL时,对PC12细胞活力显示较强的抑制作用,并呈现浓度依赖性。PC12细胞本身是一种肿瘤神经元细胞,由此可见茶叶提取物具有潜在的抗肿瘤活性。
如表2所示,模型组中PC12细胞活力为50.45%,与对照组(细胞活力为100%)相比较,p<0.001,表明模型构建成功。当加入500μM阳性对照药NAC培养后,细胞活力提高到100.80%,显示有明显的保护作用。茶叶提取物在浓度为10μg/mL和30μg/mL时,保护作用由50.45%分别提高至87.76%和94.48%,表明茶叶提取物对神经细胞具有较强的保护作用。
生物活性实施例2(抗PM 2.5作用评价)
随机选取90尾受精后2天(2dpf)转基因中性粒细胞与巨噬细胞绿色荧光斑马鱼于六孔板中,每孔(实验组)均处理30尾斑马鱼,静脉窦注射给予红色荧光染料建立斑马鱼巨噬细胞吞噬功能模型。模型斑马鱼用水溶给予茶叶提取物(C-1)浓度为31.25μg/mL,同时设置模型对照组,每孔(实验组)容量为3mL。结束后,每组随机取10尾斑马鱼在荧光显微镜下采集图片,用图像处理软件计算巨噬细胞吞噬清除剩余的荧光微球的数量(N);根据荧光微球剩余数量评价茶叶提取物对巨噬细胞吞噬功能的促进作用,计算公式为:巨噬细胞吞噬促进作用(%)=(N模型对照组-N供试品组)/N模型对照组×100%,统计学处理结果以mean±SE表示,采用方差分析和Dunnett’s T-检验,p<0.05为差异性显著,结果如表3所示。
表3斑马鱼巨噬细胞吞噬作用数据汇总(n=10)
与模型对照组比较,**p<0.01
由表3可知,茶叶提取物组斑马鱼的荧光微球数量为27个,与模型对照组(41个)比较p<0.01,对巨噬细胞吞噬促进率为34%,说明茶叶提取物对斑马鱼巨噬细胞吞噬功能均有促进作用。
Claims (9)
1.一种茶叶提取物,其特征在于:总儿茶素含量不低于85wt%,表没食子儿茶素没食子酸酯含量在65wt%以上,咖啡因含量不高于0.3%。
2.根据权利要求1所述的茶叶提取物,其特征在于:所述茶叶选自绿茶,优选都匀毛尖、信阳毛尖、碧螺春和黄山毛峰;茶叶选用地上或地下部分,其中地上部分优选自叶、茎或种子。
3.根据权利要求1或2所述的茶叶提取物的制备方法,其特征在于通过下述方法制备得到:将茶叶鲜品或干品与醇水溶液混合,依次进行提取、纯化、干燥得到茶叶提取物。
4.根据权利要求3所述茶叶提取物的制备方法,其特征在于通过下述方法制备得到:
(1)将茶叶洗净、晾干、或者粉碎获得茶叶提取原料。
(2)将一定量的茶叶提取原料与醇水溶液(醇的体积浓度为20%~50%)按质量比至少为1:5混合,恒温70~90℃提取至少30min,提取次数至少为2次;
(3)提取完成后,将步骤(2)中的药材提取液冷却至室温,过滤,滤液浓缩至无醇味;
(4)将步骤(3)中的滤液先进行1号柱层析分离,所得洗脱液浓缩至无醇味后,再进行2号柱层析分离,将所得的洗脱液浓缩至约50℃时的相对比重为1.05~1.20时,经干燥得到茶叶提取粉。
5.根据权利要求4所述的制备方法,其特征在于:所述1号柱层析的填料为聚酰胺树脂以及LXD-200、HP-20、LX-8、D101、AB-8等大孔树脂,优选地选用聚酰胺树脂;所述2号柱层析的填料为LXD-200、HP-20、LX-8、D101、AB-8等大孔树脂,优选地选用LXD-200或HP-20大孔树脂。
6.根据权利要求4所述的制备方法,其特征在于:所述1号柱层析分离方法为:将提取液加至装有聚酰胺树脂层析柱中,先使用至少2BV(柱体积)水洗脱,再使用至少2BV体积浓度至少为60%醇溶液洗脱,收集醇洗脱液;所述2号柱层析分离方法为:将1号柱层析醇水洗脱液浓缩至无醇味后,进入2号柱层析分离,先使用至少2BV水洗脱,再使用至少2BV体积浓度至少为20%醇溶液洗脱,收集醇洗脱液。
7.根据权利要求4所述的制备方法,其特征在于:所述醇为甲醇、乙醇、正丁醇或异丙醇,优选为乙醇;所述浓缩方法为真空浓缩、常压浓缩、膜浓缩法,优选地使用真空浓缩法;所述干燥方法为真空干燥、加热干燥、晾干、风干、冷冻干燥和喷雾干燥法;优选地,所述茶叶提取物的收率不低于6wt%。
8.一种组合物,其含有1或2所述的茶叶提取物,以及药物或食品上可接受的助剂;优选地,所述组合物的剂型选自素片、薄膜包衣片、糖衣片、肠衣片、分散片、胶囊、颗粒剂、口服溶液或口服混悬液,以及液体、乳液、膏霜、粉、块状等化妆品剂型。
9.根据权利要求1所述的茶叶提取物,权利要求8所述的组合物用于制备预防、改善或者治疗神经相关疾病的食品、保健食品或者药品的应用;用于制备治疗或预防PM 2.5颗粒物引起的症状的的食品、保健食品或者药品的应用。
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