CN110887713A - 一种黑茶茶褐素对照品及其应用 - Google Patents
一种黑茶茶褐素对照品及其应用 Download PDFInfo
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Abstract
一种黑茶茶褐素对照品,是取黑茶,用沸水浸提取得到黑茶水提取物,将所得黑茶水提取物过滤、浓缩至膏状;将膏状物用乙醇进行超声提取,反复多次直至乙醇无色,乙醇不溶物用60‑100℃热水溶解,旋转蒸发至无醇味,再加入等体积的乙酸乙酯萃取多次,至乙酸乙酯层无色;将水相旋转蒸发去除残留的乙酸乙酯,然后用离心滤膜离心,截留液加水再次离心,反复多次,直至离心透过液无色,将截留液真空冷冻干燥,所得粉末即为茶褐素对照品。从而建立检测黑茶茶褐素含量的高效液相色谱法和紫外分光光度法,以对黑茶茶褐素含量作出较为准确、快速的检测,有利于黑茶茶褐素的分析测定。
Description
技术领域
本发明涉及黑茶茶褐素含量检测用试剂,具体涉及一种黑茶茶褐素含量检测用对照品,同时涉及该对照品在检测黑茶茶褐素含量的高效液相色谱法和紫外分光光度法中的应用。
背景技术
黑茶茶褐素是指一类性质类似于红茶茶褐素的高聚合物,能溶于水而不溶于乙醇、二氯甲烷、乙酸乙酯和正丁醇等有机溶剂的褐色色素,它们是一类十分复杂的化合物,主要由多酚与蛋白质或多糖形成的复合物,现代研究表明,茶褐素具有降脂减肥、抗氧化、降血脂等多种生理活性。
传统的茶褐素含量检测采用红茶茶褐素检测方法,很多学者在研究中发现,将红茶与普洱茶、茯砖茶的色谱图进行对比,发现红茶与黑茶在380nm处具有截然不同的色谱图及化学组成成分,因此,红茶茶褐素检测方法并不适用于黑茶茶褐素检测方法。
发明内容
本发明的目的是针对上述现有技术的不足,提供一种黑茶茶褐素含量检测用对照品,同时提供根据该对照品的光谱性质,建立检测黑茶茶褐素含量的高效液相色谱法和紫外分光光度法,以对黑茶茶褐素含量作出较为准确、快速的检测,而利于黑茶茶褐素的分析测定。
为达上述目的,本发明所采用的技术方案是:一种黑茶茶褐素对照品,该茶褐素对照品是按如下步骤制备得到:
a.以黑茶为原料,用沸水浸提得到黑茶水提取物,将所得黑茶水提取物过滤、浓缩至膏状;
b.将膏状物用95%质量浓度的乙醇进行超声提取,重复多次直至乙醇无色,乙醇不溶物用热水溶解,旋转蒸发至无醇味,再加入等体积的乙酸乙酯萃取多次,至乙酸乙酯层无色;
c.将水相旋转蒸发去除残留的乙酸乙酯,然后用离心滤膜离心,截留液加水再次离心,反复多次,直至离心透过液无色,将截留液真空冷冻干燥,所得粉末即为茶褐素对照品。
上述步骤a中黑茶水提取物是于黑茶中加入为黑茶重量8-10倍的沸水,80-100℃水浴浸提2-3次,每次30-60min,合并浸提液,即得。
上述步骤b中超声提取时的温度为25-60℃,每次提取时间为20-30min,乙醇用量为膏状物重量的2-3倍;乙醇不溶物用为乙醇不溶物重量1-2倍的60-100℃热水溶解。
上述步骤c中离心滤膜孔径为3000Da。
上述黑茶为普洱茶或茯砖茶。
茶褐素是一类能溶于水而不溶于乙醇、二氯甲烷、乙酸乙酯和正丁醇等有机溶剂的褐色色素。本试验根据茶褐素的定义和性质采用沸水提取,然后用95%乙醇和乙酸乙酯除去儿茶素类、咖啡因、茶红素等杂质,这些杂质在280nm下具有较强吸收;再采用低分子量的滤膜除去小分子量的化合物,从而截留分子量大的色素,而获得茶褐素对照品。
本发明同时提供上述黑茶茶褐素对照品在建立黑茶茶褐素的高效液相色谱测定方法和紫外分光光度测定方法中的应用。该高效液相色谱测定方法和紫外分光光度测定方法的检测波长皆为440nm。
采用本发明的黑茶茶褐素对照品建立的高效液相色谱法和紫外分光光度法对不同黑茶样品进行检测,结果显示,该两种方法检测的茶褐素含量无显著性差异,但与传统罗伯特方法检测的茶褐素含量具有极显著性差异。本紫外分光光度法制样简单,只需提取直接检测,可代替高效液相色谱法进行快速检测。
附图说明
图1是茶褐素对照品及黑茶水提取物的色谱图(280nm)。
图2是茶褐素对照品的UV-VIS光谱图。
图3是HPLC测定方法的茶褐素对照品标准曲线。
图4是黑茶水提取物与茶褐素对照品的HPLC图(440nm)。
图5是茶褐素对照品与吸光度的关系图。
具体实施方式
实施例1
以普洱茶为原料,于普洱茶中加入为普洱茶重量8倍的沸水,室温下浸提2-3次,每次60min,合并浸提液,得普洱茶水提取物,将该普洱茶水提取物过滤、浓缩到至膏状;将膏状物用2倍重量的95%质量浓度的乙醇于25℃超声提取30min,反复多次,直至乙醇无色,去除乙醇提取液,乙醇不溶物用1倍重量的60℃热水溶解后旋转蒸发至无醇味,再加入等体积的乙酸乙酯萃取多次,至乙酸乙酯层无色;将水相旋转蒸发去除残留的乙酸乙酯,然后用3000Da的离心滤膜离心,截留液加水再次离心,反复多次,直至离心透过液无色,将截留液真空冷冻干燥,所得粉末即为茶褐素对照品。
实施例2
以茯砖茶为原料,于茯砖茶中加入为茯砖茶重量10倍的沸水,室温下浸提2-3次,每次30min,合并浸提液,得茯砖茶水提取物,将该茯砖茶水提取物过滤、浓缩到至膏状;将膏状物用3倍重量的95%质量浓度的乙醇于60℃超声提取20min,反复多次,直至乙醇无色,去除乙醇提取液,乙醇不溶物用2倍重量的100℃热水溶解后旋转蒸发至无醇味,再加入等体积的乙酸乙酯萃取多次,至乙酸乙酯层无色;将水相旋转蒸发去除残留的乙酸乙酯,然后用3000Da的离心滤膜离心,截留液加水再次离心,反复多次,直至离心透过液无色,将截留液真空冷冻干燥,所得粉末即为茶褐素对照品。
实施例3
以普洱茶为原料,于普洱茶中加入为普洱茶重量9倍的沸水,室温下浸提2-3次,每次45min,合并浸提液,得普洱茶水提取物,将该普洱茶水提取物过滤、浓缩到至膏状;将膏状物用2倍重量的95%质量浓度的乙醇于40℃超声提取25min,反复多次,直至乙醇无色,去除乙醇提取液,乙醇不溶物用2倍重量的80℃热水溶解后旋转蒸发至无醇味,再加入等体积的乙酸乙酯萃取多次,至乙酸乙酯层无色;将水相旋转蒸发去除残留的乙酸乙酯,然后用3000Da的离心滤膜离心,截留液加水再次离心,反复多次,直至离心透过液无色,将截留液真空冷冻干燥,所得粉末即为茶褐素对照品。
实施例4
1.茶褐素对照品的色谱峰
结合参见图1,黑茶水提取物(图中用黑茶水提物表示)在0-40min内具有多个色谱峰,这些色谱峰为茶叶中一些有机酸、儿茶素、咖啡因、黄酮等成分,而制备的茶褐素对照品在25-45min内形成一个呈正态分布的色谱峰,无其他峰。
2.茶褐素对照品紫外吸收特征
由图2可知,茶褐素对照品在280nm、340nm处有较大吸收峰,在440nm也有一个平稳吸收。280nm、340nm处的吸收说明分子中含有苯环,并且苯环上带酚羟基,表明茶褐素为多酚类聚合物,同时蛋白质、核酸等在该波长下也有一定吸收。440nm为可见光区,表明茶褐素含有一定助色基团和生色基团,应为茶褐素的特征吸收。故选择440nm作为高效液相色谱法和紫外分光光度法的检测波长。
3.茶褐素对照品含量的确定
3.1.多糖的含量测定
采用GC-三甲基硅烷柱前衍生方法测定。未水解样中未有葡萄糖、果糖、蔗糖、麦芽糖色谱峰,表明茶褐素对照品中已无游离糖。水解样中单糖组成及含量见表1,可见茶褐素对照品中多糖含量为9.99%。
表1茶褐素对照品多糖含量(%)
3.2.氨基酸及蛋白质含量测定
采用Waters公司专用氨基酸测定方法,绘制18种氨基酸的标准曲线,根据标准曲线计算样品中氨基酸含量。未水解样中未检测到游离氨基酸的存在,表明茶褐素对照品中已无游离氨基酸。蛋白质水解样中各氨基酸的含量见表2,可见茶褐素对照品中蛋白质总量为2.64%。
表2茶褐素对照品中蛋白质水解后氨基酸含量(%)
编号 | 氨基酸 | 含量 | 编号 | 氨基酸 | 含量 |
1 | 天冬氨酸 | 0.226 | 10 | 茶氨酸 | / |
2 | 丝氨酸 | 0.138 | 11 | 胱氨酸 | 0.606 |
3 | 谷氨酸 | 0.265 | 12 | 酪氨酸 | 0.033 |
4 | 甘氨酸 | 0.328 | 13 | 缬氨酸 | 0.208 |
5 | 组氨酸 | / | 14 | 蛋氨酸 | 0.066 |
6 | 精氨酸 | 0.051 | 15 | 赖氨酸 | 0.133 |
7 | 苏氨酸 | 0.055 | 16 | 异亮氨酸 | 0.015 |
8 | 丙氨酸 | 0.341 | 17 | 亮氨酸 | 0.086 |
9 | 脯氨酸 | 0.060 | 18 | 苯丙氨酸 | 0.029 |
合计 | 2.639 |
3.3.茶褐素对照品含量确定
茶褐素主要是茶多酚与多糖、蛋白质组成的大分子聚合物,因此,总量减去多糖与蛋白质后可视为茶褐素的含量。即茶褐素含量(%)=(1-多糖含量-蛋白质含量)×100%=(1-9.99%-2.64%)×100%=87.37%。
实施例5
1.HPLC检测方法
1.1色谱条件:色谱柱C18色谱柱,二极管阵列检测器(PDA),检测器温度30℃,进样量20μL,流速1mL/min;流动相A相为0.2%磷酸水,B相为乙腈,梯度洗脱:0-10min,B:3%→5%;10-50min,B:5%→60%;50-60min,B:60%→60%,柱温30℃。
1.2标准曲线的绘制:
准确称取21.35mg茶褐素对照品用蒸馏水定容至10mL,分别取5μL、10μL、25μL、50μL、100μL进样,绘制峰面积与茶褐素对照品质量的标准曲线(440nm下)。标准曲线见图3,线性回归方程为:y=27466013.2x+128564.7,R2=1.000,表明在茶褐素对照品在0.0098-0.196mg范围内,浓度与峰面积具有良好的相关性。
1.3样品测定:
取3.0g茶样,用100mL沸水冲泡10min,取5mL样液,用等体积乙酸乙酯萃取至乙酸乙酯层无色(5-8次),萃取下相用水补充至5ml,取20μL进高效液相色谱仪,记录440nm下的峰面积。黑茶水提取物(图4中用黑茶水提物表示)及茶褐素对照品的HPLC见图4,由图可知,黑茶水提取物在440nm下与茶褐素对照品在相同的保留时间内也具有相似的正态分布色谱峰,根据标准曲线计算黑茶中茶褐素的含量。
2.紫外检测方法(紫外分光光度法)
2.1标准曲线的绘制:
准确称取26.68mg茶褐素对照品用蒸馏水定容至25mL,分别准确移取4mL、3mL、2mL、1mL、0.5mL,加蒸馏水至5mL。用紫外分光光度计在440nm下,用1cm厚的比色皿,以蒸馏水做空白对照,测定其吸光度,结合参见图5。茶褐素对照品的标准曲线为:y=1.0003x+0.03,R2=0.998,可见茶褐素对照品浓度和吸光度具有良好的线性关系。
2.2样品测定:
取3.0g茶样,用100mL沸水在沸水浴中浸提10min,过滤,滤液用紫外分光光度计在440nm下,用1cm厚的比色皿,以蒸馏水做空白对照,测定不同茶样的吸光度。根据标准曲线所得公式,计算不同黑茶的吸光度所得的茶褐素含量。
3.传统罗伯特检测方法
根据茶学实验指导进行:准确称取9.00g茶样(不需磨碎),加入沸水375mL,沸水浴中浸提10min,过滤,滤液冷却至室温。用移液管分别吸取25ml茶汤滤液和25ml正丁醇放入分液漏斗中,摇振3min,待分层后将水层放于50ml三角瓶中,取水层液2ml加饱和草酸2ml,加入6ml蒸馏水,加95%乙醇定容至25ml,在380nm波长下进行测定(95%乙醇做空白对照)。
计算公式:茶褐素(%)=(吸光值×7.06)×100/样品干物重。
4.不同黑茶样品三种方法含量测定结果
不同黑茶样品采用上述三种方法测得的茶褐素含量结果见表3,从表3可知,罗伯特检测方法结果普遍偏高,是HPLC检测方法的1.2~2.2倍,是紫外检测方法的1.4~2倍,可能是由于检测波长为380nm,在该波长下存在杂质的干扰吸收。HPLC检测不同年份茯砖茶茶褐素的含量在1%~5%,有随储存年份延长含量增大的趋势;普洱熟茶茶褐素含量在7%~11%范围内,不同品牌产品间有一定差异性。推测茶褐素含量主要与加工工艺和原料组成密切相关,普洱熟茶的茶褐素含量要高于茯砖茶。采用SPSS软件进行方差分析,结果见表4,由表4可知,HPLC检测方法与紫外检测方法无显著性差异,二者与罗伯特检测方法均具有极显著性差异。
表3不同黑茶品种、不同检测方法的茶褐素含量(%)
注:小写字母表示品种间差异达显著水平(P<0.05)。
表4三种检测方法的方差分析结果
注:*、**分别表示在p<0.05、p<0.01水平下达显著水平。
茶褐素为主要由多酚与蛋白质或多糖形成的复合物,本试验根据茶褐素的定义和性质采用沸水提取,然后用95%乙醇和乙酸乙酯除去儿茶素类、咖啡因、茶红素、黄酮等杂质获得纯净的茶褐素复合物(参见图1);同时,通过测定该复合物中糖及蛋白质含量,从而确定复合物中茶褐素的含量。该复合物在440nm可见光区具有特征吸收,而蛋白质、多糖在该波长下无吸收,表明该波长是茶褐素产生的特征吸收,能作为检测波长,可以避免蛋白质、多糖、多酚、儿茶素、黄酮等的干扰,准确检测茶褐素含量。而且,现有文献多次质疑传统罗伯特检测方法的不适用性,主要在于380nm下还有黄酮等杂质造成一定的吸收,使结果偏高,本检测方法检测结果均低于传统罗伯特方法,且具有显著性差异,从上述三点可知,本方法具有一定的科学和适用性。
Claims (10)
1.一种黑茶茶褐素对照品,其特征在于,该茶褐素对照品是按如下步骤制备得到:
a.以黑茶为原料,用沸水浸提得到黑茶水提取物,将所得黑茶水提取物过滤、浓缩至膏状;
b.将膏状物用95%质量浓度的乙醇进行超声提取,反复多次直至乙醇无色,乙醇不溶物用热水溶解,旋转蒸发至无醇味,再加入等体积的乙酸乙酯萃取多次,至乙酸乙酯层无色;
c.将水相旋转蒸发去除残留的乙酸乙酯,然后用离心滤膜离心,截留液加水再次离心,反复多次,直至离心透过液无色,将截留液真空冷冻干燥,所得粉末即为茶褐素对照品。
2.如权利要求1所述的一种黑茶茶褐素对照品,其特征在于,所述步骤a中黑茶水提取物是于黑茶中加入为黑茶重量8-10倍的沸水,80-100℃水浴浸提2-3次,每次30-60min,合并浸提液,即得。
3.如权利要求1所述的一种黑茶茶褐素对照品,其特征在于,所述步骤b中超声提取时的温度为25-60℃,每次提取时间为20-30min,乙醇用量为膏状物重量的2-3倍。
4.如权利要求1所述的一种黑茶茶褐素对照品,其特征在于,所述步骤b中乙醇不溶物用1-2倍重量的60-100℃热水溶解。
5.如权利要求1所述的一种黑茶茶褐素对照品,其特征在于,所述步骤c中离心滤膜孔径为3000Da。
6.如权利要求1所述的一种黑茶茶褐素对照品,其特征在于,所述黑茶为普洱茶或茯砖茶。
7.权利要求1-6中任一项所述的黑茶茶褐素对照品在建立黑茶茶褐素高效液相色谱测定方法中的应用。
8.如权利要求7所述的应用,其中,该高效液相色谱测定方法的检测波长为440nm。
9.权利要求1-6中任一项所述的黑茶茶褐素对照品在建立黑茶茶褐素紫外分光光度测定方法中的应用。
10.如权利要求9所述的应用,其中,该紫外分光光度测定方法的检测波长为440nm。
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