CN113125581A - 一种测定花青素单体含量的hplc方法 - Google Patents
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Abstract
本发明公开了一种测定花青素单体含量的HPLC方法,涉及植物提取技术领域;该方法可简便、快速、准确对花青素单体含量进行测定。所述HPLC方法检测法的参数为:流动相:A‑含0.1%甲酸的水溶液,B‑含0.1%甲酸的乙腈溶液;检测波长:520nm;色谱柱:十八烷基梯度洗脱程序0~55min,90%~75%A;55~57min,75%~95%A;57~60min,95%A。
Description
技术领域
本发明涉及植物提取技术领域,尤其是一种测定花青素单体含量的HPLC方法。
背景技术
花青素又称花色素或花色苷,是自然界一类广泛存在于植物中的水溶性天然色素。水果、蔬菜、谷类、深色花卉植物中的主要呈色物质大部分与之有关。在植物细胞液泡不同的pH条件下,花青素使花瓣呈现五彩缤纷的颜色。花青素主要用于食品着色、染料、医药、化妆品等方面。花青素是一种很强的抗氧化剂,对人体的衰老有预防和缓解作用;花青素可以促进视网膜细胞中的视紫质再生,预防近视,缓解眼疲劳、增进视力;花青素通过对弹性蛋白酶和胶原蛋白酶的抑制使皮肤变得光滑和抗辐射的作用;花青素具有深入细胞保护细胞膜不被自由基氧化的作用,具有强力抗氧化和抗过敏功能,能穿越血脑屏障,保护脑神经不被氧化,具有稳定脑组织功能、保护大脑不受毒素伤害的作用。
基于此,建立一种简便、快速、准确测定花青素单体含量的方法很有必要,从而进一步对花青素的质量控制方法进行完善。
发明内容
本发明旨在提供一种测定花青素单体含量的HPLC方法,该方法可简便、快速、准确对花青素单体含量进行测定。
为实现上述目的,本发明提供的技术方案是:一种测定花青素单体含量的HPLC方法,所述HPLC检测法的参数为:
流动相:A-含0.1%甲酸的水溶液,B-含0.1%甲酸的乙腈溶液;
检测波长:520nm;
色谱柱:十八烷基键合硅胶
梯度洗脱程序0~55min,90%~75%A;55~57min,75%~95%A;57~60min,95%A。
所述方法是从如下混合物中测定花青素:
所述混合物是将多酚类化合物与花青素按照0~1:0~10浓度比进行混合;进一步地,所述多酚类化合物与花青素的混合浓度比分别为1:0、1:1、1:5、1:10和/或0:1。
混合的温度是在100℃以内;进一步地,混合的温度可以选自0~90℃;更进一步地,混合的温度可以选自80~90℃。
所述混合时间大于等于0h;进一步地,混合时间选自0h、5h、10h、15h、20h、25h、30h或35h。
所述多酚类化合物的提取方法为:将浆果粉碎后用乙醇提取,所述乙醇体积浓度优选为60%;进一步地,所述浆果与乙醇的料液比为1:3~9g/mL,优选为1:6.7g/mL。在上述提取过程中利用醋酸钠缓冲液将pH稳定在3.6。
所述提取温度为40~50℃,提取时间为20~40min;优选地,所述提取温度为45℃,提取时间为30min。
所述多酚类化合物为从蓝靛果、树莓和/或黑加仑中提取而得。
所述花青素为从黑果枸杞中提取的天然花青素。
所述提取方法为:将黑果枸杞加至甲醇溶液中后,超声波提取,即得;优选地,所述黑果枸杞与甲醇溶液的料液比为5g:75mL;所述甲醇溶液的体积浓度优选为50%。
所述提取温度为60~80℃,提取时间为80~100min;优选地,所述提取温度为70℃,提取时间为90min。
本发明与现有技术相比,具有以下优点:本发明所述HPLC检测方法具体良好的重复性和分离度,且简单快速、灵敏度高、结果准确可靠。因此,本发明所述的测定方法可对黑果枸杞中提取的花青素单体含量进行快速常规分析测定。
附图说明
图1为浆果多酚添加前后黑果枸杞花青素HPLC色谱的比较图。
图2为黑果枸杞花青素提取物中鉴定得到的8种花色苷单体化合物结构式图;
图3为天然多酚提取物存在下黑果枸杞总花色苷和主要花色苷单体半衰期的变化图。
具体实施方式
下面结合具体实施方式对本发明的权利要求做进一步的详细说明。
以下实施例中所用材料、仪器及试剂如下所示:
材料:树莓,黑加仑以及蓝靛果由青海瑶池生物科技有限公司提供。
仪器:UPLC-Triple-TOF/MS系统:AcquityTM ultra型高效液相色谱仪(美国Waters公司),Triple TOF 5600+型飞行时间质谱,配有电喷雾离子源(美国AB SCIEX公司);BioTek Epoch全波长酶标仪;Eppendorf minispan离心机(德国Eppendorf公司)。
试剂:色谱用水为屈臣氏纯净水;色谱级乙腈购于上海化学试剂有限公司;其他所用试剂均为分析级试剂。
实施例1黑果枸杞花青素提取及含量测定
准确称取黑果枸杞5.0g于棕色试剂瓶中,加入75mL50%甲醇,在超声波功率为150W、频率为20kHz、70℃下超声90min提取,即得。
将上述提取所得的花青素采用pH示差法测得其含量为16.24±0.16mg/mL。pH示差法测定花青素的含量:用蒸馏水作为空白对照,分别在520nm和700nm处测定用氯化钾缓冲溶液(0.025mol/L,pH 1.0)根据不同样品中花青素的含量,稀释成10-100倍的样品溶液。同理在520nm和700nm处测定用醋酸钠缓冲溶液(0.4mol/L,pH 4.5)根据不同样品中花青素的含量,稀释成10~100倍的样品,注意此处要在20~50min内测定完样品的吸光度。
以锦葵色素-3,5-二葡萄糖苷计算花青素的浓度,公式如式1所示:
其中:A=(Aλmax–A700nm)pH 1.0–(Aλmax–A700nm)pH 4.5;MW(分子量)=锦葵色素-3,5-二葡萄糖苷的摩尔质量655.2g/mol;DF为稀释因子;103是将单位g换算为mg;ε为锦葵色素-3,5-二葡萄糖苷的消光系数,为20 500L/mol/cm;l为比色杯厚度,cm。
利用以下参数的HPLC-MS联用技术对黑枸杞花青素进行定量定性分析:
液质中液相的洗脱条件:色谱条件色谱柱为安捷伦ZORBAX-SB C18(100mm×4.6mmi.d.,1.8μm);以含0.1%甲酸的水溶液为流动相A,以含0.1%甲酸的乙腈溶液为流动相B,线性梯度洗脱,0min 5%B;2min 5%B;25min 50%B;35min 95%B;37min 95%B,38min5%B,流速为0.8mL/min;柱温为30℃;检测波长为520nm;进样量:5μL。
UPLC-Triple-TOF 5600+飞行时间液质联用仪:正负离子扫描模式;扫描范围:100-1500m/z;雾化气(GS1):50psi,雾化气(GS2):50psi,气帘气(CUR):35psi;离子源温度(TEM):550℃(负)600℃(正);离子源电压(IS):-4500V(负)5500V(正);一级扫描:去簇电压(DP):100V;聚焦电压(CE):10V;二级扫描:使用TOF MS-Product Ion-IDA模式采集质谱数据,CID能量为-20、-40和-60V,进样前,用CDS泵做质量轴校正,使质量轴误差小于2ppm。
表1黑果枸杞花青素鉴定
上表中所述黑果枸杞花青素提取物中鉴定到的8种花色苷单体化合物结构式图详见图2。
实施例2浆果多酚类化合物的提取及含量测定
称取新鲜浆果300g,使用料理机将其粉碎打浆,用60%的乙醇按照1:6.7的料液比在45℃、100W超声波下超声30min后,在2500r/min的条件下离心10min,收集上清,残渣用同样的方法重复提取3次,将3次提取液合并抽滤除去多余的残渣,使用旋转蒸发仪在40℃条件下真空浓缩,分别浓缩至100mL,即得。
上述浆果为蓝靛果、树莓以及黑加仑中的任一种。
测定浆果提取的多酚类化合物:用95%乙醇将AB-8大孔树脂浸泡24h,然后使用无水乙醇洗涤树脂至加水无白色浑浊出现,之后用蒸馏水冲洗至无醇味。用2-3倍柱体积的3%的HCl溶液浸泡树脂4-6h,用蒸馏水冲洗直至pH值为中性,最后用2-3倍柱体积的3%的NaOH溶液浸泡树脂4-6h,用蒸馏水冲洗直至pH值为中性后备用。量取四种浆果多酚提取液100m L进行上样,吸附12h后用1%酸化乙醇冲洗上样柱,以除去其中的非酚酸物质,再用一定体积的60%乙醇将多酚进行分配,每个色谱柱用60%的乙醇洗脱两个柱体积。分装后保存于-20℃冰箱,用于下一步的分析。
多酚含量是使用Folin-Ciocalteu方法并经过一些修改进行测定。称取0.5g的没食子酸,使用10mL蒸馏水进行溶解,定容到100mL,再使用蒸馏水将其稀释50倍,得到0.1mg/mL的没食子酸标准溶液。吸取没食子酸标准溶液分别为0、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0mL加入到10mL比色管中,并用蒸馏水定容到2mL,摇匀后每管分别加入1.0mL福林酚试剂,反应4min后加入1.0mL 10%的Na2CO3溶液,在25℃条件下水浴2h,使用分光光度计测定反应溶液在765nm波长处的吸光度,并做三次平行试验,按照式2绘制标准曲线:
Y(没食子酸当量含量)=X(吸光度)/4.4115-0.0112 (式2)
将天然多酚提取液用水稀释至合适的浓度后,分别准确吸取1mL稀释液于10mL比色管中,按照上述标准曲线(式2)测定方法,在765nm波长下测定反应溶液的吸光度值。使用上述标准曲线计算提取液中相应的多酚含量,按照式3公式进行换算(多酚含量以没食子酸当量表示):
W=C×V×N/M (式3)
式中:W—多酚含量(mg/g);C—没食子酸质量浓度(mg/L);V—提取液体积(mL);N—稀释倍数;M—取样量(g)。
表2各种浆果多酚提取物中多酚的含量
实施例3花青素与多酚混合液的制备与处理
将实施例2提取的多酚类化合物与实施例1提取的花青素分别按照1:0、1:1、1:5、1:10、0:1共计5个不同浓度比进行混合,将混合液在85℃水浴分别处理0h、5h、10h、15h、20h、25h、30h、35h后,分别利用pH示差法和HPLC检测混合液中总花青素以及花青素单体含量的变化。其中,计算公式为式1,花青素的含量测定参照的是国标,测定波长分别是520和700nm。
所述HPLC检测法的参数为:
流动相:A-含0.1%甲酸的水溶液,B-含0.1%甲酸的乙腈溶液;流速:1mL/min;检测波长:520nm;色谱柱:C18 column(4.6×250mm,CAPCELL PAK ACR,Shiseido Company,Limited,东京,日本Japan);进样量:10μL;柱温箱:30℃;梯度洗脱程序0~55min,90%~75%A;55~57min,75%~95%A;57~60min,95%A。
HPLC检测结果分析:HPLC的分析结果具有良好的重复性和分离度,HPLC图谱见图1,该图比较了树莓、沙棘、蓝靛果和黑加仑多酚添加前后黑果枸杞花青素HPLC色谱图。其中,图a为黑果枸杞花青素高效液相色谱图;图b1、c1、d1和e1分别为树莓、沙棘、蓝靛果和黑加仑多酚提取物中所含花青素高效液相色谱图;图b2、c2、d2和e2分别为黑果枸杞花青素树莓、沙棘、蓝靛果和黑加仑多酚提取物混合液的高效液相色谱图。
根据pH示差法和HPLC-MS结果显示花青素的半衰期在添加浆果多酚类化合物前后有以下变化:
在添加树莓多酚类化合物的情况下,黑果枸杞花青素的半衰期由25.68h延长至75.90h,主要成分矮牵牛素-3-O-rutinoside(trans-p-coumaroyl)-5-O-glucoside的半衰期由自然状态下的20.69h最高延长至56.37h,矮牵牛素-3-O-galactoside-5-O-glucoside由25.21h最高延长至48.38h,详见表3。
在添加蓝靛果多酚类化合物的情况下,花青素的半衰期最高延长至96.13h,主要成分矮牵牛素-3-O-rutinoside(trans-p-coumaroyl)-5-O-glucoside的半衰期由自然状态下的20.43h延长至58.82h,其他飞燕草素类和锦葵素类花青素单体的半衰期也得到明显延长,详见表3。
在添加黑加仑多酚类化合物的情况下,花青素的半衰期最高延长至44.64h,主要成分矮牵牛素-3-O-rutinoside(trans-p-coumaroyl)-5-O-glucoside的半衰期由自然状态下的20.00延长至24.87h,详见表3。
综上所述,在添加蓝靛果、树莓以及黑加仑多酚类化合物的情况下,黑果枸杞花青素的半衰期明显延长,主要成分矮牵牛素-3-O-rutinoside(trans-p-coumaroyl)-5-O-glucoside的半衰期由自然状态下的20.43h最高可延长至58.82h,其他飞燕草素类和锦葵素类花青素单体的半衰期也得到明显延长。
下述表3中,比例关系为黑果枸杞提取的花青素与三种浆果提取的多酚类化合物的混合浓度比例。
表3添加不同比例的天然多酚提取物前后黑果枸杞花青素半衰期的变化
Peak 1Petunidin-3-O-galactoside-5-O-glucoside;
Peak 2Petunidin 3,5-O-diglucopyranoside;
Peak 3Petunidin 3-O-β-D-galactopyranoside;
Peak 4Petunidin-3-O-glucoside;
Peak 5Petunidin3-O-[6-O-(4-O-(4-O-(beta-D-glucopyranosyl)-trans-p-coumaroyl)-alpha-L-rhamnopyranosyl)-beta-D-glucopyranoside]-5-O-[beta-D-glucopyranoside];
Peak 6Petunidin3-O-[6-O-(4-O-cis-p-coumaroyl-alpha-L-rhamnopyranosyl)-beta-D-glucopyranoside]-5-O-[beta-D-glucopyranoside];
Peak 7Petunidin3-O-[6-O-(4-O-trans-p-coumaroyl-alpha-L-rhamnopyranosyl)-beta-D-glucopyranoside]-5-O-[beta-D-glucopyranoside];
Peak 8Malvidin3-O-[6-O-(4-O-p-coumaroyl-α-L-rhamnosyl)-β-D-glucopyranoside]-5-O-β-D-glucopyranoside.
在天然多酚提取物存在下黑果枸杞总花色苷和主要花色苷单体半衰期的变化图详见图3,其中a-d为天然多酚提取物的存在下总花色苷半衰期的变化,(a)树莓,(b)沙棘,(c)蓝靛果,(d)黑加仑;e-h为天然多酚提取物存在下主要花色苷单体的半衰期变化,(e)树莓,(f)沙棘,(g)蓝靛果,(h)黑加仑。
本发明将从蓝靛果、树莓以及黑加仑中提取的多酚类化合物与花色苷分别按照不同浓度比进行混合,将混合液在85℃水浴分别处理0,5,10,15,20,25,30,35h,分别利用pH示差法和HPLC检测总花色苷以及花色苷单体含量的变化,最后利用HPLC-MS联用技术对黑枸杞花青素进行定量定性分析,以期明确每种花青素单体的稳定性。结果显示,在添加蓝靛果、树莓以及黑加仑多酚类化合物的情况下,黑果枸杞花青素的半衰期明显延长,其中主要成分矮牵牛素-3-O-rutinoside(trans-p-coumaroyl)-5-O-glucoside的半衰期由自然状态下的20.43h最高可延长至58.82h;飞燕草素类和锦葵素类花青素单体的半衰期也得到明显延长。由此可见,本发明所述组合物相较于未与多酚类化合物混合的花青素,可以显著增强黑果枸杞花青素的半衰期,从而提高其稳定性。
Claims (10)
1.一种测定花青素单体含量的HPLC方法,其特征在于,所述HPLC检测法的参数为:
流动相:A-含0.1%甲酸的水溶液,B-含0.1%甲酸的乙腈溶液;
检测波长:520nm;
色谱柱:十八烷基键合硅胶
梯度洗脱程序0~55min,90%~75%A;55~57min,75%~95%A;57~60min,95%A。
2.根据权利要求1所述的HPLC方法,其特征在于,所述方法是从如下混合物中测定花青素:
所述混合物是将多酚类化合物与花青素按照0~1:0~10浓度比进行混合;进一步地,所述多酚类化合物与花青素的混合浓度比分别为1:0、1:1、1:5、1:10和/或0:1。
3.根据权利要求2所述测定花青素单体含量的HPLC方法,其特征在于,混合的温度是在100℃以内;进一步地,混合的温度可以选自0~90℃;更进一步地,混合的温度可以选自80~90℃。
4.根据权利要求1所述测定花青素单体含量的HPLC方法,其特征在于,所述混合时间大于等于0h;进一步地,混合时间选自0h、5h、10h、15h、20h、25h、30h或35h。
5.根据权利要求1所述测定花青素单体含量的HPLC方法,其特征在于,所述多酚类化合物的提取方法为:将浆果粉碎后用乙醇提取,所述乙醇体积浓度优选为60%;进一步地,所述浆果与乙醇的料液比为1:3~9g/mL,优选为1:6.7g/mL。
6.根据权利要求5所述测定花青素单体含量的HPLC方法,其特征在于,所述提取温度为40~50℃,提取时间为20~40min;优选地,所述提取温度为45℃,提取时间为30min。
7.根据权利要求5所述测定花青素单体含量的HPLC方法,其特征在于,所述多酚类化合物为从蓝靛果、树莓和/或黑加仑中提取而得。
8.根据权利要求1所述测定花青素单体含量的HPLC方法,其特征在于,所述花青素为从黑果枸杞中提取的天然花青素。
9.根据权利要求8所述测定花青素单体含量的HPLC方法,其特征在于,所述提取方法为:将黑果枸杞加至甲醇溶液中后,超声波提取,即得;优选地,所述黑果枸杞与甲醇溶液的料液比为5g:75mL;所述甲醇溶液的体积浓度优选为50%。
10.根据权利要求8所述测定花青素单体含量的HPLC方法,其特征在于,所述提取温度为60~80℃,提取时间为80~100min;优选地,所述提取温度为70℃,提取时间为90min。
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