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  药物分析杂志   2018, Vol. 38 Issue (1): 34-40.  DOI: 10.16155/j.0254-1793.2018.01.05
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糖类分析专栏

引用本文 [复制中英文]

陈凌霄, 吴定涛, 赵静, 李绍平. 高效液相色谱联用电喷雾检测器分析不同植物中棉子糖系列寡糖[J]. 药物分析杂志, 2018, 38(1): 34-40. DOI: 10.16155/j.0254-1793.2018.01.05.
[复制中文]
CHEN Ling-xiao, WU Ding-tao, ZHAO Jing, LI Shao-ping. Analysis of raffinose family oligosaccharides in selected plants using high-performance liquid chromatography with charged aerosol detector[J]. Chinese Journal of Pharmaceutical Analysis, 2018, 38(1): 34-40. DOI: 10.16155/j.0254-1793.2018.01.05.
[复制英文]

基金项目

国家自然科学基金(No.81673389和No.81603069);澳门科学技术发展基金项目(074/2016/A2、034/2017/A1和040/2016/A);澳门大学研究基金(MYRG2015-00202和MYRG2015-00122)

第一作者

陈凌霄, Tel:+853-88222625, E-mail:yb47517@umac.mo;
吴定涛, Tel:+853-88222625, E-mail:yb27502@umac.mo

通信作者

赵静,Tel:+853-88224873;E-mail:zhaojing.cpu@163.com
李绍平,Tel:+853-88224692;E-mail:lishaoping@hotmail.com

文章历史

收稿日期:2017-10-16
高效液相色谱联用电喷雾检测器分析不同植物中棉子糖系列寡糖
陈凌霄 , 吴定涛 , 赵静 , 李绍平     
澳门大学中药质量研究国家重点实验室, 中国澳门 999078
摘要目的:应用高效液相色谱联用电喷雾检测器(HPLC-CAD)分析不同植物中棉子糖系列寡糖(RFOs)。方法:采用微波辅助提取法制备RFOs提取物,再应用HPLC-CAD进行定性、定量分析。色谱条件:采用氨基色谱柱(4.6 mm×250 mm,3.5 μm),流动相为水(A)-乙腈(B),梯度洗脱(0~20 min,75% B→45% B,20-22 min,45% B→75% B),流速为1 mL·min-1,柱温为30℃。外标一点法定量。结果:供试品溶液在48 h内稳定性较好,RSD小于2.8%;低、中、高浓度供试品溶液的重复性RSD分别小于3.6%、3.8%和3.1%。RFOs在豆科植物种子中分布广泛,其在水苏(总量426.19 mg·g-1)和生地黄(总量373.40 mg·g-1)中含量较高,而在车前草(总量1.08 mg·g-1)中含量极低。结论:水苏和地黄有潜力成为开发RFOs新资源。
关键词棉子糖    水苏糖    毛蕊花糖    水苏    生地黄    车前草    定量分析    质量控制    高效液相色谱    电喷雾检测器    
Analysis of raffinose family oligosaccharides in selected plants using high-performance liquid chromatography with charged aerosol detector
CHEN Ling-xiao, WU Ding-tao, ZHAO Jing, LI Shao-ping    
State Key Laboratory of Quality Research in Chinese Medicine, University of Macau, Macao 999078, China
Abstract: Objective: To determine raffinose family oligosaccharides (RFOs) in different plants using high performance liquid chromatography coupled with charged aerosol detector (HPLC-CAD).Methods: RFOs were extracted from selected plants by using microwave assisted extraction.Then the extract was analyzed using HPLC-CAD.The separation was performed on an amide column (4.6 mm×250 mm, 3.5 μm).The mobile phase consists of water (A) and acetonitrile (B) with gradient elution(0-20 min, 75%B→45%B, 20-22 min, 45%B→75%B).The flow rate and the column temperature were 1.0 mL·min-1 and 30℃, respectively.Single point external standard method was used for quantification of RFOs.Results: Samples were stable in 48 h with RSD less than 2.83%.The repeatability presented as RSDs of sample solution in low, middle and high levels were 3.6%, 3.8% and 3.1%, respectively.RFOs were widely distributed in different seeds of edible plants from Leguminosae family, and the high amount of RFOs was found in Stachys japonica (total content 426.19 mg·g-1) and Rehmannia glutinosa (total content 426.19 mg·g-1), but only a small amount of RFOs was found in Plantago asiatica (total content 1.08 mg·g-1).Conclusion: Rehmannia glutinosa and Stachys japonica might be new potential sources of RFOs.
Key words: raffinose    stachyose    verbascose    Stachys japonica    Rehmannia glutinosa    Plantago asiatica    quantitative analysis    quality control    HPLC    CAD    

棉子糖系列寡糖(RFOs)作为一种天然食品添加剂和益菌因子,已被广泛应用于食品及功能性保健食品领域[1]。RFOs是由数个半乳糖以α-(1,6)糖苷键连接在蔗糖上,所构成的一系列水溶性非还原糖。RFOs主要包括棉子糖(三糖)、水苏糖(四糖)和毛蕊花糖(五糖)等,具有改善葡萄糖耐受[2],抑制肝胆固醇[3],增强免疫[4]和改善肠道菌群[5]等多种生物活性。

RFOs广泛存在于植物界,是含量仅次于蔗糖的一类可溶性糖类成分[6]。葫芦科、豆科、唇形科、木犀科、玄参科等多种植物均利用RFOs来转运和储存能量[7]。在不同科属植物中,豆科植物种子如大豆[8]、绿豆[9]、三角豆[10]、芸豆[11]、豌豆[12]、红豆[13]等含有丰富RFOs。此外,RFOs也存在于一些药食两用植物如地黄、水苏等[14]。为了开发富含RFOs新资源,建立定性、定量分析RFOs方法,明确其在不同科属植物中的分布具有重要意义。

目前,RFOs常用分析方法包括比色法[15]、高效阴离子色谱联用脉冲安培检测器法(HPAEC-PAD)[8]、亲水相互作用色谱联用蒸发光散射检测器法(HILIC-ELSD)[16],以及亲水相互作用色谱联用示差折光检测器法(HILIC-RID)[17]等。其中,比色法简单、方便,但只能测定总糖含量,并且测定结果易受单糖类型影响。虽然HPAEC-PAD法分离度好,但糖类物质在碱性条件下易异构化和降解[18]。HILIC虽有较好的分辨率,但RID和ELSD灵敏度均较低[16, 19],并且RID不适宜梯度洗脱,影响分离能力[17]。近年来,电喷雾式检测器(CAD)作为一种新型通用检测器,已被成功应用于寡糖类化合物的分析检测[20-21]。与ELSD检测器相似,CAD响应值不依赖于分析物的结构[22],并且CAD检测器灵敏度远高于ELSD和RID检测器[23]。在前期工作中,本课题组成功应用HPLC-CAD法对菊型果寡糖[20]、木寡糖[24]等进行过分析,并研究了菊型果寡糖在不同植物中的含量。本研究应用HPLC-CAD结合高通量微波辅助提取技术,实现对不同生物来源样品中RFOs定性、定量分析。

1 仪器与试药

Multiwave 3000微波消解仪(Anton Paar GmbH公司),Ultimate 3000超高效液相系统,Chromeleon 6.8色谱工作站(Dionex公司),Waters Amide色谱柱(4.6 mm×250 mm,3.5 μm;填料:亚乙基桥杂化颗粒三键键合酰胺基;Waters公司)AG135与ARB120电子分析天平(Mettler Toledo公司),Millipore Milli-Q超纯水制备系统(Millipore公司)。

样品共16批,详细信息见表 1。豆科植物种子收集自澳门与珠海当地市场,生地黄、车前草和水苏主要购买于珠海当地药店。各批药材由澳门大学乔春峰博士鉴定确认。

表 1 样品信息 Table 1 Characteristics of the investigated samples

棉子糖系列寡糖,主要包括棉子糖、水苏糖、毛蕊花糖对照品由本实验室纯化制备[25],其纯度大于97%(w/w,HPLC-DAD-CAD法测定),结构信息见图 1。色谱级乙腈购于RCI Labscan公司,超纯水由Milli-Q超纯水制备系统制备,分析级乙醇购于天津市富宇精细化工有限公司。

图 1 棉子糖系列寡糖(棉子糖、水苏糖和毛蕊花糖)与蔗糖结构 Figure 1 Chemical structures of raffinose family oligosaccharides (raffinose, stachyose and verbascose) and sucrose
2 方法与结果 2.1 对照品溶液制备

精密称取对照品(蔗糖、棉子糖、水苏糖、毛蕊花糖)适量,置5 mL量瓶中。加60%乙醇溶解并定容至刻度,摇匀,配制成质量浓度为2.0 mg·mL-1混合对照品溶液,4 ℃储存备用。

2.2 供试品溶液制备

所有样品研磨成均匀细粉,过40目筛。参照本课题组已建立的方法[20],应用Multiwave 3000微波消解仪提取寡糖。精密称取待测样品干燥细粉0.1 g,置于5 mL提取瓶中,加入60%乙醇溶液2.0 mL。微波辅助提取,功率400 W,时间4 min,温度80 ℃。提取结束后,提取液离心(5 000×g,5 min)取上清液1 mL,置于10 mL量瓶中,用60%乙醇定容至刻度,溶液过0.45 μm微孔滤膜后作HPLC-CAD分析。

2.3 HPLC-CAD分析

采用Waters Amide色谱柱(4.6 mm×250 mm,3.5 μm),流动相为水(A)-乙腈(B),梯度洗脱(0~20 min,75%B→45%B,20~22 min,45%B→75%B,流速为1.0 mL·min-1,柱温30 ℃,进样量10 μL,CAD氮气压力为0.24 MPa。

2.4 重复性及稳定性试验

精密称定豌豆样品粉末0.08、0.10、0.12 g各3份,按供试品溶液制备方法制备低、中、高浓度的供试品溶液,并测定含量,验证方法重复性。取中间浓度供试品溶液,在上述色谱条件下,分别于0、2、4、6、8、12、24、48 h进样分析,验证方法稳定性。

低、中、高浓度供试品溶液重复性RSD分别小于3.6%、3.8%和3.1%;稳定性试验结果(表 2)表明,供试品溶液在48 h内稳定性较好,RSD小于2.8%。说明该方法具有良好的重复性和稳定性。

表 2 4种糖重复性和稳定性考察结果 Table 2 Repeatability and stability of sucrose, raffinose, stachyose and verbascose
2.5 样品分析

RFOs是一类可溶性非还原糖,采用HPLC-CAD方法测定不同来源(豆科、玄参科、唇形科和车前草科)样品中RFOs和蔗糖含量(图 2表 3)。结果表明:豆科植物样品几乎都含有蔗糖和RFOs,但各种寡糖在不同来源豆科植物种子中含量差异较大,如眉豆与大豆中水苏糖和蔗糖含量最高。但在绿豆中毛蕊花糖含量最高,达总糖的45.3%。特别是RFOs在水苏和生地黄中含量非常高,总量分别达到426.19 mg·g-1和373.40 mg·g-1,但车前草中几乎检测不到。实际上,RFOs作为植物能量成分,主要储存在植物块茎(如生地黄)中,植物叶子(如车前草)中含量较少[26]

1.蔗糖(sucrose)2.棉子糖(raffinose)3.水苏糖(stachyose)4.毛蕊花糖(verbascose) 图 2 对照品(A)及样品(样品编号同表 1)HPLC-CAD色谱图 Figure 2 HPLC-CAD chromatograms of mixed standards (A) and tested samples(the sample codes were the same as in Tab. 1)

表 3 被测植物中4种糖的含量 Table 3 The contents of sucrose, raffinose, stachyose and verbascose in the investigated samples
3 讨论

HILIC法是糖类化合物常用分析技术之一。通过比较不同HILIC色谱柱,本文选择Waters氨基柱(4.6 mm×250 mm,3.5 μm)用于RFOs和蔗糖分离,该色谱柱具有良好的分辨率和信噪比。另外,考察了不同梯度洗脱条件,最终建立的HILIC-CAD方法能在20 min内对待测成分实现良好分离和分析,蔗糖、棉子糖、水苏糖、毛蕊花糖均达到基线分离。

尽管已有大量研究证实RFOs存在于豆科、玄参科、唇形科和车前科植物中,但是少有研究系统比较这些科属代表植物中RFOs含量。当前市场上(特别是日本)RFOs主要来自大豆,但大豆中RFOs含量相对较低,进而限制了RFOs在健康产品中的应用[27]。本研究结果表明:水苏和地黄富含大量RFOs,有望作为开发富含RFOs的新资源,本研究建立的HPLC-CAD方法也有利于对RFOs及其产品进行质量控制。

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