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Article(id=1153429497513763345, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153429493357203682, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20241120003, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1732032000000, receivedDateStr=2024-11-20, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1752928621891, onlineDateStr=2025-07-19, pubDate=1741968000000, pubDateStr=2025-03-15, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1752928621891, onlineIssueDateStr=2025-07-19, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1752928621891, creator=13701087609, updateTime=1752928621891, updator=13701087609, issue=Issue{id=1153429493357203682, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='5', pageStart='1', pageEnd='326', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1752928620900, creator=13701087609, updateTime=1758690311058, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1177595773500932351, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153429493357203682, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1177595773500932352, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153429493357203682, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=239, endPage=245, ext={EN=ArticleExt(id=1153429497983525396, articleId=1153429497513763345, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Effects of new deep eutectic solvent on absorbance of tea polysaccharide, columnId=1151895321388347923, journalTitle=Journal of Food Safety & Quality, columnName=Food Analysis and Detection, runingTitle=null, highlight=null, articleAbstract=

Objective To study the effects of deep eutectic solvent (DES) on the absorbance of tea polysaccharide by visible spectrophotometry, and establish a fast and efficient method for the determination of tea polysaccharide. Methods Choline chloride/DL-tartaric acid was used as the extraction solvent of tea polysaccharide. The influence of DES on the determination of tea polysaccharide content in anthrone sulfuric acid and phenol-sulfuric acid 2 kinds of detection methods was investigated. The linear relationship, precision, stability, repeatability and standard recovery rate of the 2 kinds of detection methods were compared. Results The best method for the determination of tea polysaccharides was phenol-sulfuric acid method, the optimal detection wavelength was 484 nm, and the absorbance had a good linear relationship with glucose concentration between 20 and 100 μg/mL. The precision relative standard deviation (RSD) of this method was 0.10%, the stability RSD was 0.00%, the repeatability RSD was 2.40%, and the recovery rate was 94.25%-111.19%, and the content of tea polysaccharide was 54.76 mg/g. Conclusion Phenol-sulfuric acid method is suitable for the determination of tea polysaccharide in tea with DES as extraction solvent.

, correspAuthors=Pei-Qin CAO, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Pei-Qin CAO, Yan-Fen XIAO, Yi-Ping SONG), CN=ArticleExt(id=1153429517398958923, articleId=1153429497513763345, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=新型低共熔溶剂对茶多糖吸光度的影响, columnId=1151895321958773274, journalTitle=食品安全质量检测学报, columnName=食品分析与检测, runingTitle=null, highlight=null, articleAbstract=

目的 探究新型低共熔溶剂(deep eutectic solvent, DES)在可见分光光度法检测中对茶多糖吸光度的影响, 建立快速高效的茶多糖含量检测方法。方法 氯化胆碱/DL-酒石酸DES作为茶多糖的提取溶剂, 考察蒽酮-硫酸和苯酚-硫酸两种检测方法中DES对茶多糖含量检测的影响, 并对方法学线性关系、精密度、稳定性、重复性和加标回收率进行比较。结果 茶多糖测定的最佳方法为苯酚-硫酸法, 最适检测波长为484 nm, 葡萄糖质量浓度在20~100 μg/mL之间吸光度具有良好的线性关系, 其精密度相对标准偏差(relative standard deviation, RSD)为0.10%, 稳定性RSD为0.00%, 重复性RSD为2.40%, 加标回收率为94.25%~111.19%, 测得茶多糖含量为54.76 mg/g。结论 苯酚-硫酸法适用于以DES为提取溶剂的茶叶中茶多糖含量测定。

, correspAuthors=曹佩琴, authorNote=null, correspAuthorsNote=
* 曹佩琴(1989—), 女, 博士, 讲师, 主要研究方向为茶叶功能成分利用。E-mail:
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Agriculture and Technology, 2023, 43(10): 14-19., articleTitle=Study on microwave-ultrasonic collaborative extraction process optimization and determination method of Atractylodes macrocephala Koidz. polysaccharide, refAbstract=null)], funds=[Fund(id=1177619437856375387, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497513763345, awardId=21B0587, language=CN, fundingSource=湖南省教育厅青年项目(21B0587), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1177619434182165037, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497513763345, xref=null, ext=[AuthorCompanyExt(id=1177619434224108078, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497513763345, companyId=1177619434182165037, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Department of Chemistry and Chemical Engineering, Hunan Institute of Science and Technology, Yueyang 414006, China), AuthorCompanyExt(id=1177619434232496687, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497513763345, companyId=1177619434182165037, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=湖南理工学院化学化工学院, 岳阳 414006)])], figs=[ArticleFig(id=1177619436333843019, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497513763345, language=EN, label=Fig.1, caption=Effects of wavelength on the absorbance of phenol-sulfuric acid method and anthrone-sulfuric acid method, figureFileSmall=CIr4EzMBhcFvJ1fopSMTzQ==, figureFileBig=8RJxnXfU74NOFQruhlrEww==, tableContent=null), ArticleFig(id=1177619436421923404, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497513763345, language=CN, label=图1, caption=检测波长对苯酚-硫酸法和蒽酮-硫酸法吸光度的影响

注: A. 苯酚-硫酸法; B. 蒽酮-硫酸法, 图2、3同。

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注: 图中不同字母表示组内显著性差异(P<0.05), 图3同。

, figureFileSmall=uFYzwx8wYuEyj/L3lHkRjg==, figureFileBig=DYUxzqFLXtarpLmY6cO2/g==, tableContent=null), ArticleFig(id=1177619436602278479, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497513763345, language=EN, label=Fig.3, caption=Effects of DES content on absorbance of phenol-sulfuric acid method and anthrone-sulfuric acid method, figureFileSmall=elr4mj1ytFy9pwbuEE4MCw==, figureFileBig=nHDmvWW7QiNb/A2nrJfY3w==, tableContent=null), ArticleFig(id=1177619436656804432, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497513763345, language=CN, label=图3, caption=DES含量对苯酚-硫酸法和硫酸-蒽酮法吸光度的影响, figureFileSmall=elr4mj1ytFy9pwbuEE4MCw==, figureFileBig=nHDmvWW7QiNb/A2nrJfY3w==, tableContent=null), ArticleFig(id=1177619436728107601, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497513763345, language=EN, label=Table 1, caption=

Precision of sample

, figureFileSmall=null, figureFileBig=null, tableContent=
方法 组别 RSDs
/%
1 2 3 4 5 6
蒽酮-硫酸法 81.39 81.21 81.21 81.21 81.39 81.39 0.12
苯酚-硫酸法 55.90 55.90 55.78 55.78 55.78 55.78 0.10
), ArticleFig(id=1177619436803605074, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497513763345, language=CN, label=表1, caption=

样品精密度

, figureFileSmall=null, figureFileBig=null, tableContent=
方法 组别 RSDs
/%
1 2 3 4 5 6
蒽酮-硫酸法 81.39 81.21 81.21 81.21 81.39 81.39 0.12
苯酚-硫酸法 55.90 55.90 55.78 55.78 55.78 55.78 0.10
), ArticleFig(id=1177619436862325331, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497513763345, language=EN, label=Table 2, caption=

Stability of sample

, figureFileSmall=null, figureFileBig=null, tableContent=
方法 时间/min RSDs
/%
0 10 20 30 40 50
蒽酮-硫酸法 91.08 90.54 90.35 90.35 90.35 90.54 0.30
苯酚-硫酸法 59.67 59.67 59.67 59.67 59.67 59.67 0.00
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样品稳定性

, figureFileSmall=null, figureFileBig=null, tableContent=
方法 时间/min RSDs
/%
0 10 20 30 40 50
蒽酮-硫酸法 91.08 90.54 90.35 90.35 90.35 90.54 0.30
苯酚-硫酸法 59.67 59.67 59.67 59.67 59.67 59.67 0.00
), ArticleFig(id=1177619437021708885, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497513763345, language=EN, label=Table 3, caption=

Repeatability of sample

, figureFileSmall=null, figureFileBig=null, tableContent=
方法 组别 RSDs
/%
1 2 3 4 5 6
蒽酮-硫酸法 86.88 94.19 91.08 92.36 91.45 87.79 3.07
苯酚-硫酸法 61.32 58.61 60.14 58.61 62.15 59.55 2.40
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样品重复性

, figureFileSmall=null, figureFileBig=null, tableContent=
方法 组别 RSDs
/%
1 2 3 4 5 6
蒽酮-硫酸法 86.88 94.19 91.08 92.36 91.45 87.79 3.07
苯酚-硫酸法 61.32 58.61 60.14 58.61 62.15 59.55 2.40
), ArticleFig(id=1177619437202063959, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497513763345, language=EN, label=Table 4, caption=

Recovery rate of sample

, figureFileSmall=null, figureFileBig=null, tableContent=
方法 加入量/μg 平均回收率/% RSDs/%
蒽酮-硫酸法 10 103.88 10.55
20
30
苯酚-硫酸法 15 100.96 8.92
30
45
), ArticleFig(id=1177619437336281688, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497513763345, language=CN, label=表4, caption=

样品加标回收率

, figureFileSmall=null, figureFileBig=null, tableContent=
方法 加入量/μg 平均回收率/% RSDs/%
蒽酮-硫酸法 10 103.88 10.55
20
30
苯酚-硫酸法 15 100.96 8.92
30
45
), ArticleFig(id=1177619437508248153, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497513763345, language=EN, label=Table 5, caption=

Result of the content of polysaccharide in DES tea polysaccharide samples by 2 kinds of methods

, figureFileSmall=null, figureFileBig=null, tableContent=
方法 平均值/(mg/g) RSDs/%
蒽酮-硫酸法 83.22 6.83
苯酚-硫酸法 54.76 3.12
), ArticleFig(id=1177619437629882970, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429497513763345, language=CN, label=表5, caption=

两种方法测定DES茶多糖样品中多糖的含量结果

, figureFileSmall=null, figureFileBig=null, tableContent=
方法 平均值/(mg/g) RSDs/%
蒽酮-硫酸法 83.22 6.83
苯酚-硫酸法 54.76 3.12
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新型低共熔溶剂对茶多糖吸光度的影响
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曹佩琴 * , 肖艳芬 , 宋一平
食品安全质量检测学报 | 食品分析与检测 2025,16(5): 239-245
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食品安全质量检测学报 | 食品分析与检测 2025, 16(5): 239-245
新型低共熔溶剂对茶多糖吸光度的影响
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曹佩琴* , 肖艳芬, 宋一平
作者信息
  • 湖南理工学院化学化工学院, 岳阳 414006

通讯作者:

* 曹佩琴(1989—), 女, 博士, 讲师, 主要研究方向为茶叶功能成分利用。E-mail:
Effects of new deep eutectic solvent on absorbance of tea polysaccharide
Pei-Qin CAO* , Yan-Fen XIAO, Yi-Ping SONG
Affiliations
  • Department of Chemistry and Chemical Engineering, Hunan Institute of Science and Technology, Yueyang 414006, China
出版时间: 2025-03-15 doi: 10.19812/j.cnki.jfsq11-5956/ts.20241120003
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摘要
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目的 探究新型低共熔溶剂(deep eutectic solvent, DES)在可见分光光度法检测中对茶多糖吸光度的影响, 建立快速高效的茶多糖含量检测方法。方法 氯化胆碱/DL-酒石酸DES作为茶多糖的提取溶剂, 考察蒽酮-硫酸和苯酚-硫酸两种检测方法中DES对茶多糖含量检测的影响, 并对方法学线性关系、精密度、稳定性、重复性和加标回收率进行比较。结果 茶多糖测定的最佳方法为苯酚-硫酸法, 最适检测波长为484 nm, 葡萄糖质量浓度在20~100 μg/mL之间吸光度具有良好的线性关系, 其精密度相对标准偏差(relative standard deviation, RSD)为0.10%, 稳定性RSD为0.00%, 重复性RSD为2.40%, 加标回收率为94.25%~111.19%, 测得茶多糖含量为54.76 mg/g。结论 苯酚-硫酸法适用于以DES为提取溶剂的茶叶中茶多糖含量测定。

低共熔溶剂  /  茶多糖  /  检测方法

Objective To study the effects of deep eutectic solvent (DES) on the absorbance of tea polysaccharide by visible spectrophotometry, and establish a fast and efficient method for the determination of tea polysaccharide. Methods Choline chloride/DL-tartaric acid was used as the extraction solvent of tea polysaccharide. The influence of DES on the determination of tea polysaccharide content in anthrone sulfuric acid and phenol-sulfuric acid 2 kinds of detection methods was investigated. The linear relationship, precision, stability, repeatability and standard recovery rate of the 2 kinds of detection methods were compared. Results The best method for the determination of tea polysaccharides was phenol-sulfuric acid method, the optimal detection wavelength was 484 nm, and the absorbance had a good linear relationship with glucose concentration between 20 and 100 μg/mL. The precision relative standard deviation (RSD) of this method was 0.10%, the stability RSD was 0.00%, the repeatability RSD was 2.40%, and the recovery rate was 94.25%-111.19%, and the content of tea polysaccharide was 54.76 mg/g. Conclusion Phenol-sulfuric acid method is suitable for the determination of tea polysaccharide in tea with DES as extraction solvent.

deep eutectic solvents  /  tea polysaccharide  /  detection methods
曹佩琴, 肖艳芬, 宋一平. 新型低共熔溶剂对茶多糖吸光度的影响. 食品安全质量检测学报, 2025 , 16 (5) : 239 -245 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20241120003
Pei-Qin CAO, Yan-Fen XIAO, Yi-Ping SONG. Effects of new deep eutectic solvent on absorbance of tea polysaccharide[J]. Journal of Food Safety & Quality, 2025 , 16 (5) : 239 -245 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20241120003
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0 引言
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茶多糖(tea polysaccharides, TPS)是茶叶中非常重要的一类具有生物活性的大分子物质。茶多糖在茶叶中的含量为1.5%~13.0%, 是一类与蛋白质结合在一起的酸性多糖或酸性糖蛋白[1]。茶多糖由2~10种单糖组成, 包括鼠李糖、阿拉伯糖、葡萄糖、半乳糖、木糖、甘露糖、岩藻糖、半乳糖酸、葡萄糖酸和核糖, 糖苷键的链接方式为(1→2)、(1→3)、(1→4)和(1→6), 其平均分子量范围从1.02 kDa至4940 kDa[2-3]。体外和体内研究显示, 茶多糖具有多种生物活性和药理学潜力, 如抗氧化、保肝、免疫调节、抗病毒、抗菌、降糖和抗肿瘤活性[4-6]。
低共熔溶剂(deep eutectic solvent, DES)是由氢键供体与氢键受体通过氢键相互作用形成的共熔混合物[7]。DESs作为可替代有机溶剂的新一代环保“绿色”溶剂, 具有绿色环保可降解、无毒、挥发性低等优点, 受到了广泛的关注[8]。研究显示DESs在生物质原料前处理中有利于细胞破损, 活性成分的溶出, 能获得较高的提取率[9-11]。GAO等[12]使用氢受体为氯化胆碱、甜菜碱、甘氨酸和L-脯氨酸与氢受体为尿素、酸类和醇类等制备17种DES, 甜菜碱/乙二醇DES对油茶籽多糖的提取率最高为121.9 mg/g, 显著高于水和碱液, 工艺优化后最大提取率为152.37  mg/g。CAI等[13]用氢键受体(乙醇胺、二乙醇胺和三乙醇胺)与氢键供体(邻甲酚、间甲酚和对甲酚)制备9种DES提取灵芝多糖, 多糖得率均显著高于水。GUO等[14]采用热水提取、微波提取、超声-微波辅助提取、加压水提取、碱液提取、高速剪切均质提取、DES和超声辅助DES提取8种工艺提取甜茶粗多糖, 结果表明不同的提取工艺对茶多糖的组成、结构和生物学特性有不同的影响, 用DESs提取的茶多糖具有较好的生物学性能。目前, DESs提取茶叶中的多糖研究还较少。
DESs因其具有极强的可设计性, 组成多变复杂, 理化性质差异大[15-16], 对于茶多糖的提取率也有着极大的差异[17-18]。因此, 为了筛选出最优DES用于茶多糖的提取, 首先要确定检测方法。植物多糖因无变旋现象和还原性, 一般需要经过处理后才能检测出其含量。目前常见的检测方法有滴定法、比色法和色谱法等, 不同来源的多糖所适用的检测方法不同[19-21]。高效液相色谱法进行多糖检测时, 样品前处理工序比较烦琐, 测定时间较长, 且检测费用较高, 不便快速测定[22]。比色法利用组成多糖的单糖缩合反应而建立的方法, 如蒽酮-硫酸法、苯酚-硫酸法等, 相比于高效液相色谱法, 这两种方法操作简单、样品无需烦琐前处理、实用性强, 具有更好的使用价值[23-24]。因此蒽酮-硫酸法和苯酚-硫酸法可见分光光度法常用于茶多糖的含量检测。然而, 前期研究显示DESs的存在可能会影响茶多糖可见分光光度法含量检测结果的准确性。因此本研究比较了苯酚-硫酸法和蒽酮-硫酸法两种检测方法中, DESs在可见分光光度法检测中对茶多糖吸光度的影响, 考察了两种检测方法在线性关系、精密度、稳定性、重复性和加标回收率的优劣, 旨在建立最佳的茶多糖测定方法。
1 材料与方法
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1.1 试剂与材料
氯化胆碱、DL-酒石酸、草酸、L-脯氨酸、乙二醇、甜菜碱、尿素、苯酚、硫酸(分析纯, 上海泰坦科技股份有限公司); 葡萄糖(分析纯, 国药集团化学试剂有限公司); 蒽酮(分析纯, 北京酷来博科技有限公司)。
紧压金花黄茶粉末(30目, 岳阳市那山那水茶业有限责任公司)。
1.2 仪器与设备
IKA RH digital磁力搅拌器(德国艾卡公司); KQ3200E超声波清洗机(昆山市超声仪器有限公司); A580型紫外分光光度计(上海翱艺仪器有限公司); FA1104万分之一电子分析天平(上海梅特勒-托利多仪器有限公司)。
1.3 实验方法
1.3.1 DES溶剂的制备
按照摩尔比1:2称取氯化胆碱和酒石酸混合在密闭离心管中, 加入30 wt%的水, 在80 ℃下磁力搅拌加热, 直至溶液澄清透明。
1.3.2 茶多糖DES提取液制备
以含水量30 wt%的氯化胆碱-酒石酸(摩尔比1:2)为低共熔提取溶剂, 称取0.1 g茶粉, 加入2 mL DES提取剂, 60 ℃下超声30 min, 过滤, 滤液加水定容于100 mL容量瓶, 获得茶多糖DES提取液。按照相同条件, 水作为提取溶剂对茶粉进行提取, 过滤, 滤液加水定容于100 ml容量瓶, 获得到茶多糖水提液, 作为参比溶液。
1.3.3 相关标准溶液的配制
(1)葡萄糖对照品溶液的配制
配制成5 mg/mL的葡萄糖母液, 吸取2 mL加水定容至100 mL容量瓶, 作为100 μg/mL葡萄糖对照品溶液。按照上述条件, 配制5 mg/mL的葡萄糖DES母液, 吸取2 mL加水定容至100 mL容量瓶, 作为100 μg/mL葡萄糖DES对照品溶液。
(2) DES空白溶液的配制
取2 mL 1.3.1中的DES溶液于100 mL容量瓶中, 加水定容, 获得DES空白溶液。
1.3.4 DES对蒽酮-硫酸和苯酚-硫酸可见分光光度检测方法的影响
(1) DES对检测波长的影响
蒽酮-硫酸法: 分别取葡萄糖对照品溶液、茶多糖水提液、葡萄糖DES对照品溶液和茶多糖DES提取液各0.5 mL, 加入4 mL 6%蒽酮-硫酸混匀, 沸水浴3 min后冷却以超纯水作为试剂空白, 溶液于450~800 nm范围内扫描, 绘制可见吸收光谱图, 确定其最大吸收波长。
苯酚-硫酸法: 分别取葡萄糖对照品溶液、茶多糖水提液、葡萄糖DES对照品溶液和茶多糖DES提取液各0.5 mL, 加入0.5 mL水、0.5 mL 5%苯酚溶液, 振荡后加入2.5 mL硫酸, 混匀, 沸水浴15 min后冷却。以超纯水作为试剂空白, 于400~800 nm范围内扫描, 绘制可见吸收光谱图, 确定其最大吸收波长。
(2)水浴时间对吸光度的影响
蒽酮-硫酸法: 分别取葡萄糖对照品溶液和DES空白溶液与蒽酮-硫酸和苯酚-硫酸反应沸水浴分别反应3、6、9、12、15 min后冷却, 于最大吸收波长处测定吸光度值。
苯酚-硫酸法: 分别取葡萄糖对照品溶液和DES空白溶液与蒽酮-硫酸和苯酚-硫酸反应后, 沸水浴分别反应5、10、15、20、25 min后冷却, 于最大吸收波长处测定吸光度值。
(3) DES含量对吸光度的影响
分别移取2、4、6、8、10 mL DES溶液定容至100 mL容量瓶, 移取各溶液与蒽酮-硫酸反应后沸水浴3 min后冷却, 与苯酚-硫酸反应后沸水浴15min, 冷却后于最大吸收波长处测定吸光度值。
1.3.5 方法学研究
(1)线性关系考察
蒽酮-硫酸法: 吸取葡萄糖母液, 配成质量浓度为25、50、100、150、200、250 μg/mL的葡萄糖溶液。溶液按照最佳检测条件反应后, 在最大吸收波长测定吸光度, 以吸光度值为横坐标(X), 葡萄糖标准溶液质量浓度为纵坐标(Y, μg/mL), 制作标准曲线, 得到线性回归方程。
苯酚-硫酸法: 吸取葡萄糖母液, 配成质量浓度为20、40、60、80、100 μg/mL的葡萄糖溶液。溶液按照最佳检测条件反应后, 在最大吸收波长测定吸光度, 以吸光度值为横坐标(X), 葡萄糖标准溶液质量浓度为纵坐标(Y, μg/mL), 制作标准曲线, 得到线性回归方程。
(2)精密度
吸取0.5 mL茶多糖提取液, 按照蒽酮-硫酸和苯酚-硫酸两种方法分别操作, 连续测定6次。按照公式(1)计算RSD值。
RSD/%=(标准偏差/算术平均值)×100%
(3)稳定性
参照蒽酮-硫酸和苯酚-硫酸的方法, 精确量取0.5 mL茶多糖提取液, 加热后冷却, 分别在冷却后0、10、20、30、40、50 min时测定吸光度, 考察50 min内显色液及方法的稳定性。计算RSD值。
(4)重复性
吸取相同的茶多糖提取液6份, 尽量保证每份溶液完全相同, 参照蒽酮-硫酸和苯酚-硫酸中的方法, 测定吸光度, 计算RSD值。
(5)加标回收率
首先吸取0.5 mL茶多糖提取液, 照蒽酮-硫酸和苯酚-硫酸中的方法, 测定吸光度, 代入标准曲线方程中, 计算出茶多糖的含量, 再准备6份相同的茶多糖粗提液, 分为2组, 加入0.5 mL已知不同浓度的葡萄糖溶液, 分别按照苯酚-硫酸法和蒽酮-硫酸法测定吸光度。计算加标回收率和RSD值。加标回收率的计算如公式(2)所示。
加标回收率/%=(加标样品测定值-样品测定值)/加标量×100%
1.3.6 茶多糖含量的测定
称取6份相同的茶粉, 按照1.3.2中的方法提取, 提取后取3份用蒽酮-硫酸法测定吸光值, 另3份用苯酚-硫酸法测定, 再通过线性回归方程计算多糖含量。
1.4 数据处理
所有实验数据均进行3次平行实验, 平均值±相对标准偏差(relative standard deviation, RSD)。表示结果。采用SPSS 21软件进行显著性分析, 采用方差分析和邓肯a分析, 当P<0.05时具有统计学上显著性差异, 使用Origin 2022软件处理实验数据并绘制成图。
2 结果与分析
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2.1 DES对苯酚-硫酸和蒽酮-硫酸检测方法的影响
苯酚-硫酸法的原理, 多糖在硫酸的作用下生成糖醛衍生物, 然后与苯酚生成橙黄色化合物, 在490 nm处有一最大紫外吸收峰, 随着多糖含量的增加, 其颜色和吸光度也会递增[25]。蒽酮-硫酸法的原理是多糖在硫酸的作用下生成糖醛衍生物(羟甲基糖醛), 再与蒽酮脱和水缩合成蓝绿色化合物, 其颜色的深浅和吸光值与多糖含量呈正比[26]。
2.1.1 检测波长对苯酚-硫酸和蒽酮-硫酸法吸光度的影响
葡萄糖对照品溶液、葡萄糖DES对照品溶液、茶多糖水提液和茶多糖DES提取液与苯酚-硫酸和蒽酮-硫酸反应后在400~800 nm的可见光扫描结果如图1所示。如图1A所示, 4种溶液均只有一个特征吸收峰, 峰形一致, 对称性好。茶多糖水提液和茶多糖DES提取液与苯酚-硫酸反应后的最大吸收波长均在484 nm, 与文献中的490 nm最大吸收波长相吻合[27-28]。因此选择484 nm作为茶多糖苯酚-硫酸法含量测定波长。如图1B所示, 葡萄糖DES对照品溶液和茶多糖DES提取液与蒽酮-硫酸反应后, 在465 nm处存在一个最大的吸收峰, 而茶多糖水提液和葡萄糖对照品溶液在此处没有特征吸收, 这可能是DES与蒽酮-硫酸的特征吸收峰。同时, 4种溶液在567 nm波长处均具有明显的特征吸收峰, 可作为后续茶多糖含量测定的最大吸收波长。综上, 以绿色溶剂DES为提取溶剂提取茶叶中茶多糖, 茶多糖在苯酚-硫酸作用下生成络合衍生物组分单一, 显色效果良好, 与蒽酮-硫酸相比较稳定性较好。
2.1.2 水浴时间对蒽酮-硫酸和苯酚-硫酸法吸光度的影响
葡萄糖对照品溶液和DES空白溶液与苯酚-硫酸和蒽酮-硫酸沸水浴时间对吸光度的影响结果如图2所示。图2A所示, 苯酚-硫酸水浴时间对葡萄糖对照品溶液吸光度0.428~0.485之间波动; 对DES空白溶液吸光度在0.007~0.009之间波动, 对吸光度的影响基本可以忽略。根据其他参考文献的最佳水浴时间, 选择水浴时间15 min作为后续反应条件。图2B所示, 随着加热时间的延长, 葡萄糖对照品溶液的吸光度由0.538增至0.727, 加热9 min显著高于3 min和6 min的吸光度(P<0.05)。同时, 加热时间对DES空白溶液的影响更加明显, 在加热3 min时, 吸光度仅有0.024, 而在加热15 min时显著增加(P<0.05), 吸光度变为0.466。因此, 水浴时间会对葡萄糖和DES溶液的吸光度产生较大影响, 考虑到DES茶多糖粗提液中含有DES, 选择加热时间3 min作为后续研究。综上, 苯酚-硫酸法在显色反映过程中水浴时间对吸光度的影响小于蒽酮-硫酸法, 更加适合于以DES为提取溶剂的茶多糖含量检测。
2.1.3 DES含量对苯酚-硫酸和蒽酮-硫酸法吸光度的影响
DES加入量对苯酚-硫酸和蒽酮-硫酸法吸光度的影响结果如图3所示。如图3A所示, 随着DES含量的增加, 苯酚-硫酸法吸光度呈现出增加的趋势。在DES含量为2 mL时, 吸光度为0.0137, 而当DES的含量为10 mL时, 吸光度显著增加至0.113 (P<0.05)。如图3B所示, 随着DES含量的增加, 蒽酮-硫酸法吸光度呈现增加的趋势, 在DES含量为2 mL时吸光度为0.015, 而当DES的含量为10 mL时, 吸光度为0.051 (P<0.05)。综上, DES与苯酚-硫酸反应后会有一定的吸光度, 且会随着DES含量的增加而累加, 变化趋势较蒽酮-硫酸法大。因此, 选择苯酚-硫酸和蒽酮-硫酸法检测茶多糖含量需要考虑溶液中DES含量对吸光度的影响, 消除溶剂误差。
2.2 方法学研究
2.2.1 线性关系考察
硫酸-蒽酮法葡萄糖溶液的回归方程为Y=0.00547X+0.02077, r2=0.9996, 说明葡萄糖质量浓度在25~175 μg/mL之间, 吸光度具有线性关系良好。苯酚-硫酸法葡萄糖溶液的回归方程为Y=0.00848X+0.015, r2=0.9996, 说明葡萄糖质量浓度在20~100 μg/mL之间, 吸光度具有线性关系良好。
2.2.2 精密度
苯酚-硫酸法和蒽酮-硫酸法精密度的结果如表1所示, 蒽酮-硫酸法的RSD为0.12%, 苯酚-硫酸法的RSD为0.10%, 两种方法的RSD值均较小, 说明仪器的精密度良好。
2.2.3 稳定性
苯酚-硫酸法和蒽酮-硫酸法稳定性结果见表2。两种方法50 min内均表现出较高的稳定性, 其中, 蒽酮-硫酸法吸光度稳定性较低, RSD为0.30%, 而苯酚-硫酸法RSD为0.00%, 在50 min内吸光度没有波动, 说明该溶液50 min内稳定。
2.2.4 重复性
苯酚-硫酸法和蒽酮-硫酸法稳定性结果表3。结果表明, 蒽酮-硫酸法和苯酚-硫酸法的RSD分别为3.07%和2.40%, RSD均小于5.0%, 说明这两种方法的重现性良好。苯酚-硫酸法的RSD值略小于蒽酮-硫酸法, 苯酚-硫酸法的重现性优于蒽酮-硫酸法。
2.2.5 加标回收率
蒽酮-硫酸和苯酚-硫酸法的加标回收率结果见表4。按照相关标准, 加标回收率的合适范围为80%~120%, 蒽酮-硫酸法的加标回收率为95.23%~116.20%, 平均加标回收率为103.88%; 苯酚-硫酸法的加标回收率为94.25%~ 111.19%, 平均加标回收率为100.96%, 两种方法的加标回收率均在标准范围内, 表明这两种方法均适用于多糖的含量测定。其中苯酚-硫酸法的RSD值较蒽酮-硫酸法较低, 说明苯酚-硫酸法测定DES茶多糖粗提液中多糖含量精确度更高。
2.2.6 茶多糖的含量
苯酚-硫酸和蒽酮硫酸法对茶多糖含量测定结果如表5所示。蒽酮-硫酸法测定茶多糖含量为83.22 mg/g, 其RSD为6.83%。苯酚-硫酸法测定茶多糖含量为54.76 mg/g, 其RSD为3.12%, 由此看出, 苯酚-硫酸法与蒽酮-硫酸法测定茶多糖含量存在一定的差异, 因为苯酚-硫酸法多用于样品中甲基化的糖、戊糖和多聚糖的测定, 基本不受蛋白质的影响; 而蒽酮-硫酸法几乎可以测定所有可溶性碳水化合物的含量, 但其显色易受干扰, 稳定性较差[28-30]。综合比较, 苯酚-硫酸法测定茶多糖含量的RSD较小, 故苯酚-硫酸法优于蒽酮-硫酸法。
3 结论
收起
本研究建立了一种基于绿色新型溶剂DES的茶多糖分光光度检测方法。该法以氯化胆碱/DL-酒石酸DES为提取溶剂提取茶叶中茶多糖。目前, 常用的茶多糖检测方法为蒽酮-硫酸法和苯酚-硫酸法, 研究中发现苯酚-硫酸与DES茶叶提取液中茶多糖反应后全波长扫描有明显的特征吸收峰, 操作条件水浴时间差异对吸光度的影响较小, 有利于茶多糖含量检测, 而且苯酚-硫酸法检测方法得出的茶多糖含量远低于蒽酮-硫酸法, 与其他文献中多糖检测方法比较结果一致。然而, 在研究DES含量对吸光度的影响时, 随着检测溶液中DES含量增加吸光度增加。因此, 在检测过程中应对反应条件严格控制, 对于样品体系中DES含量保持一致, 或者检测时需通过DES空白溶液扣除溶剂影响, 获得准确稳定的数据。
基金
收起
  • 湖南省教育厅青年项目(21B0587)
文献
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20241120003
  • 接收时间：2024-11-20
  • 首发时间：2025-07-19
  • 出版时间：2025-03-15
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  • 收稿日期：2024-11-20
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湖南省教育厅青年项目(21B0587)
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    湖南理工学院化学化工学院, 岳阳 414006

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* 曹佩琴(1989—), 女, 博士, 讲师, 主要研究方向为茶叶功能成分利用。E-mail:
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2种不同金属材料的力学参数
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Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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