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Article(id=1198652618005377950, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1198652605778985059, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2023-0294, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1678377600000, receivedDateStr=2023-03-10, revisedDate=1688486400000, revisedDateStr=2023-07-05, acceptedDate=null, acceptedDateStr=null, onlineDate=1763710654020, onlineDateStr=2025-11-21, pubDate=1691769600000, pubDateStr=2023-08-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1763710654020, onlineIssueDateStr=2025-11-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1763710654020, creator=13701087609, updateTime=1763710654020, updator=13701087609, issue=Issue{id=1198652605778985059, tenantId=1146029695717560320, journalId=1189982191388893191, year='2023', volume='58', issue='8', pageStart='0', pageEnd='2540', issueExtLink='null', onlineDate='null', pubDate='1691769600000', pubDateStr='2023-08-12', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1763710651106, creator='13701087609', updateTime=1763710739504, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1198652976601596347, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1198652605778985059, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1198652976601596348, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1198652605778985059, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=2476, endPage=2482, ext={EN=ArticleExt(id=1198652618575803322, articleId=1198652618005377950, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Determination of monosaccharide composition of polysaccharide and free monosaccharides in Cistanches Herba by ion chromatography-integral pulsed amperometry, columnId=null, journalTitle=Acta Pharmaceutica Sinica, columnName=null, runingTitle=null, highlight=null, articleAbstract=

Polysaccharides and free monosaccharides are important active components in Cistanches Herba, which have functions of anti-aging and immunological activity regulation. The study of monosaccharide composition in polysaccharide and free monosaccharide can lay a foundation for the study of primary structure, spatial structure of Cistanche polysaccharide and biological activity of Cistanches Herba. In this study, a method of water extraction and alcohol precipitation was used to extract Cistanche polysaccharide. Trifluoroacetic acid was selected as the hydrolytic acid for polysaccharide hydrolysis. An orthogonal experimental method is established. Three levels of acid concentration, hydrolysis temperature and hydrolysis time were selected to investigate the optimal hydrolysis condition. The optimal hydrolysis condition was 0.08 mol·L-1 trifluoroacetic acid hydrolysis at 100 ℃ for 3 h. The free monosaccharides of Cistanches Herba were extracted by water extraction. The established ion chromatogram integrated pulsed amperometry method can efficiently separate 11 monosaccharides in a short time. The method has good repeatability and high sensitivity, methodological experiment results meet the requirements of quantitative determination. It can accurately determine the monosaccharide composition of Cistanche polysaccharide and free monosaccharide content. Ion chromatography does not require derivatization operation and the pre-treatment steps are simple. This method can measure fructose, but PMP derivation-HPLC method can't. The monosaccharide composition of Cistanche polysaccharide include fucose, arabinose, rhamnose-galactose, glucose, xylose, mannose, fructose, ribose and glucuronic acid, among which the contents of glucose and fructose are relatively high. The free monosaccharides in the water extract of Cistanches Herba include glucose, fructose and mannose.

, authors=null, authorsList=Wei-yi XU, Zhen-bang JIANG, Jing FAN, Hong-yu JIN, Shuang-cheng MA, authorCompany=null, correspAuthors=Hong-yu JIN, Shuang-cheng MA, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2023 Acta Pharmaceutica Sinica. All rights reserved., 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, fund=null), CN=ArticleExt(id=1198652619481772043, articleId=1198652618005377950, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=离子色谱-积分脉冲安培法测定肉苁蓉多糖的单糖组成及游离单糖含量, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

肉苁蓉多糖和游离态单糖是肉苁蓉中的重要活性成分, 具有抗衰老、免疫活性调节等作用。多糖的单糖组成和游离态单糖研究可以为肉苁蓉多糖一级结构、空间结构及肉苁蓉生物活性的研究奠定基础。本研究采用水提醇沉法提取肉苁蓉多糖, 多糖水解选取三氟乙酸作为水解酸, 建立正交实验考察三氟乙酸浓度、温度、水解时间对多糖裂解的影响, 确定最佳水解条件: 0.08 mol·L-1三氟乙酸在100 ℃水解3 h; 采用水提法提取肉苁蓉游离态单糖。建立的离子色谱-积分脉冲安培法可在短时间内高效分离11种单糖, 方法重复性好, 灵敏度高, 方法学结果符合定量测定要求, 可准确测定肉苁蓉多糖的单糖组成及游离单糖含量。离子色谱法不需要衍生化操作, 前处理步骤简单, 填补了PMP衍生-HPLC法无法测定果糖的缺陷。肉苁蓉多糖的单糖组成有岩藻糖、阿拉伯糖、鼠李糖、半乳糖、葡萄糖、木糖、甘露糖、果糖、核糖、葡萄糖醛酸, 其中葡萄糖、果糖含量相对较高; 肉苁蓉水提液中游离态单糖有葡萄糖、果糖、甘露糖。

, authors=null, authorsList=许玮仪, 姜振邦, 范晶, 金红宇, 马双成, authorCompany=null, correspAuthors=金红宇, 马双成, authorNote=null, correspAuthorsNote=
*金红宇, Tel: 86-10-53851413, E-mail: ;
马双成, Tel: 86-10-53852072, E-mail:
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Pharm Today (今日药学), 2016, 26: 304-307., articleTitle=Determination of monosaccharides in polysaccharide extracted from Sophorae tonkinensis Gagnep. by PMP derivatization-high performance liquid chromatography, refAbstract=null)], funds=[Fund(id=1198960118189621593, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652618005377950, awardId=2022YFC3501505, language=CN, fundingSource=国家重点研发计划(2022YFC3501505), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1198960111965274807, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652618005377950, xref=null, ext=[AuthorCompanyExt(id=1198960111994634942, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652618005377950, companyId=1198960111965274807, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1. 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Fuc: Fucose; Ara: Arabinose; Rha: Rhamnose; Gal: Galactose; Glc: Glucose; Xyl: Xylose; Man: Mannose; Fru: Fructose; Rib: Ribose; Glc-A: Glucuronic acid , figureFileSmall=nL+h8rVh+Cc49/57lMW/rw==, figureFileBig=anWg1qkteEHyAihR1B0toQ==, tableContent=null), ArticleFig(id=1198960116662894762, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652618005377950, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
No. Name Harvest site Harvest time Processing method
1 Herb of Cistanche deserticola Alxa Ejina Banner, Inner Mongolia 2019, autumn Dry in the sun
2 Herb of Cistanche deserticola Bayannur City, Inner Mongolia 2019, autumn Dry in the sun
3 Herb of Cistanche deserticola Bayannur City, Inner Mongolia 2019, autumn Dry in the shade
4 Herb of Cistanche salsa Si zi wang qi, Inner Mongolia 2020, spring Dry in the shade
5 Herb of Cistanche salsa Sonite Left Banner, Ximeng, Inner Mongolia 2020, spring Dry in the sun
6 Herb of Cistanche salsa A zuo qi, Inner Mongolia 2020, spring Dry in the sun
7 Herb of Cistanche sinensis A zuo qi, Inner Mongolia 2020, spring Dry in the shade
8 Herb of Cistanche sinensis Alxa Left Banner, Inner Mongolia 2020, spring Dry in the sun
9 Herb of Cistanche deserticola A Meng, Inner Mongolia 2020, spring Dry in the sun
10 Herb of Cistanche deserticola A Meng, Inner Mongolia 2020, autumn Dry in the sun
11 Herb of Cistanche deserticola Ba Meng, Inner Mongolia 2020, spring Dry in the sun
12 Herb of Cistanche deserticola Ba Meng, Inner Mongolia 2020, autumn Dry in the sun
13 Herb of Cistanche deserticola A Meng, Inner Mongolia 2020, autumn Dry in the sun
), ArticleFig(id=1198960116813889726, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652618005377950, language=CN, label=Table 1, caption=

Cistanches Herba sample information

, figureFileSmall=null, figureFileBig=null, tableContent=
No. Name Harvest site Harvest time Processing method
1 Herb of Cistanche deserticola Alxa Ejina Banner, Inner Mongolia 2019, autumn Dry in the sun
2 Herb of Cistanche deserticola Bayannur City, Inner Mongolia 2019, autumn Dry in the sun
3 Herb of Cistanche deserticola Bayannur City, Inner Mongolia 2019, autumn Dry in the shade
4 Herb of Cistanche salsa Si zi wang qi, Inner Mongolia 2020, spring Dry in the shade
5 Herb of Cistanche salsa Sonite Left Banner, Ximeng, Inner Mongolia 2020, spring Dry in the sun
6 Herb of Cistanche salsa A zuo qi, Inner Mongolia 2020, spring Dry in the sun
7 Herb of Cistanche sinensis A zuo qi, Inner Mongolia 2020, spring Dry in the shade
8 Herb of Cistanche sinensis Alxa Left Banner, Inner Mongolia 2020, spring Dry in the sun
9 Herb of Cistanche deserticola A Meng, Inner Mongolia 2020, spring Dry in the sun
10 Herb of Cistanche deserticola A Meng, Inner Mongolia 2020, autumn Dry in the sun
11 Herb of Cistanche deserticola Ba Meng, Inner Mongolia 2020, spring Dry in the sun
12 Herb of Cistanche deserticola Ba Meng, Inner Mongolia 2020, autumn Dry in the sun
13 Herb of Cistanche deserticola A Meng, Inner Mongolia 2020, autumn Dry in the sun
), ArticleFig(id=1198960116893581514, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652618005377950, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Time/min Voltage/V Integration
0.000 0.100
0.200 0.100 On
0.400 0.100 Off
0.410-0.420 -2.000
0.430 0.600
0.440-0.500 -0.100
), ArticleFig(id=1198960116985856214, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652618005377950, language=CN, label=Table 2, caption=

Waveform of carbohydrates

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Time/min Voltage/V Integration
0.000 0.100
0.200 0.100 On
0.400 0.100 Off
0.410-0.420 -2.000
0.430 0.600
0.440-0.500 -0.100
), ArticleFig(id=1198960117120073956, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198652618005377950, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Compound Linear range/μg·mL-1 Regression equation R2 LOQ/μg·mL-1 Precision/%
Fuc 0.5-20.0 Y = 0.21x + 0.292 0 0.997 6 0.008 3.7
Ara 0.5-20.0 Y = 0.267 5x + 0.320 1 0.998 0 0.015 3.8
Rha 0.5-20.0 Y = 0.178 5x + 0.078 6 0.999 4 0.012 3.3
Gal 0.5-20.0 Y = 0.271 1x + 0.534 1 0.999 7 0.014 3.9
Glc 0.5-20.0 Y = 0.430 9x + 0.057 9 0.999 8 0.014 2.6
Xyl 0.5-20.0 Y = 0.457 6x + 0.067 6 0.999 8 0.020 2.1
Man 0.5-20.0 Y = 0.318 9x + 0.062 6 0.999 5 0.030 3.4
Fru 0.5-20.0 Y = 0.238 9x - 0.000 7 0.999 8 0.077 4.1
Rib 0.5-20.0 Y = 0.351 1x - 0.057 1 0.999 5 0.060 2.5
Glc-A 0.5-50.0 Y = 1.129 5x + 0.347 3 0.999 8 0.040 3.0
Gal-A 0.5-50.0 Y = 0.529 4x + 0.087 7 0.999 9 0.080 3.1
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Linear ranges, regression equations, correlation coefficients, LOQs and precisions of the 9 monosaccharides and 2 uronic acids. Gal-A: Galacturonic acid; Y: Peak area; x: Mass concentration, μg·mL-1; LOQ: Limit of quantitation

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Compound Linear range/μg·mL-1 Regression equation R2 LOQ/μg·mL-1 Precision/%
Fuc 0.5-20.0 Y = 0.21x + 0.292 0 0.997 6 0.008 3.7
Ara 0.5-20.0 Y = 0.267 5x + 0.320 1 0.998 0 0.015 3.8
Rha 0.5-20.0 Y = 0.178 5x + 0.078 6 0.999 4 0.012 3.3
Gal 0.5-20.0 Y = 0.271 1x + 0.534 1 0.999 7 0.014 3.9
Glc 0.5-20.0 Y = 0.430 9x + 0.057 9 0.999 8 0.014 2.6
Xyl 0.5-20.0 Y = 0.457 6x + 0.067 6 0.999 8 0.020 2.1
Man 0.5-20.0 Y = 0.318 9x + 0.062 6 0.999 5 0.030 3.4
Fru 0.5-20.0 Y = 0.238 9x - 0.000 7 0.999 8 0.077 4.1
Rib 0.5-20.0 Y = 0.351 1x - 0.057 1 0.999 5 0.060 2.5
Glc-A 0.5-50.0 Y = 1.129 5x + 0.347 3 0.999 8 0.040 3.0
Gal-A 0.5-50.0 Y = 0.529 4x + 0.087 7 0.999 9 0.080 3.1
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Cistanche Compound Total/μg·mL-1 Background/μg·mL-1 Added/μg·mL-1 Recovery/% RSD/%
Monosaccharides after hydrolysis of polysaccharide Fuc 18.02 0 19.80 91.0 3.0
Ara 107.36 33.68 74.73 98.6 2.8
Rha 34.43 16.08 20.02 91.7 6.6
Gal 25.77 7.64 20.00 90.7 4.3
Glc 583.20 101.08 563.22 85.6 1.4
Xyl 9.34 1.24 7.55 107.3 3.0
Man 11.17 3.07 7.80 103.8 7.0
Fru 444.46 148.43 255.20 116.0 2.1
Rib 1.79 1.11 0.73 93.2 7.1
Glc-A 3.09 1.39 1.89 89.9 2.0
Gal-A 1.94 0 2.03 95.6 3.2
Free monosaccharides Glc 79.59 34.77 49.60 90.4 9.3
Man 2.88 1.67 1.30 93.1 1.6
Fru 94.51 43.73 52.69 96.4 4.8
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Spiked recoveries and RSDs of the monosaccharides after hydrolysis of polysaccharide and the free monosaccharides in Cistanche

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Cistanche Compound Total/μg·mL-1 Background/μg·mL-1 Added/μg·mL-1 Recovery/% RSD/%
Monosaccharides after hydrolysis of polysaccharide Fuc 18.02 0 19.80 91.0 3.0
Ara 107.36 33.68 74.73 98.6 2.8
Rha 34.43 16.08 20.02 91.7 6.6
Gal 25.77 7.64 20.00 90.7 4.3
Glc 583.20 101.08 563.22 85.6 1.4
Xyl 9.34 1.24 7.55 107.3 3.0
Man 11.17 3.07 7.80 103.8 7.0
Fru 444.46 148.43 255.20 116.0 2.1
Rib 1.79 1.11 0.73 93.2 7.1
Glc-A 3.09 1.39 1.89 89.9 2.0
Gal-A 1.94 0 2.03 95.6 3.2
Free monosaccharides Glc 79.59 34.77 49.60 90.4 9.3
Man 2.88 1.67 1.30 93.1 1.6
Fru 94.51 43.73 52.69 96.4 4.8
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Compound Herb of Cistanche salsa Herb of Cistanche sinensis Herb of Cistanche deserticola
Content/μg·g-1 Molar ratio Content/μg·g-1 Molar ratio Content/μg·g-1 Molar ratio
Fuc 60.34 1.00 36.25 1.00 45.60 1.00
Ara 1 679.35 30.43 1 547.45 46.67 1 875.56 44.97
Rha 1 479.66 22.10 2 676.81 66.54 1 595.74 31.53
Gal 343.41 5.19 287.51 7.23 347.40 6.94
Glc 11 555.63 174.49 5 596.14 140.66 12 800.92 255.78
Xyl 305.59 5.54 350.04 10.56 190.60 4.57
Man 163.23 2.46 89.97 2.26 169.15 3.38
Fru 7 316.44 110.48 3 196.96 80.35 9 435.65 188.53
Rib 71.50 1.30 65.70 1.98 78.88 1.89
Glc-A 68.70 0.96 59.70 1.39 56.60 1.05
Total 23 043.84 / 13 906.53 / 26 596.09 /
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The content and molar ratios of monosaccharides after polysaccharide hydrolysis in 3 kinds of Cistanche

, figureFileSmall=null, figureFileBig=null, tableContent=
Compound Herb of Cistanche salsa Herb of Cistanche sinensis Herb of Cistanche deserticola
Content/μg·g-1 Molar ratio Content/μg·g-1 Molar ratio Content/μg·g-1 Molar ratio
Fuc 60.34 1.00 36.25 1.00 45.60 1.00
Ara 1 679.35 30.43 1 547.45 46.67 1 875.56 44.97
Rha 1 479.66 22.10 2 676.81 66.54 1 595.74 31.53
Gal 343.41 5.19 287.51 7.23 347.40 6.94
Glc 11 555.63 174.49 5 596.14 140.66 12 800.92 255.78
Xyl 305.59 5.54 350.04 10.56 190.60 4.57
Man 163.23 2.46 89.97 2.26 169.15 3.38
Fru 7 316.44 110.48 3 196.96 80.35 9 435.65 188.53
Rib 71.50 1.30 65.70 1.98 78.88 1.89
Glc-A 68.70 0.96 59.70 1.39 56.60 1.05
Total 23 043.84 / 13 906.53 / 26 596.09 /
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离子色谱-积分脉冲安培法测定肉苁蓉多糖的单糖组成及游离单糖含量
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许玮仪 1 , 姜振邦 2 , 范晶 1 , 金红宇 1, * , 马双成 1, *
药学学报 | 研究论文 2023,58(8): 2476-2482
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药学学报 |研究论文 2023 , 58 (8) : 2476 -2482
离子色谱-积分脉冲安培法测定肉苁蓉多糖的单糖组成及游离单糖含量
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许玮仪1, 姜振邦2, 范晶1, 金红宇1, * , 马双成1, *
作者信息
  • 1.中国食品药品检定研究院, 北京 102629
  • 2.赛默飞世尔科技(中国)有限公司, 上海 201206
通讯作者:
*金红宇, Tel: 86-10-53851413, E-mail: ;
马双成, Tel: 86-10-53852072, E-mail:
Determination of monosaccharide composition of polysaccharide and free monosaccharides in Cistanches Herba by ion chromatography-integral pulsed amperometry
Wei-yi XU1, Zhen-bang JIANG2, Jing FAN1, Hong-yu JIN1, * , Shuang-cheng MA1, *
Affiliations
  • 1. National Institutes for Food and Drug Control, Beijing 102629, China
  • 2. Thermo Fisher Scientific China, Shanghai 201206, China
出版时间: 2023-08-12 doi: 10.16438/j.0513-4870.2023-0294
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摘要
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肉苁蓉多糖和游离态单糖是肉苁蓉中的重要活性成分, 具有抗衰老、免疫活性调节等作用。多糖的单糖组成和游离态单糖研究可以为肉苁蓉多糖一级结构、空间结构及肉苁蓉生物活性的研究奠定基础。本研究采用水提醇沉法提取肉苁蓉多糖, 多糖水解选取三氟乙酸作为水解酸, 建立正交实验考察三氟乙酸浓度、温度、水解时间对多糖裂解的影响, 确定最佳水解条件: 0.08 mol·L-1三氟乙酸在100 ℃水解3 h; 采用水提法提取肉苁蓉游离态单糖。建立的离子色谱-积分脉冲安培法可在短时间内高效分离11种单糖, 方法重复性好, 灵敏度高, 方法学结果符合定量测定要求, 可准确测定肉苁蓉多糖的单糖组成及游离单糖含量。离子色谱法不需要衍生化操作, 前处理步骤简单, 填补了PMP衍生-HPLC法无法测定果糖的缺陷。肉苁蓉多糖的单糖组成有岩藻糖、阿拉伯糖、鼠李糖、半乳糖、葡萄糖、木糖、甘露糖、果糖、核糖、葡萄糖醛酸, 其中葡萄糖、果糖含量相对较高; 肉苁蓉水提液中游离态单糖有葡萄糖、果糖、甘露糖。

离子色谱-积分脉冲安培  /  肉苁蓉  /  单糖组成  /  肉苁蓉多糖  /  游离单糖

Polysaccharides and free monosaccharides are important active components in Cistanches Herba, which have functions of anti-aging and immunological activity regulation. The study of monosaccharide composition in polysaccharide and free monosaccharide can lay a foundation for the study of primary structure, spatial structure of Cistanche polysaccharide and biological activity of Cistanches Herba. In this study, a method of water extraction and alcohol precipitation was used to extract Cistanche polysaccharide. Trifluoroacetic acid was selected as the hydrolytic acid for polysaccharide hydrolysis. An orthogonal experimental method is established. Three levels of acid concentration, hydrolysis temperature and hydrolysis time were selected to investigate the optimal hydrolysis condition. The optimal hydrolysis condition was 0.08 mol·L-1 trifluoroacetic acid hydrolysis at 100 ℃ for 3 h. The free monosaccharides of Cistanches Herba were extracted by water extraction. The established ion chromatogram integrated pulsed amperometry method can efficiently separate 11 monosaccharides in a short time. The method has good repeatability and high sensitivity, methodological experiment results meet the requirements of quantitative determination. It can accurately determine the monosaccharide composition of Cistanche polysaccharide and free monosaccharide content. Ion chromatography does not require derivatization operation and the pre-treatment steps are simple. This method can measure fructose, but PMP derivation-HPLC method can't. The monosaccharide composition of Cistanche polysaccharide include fucose, arabinose, rhamnose-galactose, glucose, xylose, mannose, fructose, ribose and glucuronic acid, among which the contents of glucose and fructose are relatively high. The free monosaccharides in the water extract of Cistanches Herba include glucose, fructose and mannose.

ion chromatography-integrated pulse amperometry  /  Cistanches Herba  /  monosaccharide composition  /  polysaccharide  /  free monosaccharide
许玮仪, 姜振邦, 范晶, 金红宇, 马双成. 离子色谱-积分脉冲安培法测定肉苁蓉多糖的单糖组成及游离单糖含量. 药学学报, 2023 , 58 (8) : 2476 -2482 . DOI: 10.16438/j.0513-4870.2023-0294
Wei-yi XU, Zhen-bang JIANG, Jing FAN, Hong-yu JIN, Shuang-cheng MA. Determination of monosaccharide composition of polysaccharide and free monosaccharides in Cistanches Herba by ion chromatography-integral pulsed amperometry[J]. Acta Pharmaceutica Sinica, 2023 , 58 (8) : 2476 -2482 . DOI: 10.16438/j.0513-4870.2023-0294
正文
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肉苁蓉原植物是列当科植物肉苁蓉属的寄生植物, 经过调研, 我国有5种肉苁蓉, 分别是荒漠肉苁蓉、沙苁蓉、管花肉苁蓉、盐生肉苁蓉、白花盐苁蓉[1-3]。肉苁蓉在我国有2 000多年的药用历史, 在处方中应用广泛, 肉苁蓉属植物具有多种生物活性, 如补肾壮阳、抗氧化、抗疲劳、利尿通便、增强记忆等作用[4]。
研究表明, 肉苁蓉多糖是肉苁蓉属植物最重要的活性成分之一[5]。肉苁蓉多糖具有抗衰老、免疫活性调节、改善脾虚、抗病毒抗肿瘤、促进造血等药理作用[6-10]。多糖的生物活性与其理化性质、相对分子质量范围、多糖的组成成分等有关, 而能准确测定肉苁蓉多糖中单糖组成, 则对研究其化学结构和性质具有重要意义[11, 12]。一级结构是空间结构的基础, 单糖组成是多糖一级机构的主要内容, 游离态单糖也是肉苁蓉糖组成的重要部分, 因此测定肉苁蓉多糖的单糖组成和游离单糖具有重要意义[13]。
本文考察了肉苁蓉多糖的水解条件, 研究了离子色谱-积分脉冲安培法测定9种单糖及2种糖醛酸, 为肉苁蓉多糖的深入研究与应用奠定基础。离子色谱-积分脉冲安培法, 采用不同浓度氢氧化钠淋洗液, 无需衍生即可分离测定样品中的糖类化合物, 分离度好, 检测灵敏度高, 低消耗、环保[14]。
材料与方法
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仪器与试剂  ICS-5000型离子色谱仪, Au工作电极, AgCl参比电极, Chromeleon7色谱工作站, AS自动进样器。分析柱: Dionex Carbopac PA210-Fast-4 μm, 150 mm × 4 mm; 保护柱: Dionex Carbopac PA210-Fast-4 μm, 30 mm × 4 mm。以上均购于赛默飞世尔科技中国有限公司。
恒温水浴振荡器, 购于上海博迅实业有限公司医疗设备厂; 电热鼓风干燥箱, 购于上海一恒科学仪器有限公司; 50%氢氧化钠溶液(w/w), 购于美国Fisher Chemical公司, 批号166472。水为屈臣氏蒸馏水。
岩藻糖(Fuc), 批号112014-201601, 纯度99.7%; 葡萄糖(Glc), 批号110833-201506, 纯度99.9%; 木糖(Xyl), 批号111508-200404, 纯度99.9%; 甘露糖(Man), 批号140651-201504, 纯度99.4%; 鼠李糖(Rha), 批号111683-201502, 鉴别用; 果糖(Fru), 批号100231-201305, 纯度99.4%; 核糖(Rib), 批号140668-201302, 纯度99.6%; 葡萄糖醛酸(Glc-A), 批号140648-201403, 纯度99.1%; 半乳糖醛酸(Gal-A), 批号111646-201702, 纯度95.1%; 以上均购于中国食品药品检定研究院。阿拉伯糖(Ara), 批号20601, 纯度99.0%; 半乳糖(Gal), 批号91459, 纯度99.9%; 以上均购于德国Dr. Ehrenstorfer公司。
研究中共收集肉苁蓉样品13批, 样品信息见表 1。粉碎, 过80目筛, 备用。
水解条件的优化  肉苁蓉多糖的水解效果与所使用酸的种类、浓度、水解温度和水解时间有关。文献[15]报道多糖水解可使用硫酸、盐酸和三氟乙酸, 其中三氟乙酸具有更易挥发的特性, 易通过氮吹等手段去除, 并且其酸性相对较弱, 水解过程中单糖降解的副反应小, 因此使用较为广泛。本方法使用三氟乙酸水解多糖后通过氮吹除酸, 再以水溶解水解产物, 所得溶液组成简单, 容易控制稀释倍数, 操作过程简单易行。经实验室多次预实验考察, 三氟乙酸在80~120 ℃、浓度范围0.08~0.80 mol·L-1、水解1~3 h条件下可以破坏糖苷键, 利于多糖降解为单糖。因此, 以肉苁蓉多糖水解后单糖总量为考察指标, 选取三氟乙酸浓度、温度和水解时间三个因素各选择3个水平进行试验, 选用L9 (34) 正交表安排试验, 获得最优水解条件。
标准溶液的配制  精密称取岩藻糖、阿拉伯糖、鼠李糖、半乳糖、葡萄糖、木糖、甘露糖、果糖、核糖对照品各10 mg, 加水定容至25 mL, 作为400 μg·mL-1单糖混标储备液。精密吸取储备液适量, 以水逐级稀释成质量浓度分别为20、10、5、2、1、0.5 μg·mL-1的单糖对照品混标溶液。精密称取葡萄糖醛酸和半乳糖醛酸对照品各10 mg, 加水定容至25 mL, 作为400 μg·mL-1糖醛酸混标储备液。精密吸取储备液适量, 以水逐级稀释成质量浓度分别为50、20、10、5、2、1、0.5 μg·mL-1的糖醛酸对照品混标溶液。
样品处理  取肉苁蓉样品约5 g, 精密称定, 置于三角瓶中, 加水100 mL, 于恒温水浴振荡器中70 ℃振荡1 h, 离心(6 000 r·min-1, 10 min), 上清液倒入烧杯中; 药渣加水100 mL, 振荡30 min, 离心(6 000 r·min-1, 10 min), 上清液倒入烧杯中; 按以上方法再提取1次, 合并3次提取液, 浓缩至约15 mL, 放凉, 用水定容至25 mL。作为糖提取液。
多糖水解液配制  取糖提取液0.5 mL, 加无水乙醇15 mL (含醇量达到96.7%), 摇匀, 冷藏过夜。离心(6 000 r·min-1, 10 min), 上清液弃去, 沉淀用无水乙醇5 mL洗两次, 离心。沉淀用水2 mL溶解。取1.0 mL, 加0.08 mol·L-1三氟乙酸0.5 mL, 于100 ℃烘箱中放置3 h。取出后, 氮吹干, 加水2 mL, 过滤后, 可进样。
游离单糖液配制  取糖提取液0.5 mL, 加水稀释到50 mL, 摇匀, 过滤后, 可进样。
色谱条件  柱温: 15 ℃, 流速: 0.6 mL·min-1; 配制20、200 mmol·L-1氢氧化钠淋洗液; 洗脱条件1: 0~6 min, 1.6 mmol·L-1; 6~10 min, 1.6~6 mmol·L-1; 10~14.5 min, 6~16 mmol·L-1; 14.5~16 min, 16 mmol·L-1; 16~17 min, 16~1.6 mmol·L-1; 17~27 min, 200 mmol·L-1; 27~28 min, 200~1.6 mmol·L-1; 28~31 min, 1.6 mmol·L-1; 洗脱条件2: 0~13 min, 176 mmol·L-1。
检测方式  积分安培检测, Au工作电极, AgCl参比电极模式, 糖四位电位波形, 电位波形见表 2。
结果与讨论
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1 色谱柱的选择和淋洗液梯度条件的优化
Dionex Carbopac PA210-Fast-4 μm色谱柱对单糖分离效率高, 可预测单糖、二糖、三糖、四糖、五糖的分离和准确定量, 是一种阴离子交换柱, 分离时间短, 效率高, 具有高分辨率、高重现性的特点, 4 μm树脂颗粒分离效率更高, 峰积分更准确, 结果更可靠。Dionex CarboPac PA 210-4 μm色谱柱与脉冲安培检测(PAD) 系统一起使用, 允许在最小的样品制备过程中直接定量非衍生碳水化合物。
肉苁蓉糖提取液中有多糖和游离单糖, 多糖水解后测出多种单糖及糖醛酸成分。研究中使用两种色谱淋洗条件, 对肉苁蓉中9种单糖和2种糖醛酸进行了定性和定量分析。其中条件1用于测定岩藻糖、阿拉伯糖、鼠李糖、半乳糖、葡萄糖、木糖、甘露糖、果糖、核糖, 以上9种单糖所有实验数据均采用淋洗液条件1获得。条件2用于测定半乳糖醛酸、葡萄糖醛酸, 以上两种糖醛酸的所有实验数据均采用淋洗液条件2获得。两种淋洗液条件下样品中单糖及糖醛酸分离色谱图见图 1。
使用阴离子交换色谱柱分离单糖, 淋洗液中OH-有两种作用: 作为淋洗离子, OH-浓度增大, 淋洗强度增大, 使单糖在色谱柱上的保留减弱。同时OH-提供糖解离所需的高pH环境, 常见单糖的pKa基本在12~14之间, 当糖还未完全解离为离子时, 单糖的离子化程度随pH增加而增大, 其在色谱柱上保留能力也会随之增强。单糖的保留行为是上述两种作用共同影响的结果[16]。
淋洗液浓度越低, 单糖与离子交换基团相互作用时间长, 可获得更大分离度。而糖醛酸的分离需要相对较高的淋洗液浓度。因此研究中使用两种淋洗液条件, 分别分离单糖组和糖醛酸组。
2 水解条件的优化
正交试验结果表明, 在不同浓度三氟乙酸、不同温度、不同水解时间条件下, 肉苁蓉水解程度不同。以单糖总量为衡量指标, 三氟乙酸浓度对多糖水解影响最大, 其次为温度和时间, 最终确定肉苁蓉多糖水解条件为0.08 mol·L-1三氟乙酸, 100 ℃, 水解时间3 h。
3 方法学验证
3.1 方法的线性范围、定量限和精密度
在优化的色谱条件下, 以单糖或糖醛酸的峰面积(Y) 对其质量浓度(x) 进行线性回归, 得到回归方程和相关系数。以信噪比为10时的质量浓度为定量限。取5 μg·mL-1单糖及糖醛酸混合对照品溶液进样6次并计算RSD, 考察方法的精密度, 结果见表 3。
3.2 方法重复性
肉苁蓉样品取6份, 进行重复性试验。肉苁蓉多糖水解后, 测得9种单糖和1种糖醛酸, 分别是岩藻糖、阿拉伯糖、鼠李糖、半乳糖、葡萄糖、木糖、甘露糖、果糖、核糖和葡萄糖醛酸, 含量结果重复性良好, RSD < 5%; 肉苁蓉游离单糖测得3种, 分别是葡萄糖、甘露糖、果糖, 含量结果重复性良好, RSD < 5%; 以上均符合方法学定量要求。
3.3 加标回收率
肉苁蓉样品按上文处理后进行测定, 计算出样品中水解单糖及游离单糖含量。进行加标回收试验, 每个浓度平行测定6次。
结果显示, 肉苁蓉多糖水解后, 9种单糖和2种糖醛酸回收率在85.6%~116.0%之间, 相对标准偏差在1.4%~7.1%之间; 肉苁蓉游离单糖回收率在90.4%~96.4%之间, 相对标准偏差在1.6%~9.3%之间, 结果见表 4。
4 样品测定
对收集到的13批肉苁蓉按上述方法进行测定。
盐生肉苁蓉、沙苁蓉、荒漠肉苁蓉的多糖提取液水解后, 可以测得9种单糖和1种糖醛酸: 葡萄糖醛酸。9种单糖为岩藻糖、阿拉伯糖、鼠李糖、半乳糖、葡萄糖、木糖、甘露糖、果糖、核糖。计算三种肉苁蓉中各单糖及糖醛酸浓度, 浓度值为不同来源样品平均值; 以成分的物质的量进行计算, 以岩藻糖摩尔数为基准, 计算各种单糖与岩藻糖的摩尔数之比, 3种肉苁蓉中单糖及醛酸摩尔比为不同批次样品平均值, 结果见表 5。肉苁蓉多糖水解液中, 葡萄糖含量最高, 果糖含量次之, 阿拉伯糖、鼠李糖含量也相对较高, 岩藻糖、半乳糖、木糖、甘露糖、核糖含量偏低。盐生肉苁蓉、沙苁蓉、荒漠肉苁蓉中游离单糖为葡萄糖、甘露糖、果糖, 以甘露糖摩尔数为基准, 计算各单糖与甘露糖的摩尔数之比。盐生肉苁蓉3种单糖物质的量比为94.6∶1∶104.4; 沙苁蓉3种单糖物质的量比为46.4∶1∶65.3; 荒漠肉苁蓉3种单糖物质的量比为65.4∶1∶66.8。3种肉苁蓉中游离单糖及醛酸摩尔比为不同批次样品平均值。
从测定结果可以看出, 三种肉苁蓉中含有丰富的游离单糖, 其多糖水解后单糖种类更加多样。从多糖的单糖组成分析看, 盐生肉苁蓉、荒漠肉苁蓉中单糖总量明显高于沙苁蓉, 盐生肉苁蓉、荒漠肉苁蓉的葡萄糖、果糖含量极高, 沙苁蓉中鼠李糖、木糖含量稍高于另外两种。肉苁蓉中游离单糖和多糖的单糖组成研究, 为肉苁蓉大分子多糖的化学结构性质和其生物活性研究奠定了基础。
5 PMP衍生-HPLC法
1-苯基-3-甲基-5-吡唑啉酮柱前衍生-高效液相色谱法(PMP衍生-HPLC法) 是常用的测定多糖中单糖组成的方法, 单糖因结构原因紫外吸收非常弱, 但经过PMP衍生化操作后, 在245 nm处有强紫外吸收。常用的典型衍生方法为: 向100 μL样品溶液中加入100 μL 0.3 mol·L-1氢氧化钠和120 μL 0.3 mol·L-1 PMP溶液, 充分混合, 在70 ℃避光1 h, 冷却至室温后用100 μL 0.3 mol·L-1盐酸中和, 再用500 μL氯仿反复萃取3次, 离心后取上清液即可用于色谱或质谱分析[17]。
通过采用PMP衍生法可实现对中性糖如甘露糖、葡萄糖、半乳糖、木糖、阿拉伯糖、鼠李糖、岩藻糖, 酸性糖如葡萄糖醛酸、半乳糖醛酸, 碱性糖如氨基葡萄糖、氨基半乳糖等单糖的同时测定。PMP衍生适合于醛糖衍生化, 而果糖属于酮糖, 衍生化操作后无法测得, 大量文献[18-24]也证实, 使用此法测定多糖中单糖组成时, 都无果糖。
综上所述, PMP衍生-HPLC法前处理过程复杂且无法测定果糖, 离子色谱法前处理简单且肉苁蓉中果糖含量非常高, 在肉苁蓉多糖组成测定时选用离子色谱法更优。
结论
收起
肉苁蓉多糖最佳水解条件为0.08 mol·L-1三氟乙酸, 在100 ℃水解3 h。离子色谱-积分脉冲安培法不需衍生化操作, 分离度好, 灵敏度高, 克服了PMP衍生-HPLC法无法测定果糖的缺陷。方法学表明此方法线性、精密度、重复性和回收率良好。肉苁蓉多糖组成主要含岩藻糖、阿拉伯糖、鼠李糖、半乳糖、葡萄糖、木糖、甘露糖、果糖、核糖、葡萄糖醛酸, 其中葡萄糖、果糖含量相对较高。肉苁蓉水提液中游离单糖有葡萄糖、果糖、甘露糖。
作者贡献: 许玮仪负责实验、数据分析和文章撰写; 姜振邦负责方法和仪器使用指导; 范晶共同完成样品实验部分; 金红宇和马双成指导并修改论文。
利益冲突: 所有作者声明无利益冲突。
基金
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  • 国家重点研发计划(2022YFC3501505)
文献
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2023年第58卷第8期
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doi: 10.16438/j.0513-4870.2023-0294
  • 接收时间：2023-03-10
  • 首发时间：2025-11-21
  • 出版时间：2023-08-12
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  • 收稿日期：2023-03-10
  • 修回日期：2023-07-05
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    1.中国食品药品检定研究院, 北京 102629
    2.赛默飞世尔科技(中国)有限公司, 上海 201206

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2种不同金属材料的力学参数
科
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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