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Article(id=1208489267597263299, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1208489265789518263, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2021-0266, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1624377600000, receivedDateStr=2021-06-23, revisedDate=1631635200000, revisedDateStr=2021-09-15, acceptedDate=null, acceptedDateStr=null, onlineDate=1766055894039, onlineDateStr=2025-12-18, pubDate=1636646400000, pubDateStr=2021-11-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1766055894039, onlineIssueDateStr=2025-12-18, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1766055894039, creator=13701087609, updateTime=1766055894039, updator=13701087609, issue=Issue{id=1208489265789518263, tenantId=1146029695717560320, journalId=1189982191388893191, year='2021', volume='56', issue='11', pageStart='2881', pageEnd='3202', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1766055893609, creator=13701087609, updateTime=1766137012710, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1208829504026448153, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1208489265789518263, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1208829504026448154, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1208489265789518263, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=3112, endPage=3117, ext={EN=ArticleExt(id=1208489271078535705, articleId=1208489267597263299, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Optimization of the extraction process for polysaccharides from Cistanche and their molecular weight distribution, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

A simple and efficient method for extracting Cistanche polysaccharides was obtained and used to extract four Cistanche polysaccharides. The molecular weight, distribution and polysaccharide mass fraction of the four Cistanche polysaccharides were measured by High performance size exclusion chromatography-multiangle laser light scatter-refractive index detector (HPSEC-MALLS-RID) and differences between the four Cistanche polysaccharides were determined. The results of a single factor test and an orthogonal test showed that the best process parameters for extracting Cistanche polysaccharides used a mixture of enzyme (4%), a ratio of complex enzyme (cellulase∶pectinase) 1∶1, ultrasonic power at 350 W (40 kHz), for 20 min, enzymolysis temperature of 50 ℃, and pH 6. The average yield of three repeated extractions was 5.14%, the mass fraction was 90.78%, the comprehensive index was 47.96%, and the RSD = 1.51%, indicating good reproducibility. The four Bossica Cistanche polysaccharides are all spherical structures but their molecular weights and distributions differ. The optimized compounded enzyme combined with ultrasonic extraction increases the yield of Cistanche polysaccharides. It is a simple, convenient, economical and environmentally friendly extraction method, which lays the foundation for future polysaccharide research.

, correspAuthors=Lei GAO, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2021 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, authorCompany=null, fund=null, authors=null, authorsList=Wen YANG, Hai-yan YU, Dong WANG, Xue-mei ZHOU, Jian-ping ZHANG, Lei GAO), CN=ArticleExt(id=1208489272135500370, articleId=1208489267597263299, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=肉苁蓉类药材多糖提取工艺优化及多糖的平均分子质量分布, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

本文以肉苁蓉多糖的得率以及质量分数为评价指标，通过单因素考察以及正交试验确定复合酶联合超声提取工艺的最适提取条件，从而得到一种简便、高效提取肉苁蓉多糖的方法。用此方法提取荒漠肉苁蓉、管花肉苁蓉、盐生肉苁蓉、沙苁蓉中的多糖，通过高分子排阻色谱-十八角度激光光散射仪配置示差检测器测定4种肉苁蓉多糖的平均分子质量、分布以及多糖的质量分数，分析4种肉苁蓉多糖间存在的差异。最终确定提取肉苁蓉多糖的最佳工艺参数为复合酶用量4%、复合酶配比（纤维素酶∶果胶酶）1∶3、超声功率350 W（40 kHz）、超声时间20 min、酶解温度50 ℃、pH值为6。在此提取工艺下重复提取3次平均得率为5.14%，质量分数为90.78%，综合指标为47.96%，RSD为1.51%，重现性较好。通过软件计算可知4种肉苁蓉类多糖均为球形结构，但其平均分子质量及其分布存在差异，优化后的复合酶联合超声提取工艺能够有效地增大肉苁蓉多糖的得率，是一种简单方便、经济环保的提取方法，为日后多糖的研究奠定了基础。

, correspAuthors=高磊, authorNote=null, correspAuthorsNote=
*高磊, Tel: 13848919268, E-mail:
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LS: Light scattering; DRI: Differential refractive index , figureFileSmall=X479uv2aJ6swkLT7i5viQQ==, figureFileBig=gD5KuC0hCDKL2WxFW6FR5A==, tableContent=null), ArticleFig(id=1208489278548591584, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1208489267597263299, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
No.Sample codeSource
1C. deserticola-1Byannaoer, Inner Mongolia Autonomous Region
2C. deserticola-2Dko, Inner Mongolia Autonomous Region
3C. deserticola-3Alashan, Inner Mongolia Autonomous Region
4C. deserticola-4Alashan, Inner Mongolia Autonomous Region
5C. deserticola-5Alashan, Inner Mongolia Autonomous Region
6C. salsa-1Alashan, Inner Mongolia Autonomous Region
7C. salsa-2Wlanchabu, Inner Mongolia Autonomous Region
8C. salsa-3Alashan, Inner Mongolia Autonomous Region
9C. salsa-4Xlinhaote, Inner Mongolia Autonomous Region
10C. salsa-5Sziwang, Inner Mongolia Autonomous Region
11C. tubulosa-1Yingbage, Xinjiang Uigur Autonomous Region
12C. tubulosa-2Yutian, Xinjiang Uigur Autonomous Region
13C. tubulosa-3Dliyaboyi, Xinjiang Uigur Autonomous Region
14C. tubulosa-4Lganboziya, Xinjiang Uigur Autonomous Region
15C. tubulosa-5Yutian, Xinjiang Uigur Autonomous Region
16C. sinensis-1Alashan, Inner Mongolia Autonomous Region
17C. sinensis-2Alashan, Inner Mongolia Autonomous Region
18C. sinensis-3Alashan, Inner Mongolia Autonomous Region
19C. sinensis-4Alashan, Inner Mongolia Autonomous Region
20C. sinensis-5Alashan, Inner Mongolia Autonomous Region
), ArticleFig(id=1208489278699586551, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1208489267597263299, language=CN, label=Table 1, caption=

Sample information of four kinds of Cistanche

, figureFileSmall=null, figureFileBig=null, tableContent=
No.Sample codeSource
1C. deserticola-1Byannaoer, Inner Mongolia Autonomous Region
2C. deserticola-2Dko, Inner Mongolia Autonomous Region
3C. deserticola-3Alashan, Inner Mongolia Autonomous Region
4C. deserticola-4Alashan, Inner Mongolia Autonomous Region
5C. deserticola-5Alashan, Inner Mongolia Autonomous Region
6C. salsa-1Alashan, Inner Mongolia Autonomous Region
7C. salsa-2Wlanchabu, Inner Mongolia Autonomous Region
8C. salsa-3Alashan, Inner Mongolia Autonomous Region
9C. salsa-4Xlinhaote, Inner Mongolia Autonomous Region
10C. salsa-5Sziwang, Inner Mongolia Autonomous Region
11C. tubulosa-1Yingbage, Xinjiang Uigur Autonomous Region
12C. tubulosa-2Yutian, Xinjiang Uigur Autonomous Region
13C. tubulosa-3Dliyaboyi, Xinjiang Uigur Autonomous Region
14C. tubulosa-4Lganboziya, Xinjiang Uigur Autonomous Region
15C. tubulosa-5Yutian, Xinjiang Uigur Autonomous Region
16C. sinensis-1Alashan, Inner Mongolia Autonomous Region
17C. sinensis-2Alashan, Inner Mongolia Autonomous Region
18C. sinensis-3Alashan, Inner Mongolia Autonomous Region
19C. sinensis-4Alashan, Inner Mongolia Autonomous Region
20C. sinensis-5Alashan, Inner Mongolia Autonomous Region
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LevelABCD
Compound enzyme ratioUltrasonic power /W (40 kHz)Enzymatic hydrolysis pHUltrasound time/min
11∶1350420
21∶2400530
31∶3500640
), ArticleFig(id=1208489278938660881, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1208489267597263299, language=CN, label=Table 2, caption=

L9(34) orthogonal test factors

, figureFileSmall=null, figureFileBig=null, tableContent=
LevelABCD
Compound enzyme ratioUltrasonic power /W (40 kHz)Enzymatic hydrolysis pHUltrasound time/min
11∶1350420
21∶2400530
31∶3500640
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No.ABCDYield/%Quality score/%Comprehensive index/%
111114.6387.6646.15
212223.5978.9641.28
313333.4675.5639.51
421233.7685.5544.67
522313.5191.4547.48
623122.9867.7835.38
731323.5091.6447.57
832132.7977.3640.08
933213.8081.2842.54
K1126.94138.39121.61136.17
K2127.53128.84128.49124.23
K3130.19117.43134.56124.26
R3.2520.9612.9511.94
), ArticleFig(id=1208489279215484981, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1208489267597263299, language=CN, label=Table 3, caption=

Results L9(34) orthogonal test

, figureFileSmall=null, figureFileBig=null, tableContent=
No.ABCDYield/%Quality score/%Comprehensive index/%
111114.6387.6646.15
212223.5978.9641.28
313333.4675.5639.51
421233.7685.5544.67
522313.5191.4547.48
623122.9867.7835.38
731323.5091.6447.57
832132.7977.3640.08
933213.8081.2842.54
K1126.94138.39121.61136.17
K2127.53128.84128.49124.23
K3130.19117.43134.56124.26
R3.2520.9612.9511.94
), ArticleFig(id=1208489279316148288, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1208489267597263299, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Sample codePeak 1Peak 2Peak 3
MwMass fraction/%MwMass fraction/%MwMass fraction/%
C. deserticola5.137×105-9.426×10520.324.66×104-8.816×10467.783.074×104-6.140×10411.89
C. salsa8.636×105-3.568×10630.565.876×104-1.536×10558.023.800×104-1.519×10511.38
C. tubulosa1.354×106-3.165×10619.566.916×104-1.398×10563.544.480×104-1.086×10514.08
C. Sinensis5.991×105-3.144×10610.522.766×104-1.654×10573.201.350×104-1.709×10515.80
), ArticleFig(id=1208489279475531858, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1208489267597263299, language=CN, label=Table 4, caption=

Molecular weight and distribution of polysaccharides of four kinds of Cistanche. Mw: Molecular weight

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Sample codePeak 1Peak 2Peak 3
MwMass fraction/%MwMass fraction/%MwMass fraction/%
C. deserticola5.137×105-9.426×10520.324.66×104-8.816×10467.783.074×104-6.140×10411.89
C. salsa8.636×105-3.568×10630.565.876×104-1.536×10558.023.800×104-1.519×10511.38
C. tubulosa1.354×106-3.165×10619.566.916×104-1.398×10563.544.480×104-1.086×10514.08
C. Sinensis5.991×105-3.144×10610.522.766×104-1.654×10573.201.350×104-1.709×10515.80
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肉苁蓉类药材多糖提取工艺优化及多糖的平均分子质量分布
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杨雯 1 , 于海艳 1 , 王栋 2 , 周雪梅 2 , 张建平 2 , 高磊 3, *
药学学报 | 研究论文 2021,56(11): 3112-3117
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药学学报 | 研究论文 2021, 56(11): 3112-3117
肉苁蓉类药材多糖提取工艺优化及多糖的平均分子质量分布
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杨雯1, 于海艳1, 王栋2, 周雪梅2, 张建平2, 高磊3, *
作者信息
  • 1.内蒙古医科大学药学院, 内蒙古 呼和浩特 010000
  • 2.内蒙古自治区药品检验研究院, 内蒙古自治区中蒙药标准研究重点实验室, 内蒙古 呼和浩特 010000
  • 3.内蒙古自治区药物警戒中心, 内蒙古 呼和浩特 010000

通讯作者:

*高磊, Tel: 13848919268, E-mail:
Optimization of the extraction process for polysaccharides from Cistanche and their molecular weight distribution
Wen YANG1, Hai-yan YU1, Dong WANG2, Xue-mei ZHOU2, Jian-ping ZHANG2, Lei GAO3, *
Affiliations
  • 1. School of Pharmacy, Inner Mongolia Medical University, Hohhot 010000, China
  • 2. Inner Mongolia Key Laboratory of Chinese and Mongolian Medicines Standard Research, Inner Mongolia Pharmaceutical Inspection Institute, Hohhot 010000, China
  • 3. Inner Mongolia Pharmacovigilance Center, Hohhot 010000, China
出版时间: 2021-11-12 doi: 10.16438/j.0513-4870.2021-0266
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本文以肉苁蓉多糖的得率以及质量分数为评价指标，通过单因素考察以及正交试验确定复合酶联合超声提取工艺的最适提取条件，从而得到一种简便、高效提取肉苁蓉多糖的方法。用此方法提取荒漠肉苁蓉、管花肉苁蓉、盐生肉苁蓉、沙苁蓉中的多糖，通过高分子排阻色谱-十八角度激光光散射仪配置示差检测器测定4种肉苁蓉多糖的平均分子质量、分布以及多糖的质量分数，分析4种肉苁蓉多糖间存在的差异。最终确定提取肉苁蓉多糖的最佳工艺参数为复合酶用量4%、复合酶配比（纤维素酶∶果胶酶）1∶3、超声功率350 W（40 kHz）、超声时间20 min、酶解温度50 ℃、pH值为6。在此提取工艺下重复提取3次平均得率为5.14%，质量分数为90.78%，综合指标为47.96%，RSD为1.51%，重现性较好。通过软件计算可知4种肉苁蓉类多糖均为球形结构，但其平均分子质量及其分布存在差异，优化后的复合酶联合超声提取工艺能够有效地增大肉苁蓉多糖的得率，是一种简单方便、经济环保的提取方法，为日后多糖的研究奠定了基础。

肉苁蓉多糖  /  复合酶  /  平均分子质量

A simple and efficient method for extracting Cistanche polysaccharides was obtained and used to extract four Cistanche polysaccharides. The molecular weight, distribution and polysaccharide mass fraction of the four Cistanche polysaccharides were measured by High performance size exclusion chromatography-multiangle laser light scatter-refractive index detector (HPSEC-MALLS-RID) and differences between the four Cistanche polysaccharides were determined. The results of a single factor test and an orthogonal test showed that the best process parameters for extracting Cistanche polysaccharides used a mixture of enzyme (4%), a ratio of complex enzyme (cellulase∶pectinase) 1∶1, ultrasonic power at 350 W (40 kHz), for 20 min, enzymolysis temperature of 50 ℃, and pH 6. The average yield of three repeated extractions was 5.14%, the mass fraction was 90.78%, the comprehensive index was 47.96%, and the RSD = 1.51%, indicating good reproducibility. The four Bossica Cistanche polysaccharides are all spherical structures but their molecular weights and distributions differ. The optimized compounded enzyme combined with ultrasonic extraction increases the yield of Cistanche polysaccharides. It is a simple, convenient, economical and environmentally friendly extraction method, which lays the foundation for future polysaccharide research.

Cistanche deserticola polysaccharide  /  complex enzyme  /  molecular weight
杨雯, 于海艳, 王栋, 周雪梅, 张建平, 高磊. 肉苁蓉类药材多糖提取工艺优化及多糖的平均分子质量分布. 药学学报, 2021 , 56 (11) : 3112 -3117 . DOI: 10.16438/j.0513-4870.2021-0266
Wen YANG, Hai-yan YU, Dong WANG, Xue-mei ZHOU, Jian-ping ZHANG, Lei GAO. Optimization of the extraction process for polysaccharides from Cistanche and their molecular weight distribution[J]. Acta Pharmaceutica Sinica, 2021 , 56 (11) : 3112 -3117 . DOI: 10.16438/j.0513-4870.2021-0266
正文
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肉苁蓉始载于《神农本草经》[1]。近年来, 国内外对肉苁蓉的研究逐渐深入, 肉苁蓉具有填精益髓、润肠通便、保肝、提高记忆力等功效[2, 3], 多糖作为肉苁蓉主要的成分, 具有重要的研究价值。但由于提取多糖的工艺复杂, 得率低, 目前对肉苁蓉多糖的研究有限。提取多糖主要的方法为热水浸提法[4-6], 但此法操作复杂, 耗时长, 且长时间的加热有可能造成多糖的褐变及其他水溶性成分的溶解, 导致纯度降低[7]。近年来微波提取[8-10]、超声提取[11, 12]、酶提取法[13, 14]也被广泛应用。微波提取法是用微波进行样品预处理, 利用其波动性和高频特性, 借助微波的电磁场, 加速目标成分进入溶剂, 促进提取[8]。超声波提取是利用强烈空化效应及击碎和搅拌作用等多级效应, 使多糖释放、扩散和溶解加快, 从而显著增大提取效率[7]。酶通过高度的专一性, 破坏细胞壁, 加速胞内多糖溶出, 达到增大得率的目的[15], 近年来被广泛应用于中药有效成分的提取。本文采用复合酶联合超声提取工艺并对其进行优化, 通过超声波配合复合酶加剧对细胞壁的破坏, 从而增大多糖的得率以及质量分数, 对多糖的研究具有重大参考价值。
多角度激光检测器常与黏度检测器、紫外检测器以及示差检测器等设备联合应用于多分散系数、第二维利系数、平均分子质量分布等的检测分析工作, 是现代化的光散射技术在高分子材料分析中的开发与利用的重要成果[16], 能够测定聚合物、多糖、蛋白质、核酸等生物大分子的平均分子质量[17]。多糖作为生物高分子化合物, 其药理活性, 不但取决于多糖链的化学结构, 与多糖本身的溶解度、相对分子质量及分布、支化度、链构象等物理性质也有着密切关系[18]。本文所应用的高效分子排阻色谱-十八角度激光光散射仪联用法(HPSEC-MALLS-RID) 是通过激光检测器(MALLS) 和示差检测器(RID) 同时测定, 通过计算可以直接得到样品中每个点的平均分子质量及其组分分布[19, 20], 目前用此项技术测定肉苁蓉多糖分子质量及其分布的研究报道较少。
材料与方法
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材料   从2018年至2021年, 共收集到荒漠肉苁蓉、盐生肉苁蓉、管花肉苁蓉以及沙苁蓉样品共计20批, 均为春季采集, 阴干方式处理。经内蒙古自治区药品检验研究院检定H1~H5为荒漠肉苁蓉(Cistanche deserticola Y.C.Ma) 干燥带鳞叶的肉质茎, Y1~Y5为盐生肉苁蓉[Cistanche salsa (C.A.Mey.) G.Beck] 干燥带鳞叶的肉质茎, G1~G5为管花肉苁蓉[C.tubulosa (Schenk) Wight] 干燥带鳞叶的肉质茎, S1~S5为沙苁蓉(Cistanche sinensis G.Beck) 干燥带鳞叶的肉质茎, 见表 1。
仪器与试剂  Waters高效液相色谱仪、DAWN HELEOS II十八角度激光光散射仪(美国怀雅特公司)、示差折光检测器、KQ-500DE型数控超声波清洗器(昆山市超声仪器有限公司)、AE100型万分之一电子天平(瑞士梅特勒-托利多公司)、ME5型百万分之一天平(德国赛多利斯公司)、TDL-5A型离心机(上海菲恰尔分析仪器有限公司)、PB-10型pH计(北京赛多利斯科学仪器有限公司)。
柠檬酸(天津市科密欧化学试剂有限公司)、氯化钠(国药集团化学试剂有限公司)、95%乙醇(国药集团化学试剂有限公司)、右旋糖酐5000 (Solarbio试剂公司)、纯化水。
HPSEC-MALLS-RID分析条件  色谱柱: TSKgel G4000 Wxl与TSKgel G6000 Wxl串联; 流动相: 0.1 mol·L-1氯化钠溶液; 流速: 0.5 mL·min-1; 进样量: 100 μL; 柱温: 30 ℃; dn/dc: 0.145 0。
复合酶联合超声提取法提取多糖  取肉苁蓉粉末(过4号筛) 约10 g, 加10倍体积的乙醇回流脱脂3 h, 离心, 取滤渣加30倍水, 超声提取, 使用柠檬酸调节pH值后, 加入复合酶(纤维素酶: 果胶酶), 水浴恒温50 ℃提取2 h, 90 ℃灭酶10 min, 离心, 将滤液旋蒸至约100 mL, 加1/4 sevge试剂(三氯甲烷∶正丁醇4∶1), 振摇30 min后离心10 min, 弃去沉淀, 取上清液再重复操作2次, 上清液用4倍量乙醇沉淀多糖, 4 ℃静置过夜, 离心得沉淀, 分别用无水乙醇、丙酮冲洗沉淀三次, 60 ℃烘干得肉苁蓉多糖。
单因素考察  在脱脂后的料液比为1∶30、复合酶为纤维素酶和果胶酶、酶解温度50 ℃、酶解2 h、四倍量乙醇沉淀为确定因素的提取条件下, 分别考察超声时间(20、30、40、50和60 min), 超声功率[250、300、350、400和500 W (40 kHz)], 复合酶用量(1.5%、2.0%、2.5%、4.0%、5.0%), 复合酶配比(纤维素酶∶果胶酶, 3∶1、2∶1、1∶1、1∶2、1∶3), 酶解pH值(3、4、5、6、7) 对得率的影响。
正交试验  在单因素试验的基础上进行L9(34) 正交试验(表 2), 以肉苁蓉多糖得率以及质量分数的综合评分为指标, 对超声时间、超声功率、复合酶配比以及酶解pH值4个因素进行优选。采用综合加权评分法处理数据, 权重系数均为0.5, 以下面公式计算综合评分: 综合评分= 0.5 ×多糖得率+ 0.5 ×多糖质量分数。
通过正交试验的直观分析和方差分析优化最佳制备工艺。
多糖平均分子质量及质量分数的测定  供试品溶液的制备: 精密称取4种苁蓉多糖样品, 用0.1 mol·L-1氯化钠溶液配成3 mg·mL-1的溶液, 待样品完全溶解后用0.22 μm滤膜过滤, 滤液经HPSEC-MALLS-RID进行分析。
多糖得率= m / M × 100%, 式中m为粗多糖样品的质量(g); M为药材的取样量(g)。
多糖的质量分数= m1 / M1 × 100%, 式中m1为多糖实测值(μg); M1为粗多糖理论进样量(μg)。
方法学考察
精密度  由于目前无肉苁蓉多糖对照品, 故选用相对分子质量已知的葡聚糖即右旋糖酐进行精密度试验。取右旋糖酐加适量的流动相制成1 mL约含3 mg的对照品溶液, 精密吸取同一对照品溶液100 μL, 连续进样6次, 测定右旋糖酐的实测值计算RSD值。
稳定性  精密吸取同一供试品溶液(H3) 100 μL, 于制备后0、2、4、8、12、24 h分别进样分析, 以实测值计算RSD。
重复性  取同一供试品荒漠肉苁蓉(H3) 各6份, 按照拟定分析方法进行测定, 计算荒漠肉苁蓉多糖的质量分数, 计算RSD。
准确度  取已知质量分数为99%的右旋糖酐, 平行取样6份, 加适量的流动相制成1 mL约含3 mg的对照品溶液, 计算右旋糖酐的平均回收率, 计算RSD。
数据处理  多糖平均分子质量及质量分数的测定采用ASTRA 7.3.1软件收集和处理数据。
结果
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1 复合酶联合超声提取肉苁蓉多糖的单因素试验
1.1 超声时间对肉苁蓉多糖得率的影响
在20~60 min考察范围内, 肉苁蓉多糖的得率随着时间的增大先增大后减小, 40 min达到最大值, 推测超声时间过长对多糖有破坏作用, 导致糖苷键断裂, 故选择20、30和40 min作为三个水平进行正交试验。
1.2 超声功率对肉苁蓉多糖得率的影响
在250~500 W的范围内, 多糖的得率与超声功率呈正相关, 500 W时多糖的得率最高。推测加大超声功率对细胞壁的破坏力增大, 促进多糖的释放, 故选择350、400和500 W (40 kHz) 进行正交试验。
1.3 复合酶用量对肉苁蓉多糖得率的影响
在复合酶为纤维素酶和果胶酶的条件下, 复合酶用量在1.5%~5.0%的范围内, 多糖的提取率随着酶用量的增加而增加。在当复合酶的用量增加为底物质量浓度的4.0%时, 肉苁蓉多糖的得率不再上升, 推测增大复合酶用量可以增加反应浓度, 使细胞壁软化、膨胀和崩溃, 使得率升高, 由于酶存在竞争性抑制的特点, 随着酶浓度的增加, 溶液趋于饱和。在考察范围内, 选择4%为最适复合酶用量。由于酶用量对试验结果影响较小, 所以在正交试验中将不再考察此因素对多糖综合指标的影响。
1.4 复合酶配比对肉苁蓉多糖得率的影响
在复合酶用量确定为4%的条件下, 对纤维素酶和果胶酶的配比进行了考察, 在考察范围内, 纤维素酶∶果胶酶为1∶2时, 多糖的得率达到最大值, 故选择纤维素酶∶果胶酶分别为1∶1、1∶2、1∶3进行正交试验。
1.5 pH值对肉苁蓉多糖得率的影响
合适的pH值可以增大多糖的得率, 在考察范围内, 多糖的得率随着pH值的变化而变化, 当pH值为5时, 多糖的得率达到最大值, 随即下降, 故选择pH值分别为4、5、6进行正交试验。
2 复合酶联合超声提取肉苁蓉多糖的正交试验
根据正交试验结果可知, 各因素对得率的影响不一。通过极差分析可知(表 3), 影响多糖得率和质量分数的因素依次为超声功率 > 酶解pH值> 超声时间 > 复合酶配比。根据方差分析可知, 超声功率对肉苁蓉多糖的综合指标影响最显著, 酶解pH值、超声时间以及复合酶配比对肉苁蓉多糖的综合指标没有显著影响, 故肉苁蓉多糖的最适提取条件为A3B1C3D1 (表 2), 在此提取工艺下重复提取3次平均得率为5.14%, 质量分数为90.78%, 综合指标为47.96%, RSD = 1.51%, 该提取工艺重现性较好。
本文通过复合酶联合超声提取法, 得到了肉苁蓉多糖的最适提取条件为复合酶用量4%、复合酶配比(纤维素酶∶果胶酶) 1∶3、超声功率350 W (40 kHz)、超声时间20 min、酶解温度50 ℃、pH值为6, 此方法提取率高, 重现性好。
3 方法学考察
3.1 精密度
连续进样6次测定右旋糖酐的实测值, 计算RSD为0.31%, 结果表明仪器精密度良好。
3.2 稳定性
精密吸取同一供试品溶液(H3) 100 μL分别进样测定质量分数, 计算RSD为1.26%, 结果表明供试品溶液在24 h内稳定。
3.3 重复性
测定结果显示荒漠肉苁蓉多糖的质量分数为87.75%, RSD为0.57%, 表明该方法重复性良好。
3.4 准确度
按照拟定分析方法进行测定, 计算右旋糖酐质量的平均回收率为98.38%, RSD为1.17%, 误差在允许范围内, 表明该方法测定多糖的质量分数结果准确。
4 四种苁蓉多糖平均分子质量分布及其质量分数比较分析
通过ASTRA 7.3.1软件对结果处理, 其对应的平均分子质量如表 4所示, 4种苁蓉类多糖色谱图如图 1所示。从图 1可知, MALLS和RID的色谱图存在明显差异, 出峰时间与出峰数量不一致, MALLS与样品中平均分子质量大小有关, 平均分子质量越大出峰时间越靠前, RID的信号只与浓度有关, 由此可知, 4种苁蓉类样品中平均分子质量大的多糖浓度较低, 平均分子质量小的多糖浓度较高。
4种肉苁蓉多糖的分布范围均为宽分布, 5批荒漠肉苁蓉平均分子质量为1.849×105, 平均质量分数为85.75%; 5批盐生肉苁蓉平均分子质量为8.130×105, 平均质量分数为78.92%; 5批管花肉苁蓉平均分子质量为4.676×105, 平均质量分数为64.35%; 5批沙苁蓉的平均分子质量为3.110×105, 平均质量分数为81.88%。4种肉苁蓉平均分子质量大小比较为: 盐生肉苁蓉 > 管花肉苁蓉 > 沙苁蓉 > 荒漠肉苁蓉, 4种肉苁蓉多糖的质量分数大小比较为: 荒漠肉苁蓉 > 沙苁蓉 > 盐生肉苁蓉 > 管花肉苁蓉, 其中荒漠肉苁蓉平均分子质量最小但质量分数最大。
由表 4可知, 4种肉苁蓉平均分子质量分布趋势大致相似, 均呈现两侧含量较少, 中间平均分子质量居多的趋势, 其中盐生肉苁蓉的多糖以大分子居多。进一步分析数据可知荒漠肉苁蓉平均分子质量分布范围最为狭窄, 盐生肉苁蓉平均分子质量分布范围最为广泛。通过软件计算可知, 4种苁蓉类多糖均为球形结构。不同分子段的多糖, 其生物活性存在显著差异, 通过对4种苁蓉类药材多糖的平均分子质量及其分布的研究, 可以为后续药效活性研究提供可靠依据。
讨论
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通过复合酶联合超声提取法提取肉苁蓉多糖, 并通过单因素试验以及正交试验将方法进行优化, 此方法提取多糖具有操作简便、耗时少、提取率高等特点。HPSEC-MALLS-RID由于其无需标准物质校正, 可同时获得样品分子质量、分布和样品构象信息等特点被广泛应用于多糖的平均分子质量测定中, 采用此方法测定多糖的质量分数为目前的新兴技术, 该方法与传统苯酚-硫酸法比较具有快速、简便、准确率高等优点, 避免了苯酚-硫酸法测定多糖时, 其他物质干扰而产生较大误差, 为日后多糖的开发利用奠定了基础。
作者贡献: 杨雯主要进行了本文方法建立、实验数据处理、撰写文章及对编辑部修改意见进行核修等。于海艳、王栋、周雪梅和张建平对本文的实验思路、方法建立及文章修改提供了宝贵的意见及试验帮助。高磊从选题与设计、方法建立及文章修改提供了指导。
利益冲突: 无任何利益冲突。
基金
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  • 内蒙古自治区科技计划项目(2019GG132)
文献
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2021年第56卷第11期
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doi: 10.16438/j.0513-4870.2021-0266
  • 接收时间：2021-06-23
  • 首发时间：2025-12-18
  • 出版时间：2021-11-12
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  • 收稿日期：2021-06-23
  • 修回日期：2021-09-15
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内蒙古自治区科技计划项目(2019GG132)
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    1.内蒙古医科大学药学院, 内蒙古 呼和浩特 010000
    2.内蒙古自治区药品检验研究院, 内蒙古自治区中蒙药标准研究重点实验室, 内蒙古 呼和浩特 010000
    3.内蒙古自治区药物警戒中心, 内蒙古 呼和浩特 010000

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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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