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Article(id=1196886685649781265, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1196886663541600644, articleNumber=1001-2494(2024)22-2126-06, orderNo=null, doi=10.11669/cpj.2024.22.004, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1711382400000, receivedDateStr=2024-03-26, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1763289622937, onlineDateStr=2025-11-16, pubDate=1732204800000, pubDateStr=2024-11-22, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1763289622937, onlineIssueDateStr=2025-11-16, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1763289622937, creator=13701087609, updateTime=1763289622937, updator=13701087609, issue=Issue{id=1196886663541600644, tenantId=1146029695717560320, journalId=1190317699101192196, year='2024', volume='59', issue='22', pageStart='2099', pageEnd='2196', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1763289617666, creator=13701087609, updateTime=1763292152892, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1196897297100488858, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1196886663541600644, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1196897297104683163, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1196886663541600644, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=2126, endPage=2131, ext={EN=ArticleExt(id=1196886685851107859, articleId=1196886685649781265, tenantId=1146029695717560320, journalId=1190317699101192196, language=EN, title=Application Research of Characteristic Polypeptide Technology Based on Chemical Markers in Distiguishment and Identification of Deer-derived Medicinal Materials, columnId=null, journalTitle=Chinese Pharmaceutical Journal, columnName=null, runingTitle=null, highlight=null, articleAbstract=

OBJECTIVE To accurately distinguish deer-derived medicinal materials from four different position: Cervi Cornu Pantotrichum, Cervi Cornu, Cervi Cornu Degelatinatum and Deer hide. METHODS The extraction efficiency of two extraction solvent systems for four different kinds of deer-derived medicinal materials was compared, and the optimum enzymatic hydrolysis conditions of the samples were optimized to form an efficient and simple sample pretreatment process. A specific analysis method, combining chemical markers and characteristic polypeptides was established to identify four different position of deer-derived medicinal materials by using liquid chromatography-triple quadrupole mass spectrometry and mass spectrometry multi-reaction monitoring technology (MRM). And methodological experiments were conducted as part of this analysis method. RESULTS The optimized sample pretreatment method with high extraction efficiency and good enzymatic effect was successfully applied, and the corresponding characteristic polypeptide ion pairs were successfully detected in 24 batches of samples. CONCLUSION The distinction and identification method with high-level specificity, sensitivity and robustness of deer-derived medicinal materials were established. The innovative analysis method can be effectively used to evaluate the quality of deer-derived medicinal materials, which provides a reference for ensuring and improving the quality of deer-derived medicinal materials.

, correspAuthors=Bing WANG, Yongqiang LIN, 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=Yuzhuo ZHU, Fei XUE, Yang JIAO, Weiliang CUI, Juan CHEN, Bing WANG, Yongqiang LIN), CN=ArticleExt(id=1196886840121803336, articleId=1196886685649781265, tenantId=1146029695717560320, journalId=1190317699101192196, language=CN, title=基于化学标识物的特征多肽技术在鹿源药材区分与鉴别中的应用研究, columnId=1190352405612040510, journalTitle=中国药学杂志, columnName=论著, runingTitle=null, highlight=null, articleAbstract=

目的 基于化学标识物概念,通过特征多肽分析方法,实现对鹿茸、鹿角、鹿角霜和鹿皮4种不同部位来源的鹿源药材的精准区分。方法 比较了2种提取溶剂体系对4种不同部位来源的鹿源药材提取效率,优化样品最佳酶解条件,形成高效、简便的样品前处理流程。结合化学标识物理念和特征多肽分析方法,采用液相色谱-三重四极杆质谱及质谱多反应监测技术(MRM),建立一套针对4种不同部位鹿源药材的专属性鉴别方法,并进行方法学实验。结果 优化后的样品前处理方法提取效率高、酶解效果好,24个批次的样品均成功检测到相应的特征多肽离子对。。结论 本研究建立的鹿源药材区分与鉴别方法,具有高专属性、灵敏度和耐用性,能够有效用于鹿源药材的质量评价,为确保和提升鹿源药材的质量提供参考。

, correspAuthors=汪冰, 林永强, authorNote=null, correspAuthorsNote=
* 林永强,男,博士,主任药师 研究方向:中药质量控制关键技术研究 Tel:(0531)81216503;
汪冰,女,硕士,主任药师 研究方向:中药质量控制与产业化 Tel:(0531)81216521
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祝玉卓,女,硕士研究生 研究方向:药物分析

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祝玉卓,女,硕士研究生 研究方向:药物分析

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祝玉卓,女,硕士研究生 研究方向:药物分析

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Chin Pharm J(中国药学杂志), 2022, 57(23):1998-2003., articleTitle=Discovery and specificity verification of characteristic peptides for Cervi Cornus Colla, refAbstract=null), Reference(id=1197124888252167100, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1196886685649781265, doi=null, pmid=null, pmcid=null, year=2018, volume=49, issue=13, pageStart=3166, pageEnd=3173, url=null, language=null, rfNumber=[13], rfOrder=12, authorNames=DONG H S, ZHANG J X, HU Q, journalName=Chin Tradit Herb Drug(中草药), refType=null, unstructuredReference=DONG H S, ZHANG J X, HU Q, et al. Research progress on quality control of gelatinous Chinese materia medica[J]. Chin Tradit Herb Drug(中草药), 2018, 49(13):3166-3173., articleTitle=Research progress on quality control of gelatinous Chinese materia medica, refAbstract=null), Reference(id=1197124888306693053, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1196886685649781265, doi=null, pmid=null, pmcid=null, year=2023, volume=1705, issue=2023, pageStart=464153, pageEnd=461464, url=null, language=null, rfNumber=[14], rfOrder=13, authorNames=XUE F, WANG B, GUO D X, journalName=J Chromatogr A, refType=null, unstructuredReference=XUE F, WANG B, GUO D X, et al. Discovery of species-specific peptide markers and development of quality-evaluation strategies for Deer Horn gelatin using liquid chromatography-tandem mass spectrometry and a label-free methodology[J]. J Chromatogr A, 2023, 1705(2023):464153-461464., articleTitle=Discovery of species-specific peptide markers and development of quality-evaluation strategies for Deer Horn gelatin using liquid chromatography-tandem mass spectrometry and a label-free methodology, refAbstract=null), Reference(id=1197124889388823487, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1196886685649781265, doi=null, pmid=null, pmcid=null, year=2021, volume=57, issue=3, pageStart=93, pageEnd=97, url=null, language=null, rfNumber=[15], rfOrder=14, authorNames=RUAN H N, LUO J C, WANG Z Y, journalName=Chin J Vet Med(中国兽医杂志), refType=null, unstructuredReference=RUAN H N, LUO J C, WANG Z Y, et al. Study on comprehensive extraction process of various water-soluble effective parts in Velvet Antler[J]. Chin J Vet Med(中国兽医杂志), 2021, 57(3):93-97., articleTitle=Study on comprehensive extraction process of various water-soluble effective parts in Velvet Antler, refAbstract=null)], funds=[Fund(id=1197124887128093613, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1196886685649781265, awardId=2023YFC3504102, language=CN, fundingSource=国家重点研发计划(中医药现代化)课题(2023YFC3504102), fundOrder=null, country=null), Fund(id=1197124887178425262, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1196886685649781265, awardId=202228096, language=CN, fundingSource=“新高校20条”科研带头人工作室项目(202228096), fundOrder=null, country=null), Fund(id=1197124887350391727, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1196886685649781265, awardId=MRJT2105, language=CN, fundingSource=泉城产业领军人才支持计划创新团队项目(MRJT2105), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1197124882661159779, tenantId=1146029695717560320, journalId=1190317699101192196, 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label=Fig.1, caption=Effect of solvent system(taking Pep6 asan example) on extractionefficiency of characteristic polypeptides, figureFileSmall=UAE9rk4fhFgTgRRIcYWrDw==, figureFileBig=HpISygqL8qpKDjN2ylaW7w==, tableContent=null), ArticleFig(id=1197124886075323294, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1196886685649781265, language=CN, label=图1, caption=提取溶剂对特征多肽提取效率的影响结果(以Pep 6为例), figureFileSmall=UAE9rk4fhFgTgRRIcYWrDw==, figureFileBig=HpISygqL8qpKDjN2ylaW7w==, tableContent=null), ArticleFig(id=1197124886155015071, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1196886685649781265, language=EN, label=Fig.2, caption=Effect of solvent system(taking Pep6 asan example) on extractionefficiency of characteristic polypeptides, figureFileSmall=NMtJAaL6m4/oGH8zCC30aA==, figureFileBig=fAh25gaHZT/nWYZ+558yUA==, tableContent=null), ArticleFig(id=1197124886217929632, tenantId=1146029695717560320, journalId=1190317699101192196, 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characteristic polypeptide ion pairs in Cervi Cornu Pantotrichum, Cervi Cornu, Deer hide and Cervi Cornu Degelatinatum, figureFileSmall=+HUWQmPuc18qwiiTZziZog==, figureFileBig=/zJ5fKV5iV+hnQ65Yd+I+A==, tableContent=null), ArticleFig(id=1197124886444422052, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1196886685649781265, language=CN, label=图4, caption=特征多肽离子对在鹿茸、鹿角、鹿皮、鹿角霜中的检出结果, figureFileSmall=+HUWQmPuc18qwiiTZziZog==, figureFileBig=/zJ5fKV5iV+hnQ65Yd+I+A==, tableContent=null), ArticleFig(id=1197124886494753701, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1196886685649781265, language=EN, label=Fig.5, caption=Specificity verification results of Pep 5 and Pep 6, figureFileSmall=zx5MZxEbGDE2/HkNXBBa6A==, figureFileBig=/g8+U9iIMnfWSqieGqF+WA==, tableContent=null), ArticleFig(id=1197124886561862566, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1196886685649781265, language=CN, label=图5, caption=Pep 5和Pep 6专属性验证结果, figureFileSmall=zx5MZxEbGDE2/HkNXBBa6A==, figureFileBig=/g8+U9iIMnfWSqieGqF+WA==, tableContent=null), ArticleFig(id=1197124886645748647, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1196886685649781265, language=EN, label=Tab.1, caption=

Sample information of deer-derived medicinal materials

, figureFileSmall=null, figureFileBig=null, tableContent=
No. Position of deer Place of origin(in Chinese) Species
R1 Cervi Cornu Pantotrichum Anguo Hebei (河北安国) Cervus elaphus Linnaeus
R2 Cervi Cornu Pantotrichum Anguo Hebei (河北安国) Cervus elaphus Linnaeus
R3 Cervi Cornu Pantotrichum Bozhou Anhui(安徽亳州) Cervus elaphus Linnaeus
R4 Cervi Cornu Pantotrichum Bozhou Anhui(安徽亳州) Cervus elaphus Linnaeus
R5 Cervi Cornu Pantotrichum Bozhou Anhui(安徽亳州) Cervus elaphus Linnaeus
R6 Cervi Cornu Pantotrichum Jinan Shandong(山东济南) Cervus nippon Temminck
R7 Cervi Cornu Pantotrichum Yantai Shandong(山东烟台) Cervus elaphus Linnaeus
R8 Cervi Cornu Pantotrichum Yantai Shandong(山东烟台) Cervus elaphus Linnaeus
R9 Cervi Cornu Pantotrichum Dongying Shandong(山东东营) Cervus nippon Temminck
R10 Cervi Cornu Pantotrichum Dongying Shandong(山东东营) Cervus nippon Temminck
J1 Cervi Cornu Zhumadian Henan(河南驻马店) Cervus elaphus Linnaeus
J2 Cervi Cornu Tieling Liaoning(辽宁铁岭) Cervus elaphus Linnaeus
J3 Cervi Cornu Liaocheng Shandong(山东聊城) Cervus elaphus Linnaeus
J4 Cervi Cornu Liaocheng Shandong(山东聊城) Cervus elaphus Linnaeus
J5 Cervi Cornu Huanggang Hubei(湖北黄冈) Cervus elaphus Linnaeus
J6 Cervi Cornu Dongying Shandong(山东东营) Cervus nippon Temminck
J7 Cervi Cornu Zhumadian Henan(河南驻马店) Cervus nippon Temminck
P1 Deer hide Bozhou Anhui(安徽亳州) Cervus elaphus Linnaeus
P2 Deer hide Bozhou Anhui(安徽亳州) Cervus elaphus Linnaeus
P3 Deer hide Bozhou Anhui(安徽亳州) Cervus elaphus Linnaeus
P4 Deer hide Bozhou Anhui(安徽亳州) Cervus elaphus Linnaeus
S1 Cervi Cornu Degelatinatum Bozhou Anhui(安徽亳州) None
S2 Cervi Cornu Degelatinatum Bozhou Anhui(安徽亳州) None
S3 Cervi Cornu Degelatinatum Bozhou Anhui(安徽亳州) None
), ArticleFig(id=1197124886712857512, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1196886685649781265, language=CN, label=表1, caption=

鹿源药材样品信息

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No. Position of deer Place of origin(in Chinese) Species
R1 Cervi Cornu Pantotrichum Anguo Hebei (河北安国) Cervus elaphus Linnaeus
R2 Cervi Cornu Pantotrichum Anguo Hebei (河北安国) Cervus elaphus Linnaeus
R3 Cervi Cornu Pantotrichum Bozhou Anhui(安徽亳州) Cervus elaphus Linnaeus
R4 Cervi Cornu Pantotrichum Bozhou Anhui(安徽亳州) Cervus elaphus Linnaeus
R5 Cervi Cornu Pantotrichum Bozhou Anhui(安徽亳州) Cervus elaphus Linnaeus
R6 Cervi Cornu Pantotrichum Jinan Shandong(山东济南) Cervus nippon Temminck
R7 Cervi Cornu Pantotrichum Yantai Shandong(山东烟台) Cervus elaphus Linnaeus
R8 Cervi Cornu Pantotrichum Yantai Shandong(山东烟台) Cervus elaphus Linnaeus
R9 Cervi Cornu Pantotrichum Dongying Shandong(山东东营) Cervus nippon Temminck
R10 Cervi Cornu Pantotrichum Dongying Shandong(山东东营) Cervus nippon Temminck
J1 Cervi Cornu Zhumadian Henan(河南驻马店) Cervus elaphus Linnaeus
J2 Cervi Cornu Tieling Liaoning(辽宁铁岭) Cervus elaphus Linnaeus
J3 Cervi Cornu Liaocheng Shandong(山东聊城) Cervus elaphus Linnaeus
J4 Cervi Cornu Liaocheng Shandong(山东聊城) Cervus elaphus Linnaeus
J5 Cervi Cornu Huanggang Hubei(湖北黄冈) Cervus elaphus Linnaeus
J6 Cervi Cornu Dongying Shandong(山东东营) Cervus nippon Temminck
J7 Cervi Cornu Zhumadian Henan(河南驻马店) Cervus nippon Temminck
P1 Deer hide Bozhou Anhui(安徽亳州) Cervus elaphus Linnaeus
P2 Deer hide Bozhou Anhui(安徽亳州) Cervus elaphus Linnaeus
P3 Deer hide Bozhou Anhui(安徽亳州) Cervus elaphus Linnaeus
P4 Deer hide Bozhou Anhui(安徽亳州) Cervus elaphus Linnaeus
S1 Cervi Cornu Degelatinatum Bozhou Anhui(安徽亳州) None
S2 Cervi Cornu Degelatinatum Bozhou Anhui(安徽亳州) None
S3 Cervi Cornu Degelatinatum Bozhou Anhui(安徽亳州) None
), ArticleFig(id=1197124886788354985, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1196886685649781265, language=EN, label=Tab.2, caption=

Source, sequence and ion pair information of characteristic polypeptide[14]

, figureFileSmall=null, figureFileBig=null, tableContent=
No. Sequence Protein attribution m/z(Q1) Charge m/z(Q3) m/z(Q3)
Pep 1 GETGPAGRpGEVGPpGPpGPAGEK Collagen α-1 739.36 2 938.46 1 053.52
Pep 2 SGETGASGPpGFAGE Collagen α-2 732.84 2 875.43 1 090.51
Pep 3 GPpGESGAAGPAGPIGSR Collagen α-2 775.89 2 541.26 965.44
Pep 4 TGQPGAVGPAGIR Collagen α-2 590.83 2 797.47 287.14
Pep 5 TPVGGQPSVPGGPVR α-2-HS-glycoprotein 702.88 2 865.49 1 107.59
Pep 6 FFEHFGDLSTADAVMGNPK Adult β-globin 700.32 2 562.26 244.17
Pep 7 DLSFLPQPPQEK Collagen α-1 699.87 2 598.32 501.24
), ArticleFig(id=1197124886851269546, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1196886685649781265, language=CN, label=表2, caption=

特征多肽的来源、序列和离子对信息[14]

, figureFileSmall=null, figureFileBig=null, tableContent=
No. Sequence Protein attribution m/z(Q1) Charge m/z(Q3) m/z(Q3)
Pep 1 GETGPAGRpGEVGPpGPpGPAGEK Collagen α-1 739.36 2 938.46 1 053.52
Pep 2 SGETGASGPpGFAGE Collagen α-2 732.84 2 875.43 1 090.51
Pep 3 GPpGESGAAGPAGPIGSR Collagen α-2 775.89 2 541.26 965.44
Pep 4 TGQPGAVGPAGIR Collagen α-2 590.83 2 797.47 287.14
Pep 5 TPVGGQPSVPGGPVR α-2-HS-glycoprotein 702.88 2 865.49 1 107.59
Pep 6 FFEHFGDLSTADAVMGNPK Adult β-globin 700.32 2 562.26 244.17
Pep 7 DLSFLPQPPQEK Collagen α-1 699.87 2 598.32 501.24
), ArticleFig(id=1197124886922572715, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1196886685649781265, language=EN, label=Tab.3, caption=

The results of durability test in methodology verification

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Characteristic
polypeptide
ion pairs		 Retention time of different chromatographic columns
Agilent SB
C18 RRHD		 Thermo SCIENTIFIC
AccucoreTM
C18		 Waters
CORTECSTM
UPLC® C18
739.36→938.46 3.90 ±0.5 1.95 ±0.5 4.36 ±0.5
739.36→1 053.52 3.90 ±0.5 1.95 ±0.5 4.36 ±0.5
732.84→875.43 4.76 ±0.5 2.79 ±0.5 5.77 ±0.5
732.84→1 090.51 4.76 ±0.0 2.79 ±0.5 5.77 ±0.5
775.89→541.26 5.57 ±0.5 3.13 ±0.5 6.20 ±0.5
775.89→965.44 5.57 ±0.5 3.13 ±0.5 6.20 ±0.5
590.83→797.47 6.96 ±0.5 4.08 ±0.5 7.70 ±0.5
590.83→287.14 6.96 ±0.5 4.08 ±0.5 7.70 ±0.5
702.88→865.49 9.39 ±0.5 6.13 ±0.5 9.54 ±0.5
702.88→1 107.59 9.39 ±0.5 6.13 ±0.5 9.54 ±0.5
700.32→562.26 12.58 ±0.5 11.45 ±0.5 13.08 ±0.5
700.32→244.17 12.58 ±0.5 11.45 ±0.5 13.08 ±0.5
699.87→598.32 13.37 ±0.5 12.12 ±0.5 13.91 ±0.5
699.87→501.24 13.37 ±0.5 12.12 ±0.5 13.91 ±0.5
), ArticleFig(id=1197124886993875884, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1196886685649781265, language=CN, label=表3, caption=

方法学耐用实验结果

, figureFileSmall=null, figureFileBig=null, tableContent=
Characteristic
polypeptide
ion pairs		 Retention time of different chromatographic columns
Agilent SB
C18 RRHD		 Thermo SCIENTIFIC
AccucoreTM
C18		 Waters
CORTECSTM
UPLC® C18
739.36→938.46 3.90 ±0.5 1.95 ±0.5 4.36 ±0.5
739.36→1 053.52 3.90 ±0.5 1.95 ±0.5 4.36 ±0.5
732.84→875.43 4.76 ±0.5 2.79 ±0.5 5.77 ±0.5
732.84→1 090.51 4.76 ±0.0 2.79 ±0.5 5.77 ±0.5
775.89→541.26 5.57 ±0.5 3.13 ±0.5 6.20 ±0.5
775.89→965.44 5.57 ±0.5 3.13 ±0.5 6.20 ±0.5
590.83→797.47 6.96 ±0.5 4.08 ±0.5 7.70 ±0.5
590.83→287.14 6.96 ±0.5 4.08 ±0.5 7.70 ±0.5
702.88→865.49 9.39 ±0.5 6.13 ±0.5 9.54 ±0.5
702.88→1 107.59 9.39 ±0.5 6.13 ±0.5 9.54 ±0.5
700.32→562.26 12.58 ±0.5 11.45 ±0.5 13.08 ±0.5
700.32→244.17 12.58 ±0.5 11.45 ±0.5 13.08 ±0.5
699.87→598.32 13.37 ±0.5 12.12 ±0.5 13.91 ±0.5
699.87→501.24 13.37 ±0.5 12.12 ±0.5 13.91 ±0.5
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基于化学标识物的特征多肽技术在鹿源药材区分与鉴别中的应用研究
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祝玉卓 1, 2 , 薛菲 2 , 焦阳 1, 2 , 崔伟亮 2 , 陈娟 1, 2 , 汪冰 1, 2, * , 林永强 1, 2, *
中国药学杂志 | 论著 2024,59(22): 2126-2131
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中国药学杂志 | 论著 2024, 59(22): 2126-2131
基于化学标识物的特征多肽技术在鹿源药材区分与鉴别中的应用研究
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祝玉卓1, 2, 薛菲2, 焦阳1, 2, 崔伟亮2, 陈娟1, 2, 汪冰1, 2, *, 林永强1, 2, *
作者信息
  • 1 山东中医药大学, 济南 250355
  • 2 山东省食品药品检验研究院, 国家药监局胶类产品质量评价重点实验室, 山东省中药标准创新与质量评价工程实验室, 中药配方颗粒共性技术山东省工程研究中心, 济南 250101

    • 祝玉卓,女,硕士研究生 研究方向:药物分析

通讯作者:

* 林永强,男,博士,主任药师 研究方向:中药质量控制关键技术研究 Tel:(0531)81216503;
汪冰,女,硕士,主任药师 研究方向:中药质量控制与产业化 Tel:(0531)81216521
Application Research of Characteristic Polypeptide Technology Based on Chemical Markers in Distiguishment and Identification of Deer-derived Medicinal Materials
Yuzhuo ZHU1, 2, Fei XUE2, Yang JIAO1, 2, Weiliang CUI2, Juan CHEN1, 2, Bing WANG1, 2, *, Yongqiang LIN1, 2, *
Affiliations
  • 1 Shandong University of Traditional Chinese Medicine, Jinan 250355, China
  • 2 NMPA Key Laboratory for Quality Evaluation of Gelatin Products, Shandong Engineering Laboratory for Standard Innovation and Quality Evaluation of TCM, Shandong Engineering Research Center for Generic Technologies of Traditional Chinese Medicine Formula Granules, Shandong Institute for Food and Drug Control, Jinan 250101, China
出版时间: 2024-11-22 doi: 10.11669/cpj.2024.22.004
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摘要
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目的 基于化学标识物概念,通过特征多肽分析方法,实现对鹿茸、鹿角、鹿角霜和鹿皮4种不同部位来源的鹿源药材的精准区分。方法 比较了2种提取溶剂体系对4种不同部位来源的鹿源药材提取效率,优化样品最佳酶解条件,形成高效、简便的样品前处理流程。结合化学标识物理念和特征多肽分析方法,采用液相色谱-三重四极杆质谱及质谱多反应监测技术(MRM),建立一套针对4种不同部位鹿源药材的专属性鉴别方法,并进行方法学实验。结果 优化后的样品前处理方法提取效率高、酶解效果好,24个批次的样品均成功检测到相应的特征多肽离子对。。结论 本研究建立的鹿源药材区分与鉴别方法,具有高专属性、灵敏度和耐用性,能够有效用于鹿源药材的质量评价,为确保和提升鹿源药材的质量提供参考。

化学标识物  /  特征多肽  /  鹿源药材  /  液质联用技术  /  多反应监测

OBJECTIVE To accurately distinguish deer-derived medicinal materials from four different position: Cervi Cornu Pantotrichum, Cervi Cornu, Cervi Cornu Degelatinatum and Deer hide. METHODS The extraction efficiency of two extraction solvent systems for four different kinds of deer-derived medicinal materials was compared, and the optimum enzymatic hydrolysis conditions of the samples were optimized to form an efficient and simple sample pretreatment process. A specific analysis method, combining chemical markers and characteristic polypeptides was established to identify four different position of deer-derived medicinal materials by using liquid chromatography-triple quadrupole mass spectrometry and mass spectrometry multi-reaction monitoring technology (MRM). And methodological experiments were conducted as part of this analysis method. RESULTS The optimized sample pretreatment method with high extraction efficiency and good enzymatic effect was successfully applied, and the corresponding characteristic polypeptide ion pairs were successfully detected in 24 batches of samples. CONCLUSION The distinction and identification method with high-level specificity, sensitivity and robustness of deer-derived medicinal materials were established. The innovative analysis method can be effectively used to evaluate the quality of deer-derived medicinal materials, which provides a reference for ensuring and improving the quality of deer-derived medicinal materials.

chemical marker  /  characteristic polypeptide  /  deer-derived medicinal material  /  LC-MS  /  MRM
祝玉卓, 薛菲, 焦阳, 崔伟亮, 陈娟, 汪冰, 林永强. 基于化学标识物的特征多肽技术在鹿源药材区分与鉴别中的应用研究. 中国药学杂志, 2024 , 59 (22) : 2126 -2131 . DOI: 10.11669/cpj.2024.22.004
Yuzhuo ZHU, Fei XUE, Yang JIAO, Weiliang CUI, Juan CHEN, Bing WANG, Yongqiang LIN. Application Research of Characteristic Polypeptide Technology Based on Chemical Markers in Distiguishment and Identification of Deer-derived Medicinal Materials[J]. Chinese Pharmaceutical Journal, 2024 , 59 (22) : 2126 -2131 . DOI: 10.11669/cpj.2024.22.004
正文
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鹿茸为雄性梅花鹿(Cervus nippon Temminck)或马鹿(Cervus elaphus Linnaeus)密生茸毛未骨化的幼角,鹿角为二者已骨化的角或角托盘;鹿角霜为鹿角熬去胶质干燥后剩余的角块和粉末;鹿皮为梅花鹿或马鹿的皮。上述4种药材中鹿茸、鹿角和鹿角霜是源自鹿角的药材,均为《中国药典》2020年版中收录的名贵药材[1];鹿皮首载于《本草纲目》[2]中,常作为鹿角胶制备过程中的伪品原料。由于四者采收时期和加工工艺不同,其疗效及药用活性也存在一定差别。鹿茸主要功效为强肾壮阳、益精血、强筋骨等[3],鹿角主要用于温肾壮阳、行血消肿、强筋壮骨等[4-5];鹿角霜主要侧重于调理脾肾阳虚所致白带过多,遗尿尿频等;鹿皮能治妇女白带异常、月经过多、各种疮疡。由此可见,四者不可任意添加和使用。虽然以上四种药材均来源于鹿,但流通市场的价格悬殊较大,掺伪使假现象常有发生,且其衍生产品如鹿角胶、龟鹿二仙膏、人参鹿茸丸等的投料部位难以鉴别。目前,有研究通过DNA分子鉴定技术[6-7]、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)[8]和HPLC图谱[9-10]等技术来区分鹿源药材及其加工品,但受限于药材之间的同源性,以上技术只能达到种属级别的鉴定,而无法细致区分具体部位。
2020年,Lin等[11]提出了化学标识物概念,即寻找来源某药材(饮片)特有的而不是来源于其他药材的化学物质作为化学标识物,用于中药的真伪鉴别以及质量控制。特征多肽[12]作为在特定物种中唯一存在的多肽类成分,符合作为鉴别鹿源药材的化学标识物的要求。笔者认为,基于特征多肽的分析检测技术[13]为原药材鉴定提供了一种精确的方法,使得样品部位间的鉴定成为可能。课题组在前期研究中,依托化学标识物的概念,已成功鉴定出鹿茸和鹿角胶中具有物种特异性的特征多肽。然而这些发现是否同样适用于鹿茸、鹿角、鹿角霜和鹿皮药材的鉴别,尚未进行深入探讨。本研究基于化学标识物概念,采用液质联用技术,深入考察了样品前处理方法,并进行了方法学实验,旨在建立一套简便高效的鉴别方法,实现鹿茸、鹿角、鹿皮和鹿角霜4种鹿源药材的有效区分。
1 仪器与材料
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1.1 仪器
AB SCIEX Triple Quad 6500+高效液相色谱质谱联用仪(美国AB Sciex公司);高速离心机(DTNAMICA 公司);水浴恒温振荡器(江苏金坛市中大仪器厂);Climace1222型恒温恒湿箱(德国MMM公司);XSE205电子天平(德国赛多利斯公司)。
1.2 材料
所收集样品信息见表1。所有样品均经过山东省食品药品检验研究院汪冰药师鉴定,均为正品。本实验使用的特征多肽离子信息见表2[14]。
胰蛋白酶(批号:1003494581, Sigma公司),盐酸胍、三羟甲基氨基甲烷(Tris)、乙二胺四乙酸(EDTA)、碳酸氢铵(分析纯),二硫苏糖醇(DTT)(批号:P1694590, 上海泰坦科技有限公司),碘乙酰胺(IAA)(批号:E1915029, 阿拉丁试剂有限公司),甲酸、乙腈(色谱纯),Agilent SB C18 RRHD色谱柱(2.1 mm×100 mm,1.7 μm)、Thermo SCIENTIFIC AccucoreTM C18色谱柱(2.1 mm×100 mm,2.6 μm)、Waters CORTECSTM UPLCC18色谱柱(3.0 mm×100 mm,1.6 μm)。
2 方法
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2.1 样品前处理方法考察
2.1.1 样品提取溶剂考查
将鹿茸、鹿角、鹿角霜、去毛后鹿皮样品锉成细粉,称取2份样品粉末,每份40 mg;分别精密加入变性缓冲液(6 mol·L-1盐酸胍,1 mol·L-1 Tris,2.5 mmol·L-1 EDTA)或体积分数1% NH4HCO3溶液2 mL,90 ℃水浴震摇提取4 h,冷却至室温,离心;取上清液,用0.22 μm滤头滤过,取滤液500 μL,加水500 μL混匀,置3k超滤管中,12 000 r·min-1离心10 min,上清液再次加水500 μL,重复离心一次,备用。
2.1.2 样品酶解时间考察
吸取“2.1.1”项下样品上清液100 μL和水100 μL置进样瓶中,选择酶活性>10 000 BAEE units·mg-1的胰酶,加入10 mg·mL-1胰酶5 μL,使酶底比为1∶40,吹打均匀,置于37 ℃样品进样盘中反应。
2.1.3 样品加酶量考察
取3个进样瓶,分别加入“2.1.1”项下样品上清液100 μL和水100 μL,混匀后分别加入10 mg·mL-1胰酶5、10、20 μL,分别对应酶底比为1∶40、1∶20、1∶10,将其置于37 ℃恒温恒湿箱中反应3 h。
2.2 色谱及质谱条件
液相条件:色谱柱为 Agilent SB C18 RRHD(2.1 mm×100 mm,1.7 μm),柱温43 ℃,流速0.3 mL·min-1,流动相A为体积分数0.1%甲酸溶液,B为乙腈溶液,进行梯度洗脱(0~7 min,8%→12%B;7~11 min,12%→22%B;11~18 min,22%→40%B;18~23 min,40%→90%B;23~23.5 min,90%→8%B;23.5~28 min,8%B),进样量为5 μL。
质谱条件:采用三重四极杆质谱检测器,电喷雾离子源(ESI),正离子模式下,进行多反应监测;离子源喷雾气流速 40 μL·min-1;辅助加热气流速40 μL·min-1;离子化电压5.5 kV;离子源温度550 ℃;锥孔电压47 V,碰撞电压80 V。
2.3 方法学考察
2.3.1 专属性
分别提取Pep 5和Pep 6离子流图谱对24批次鹿源药材样品进行专属性检测。
2.3.2 耐用性
采用3种不同品牌色谱柱考察Pep 1~Pep 7的耐用性,分别为Agilent SB C18 RRHD色谱柱(2.1 mm×100 mm,1.7 μm)、Thermo SCIENTIFIC AccucoreTM C18色谱柱(2.1 mm×100 mm,2.6 μm)、Waters CORTECSTM UPLC® C18色谱柱(3.0 mm×100 mm,1.6 μm)。
3 结果
收起
3.1 样品提取溶剂考察
分别在盐溶剂体系(6 mol·L-1盐酸胍,1 mol·L-1 Tris,2.5 mmol·L-1 EDTA)和水溶剂体系(体积分数1% NH4HCO3溶液)下提取样品,表2为特征多肽离子流色谱图,对其峰型、响应值和峰面积进行分析和比较。结果以Pep 6变化最为显著,体积分数1%NH4HCO3溶液提取物的Pep 6峰面积远远小于变性缓冲液提取物,结果见图1。因此,本实验选用盐溶剂体系为提取溶剂,90 ℃水浴震摇提取4 h为提取方法。
3.2 酶解条件考察
以Pep 2、Pep 5和Pep 6作为考察对象,筛选特征多肽的稳定平衡期,特征多肽在不同酶解时间、不同加酶量下的变化趋势见图2~3。结果显示,在前180 min特征多肽峰面积随酶解时间延长而逐渐增加;在180~270 min内,Pep 2、Pep 5、Pep 6峰面积有小幅上升,几乎趋于稳定。因此,为节约时间成本,确定酶解时间为180 min。随着加酶量的增加,仅有Pep 2峰面积上升,为减少蛋白酶对特征多肽的影响,因此选择加酶量为5 μL。
3.3 鹿茸、鹿角、鹿角霜、鹿皮鉴别方法的建立
各特征多肽检测结果见图4。Pep 1、Pep 2、Pep 3、Pep 4和Pep 7在鹿茸、鹿角、鹿皮均被检测到,为三者共有特征多肽。但由于鹿角霜经过长时间煎煮,因已除去大部分胶质物质,其蛋白类物质含量较少;另外,不同厂家的制备工艺存在差异,上述5个特征多肽在鹿角霜未能全部检测到。Pep 5同时在鹿茸、鹿角和鹿角霜药材中被检测到,但在鹿皮中未被检测到;表明Pep 5为鹿角类药材的专属性特征多肽;同时,鹿角霜中检测到Pep 5也说明其具有一定耐高温特征。Pep 6在梅花鹿茸和马鹿茸中被显著检测到,在未骨化完全的鹿角可被检测到,但在鹿皮和鹿角霜中均未被检测到;因此,可通过Pep 6来区分鹿茸与鹿角、鹿角霜、鹿皮药材。综上所述,4种鹿源药材的判定原则如下:若样品可检出Pep 1、Pep 2、Pep 3、Pep 4、Pep 5、Pep 6、Pep 7特征离子峰时,则认为样品为鹿茸;若可检出Pep 1、Pep 2、Pep 3、Pep 4、Pep 5、Pep 7特征离子峰而未检出Pep 6特征离子峰,则认为样品为鹿角;若可检出Pep 1、Pep 2、Pep 3、Pep 4、Pep 7特征峰,且未检出Pep 5和Pep 6特征峰时,则认为样品为鹿皮;当样品中可检出Pep 5特征峰,则认为样品为鹿角霜。
3.4 样品检测结果
将该方法应用于所有批次样品中,按“3.3”项下判定原则,分别提取各特征多肽的离子流图谱。结果发现所有批次鹿茸样品均可检测到Pep 1~Pep 7,所有批次鹿角样品中均可检测到Pep 1、Pep 2、Pep 3、Pep 4、Pep 5、Pep 7,其中J2批次鹿角可检测到Pep 6,推测其可能为未骨化完全的鹿角;所有批次的鹿茸、鹿角和鹿角霜样品均可检测到Pep 5;所有批次的鹿皮样品中均可检测到Pep 1、Pep 2、Pep 3、Pep 4和Pep 7。
3.5 方法学考察
3.5.1 专属性
Pep 5仅在鹿茸、鹿角和鹿角霜样品中被检测到,未在鹿皮样品中检测到,见图5;Pep 6仅在鹿茸样品中检测到,未在鹿角、鹿角霜和鹿皮样品中检测到;结果表明该方法专属性良好。
3.5.2 耐用性
耐用性检测结果见表3,3种色谱柱均具有明显分离差异,表明该方法耐用性良好。
4 讨论
收起
首先,本实验通过考察盐溶剂体系和水溶剂体系探索特征多肽在提取过程中的影响。盐溶液常用于蛋白变性,具有较稳定的pH,能较好维持蛋白结构和活性,但常常伴有毒性;水溶液无毒无害,普遍用于提取胶原蛋白,但水溶剂往往会破坏蛋白结构,所得蛋白含量稍低。因此,选择合适的提取溶剂至关重要。本实验结果显示,盐溶剂提取体系提取效率较高,其所得提取物的各特征多肽离子对峰型和响应值均优于水溶剂提取体系。笔者认为主要原因有以下两点:一是受到高温加热,鹿茸中大量血蛋白被破坏;二是水溶剂张力过大,血蛋白聚团凝结,蛋白结构受到破坏[15]。实验结果证明了盐溶剂提取体系能提取出鹿源药材中的大量蛋白和多肽类物质。其次,酶解条件是得到较为稳定特征多肽的关键因素,本实验考察了不同酶解时间和不同加酶量下各特征多肽的变化情况,优化所得的样品前处理方法可成功应用于4种不同部位的鹿源药材。
此外,本实验中各特征多肽专属性和耐用性良好,可通过Pep 6的检出情况判定样品是否为鹿茸,可通过Pep 5的检出情况判定样品是否为鹿角类药材,可精确高效地区分源于不同部位的4种鹿源药材。
综上所述,本实验基于化学标识物和特征多肽分析方法,采用液质联用技术建立了一种可有效区分鹿茸、鹿角、鹿角霜、鹿皮4种鹿源药材的方法。
基金
收起
  • 国家重点研发计划(中医药现代化)课题(2023YFC3504102)
  • “新高校20条”科研带头人工作室项目(202228096)
  • 泉城产业领军人才支持计划创新团队项目(MRJT2105)
文献
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2024年第59卷第22期
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doi: 10.11669/cpj.2024.22.004
  • 接收时间：2024-03-26
  • 首发时间：2025-11-16
  • 出版时间：2024-11-22
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  • 收稿日期：2024-03-26
基金
国家重点研发计划(中医药现代化)课题(2023YFC3504102)
“新高校20条”科研带头人工作室项目(202228096)
泉城产业领军人才支持计划创新团队项目(MRJT2105)
作者信息
    1 山东中医药大学, 济南 250355
    2 山东省食品药品检验研究院, 国家药监局胶类产品质量评价重点实验室, 山东省中药标准创新与质量评价工程实验室, 中药配方颗粒共性技术山东省工程研究中心, 济南 250101

通讯作者:

* 林永强,男,博士,主任药师 研究方向:中药质量控制关键技术研究 Tel:(0531)81216503;
汪冰,女,硕士,主任药师 研究方向:中药质量控制与产业化 Tel:(0531)81216521
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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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