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Article(id=1322926476043104653, tenantId=1146029695717560320, journalId=1255847919539208197, issueId=1322926217711726961, articleNumber=null, orderNo=null, doi=10.13193/j.issn.1673-7717.2025.08.034, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=null, receivedDateStr=null, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1793339850674, onlineDateStr=2026-10-30, pubDate=1754755200000, pubDateStr=2025-08-10, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1793339850674, onlineIssueDateStr=2026-10-30, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1793339850674, creator=13701087609, updateTime=1793339850674, updator=13701087609, issue=Issue{id=1322926217711726961, tenantId=1146029695717560320, journalId=1255847919539208197, year='2025', volume='43', issue='8', pageStart='1', pageEnd='258', issueExtLink='null', onlineDate='null', pubDate='1754755200000', pubDateStr='2025-08-10', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1793339789083, creator='13701087609', updateTime=1793340517761, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1322929274071679978, tenantId=1146029695717560320, journalId=1255847919539208197, issueId=1322926217711726961, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1322929274071679979, tenantId=1146029695717560320, journalId=1255847919539208197, issueId=1322926217711726961, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=178, endPage=184, ext={EN=ArticleExt(id=1322926476500283790, articleId=1322926476043104653, tenantId=1146029695717560320, journalId=1255847919539208197, language=EN, title=Study on Chemical Composition of Yinyanghuo（Epimedii Folium）and Its Processed Products and Their Effects on Proliferation and Osteogenesis of MC3T3-E1 Cells, columnId=1256183365644968140, journalTitle=Chinese Archives of Traditional Chinese Medicine, columnName=Platform of Provincial Project, runingTitle=null, highlight=null, articleAbstract=
Objective

To analyze the chemical composition of Yinyanghuo（Epimedii Folium）and its processed products and to investigate the proliferative and osteogenic effects of its extracts on mouse Embryonic Preosteoblast Cells（MC3T3-E1 cells）.

Methods

Ten batches of Yinyanghuo（Epimedii Folium）were processed into 10 batches of Yinyanghuo（Epimedii Folium）processed with lanolin oil and 10 batches of Yinyanghuo（Epimedii Folium）processed with salt，respectively.The contents of flavonoids in the samples were determined by high performance liquid chromatography（HPLC），and the changes of flavonoids in Yinyanghuo（Epimedii Folium）before and after processing were explored by HPLC fingerprint and multivariate statistical analysis.The osteogenic inhibition model of MC3T3-E1 cells induced by dexamethasone（DEX）was established.The anti-osteoporosis effect of Epimedium and its processed products were evaluated by measuring the expressions of alkaline phosphatase（ALP），osteoprotegerin（OPG）and osteocalcin（OCN）in cells，and the possible reasons for the different anti-osteoporosis effects of Yinyanghuo（Epimedii Folium）and its processed products were further explored.

Results

The HPLC fingerprints of Yinyanghuo（Epimedii Folium）and its processed products were established，and the similarity of the fingerprints was evaluated，and 16 common peaks were identified.The results of cluster analysis classified epimedin A，epimedin B and epimedin C into one group，and icariin and baohuoside I into one group.The polar coordinate bubble diagram of each flavonoid content showed that the contents of primary flavonoid glycosides（epimedin A，epimedin B and epimedin C）decreased overall after processing，and the contents of secondary flavonoid glycosides（icariin and baohuoside I）increased overall after processing.The data of anti-osteoporosis activity showed that different processed products of Yinyanghuo（Epimedii Folium）had certain osteogenic proliferation effect，and the salt-processed Yinyanghuo（Epimedii Folium）had the most obvious effect on the enhancement of osteoblast activity inhibited by DEX.

Conclusion

Fingerprint similarity analysis and cluster analysis were used to analyze the differences in the contents of five flavonoids，so as to preliminarily explore the changes of flavonoids before and after processing. Combined with the effect of its extract on the proliferation and osteogenic ability of MC3T3-E1 cells，it was found that the content of icariin increased after salt processing of Yinyanghuo（Epimedii Folium），and the proliferation of osteogenic inhibition of MC3T3-E1 cells induced by DEX was significantly enhanced，suggesting that icariin may be the material basis for different anti-osteoporosis clinical effects of different processed Yinyanghuo（Epimedii Folium）products，laying a foundation for further exploration of the processing principle of Yinyanghuo（Epimedii Folium）.

, authors=Guangyu CHENG, Pan LI, Laihui SONG, Jinling LIANG, Wenxuan CAO, Peng YU, Yulin DAI, Jia LIU, Tianyang XU, authorsList=Guangyu CHENG, Pan LI, Laihui SONG, Jinling LIANG, Wenxuan CAO, Peng YU, Yulin DAI, Jia LIU, Tianyang XU, authorCompany=null, correspAuthors=Jia LIU, Tianyang XU, 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, fund=null), CN=ArticleExt(id=1322926480098996641, articleId=1322926476043104653, tenantId=1146029695717560320, journalId=1255847919539208197, language=CN, title=淫羊藿及其炮制品化学组成及对MC3T3-E1细胞的增殖和成骨作用研究, columnId=1256183369033965799, journalTitle=中华中医药学刊, columnName=省级项目平台, runingTitle=null, highlight=null, articleAbstract=
目的

分析淫羊藿（Epimedii folium）及其炮制品化学组成，并探究其提取物对小鼠胚胎成骨细胞前体细胞（Mouse embryonic preosteoblast cells，MC3T3-E1 cells）的增殖及成骨作用。

方法

取10批淫羊藿，分别加工炮制成羊脂油炙淫羊藿和盐炙淫羊藿各10批，利用高效液相色谱法（High performance liquid chromatography，HPLC）测定样品中黄酮类成分含量，并结合液相色谱指纹图谱及多元统计分析方法，探究淫羊藿中黄酮类化合物在炮制前后变化规律；建立地塞米松（Dexamethasone，DEX）诱导的MC3T3-E1细胞的成骨抑制模型，通过测定细胞中碱性磷酸酶（Alkaline phosphatase，ALP）、骨保护素（Osteoprotegerin，OPG）和骨钙素（Osteocalcin，OCN）的表达量来评价淫羊藿及其炮制品的抗骨质疏松效果，进一步探讨淫羊藿及其炮制品抗骨质疏松作用不同可能存在的原因。

结果

建立淫羊藿及其炮制品HPLC指纹图谱，并对指纹图谱进行相似度评价，识别出共有峰16个；聚类分析结果将朝藿定A、朝藿定B和朝藿定C归为一类，淫羊藿苷和宝藿苷I归为一类；各黄酮含量的极坐标气泡图显示初级黄酮苷（朝藿定A、朝藿定B和朝藿定C）含量在炮制后整体减少，次级黄酮苷（淫羊藿苷和宝藿苷I）含量在炮制后整体增加；抗骨质疏松活性数据表明，淫羊藿不同炮制品均具有一定的成骨增殖作用，其中淫羊藿盐炙品对DEX抑制的成骨细胞活性增强作用最为明显。

结论

采用指纹图谱相似度分析和聚类分析对5个黄酮类成分含量的差异进行分析，从而初步探索了淫羊藿炮制前后黄酮类成分的变化规律。结合其提取物对MC3T3-E1细胞增殖及成骨能力的影响，发现淫羊藿经盐炙后淫羊藿苷含量增加，对DEX引起的MC3T3-E1细胞成骨抑制的增殖作用显著增强，提示淫羊藿苷可能是淫羊藿不同炮制品抗骨质疏松临床疗效不同的物质基础，为进一步探索淫羊藿炮制原理奠定基础。

, authors=成光宇, 李盼, 宋来辉, 梁金玲, 曹文轩, 于澎, 戴雨霖, 刘佳, 许天阳, authorsList=成光宇, 李盼, 宋来辉, 梁金玲, 曹文轩, 于澎, 戴雨霖, 刘佳, 许天阳, authorCompany=null, correspAuthors=刘佳, 许天阳, authorNote=

成光宇（1971-），男，吉林长春人，主任药师，硕士研究生导师，硕士，研究方向：药理学。

, correspAuthorsNote=
刘佳（1982-），女，吉林长春人，副教授，硕士研究生导师，博士，研究方向：中药药理学。E-mail：。
许天阳（1979-），男，吉林长春人，副教授，硕士，研究方向：中药分析学。E-mail：。
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成光宇（1971-），男，吉林长春人，主任药师，硕士研究生导师，硕士，研究方向：药理学。

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成光宇（1971-），男，吉林长春人，主任药师，硕士研究生导师，硕士，研究方向：药理学。

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articleTitle=淫羊藿药材的UPLC指纹图谱研究, refAbstract=null), Reference(id=1322926493797593610, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, doi=null, pmid=null, pmcid=null, year=2019, volume=37, issue=3, pageStart=325, pageEnd=330, url=null, language=null, rfNumber=［20］, rfOrder=19, authorNames=龙立梅, 宋沙沙, 曹学丽, journalName=色谱, refType=null, unstructuredReference=龙立梅，宋沙沙，曹学丽. 基于香气成分气相色谱-质谱指纹图谱的判别分析和相似度评价用于绿茶等级差异研究［J］. 色谱，2019，37（3）：325-330., articleTitle=基于香气成分气相色谱-质谱指纹图谱的判别分析和相似度评价用于绿茶等级差异研究, refAbstract=null), Reference(id=1322926493973754379, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, doi=null, pmid=null, pmcid=null, year=2020, volume=51, issue=20, pageStart=5372, pageEnd=5380, url=null, language=null, rfNumber=［21］, rfOrder=20, authorNames=曾华婷, 郭健, 陈彦, journalName=中草药, refType=null, unstructuredReference=曾华婷，郭健，陈彦. 淫羊藿素药理作用及其新型给药系统的研究进展［J］. 中草药，2020，51（20）：5372-5380., articleTitle=淫羊藿素药理作用及其新型给药系统的研究进展, refAbstract=null), Reference(id=1322926494040863244, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, doi=null, pmid=null, pmcid=null, year=2016, volume=31, issue=6, pageStart=2386, pageEnd=2389, url=null, language=null, rfNumber=［22］, rfOrder=21, authorNames=牛晓静, 鲁静, 段晓颖, journalName=中华中医药杂志, refType=null, unstructuredReference=牛晓静，鲁静，段晓颖，等. 基于聚类分析的淫羊藿黄酮类成分的质量分析研究［J］. 中华中医药杂志，2016，31（6）：2386-2389., articleTitle=基于聚类分析的淫羊藿黄酮类成分的质量分析研究, refAbstract=null), Reference(id=1322926494103777805, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, doi=null, pmid=null, pmcid=null, year=2014, volume=39, issue=3, pageStart=370, pageEnd=377, url=null, language=null, rfNumber=［23］, rfOrder=22, authorNames=孙娥, 徐凤娟, 张振海, journalName=中国中药杂志, refType=null, unstructuredReference=孙娥，徐凤娟，张振海，等. 基于化学成分转化-肠吸收屏障网络耦联作用的中药炮制机制研究体系的构建［J］. 中国中药杂志，2014，39（3）：370-377., articleTitle=基于化学成分转化-肠吸收屏障网络耦联作用的中药炮制机制研究体系的构建, refAbstract=null), Reference(id=1322926494183469582, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, doi=null, pmid=null, pmcid=null, year=2018, volume=62, issue=null, pageStart=277, pageEnd=286, url=null, language=null, rfNumber=［24］, rfOrder=23, authorNames=JIANG J, XIAO SC, XU X M, journalName=Int Immunopharmacol, refType=null, unstructuredReference=JIANG J，XIAO SC，XU X M，et al. Isomeric flavonoid aglycones derived from Epimedii Folium exerted different intensities in antiosteoporosis through OPG/RANKL protein targets［J］. Int Immunopharmacol，2018，62：277-286., articleTitle=Isomeric flavonoid aglycones derived from Epimedii Folium exerted different intensities in antiosteoporosis through OPG/RANKL protein targets, refAbstract=null), Reference(id=1322926494456099343, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, doi=null, pmid=null, pmcid=null, year=2020, volume=26, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=［25］, rfOrder=24, authorNames=ZHANG Y, HAN B, WEI Y, journalName=Med Sci Monit, refType=null, unstructuredReference=ZHANG Y，HAN B，WEI Y，et al. Icariin promotes fracture healing in ovariectomized rats［J］. Med Sci Monit，2020，26：e924554., articleTitle=Icariin promotes fracture healing in ovariectomized rats, refAbstract=null), Reference(id=1322926494523208208, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, doi=null, pmid=null, pmcid=null, year=2022, volume=2022, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=［26］, rfOrder=25, authorNames=RUILIAN Z, YING G, HONGMEI S, journalName=Comput Math Methods Med, refType=null, unstructuredReference=RUILIAN Z，YING G，HONGMEI S，et al. Exploration of the effect of icariin on nude mice with lung cancer bone metastasis via the OPG/RANKL/RANK system［J］. Comput Math Methods Med，2022，2022：2011625., articleTitle=Exploration of the effect of icariin on nude mice with lung cancer bone metastasis via the OPG/RANKL/RANK system, refAbstract=null)], funds=[Fund(id=1322926489620066806, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, awardId=20210204035YY; 20230204034YY; YDZJ202201ZYTS205, language=CN, fundingSource=吉林省科技发展计划项目(20210204035YY; 20230204034YY; YDZJ202201ZYTS205), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1322926480392597922, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, xref=null, ext=[AuthorCompanyExt(id=1322926480405180835, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, companyId=1322926480392597922, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Changchun University of Chinese Medicine，Changchun 130117，Jilin，China), AuthorCompanyExt(id=1322926480417763748, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, companyId=1322926480392597922, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=长春中医药大学，吉林 长春 130117)])], figs=[ArticleFig(id=1322926484859531740, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=EN, label=null, caption=null, figureFileSmall=hM3ZedtYha2LhXQA0+uLTw==, figureFileBig=JzF0nVOzIRwLV9mOdQKl/Q==, tableContent=null), ArticleFig(id=1322926484930834909, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=CN, label=图1, caption=混合对照品溶液（A）、生品（B）、油炙品（C）、盐炙品（D）HPLC图

注：1.朝藿定A；2.朝藿定B；3.朝藿定C；4.淫羊藿苷；5.宝藿苷Ⅰ。

, figureFileSmall=hM3ZedtYha2LhXQA0+uLTw==, figureFileBig=JzF0nVOzIRwLV9mOdQKl/Q==, tableContent=null), ArticleFig(id=1322926485123772894, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=EN, label=null, caption=null, figureFileSmall=WhCTu3gkfq9EtG1Ew1Wmlg==, figureFileBig=kKKAaU0ATcQLPpeZP3/NSg==, tableContent=null), ArticleFig(id=1322926485232824799, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=CN, label=图2, caption=淫羊藿及其炮制品的指纹图谱

注：生品S1～S10；油炙品S11～S20；盐炙品S21～S30。

, figureFileSmall=WhCTu3gkfq9EtG1Ew1Wmlg==, figureFileBig=kKKAaU0ATcQLPpeZP3/NSg==, tableContent=null), ArticleFig(id=1322926485614506464, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=EN, label=null, caption=null, figureFileSmall=wBdk1Tx2kpTgrYoO94Zu9g==, figureFileBig=QCqyWKVSBirlJ+/9kyJ6SQ==, tableContent=null), ArticleFig(id=1322926485685809633, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=CN, label=图3, caption=5个黄酮化合物的聚类分析结果, figureFileSmall=wBdk1Tx2kpTgrYoO94Zu9g==, figureFileBig=QCqyWKVSBirlJ+/9kyJ6SQ==, tableContent=null), ArticleFig(id=1322926485752918498, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=EN, label=null, caption=null, figureFileSmall=E3xsdTbZaB/D26QbT6zJRQ==, figureFileBig=SjIE7jbszGrP4hopTEeRyw==, tableContent=null), ArticleFig(id=1322926486004576739, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=CN, label=图4, caption=淫羊藿及其炮制品中5个黄酮类化合物含量测定结果图, figureFileSmall=E3xsdTbZaB/D26QbT6zJRQ==, figureFileBig=SjIE7jbszGrP4hopTEeRyw==, tableContent=null), ArticleFig(id=1322926486080074212, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=EN, label=null, caption=null, figureFileSmall=hMGxN/nU42lIh20J3l2HvA==, figureFileBig=gjdzFifOxZn/cgZephI7hQ==, tableContent=null), ArticleFig(id=1322926487795544549, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=CN, label=图5, caption=5个黄酮化合物在生品、油炙品、盐炙品中的变化趋势极坐标气泡图, figureFileSmall=hMGxN/nU42lIh20J3l2HvA==, figureFileBig=gjdzFifOxZn/cgZephI7hQ==, tableContent=null), ArticleFig(id=1322926487904596454, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=EN, label=null, caption=null, figureFileSmall=bLHpwZnsbggyYsDjd8619g==, figureFileBig=fj1CjUZtsjKHvJ2DviuDpg==, tableContent=null), ArticleFig(id=1322926488110117351, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=CN, label=图6, caption=细胞活力及细胞增殖测定结果

注：A.地塞米松造模浓度的筛选；B.生品细胞毒的筛选；C.盐炙品细胞毒的筛选；D.油炙品细胞毒的筛选；E.淫羊藿及其炮制品对DEX造成的成骨抑制的增殖作用；DEX：地塞米松；NC：空白对照组；**P<0.05；***P<0.01。

, figureFileSmall=bLHpwZnsbggyYsDjd8619g==, figureFileBig=fj1CjUZtsjKHvJ2DviuDpg==, tableContent=null), ArticleFig(id=1322926488185614824, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=EN, label=null, caption=null, figureFileSmall=GzGQKeG0RnojAlF/ktVRtg==, figureFileBig=mG/Ki+8rc17bdQQlkWQrQw==, tableContent=null), ArticleFig(id=1322926488244335081, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=CN, label=图7, caption=淫羊藿治疗对经DEX处理的MC3T3-E1细胞上清中ALP（A）、OPG（B）和OCN（C）的表达的作用

注：DEX：地塞米松；NC：空白对照组；**P<0.05；***P<0.01。

, figureFileSmall=GzGQKeG0RnojAlF/ktVRtg==, figureFileBig=mG/Ki+8rc17bdQQlkWQrQw==, tableContent=null), ArticleFig(id=1322926488303055338, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=EN, label=null, caption=null, figureFileSmall=pmoCwKynIrJZnn3Cfzatog==, figureFileBig=JpXguE0zJdkJMJHbvdUdFA==, tableContent=null), ArticleFig(id=1322926488365969899, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=CN, label=图8, caption=朝藿定A、朝藿定B、朝藿定C、淫羊藿苷、宝藿苷I的化学结构, figureFileSmall=pmoCwKynIrJZnn3Cfzatog==, figureFileBig=JpXguE0zJdkJMJHbvdUdFA==, tableContent=null), ArticleFig(id=1322926488445661676, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
批号采集地
TH190250001通化县清河镇
TH190250002通化县五道江镇
TH190250003通化县万良镇
TH190250004通化县江东乡
TH190250005通化县朝阳镇
TH190250006通化县大泉乡
TH190250007通化县富江乡
TH190250008通化县青石镇
TH190250009通化县金斗乡
TH190251001通化县快大茂镇
), ArticleFig(id=1322926488521159149, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=CN, label=表1, caption=

淫羊藿生药材批号及产地

, figureFileSmall=null, figureFileBig=null, tableContent=
批号采集地
TH190250001通化县清河镇
TH190250002通化县五道江镇
TH190250003通化县万良镇
TH190250004通化县江东乡
TH190250005通化县朝阳镇
TH190250006通化县大泉乡
TH190250007通化县富江乡
TH190250008通化县青石镇
TH190250009通化县金斗乡
TH190251001通化县快大茂镇
), ArticleFig(id=1322926488617628142, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
批号生品油炙品盐炙品
TH190250001R1O1S1
TH190250002R2O2S2
TH190250003R3O3S3
TH190250004R4O4S4
TH190250005R5O5S5
TH190250006R6O6S6
TH190250007R7O7S7
TH190250008R8O8S8
TH190250009R9O9S9
TH190251001R10O10S10
), ArticleFig(id=1322926488697319919, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=CN, label=表2, caption=

淫羊藿及其炮制品编号

, figureFileSmall=null, figureFileBig=null, tableContent=
批号生品油炙品盐炙品
TH190250001R1O1S1
TH190250002R2O2S2
TH190250003R3O3S3
TH190250004R4O4S4
TH190250005R5O5S5
TH190250006R6O6S6
TH190250007R7O7S7
TH190250008R8O8S8
TH190250009R9O9S9
TH190251001R10O10S10
), ArticleFig(id=1322926488814760432, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
成分线性方程R线性范围/（μg）
朝藿定AY=8 964.5X-258.180.999 91.04~104.00
朝藿定BY=8 808.2X+4 645.80.999 81.13~113.00
朝藿定CY=8 866.2X+1 958.90.999 91.02~102.00
淫羊藿苷Y=9 575.1X-1 098.51.000 01.58~158.00
宝藿苷IY=13 961X+8 609.70.999 21.25~125.00
), ArticleFig(id=1322926489003504113, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=CN, label=表3, caption=

5个黄酮类化合物的线性方程、相关系数和线性范围

, figureFileSmall=null, figureFileBig=null, tableContent=
成分线性方程R线性范围/（μg）
朝藿定AY=8 964.5X-258.180.999 91.04~104.00
朝藿定BY=8 808.2X+4 645.80.999 81.13~113.00
朝藿定CY=8 866.2X+1 958.90.999 91.02~102.00
淫羊藿苷Y=9 575.1X-1 098.51.000 01.58~158.00
宝藿苷IY=13 961X+8 609.70.999 21.25~125.00
), ArticleFig(id=1322926489125138930, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
图谱编号样品编号相似度
S1R10.993
S2R20.988
S3R30.984
S4R40.986
S5R50.991
S6R60.976
S7R70.990
S8R80.992
S9R90.996
S10R100.983
S11O10.995
S12O20.994
S13O30.966
S14O40.981
S15O50.995
S16O60.993
S17O70.994
S18O80.997
S19O90.996
S20O100.987
S21S10.938
S22S20.996
S23S30.972
S24S40.977
S25S50.967
S26S60.989
S27S70.994
S28S80.995
S29S90.974
S30S100.976
), ArticleFig(id=1322926489188053491, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=CN, label=表4, caption=

淫羊藿及其炮制品的指纹图谱相似度数据

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图谱编号样品编号相似度
S1R10.993
S2R20.988
S3R30.984
S4R40.986
S5R50.991
S6R60.976
S7R70.990
S8R80.992
S9R90.996
S10R100.983
S11O10.995
S12O20.994
S13O30.966
S14O40.981
S15O50.995
S16O60.993
S17O70.994
S18O80.997
S19O90.996
S20O100.987
S21S10.938
S22S20.996
S23S30.972
S24S40.977
S25S50.967
S26S60.989
S27S70.994
S28S80.995
S29S90.974
S30S100.976
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样品编号朝藿定A/（%）朝藿定B/（%）朝藿定C/（%）淫羊藿苷/（%）宝藿苷I/（%）
R11.894 83.042 62.154 60.323 90.082 4
R22.031 33.474 62.667 90.336 70.017 1
R31.588 52.542 21.611 30.311 00.049 3
R41.621 82.683 82.109 40.290 90.103 9
R51.935 63.182 92.451 90.326 30.033 2
R61.687 22.707 61.625 30.321 20.010 5
R72.259 33.702 02.704 70.366 20.023 9
R82.125 73.516 32.449 70.347 20.026 1
R91.283 22.226 11.729 90.270 10.035 2
R103.381 15.728 25.647 20.757 40.021 7
O11.748 12.885 72.026 90.374 80.041 2
O21.906 33.254 42.386 90.423 80.072 0
O31.172 31.839 32.423 20.235 20.159 2
O41.479 22.499 91.893 30.325 60.140 5
O51.678 62.714 61.855 60.394 00.048 5
O61.741 42.871 01.841 50.673 80.111 0
O71.991 63.374 82.393 70.419 60.050 0
O81.780 23.048 42.077 40.376 30.054 2
O91.736 02.928 12.184 60.375 10.056 9
O102.672 94.488 24.008 30.777 10.027 6
S11.634 42.586 50.762 10.803 40.111 6
S21.813 33.094 71.853 90.312 30.039 9
S31.157 01.819 80.712 80.330 70.108 6
S41.457 52.365 21.010 40.493 40.130 3
S51.656 32.545 50.863 20.631 40.063 0
S62.087 83.470 81.947 30.603 40.048 4
S71.734 82.900 91.357 00.488 20.068 6
S81.879 63.023 61.593 70.267 60.027 6
S91.751 63.006 51.486 60.492 40.054 6
S102.586 04.144 42.070 50.702 20.034 5
), ArticleFig(id=1322926489490043381, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322926476043104653, language=CN, label=表5, caption=

淫羊藿及其炮制品中5个黄酮类化合物含量测定结果

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样品编号朝藿定A/（%）朝藿定B/（%）朝藿定C/（%）淫羊藿苷/（%）宝藿苷I/（%）
R11.894 83.042 62.154 60.323 90.082 4
R22.031 33.474 62.667 90.336 70.017 1
R31.588 52.542 21.611 30.311 00.049 3
R41.621 82.683 82.109 40.290 90.103 9
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R61.687 22.707 61.625 30.321 20.010 5
R72.259 33.702 02.704 70.366 20.023 9
R82.125 73.516 32.449 70.347 20.026 1
R91.283 22.226 11.729 90.270 10.035 2
R103.381 15.728 25.647 20.757 40.021 7
O11.748 12.885 72.026 90.374 80.041 2
O21.906 33.254 42.386 90.423 80.072 0
O31.172 31.839 32.423 20.235 20.159 2
O41.479 22.499 91.893 30.325 60.140 5
O51.678 62.714 61.855 60.394 00.048 5
O61.741 42.871 01.841 50.673 80.111 0
O71.991 63.374 82.393 70.419 60.050 0
O81.780 23.048 42.077 40.376 30.054 2
O91.736 02.928 12.184 60.375 10.056 9
O102.672 94.488 24.008 30.777 10.027 6
S11.634 42.586 50.762 10.803 40.111 6
S21.813 33.094 71.853 90.312 30.039 9
S31.157 01.819 80.712 80.330 70.108 6
S41.457 52.365 21.010 40.493 40.130 3
S51.656 32.545 50.863 20.631 40.063 0
S62.087 83.470 81.947 30.603 40.048 4
S71.734 82.900 91.357 00.488 20.068 6
S81.879 63.023 61.593 70.267 60.027 6
S91.751 63.006 51.486 60.492 40.054 6
S102.586 04.144 42.070 50.702 20.034 5
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淫羊藿及其炮制品化学组成及对MC3T3-E1细胞的增殖和成骨作用研究
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成光宇 , 李盼 , 宋来辉 , 梁金玲 , 曹文轩 , 于澎 , 戴雨霖 , 刘佳 , 许天阳
中华中医药学刊 | 省级项目平台 2025,43(8): 178-184
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中华中医药学刊 |省级项目平台 2025 , 43 (8) : 178 -184
淫羊藿及其炮制品化学组成及对MC3T3-E1细胞的增殖和成骨作用研究
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成光宇（1971-），男，吉林长春人，主任药师，硕士研究生导师，硕士，研究方向：药理学。

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成光宇, 李盼, 宋来辉, 梁金玲, 曹文轩, 于澎, 戴雨霖, 刘佳 , 许天阳
作者信息
  • 长春中医药大学，吉林 长春 130117
通讯作者:
刘佳（1982-），女，吉林长春人，副教授，硕士研究生导师，博士，研究方向：中药药理学。E-mail：。
许天阳（1979-），男，吉林长春人，副教授，硕士，研究方向：中药分析学。E-mail：。
作者简介:

成光宇（1971-），男，吉林长春人，主任药师，硕士研究生导师，硕士，研究方向：药理学。

Study on Chemical Composition of Yinyanghuo（Epimedii Folium）and Its Processed Products and Their Effects on Proliferation and Osteogenesis of MC3T3-E1 Cells
Guangyu CHENG, Pan LI, Laihui SONG, Jinling LIANG, Wenxuan CAO, Peng YU, Yulin DAI, Jia LIU , Tianyang XU
Affiliations
  • Changchun University of Chinese Medicine，Changchun 130117，Jilin，China
出版时间: 2025-08-10 doi: 10.13193/j.issn.1673-7717.2025.08.034
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摘要
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目的

分析淫羊藿（Epimedii folium）及其炮制品化学组成，并探究其提取物对小鼠胚胎成骨细胞前体细胞（Mouse embryonic preosteoblast cells，MC3T3-E1 cells）的增殖及成骨作用。

方法

取10批淫羊藿，分别加工炮制成羊脂油炙淫羊藿和盐炙淫羊藿各10批，利用高效液相色谱法（High performance liquid chromatography，HPLC）测定样品中黄酮类成分含量，并结合液相色谱指纹图谱及多元统计分析方法，探究淫羊藿中黄酮类化合物在炮制前后变化规律；建立地塞米松（Dexamethasone，DEX）诱导的MC3T3-E1细胞的成骨抑制模型，通过测定细胞中碱性磷酸酶（Alkaline phosphatase，ALP）、骨保护素（Osteoprotegerin，OPG）和骨钙素（Osteocalcin，OCN）的表达量来评价淫羊藿及其炮制品的抗骨质疏松效果，进一步探讨淫羊藿及其炮制品抗骨质疏松作用不同可能存在的原因。

结果

建立淫羊藿及其炮制品HPLC指纹图谱，并对指纹图谱进行相似度评价，识别出共有峰16个；聚类分析结果将朝藿定A、朝藿定B和朝藿定C归为一类，淫羊藿苷和宝藿苷I归为一类；各黄酮含量的极坐标气泡图显示初级黄酮苷（朝藿定A、朝藿定B和朝藿定C）含量在炮制后整体减少，次级黄酮苷（淫羊藿苷和宝藿苷I）含量在炮制后整体增加；抗骨质疏松活性数据表明，淫羊藿不同炮制品均具有一定的成骨增殖作用，其中淫羊藿盐炙品对DEX抑制的成骨细胞活性增强作用最为明显。

结论

采用指纹图谱相似度分析和聚类分析对5个黄酮类成分含量的差异进行分析，从而初步探索了淫羊藿炮制前后黄酮类成分的变化规律。结合其提取物对MC3T3-E1细胞增殖及成骨能力的影响，发现淫羊藿经盐炙后淫羊藿苷含量增加，对DEX引起的MC3T3-E1细胞成骨抑制的增殖作用显著增强，提示淫羊藿苷可能是淫羊藿不同炮制品抗骨质疏松临床疗效不同的物质基础，为进一步探索淫羊藿炮制原理奠定基础。

淫羊藿  /  黄酮类化合物  /  指纹图谱  /  聚类分析  /  骨质疏松
Objective

To analyze the chemical composition of Yinyanghuo（Epimedii Folium）and its processed products and to investigate the proliferative and osteogenic effects of its extracts on mouse Embryonic Preosteoblast Cells（MC3T3-E1 cells）.

Methods

Ten batches of Yinyanghuo（Epimedii Folium）were processed into 10 batches of Yinyanghuo（Epimedii Folium）processed with lanolin oil and 10 batches of Yinyanghuo（Epimedii Folium）processed with salt，respectively.The contents of flavonoids in the samples were determined by high performance liquid chromatography（HPLC），and the changes of flavonoids in Yinyanghuo（Epimedii Folium）before and after processing were explored by HPLC fingerprint and multivariate statistical analysis.The osteogenic inhibition model of MC3T3-E1 cells induced by dexamethasone（DEX）was established.The anti-osteoporosis effect of Epimedium and its processed products were evaluated by measuring the expressions of alkaline phosphatase（ALP），osteoprotegerin（OPG）and osteocalcin（OCN）in cells，and the possible reasons for the different anti-osteoporosis effects of Yinyanghuo（Epimedii Folium）and its processed products were further explored.

Results

The HPLC fingerprints of Yinyanghuo（Epimedii Folium）and its processed products were established，and the similarity of the fingerprints was evaluated，and 16 common peaks were identified.The results of cluster analysis classified epimedin A，epimedin B and epimedin C into one group，and icariin and baohuoside I into one group.The polar coordinate bubble diagram of each flavonoid content showed that the contents of primary flavonoid glycosides（epimedin A，epimedin B and epimedin C）decreased overall after processing，and the contents of secondary flavonoid glycosides（icariin and baohuoside I）increased overall after processing.The data of anti-osteoporosis activity showed that different processed products of Yinyanghuo（Epimedii Folium）had certain osteogenic proliferation effect，and the salt-processed Yinyanghuo（Epimedii Folium）had the most obvious effect on the enhancement of osteoblast activity inhibited by DEX.

Conclusion

Fingerprint similarity analysis and cluster analysis were used to analyze the differences in the contents of five flavonoids，so as to preliminarily explore the changes of flavonoids before and after processing. Combined with the effect of its extract on the proliferation and osteogenic ability of MC3T3-E1 cells，it was found that the content of icariin increased after salt processing of Yinyanghuo（Epimedii Folium），and the proliferation of osteogenic inhibition of MC3T3-E1 cells induced by DEX was significantly enhanced，suggesting that icariin may be the material basis for different anti-osteoporosis clinical effects of different processed Yinyanghuo（Epimedii Folium）products，laying a foundation for further exploration of the processing principle of Yinyanghuo（Epimedii Folium）.

Yinyanghuo（Epimedii Folium）  /  flavonoids  /  fingerprinting  /  cluster analysis  /  osteoporosis
成光宇, 李盼, 宋来辉, 梁金玲, 曹文轩, 于澎, 戴雨霖, 刘佳, 许天阳. 淫羊藿及其炮制品化学组成及对MC3T3-E1细胞的增殖和成骨作用研究. 中华中医药学刊, 2025 , 43 (8) : 178 -184 . DOI: 10.13193/j.issn.1673-7717.2025.08.034
Guangyu CHENG, Pan LI, Laihui SONG, Jinling LIANG, Wenxuan CAO, Peng YU, Yulin DAI, Jia LIU, Tianyang XU. Study on Chemical Composition of Yinyanghuo（Epimedii Folium）and Its Processed Products and Their Effects on Proliferation and Osteogenesis of MC3T3-E1 Cells[J]. Chinese Archives of Traditional Chinese Medicine, 2025 , 43 (8) : 178 -184 . DOI: 10.13193/j.issn.1673-7717.2025.08.034
正文
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淫羊藿（Epimedii Folium）首载于《神农本草经》，列为中品，其味辛甘，性温，归肝、肾经，具有补肾壮阳、强健筋骨、祛风除湿的功效，常用于肾阳虚衰、阳痿遗精、筋骨痿软、风湿痹痛等症的治疗。我国是淫羊藿的地理分布中心，主要有40个淫羊藿品种，目前形成药材商品的主要种类有15种[1]。《中华人民共和国药典》（2020年版）中收载的淫羊藿为小檗科植物淫羊藿Epimedium brevicomu Maxim、箭叶淫羊藿E.sagittatum（Sieb.etZucc.）Maxim、柔毛淫羊藿E.pubescens Maxim或朝鲜淫羊藿E.koreanum Nakai的干燥叶[2]，部分地区使用其地上部分或全草供药用。其中，朝鲜淫羊藿主要分布在我国东北地区，以吉林省南部、辽宁省东部所产朝鲜淫羊藿品质最佳，其核心适生区位于吉林省南部，如通化县、柳河县和抚松县等[3]。
淫羊藿作为传统中药材，含有丰富的多糖类、萜类、生物碱类和黄酮类等化合物，具有抗炎、抗氧化、抗骨质疏松、抗肿瘤、抗凋亡等多种药理活性，对各个系统疾病均有一定的改善作用[4-7]。研究证明淫羊藿可通过多种信号通路活化成骨细胞，并对破骨细胞有直接的抑制作用，其有效成分淫羊藿苷及淫羊藿总黄酮能有效防治骨质疏松[8-9]。其中淫羊藿苷能促进成骨髓间充质干细胞（Bone marrow mesenchymal stem cells，BMSCs）、脂肪干细胞的成骨化及抑制脂化，促进骨生成；且能提高铁代谢水平进一步加强其对破骨细胞活性、分化能力的抑制来改善骨质疏松[10-12]。SUN L J等[13]研究表明，淫羊藿苷可以刺激成骨细胞的增殖，通过增加碱性磷酸酶（Alkaline phosphatase，ALP）活性、钙化结节的数量、I型胶原和骨γ-羧基谷氨酸蛋白（Bone-γ-Carboxyglutamic Acid-Containing protein，BGP）mRNA的表达水平来刺激hFOB1.19成骨细胞的分化。
根据临床需求，许多中药材需要通过炮制改变药性[14-15]。淫羊藿古今记载并应用的炮制方法很多，但目前关于淫羊藿的炮制工艺只有羊脂油炙淫羊藿和盐炙淫羊藿等炮制品收录于《中华人民共和国药典》（2020年版）和地方炮制规范中[2，16-17]。现行版药典规定淫羊藿生品中以朝藿定A、朝藿定B、朝藿定C和淫羊藿苷的总含量作为其综合质控指标，羊脂油炙的炮制品在生品的基础上又增加了宝藿苷I的含量测定。但规定的生品与炮制品的含量下限中，3个初级黄酮苷和淫羊藿苷的总含量要求不一致。说明淫羊藿在炮制后引起了黄酮类成分含量的变化，但其具体的炮制原理目前尚不明确。
因此，本文利用高效液相色谱法测定淫羊藿及其炮制品中黄酮类成分含量，结合液相色谱指纹图谱及多元统计分析方法，探究淫羊藿中黄酮类化合物在炮制前后变化规律，并通过建立地塞米松（Dexamethasone，DEX）对胚胎成骨细胞前体细胞（Mouse Embryonic Preosteoblast Cells，MC3T3-E1 cell）细胞的成骨抑制模型，探究其对MC3T3-E1细胞增殖及成骨能力的影响，对淫羊藿油炙品及盐炙品的炮制原理进行了初步探索。
1　实验部分
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1.1　主要仪器与试剂
LC-2030高效液相色谱仪（日本岛津公司）、十万分之一电子天平（梅特勒托利多公司）、KQ-250B型超声清洗仪（昆山市超声仪器有限公司）、高速万能粉碎机（浙江红景天工贸有限公司）、HY-G20E（F）微电脑电磁炉（无锡市小天鹅华印电器有限公司）、旋转蒸发仪、离心机，ALP、骨保护素（Osteoprotegerin，OPG）和骨钙素（Osteocalcin，OCN）试剂盒（江苏酶免实业有限公司）。
甲醇、乙腈为色谱纯（禹王试剂），乙醇为分析纯（北京化工厂），水为纯水。朝藿定A对照品（批号J12HB184811，纯度≥98%），朝藿定B对照品（批号J12HB184812，纯度≥98%），朝藿定C对照品（批号N18GB166250，纯度≥98%），淫羊藿苷对照品（批号T11A11B111118，纯度≥98%）和宝藿苷I（批号A20GB158231，纯度≥98%）均购于上海源叶生物科技有限公司。
实验室共采集朝鲜淫羊藿药材10批，均采自吉林省通化县，各批次生药材批号及产地见表1。上述药材经长春中医药大学中药鉴定教研室蔡广知副教授鉴定为正品朝鲜淫羊藿（E.koreanum Nakai）药材。
1.2　淫羊藿及其炮制品的制备
取“1.1”项下淫羊藿药材，按照以下方法分别制成淫羊藿生品及淫羊藿羊脂油炙品和淫羊藿盐炙品饮片，每批药材各制得生品及2种炮制品各100 g。
淫羊藿（生品）：除去杂质，摘取叶片，喷淋清水，稍润，切丝，干燥[2]。
羊脂油炙淫羊藿（油炙品）：取羊脂油加热熔化，加入淫羊藿丝，用文火炒至均匀有光泽，取出，放凉。每100 kg淫羊藿，用羊脂油（炼油）20 kg[2]。
盐炙淫羊藿（盐炙品）：取淫羊藿丝，置锅内，用文火炒，边炒边洒食盐水，炒至叶表面绿黄色至黄棕色，取出，晾凉，筛去碎片，即得。每100 kg淫羊藿，用食盐20 kg[16-17]。
淫羊藿及其炮制品代码见表2。
1.3　液相色谱分析条件
1.3.1　供试品溶液及混合对照品溶液的制备
取“1.2”项下淫羊藿及其炮制品粉末（过3号筛）约0.1 g，精密称定，置具塞锥形瓶中，精密加入50%乙醇10 mL，称定重量，超声处理1 h，冷却至室温，再称定重量，用50%乙醇补足减失的重量，摇匀，滤过，取续滤液，即得。
精密称定朝藿定A、朝藿定B、朝藿定C、淫羊藿苷和宝藿苷I对照品适量，加甲醇分别制成每1 mL含0.10 mg的对照品储备液。精密量取以上5个黄酮类化合物对照品储备液适量，加甲醇制成每1 mL含5个对照品各10μg的混合对照品溶液。
1.3.2　色谱条件
色谱柱采用Agilent ZORBAX SB-C18（4.6 mm×250 mm，5μm）色谱柱。流动相为乙腈（A）-水（B）溶液，梯度洗脱：0～29 min，24%A；29～35 min，24→42%A；35～55 min，42%A；55～65 min，42%A。检测波长270 nm，柱温30℃，进样量10μL，流速1 mL/min。
1.3.3　方法学考察及线性关系考察
参考文献［18-19］中高效液相色谱指纹图谱方法学考察实验，考察3个指标：实验仪器的精密度、样品的稳定性及加样回收率，以分离度较好且保留时间居中的淫羊藿苷的色谱峰为参照峰，记录并计算各色谱峰的相对峰面积相对标准偏差（RSD）值。
取“1.3.1”项下制备的混合对照品溶液各0.1、1、3、5、7和10 mL加甲醇定容至10 mL。精密吸取上述溶液5μL，注入高效液相色谱仪测定峰面积，以浓度（C）为横坐标，峰面积（A）为纵坐标绘制曲线。
1.4　成骨细胞增殖实验
1.4.1　细胞培养
自液氮中取出冻存的MC3T3-E1细胞复苏，接种于含10%胎牛血清（FBS）和1%青霉素及链霉素的α-MEM完全培养基，置37℃、5%CO2的培养箱中培养。2 d换液1次，观察细胞状态，待细胞生长、融合至80%～90%时，使用胰酶消化及传代，传3代后，待细胞活力恢复用于后续实验。
1.4.2　供试药液制备
取“1.2”项下淫羊藿及其炮制品粉末（过3号筛）约10 g，精密称定，置具塞锥形瓶中，精密加入50%乙醇200 mL，称定重量，超声处理1 h，冷却至室温，再称定重量，用50%乙醇补足减失的重量，摇匀，滤过。滤液旋蒸至无醇味，冻干，即得。
精密称取不同量的淫羊藿及其炮制品提取物粉末，加入完全培养基配成1、5、10、20、40、60、80、100μg/mL的淫羊藿及其炮制品给药药液。
1.4.3　CCK8检测细胞活力
MC3T3-E1细胞以5×103个/孔的密度种于96孔培养板上，细胞孵育过夜。移去培养基，为检测不同浓度DEX对MC3T3-E1细胞的细胞活性，加入25、50、100、200和400μmol/L的DEX孵育24 h。为检测不同浓度淫羊藿及其炮制品对MC3T3-E1细胞的细胞活性，加入12.5、25、50、100、200μg/mL的淫羊藿及其炮制品孵育24 d，处理结束时，每孔加入10μL CCK8溶液和90μL完全培养基，在37℃、5%CO2培养箱中培养2 h。将波长设置为450 nm，以检测吸光度。
1.4.4　成骨标志物检测
取MC3T3-E1细胞，3000 r/min离心10 min取上清。使用酶标仪，参照ELISA试剂盒说明书，分别测量各个样本的ALP、OPG和OCN的含量。
1.5　数据处理
按“1.3”项下方法，对各供试品溶液进行高效液相色谱分析测试，每个供试品平行测定3次，记录色谱图。选取基线平稳、保留时间稳定的液相色谱图，以.cdf格式文件导入中药指纹图谱相似度评价系统（2012版），以编号为R1的淫羊藿生品谱图作为参照谱图，以淫羊藿苷为基准点进行多点校正，生成指纹图谱并计算相似度数据。高效液相色谱的各原始峰面积数据导入IBM SPSS Statistics 25.0，使用系统聚类对5个黄酮类化合物进行分类，聚类方法使用中位数聚类法，组间距离使用平方欧式距离。
采用SPSS 26.0和GraphPad Prism 8统计学软件进行统计分析，多组间比较采用单因素方差分析，以P<0.05为差异有统计学意义。
2　结果
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2.1　方法学考察及线性关系考察
方法学考察的实验结果均以表示，精密性实验RSD为1.79%±0.055%，稳定性实验RSD为2.07%±0.180%，加样回收实验RSD为2.63% ±0.594%，各方法学考察实验的RSD值均小于5%且波动小，表明本实验条件的仪器精密度高、稳定性好、准确度高，符合液相色谱定性、定量及建立指纹图谱的要求。采用标准曲线法进行定量分析，各待测物的线性方程、相关系数（R）和线性范围如表3所示。混合对照品溶液、淫羊藿及其炮制品的供试品溶液色谱图如图1所示，结果表明本方法对5个黄酮类化合物的专属性良好。
2.2　淫羊藿及其炮制品液相指纹图谱分析
高效液相色谱技术可以较为全面地反映样品中化合物的种类、含量，建立液相色谱指纹图谱有利于对中药的质量进行整体描述与评价，相似度是衡量不同样品之间差异及差异成分的重要指标[20]。指纹图谱相似度数据（编号为R1的淫羊藿生品为参照）如表4所示，各批次淫羊藿及其炮制品指纹图谱相似度较高，各谱图相似度均大于0.9，表明淫羊藿在炮制过程中主要化学成分总体变化趋势基本一致。
淫羊藿及其炮制品的指纹图谱如插页ⅩLⅩ图2所示，共匹配出16个共有峰，其中有5个为已知峰。从其匹配出的峰中看出，随着炮制方法的不同，炮制品的色谱峰相对峰面积呈一定的变化，推测[21]其原因可能是淫羊藿中黄酮类成分在炮制过程中发生相互转化，因此可以利用多元统计分析结果探索这些成分在炮制前后的变化规律。
2.3　聚类分析结果
聚类分析是一种无监督的识别方法，常用于指纹图谱的统计分析[22]。使用系统聚类中R型聚类对本文中所测的5个黄酮类化合物进行分类，结果详见图3。由系统聚类结果可知，本文中的5个黄酮类化合物被分为两类：一类为朝藿定A、朝藿定B、朝藿定C，为初级黄酮苷；另一类为淫羊藿苷和宝藿苷I，为次级黄酮苷。
2.4　含量测定结果及变化趋势
5个黄酮类化合物的含量测定结果见表5和插页ⅩLⅩ图4所示。结果可以直观看出淫羊藿及其炮制品中朝藿定A、朝藿定B和朝藿定C的含量均高于淫羊藿苷和宝藿苷I的含量。
取表5淫羊藿及其炮制品5种黄酮类化合物含量测定结果作极坐标气泡图，见图5。按下式计算气泡半径R：气泡半径R＝某批次炮制品某化合物含量/对应批次生品对应化合物含量。
极坐标气泡图结果显示：朝藿定A、朝藿定B和朝藿定C的气泡半径R<1，表明在炮制后整体呈现含量减少的趋势，可能在炮制过程中转变为其他化学成分；淫羊藿苷和宝藿苷I的气泡半径R>1，表明炮制后呈现含量增加的趋势，可能有与其结构相似的化合物脱去糖基转化为此类成分。
2.5　细胞活力和增殖测定结果
为检测不同浓度DEX对MC3T3-E1细胞的细胞活性，加入25、50、100、200和400μmol/L的DEX孵育24 h（A），结果发现在DEX浓度为100μmol/L时，细胞存活率为70%左右，故选用DEX浓度为100μmol/L作为本实验的造模浓度。为检测不同浓度淫羊藿（B.生品、C.盐炙品、D.油炙品）对MC3T3-E1细胞的细胞毒性，加入12.5、25、50、100、200μg/mL的淫羊藿孵育24 h，结果发现淫羊藿及其炮制品在200μg/mL时对MC3T3-E1的细胞活性均表现出显著的抑制作用，其他浓度均未表现出显著的细胞毒性，故后续使用100μg/mL淫羊藿作为治疗浓度。为检测淫羊藿及其炮制品对DEX造成的成骨抑制的增殖作用，本研究利用100μmol/L DEX和100 μg/mL淫羊藿及其炮制品共同给药后（E），发现各淫羊藿组均具有显著的增殖作用，其中，盐炙淫羊藿组的增殖作用最为显著。见插页LI图6。
2.6　成骨标志物检测结果
ALP在骨形成期间由成骨细胞分泌产生，具有水解磷酸酯的作用，在沉积羟基磷灰石的过程中提供必要的磷酸，同时能够通过水解减轻焦磷酸盐对骨盐形成的抑制作用，其水平是骨形成功能增强或减弱的一个反映，在本研究中，淫羊藿治疗后改善了由DEX降低的细胞上清中ALP的含量（A）。OPG属于可溶性蛋白质，可抑制破骨细胞分化、成熟，能够减少骨量丢失，保护骨组织，与NC组相比，DEX组OPG水平降低明显，骨保护素显著降低，淫羊藿给药后OPG水平明显升高，且差异具有显著性，说明淫羊藿给药后能够调节机体骨代谢，促进OPG的形成及释放，继而促进新骨形成，改善骨质，提升骨密度（B）。除此之外，OCN比ALP对骨形成功能的影响更为敏感，它是一种来自成骨细胞的非胶原蛋白，大多数嵌入在骨基质中，含有3种γ-羧基谷氨酸残基，它们与骨羟基磷灰石基质具有高亲和力，测定OCN既能反映成骨细胞活性，又能反映骨转换的程度，且OCN已被发现是骨质疏松症的重点生物标志物。结果显示，淫羊藿组细胞上清OCN水平明显高于DEX组，且差异具有统计学意义（C）。三者结果均表明淫羊藿能够改善骨基质矿化水平、提高骨转换和增强骨形成功能，且盐炙淫羊藿组对比其他组在ALP和OPG水平上具有更为显著的治疗作用。见插页LI图7。
3　结果与讨论
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本研究利用HPLC测定了淫羊藿及其炮制品中5个黄酮类化合物的含量，方法简便、稳定。采用指纹图谱相似度分析和聚类分析对5个黄酮类化合物含量的差异进行分析，从而对淫羊藿中黄酮类化合物的变化进行研究。5个黄酮类化合物的结构如图8所示，结合聚类分析和含量测定的结果，发现淫羊藿中的初级黄酮苷由于糖苷键不稳定，受热易断裂，经炮制后脱去部分糖基后成为次级黄酮苷，因此初级黄酮苷含量降低，次级黄酮苷含量增加。但是辅料的加入使得各炮制品之间的指纹图谱相似度出现差异：一是淫羊藿油炙品指纹图谱相似度数据稳定性更好；二是含量测定结果显示，次级黄酮苷中脂溶性更强的宝藿苷I在油炙品中比在盐炙品中含量高，淫羊藿苷则相反。造成上述结果的原因可能是：羊脂油属于脂溶性辅料，食盐水属于水溶离子性辅料；炮制过程中得到的宝藿苷I不断地向辅料中聚集，使得化学平衡被打破，正向反应不断进行，因此初级黄酮苷和次级黄酮苷在炮制前后变化规律截然相反。
以上结论提示这两类化合物含量炮制后变化趋势不同可以为寻找淫羊藿炮制改变药性的潜在物质基础奠定基础，如孙娥等[23]从中药的“ADME”角度对炙淫羊藿的炮制机理进行探索，提出了胶束假说，认为淫羊藿在炮制及服用的过程中，淫羊藿中的次级黄酮苷类化合物与胆酸盐、脂肪酸形成热力学和动力学稳定的胶束，从而提高生物利用度。
以往的大量研究结果表明，淫羊藿苷在骨质疏松症的治疗上具有显著的作用，能够提高斑马鱼和去卵巢大鼠的骨密度、OPG水平，降低核因子κB受体活化因子配体（Receptor activator of nuclear factor-κB ligand，RANKL）水平[24]。Ⅰ型胶原在骨基质中含量丰富，胶原蛋白Ⅰ型α2链（collagen type I alpha 2 chain，COL1A2）对骨形成至关重要，淫羊藿苷可以通过提高COL1A2水平、降低骨吸收标志物抗酒石酸酸性磷酸酶5b（tartrate-resistant acidphosphatase 5b，TRAP5b）水平，促进卵巢切除大鼠骨质疏松症后骨折模型的骨形成[25]。淫羊藿苷还可以通过降低RANKL/OPG的比例[26]，抑制肺癌的骨转移，减轻其导致的溶骨性骨破坏。淫羊藿经盐炙后淫羊藿苷含量增加，对DEX引起的MC3T3-E1细胞成骨抑制的增殖作用显著增强，这提示淫羊藿不同炮制品可以产生不同的作用趋势或者临床疗效可能与黄酮类化合物含量的变化有关，也为深入研究淫羊藿炮制原理提供了思路。
基金
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  • 吉林省科技发展计划项目(20210204035YY; 20230204034YY; YDZJ202201ZYTS205)
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2025年第43卷第8期
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doi: 10.13193/j.issn.1673-7717.2025.08.034
  • 首发时间：2026-10-30
  • 出版时间：2025-08-10
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吉林省科技发展计划项目(20210204035YY; 20230204034YY; YDZJ202201ZYTS205)
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    长春中医药大学，吉林 长春 130117

通讯作者:

刘佳（1982-），女，吉林长春人，副教授，硕士研究生导师，博士，研究方向：中药药理学。E-mail：。
许天阳（1979-），男，吉林长春人，副教授，硕士，研究方向：中药分析学。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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