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Article(id=1201177208002933665, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1201177206518145841, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2023-0338, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1679241600000, receivedDateStr=2023-03-20, revisedDate=1685116800000, revisedDateStr=2023-05-27, acceptedDate=null, acceptedDateStr=null, onlineDate=1764312563181, onlineDateStr=2025-11-28, pubDate=1704988800000, pubDateStr=2024-01-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1764312563181, onlineIssueDateStr=2025-11-28, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1764312563181, creator=13701087609, updateTime=1764312563181, updator=13701087609, issue=Issue{id=1201177206518145841, tenantId=1146029695717560320, journalId=1189982191388893191, year='2024', volume='59', issue='1', pageStart='1', pageEnd='268', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1764312562826, creator=13701087609, updateTime=1764312760268, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1201178034725417827, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1201177206518145841, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1201178034725417828, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1201177206518145841, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=198, endPage=201, ext={EN=ArticleExt(id=1201177208330089380, articleId=1201177208002933665, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Establishment and validation of bioactivity measurement method for recombinant human midkine, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

To establish and optimize a method for the detection of recombinant human midkine (rhMK) activity and verify its methodology, cell counting kit-8 (cck-8) method was used to measure the proliferation activity of rat knee chondrocytes. The specificity, accuracy, precision, linearity and robustness of the method were also verified in this study. The established method was proven to have good specificity because the buffer of rhMK and recombinant human interleukin-1 receptor antagonist have no obvious active effect; the recoveries of the samples with relative activities of 50%, 75%, 100%, 125%, 150% were in the range of 80.0% to 124.0% by statistical analysis, the relative standard deviations (RSD) of relative potency were all within 20%, the linear correlation coefficient, R2 ≥ 0.98, suggesting that the accuracy, precision and linearity of the method were good; the robustness correlation coefficient, R2 ≥ 0.92 and the ratio of maximum to minimum of sigmoidal dose-response were no less than 1.5, indicating that robustness of the methods was good. In conclusion, a bioactivity measurement method for rhMK was established and fully validated in this study and it provides a reliable method for the bioactivity analysis of rhMK routine samples during the development. This study was approved by the Animal Care and Use Committee of Shanghai Model Organisms Center, Inc. (approval number: 2019-0008-06).

, correspAuthors=Sheng-bin PENG, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2024 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=Ying ZHANG, Ting HE, Ming-feng QIU, Sheng-bin PENG), CN=ArticleExt(id=1201177209164755891, articleId=1201177208002933665, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=重组人源中期因子生物活性测定方法的建立与验证, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

建立和优化重组人源中期因子(recombinant human midkine, rhMK) 活性检测方法, 并对其方法进行验证。本文采用细胞计数试剂盒-8 (cell counting kit-8, cck-8) 法检测rhMK作用大鼠膝软骨细胞的促增殖活性, 并对方法进行专属性、准确性、精密性、线性、耐用性验证。RhMK的缓冲液及重组人源白介素-1受体拮抗剂无明显活性效力, 该方法专属性良好; 相对活性为50%、75%、100%、125%、150%的样品效价回收率(n = 6) 统计在80.0%~124.0%之间, 各相对效价的相对标准偏差均在20%以内, 线性相关系数R2 ≥ 0.98, 表明方法的准确性、精密性、线性均良好; 耐用性相关系数R2 ≥ 0.92, 量效曲线的最大值与最小值的比值≥ 1.5, 方法的耐用性良好。研究建立的rhMK的生物学活性检测方法, 其方法验证良好, 为rhMK开发过程中常规样品的生物学活性分析提供了可靠的检测方法。本研究已获得上海南方模式生物科技股份有限公司动物看护与使用委员会批准(批准号: 2019-0008-06)。

, correspAuthors=彭生斌, authorNote=null, correspAuthorsNote=
*彭生斌, Tel: 86-21-53308801, E-mail:
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Beijing: China Medical Science Press, 2020: 530-535., articleTitle=null, refAbstract=null), Reference(id=1201177215439434055, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208002933665, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[15], rfOrder=14, authorNames=null, journalName=null, refType=null, unstructuredReference=Liu LZ, Wang C, Shao H. Application of reporter gene assay in bioactivity detection of biotechnological drugs [J]. 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A: Proliferation rate; B: Dose-response curves. rhMK: Recombinant human midkine , figureFileSmall=64gvzAbw/gPm34ewVOe4hg==, figureFileBig=q7U/GYXR+WxcZ8njoGMyfA==, tableContent=null), ArticleFig(id=1201177212893491297, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208002933665, language=EN, label=null, caption=null, figureFileSmall=97LibkojL2R3IFccOkZKzw==, figureFileBig=j2t5bArznPsJlpg4m18CXg==, tableContent=null), ArticleFig(id=1201177213040291948, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208002933665, language=CN, label=Figure 2, caption= Specificity validation result. RhIL-1Ra: Recombinant human interleukin-1 receptor antagonist , figureFileSmall=97LibkojL2R3IFccOkZKzw==, figureFileBig=j2t5bArznPsJlpg4m18CXg==, tableContent=null), ArticleFig(id=1201177213170315384, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208002933665, language=EN, label=null, caption=null, figureFileSmall=7JZCaMnEcImQh9pH21ICsg==, figureFileBig=8zib3+Gnfmt2SbiUJ81lBw==, tableContent=null), ArticleFig(id=1201177213333893255, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208002933665, language=CN, label=Figure 3, caption= Robustness validation result , figureFileSmall=7JZCaMnEcImQh9pH21ICsg==, figureFileBig=8zib3+Gnfmt2SbiUJ81lBw==, tableContent=null), ArticleFig(id=1201177213468110997, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208002933665, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Target potency/%Relative potency/%Recovery rate/%RSD/%
A1A2A1A2A1A2
50414582.090.02.46.2
424484.088.0
404080.080.0
75666388.084.011.92.7
8066106.788.0
656386.784.0
100102103102.0103.012.37.6
114119114.0119.0
8910789.0107.0
125127135101.6108.06.97.6
126128100.8102.4
11211689.692.8
150186141124.094.06.95.6
165154110.0102.7
166157110.7104.7
), ArticleFig(id=1201177213593940125, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208002933665, language=CN, label=Table 1, caption=

Accuracy validation results. A1: Analyst 1; A2: Analyst 2

, figureFileSmall=null, figureFileBig=null, tableContent=
Target potency/%Relative potency/%Recovery rate/%RSD/%
A1A2A1A2A1A2
50414582.090.02.46.2
424484.088.0
404080.080.0
75666388.084.011.92.7
8066106.788.0
656386.784.0
100102103102.0103.012.37.6
114119114.0119.0
8910789.0107.0
125127135101.6108.06.97.6
126128100.8102.4
11211689.692.8
150186141124.094.06.95.6
165154110.0102.7
166157110.7104.7
), ArticleFig(id=1201177213707186340, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208002933665, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Assay123456RSD (n = 6)
Relative potency/%A1988410810612110811.8%
A2101122123868110117.1%
), ArticleFig(id=1201177213795266730, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208002933665, language=CN, label=Table 2, caption=

Repeatability validation results

, figureFileSmall=null, figureFileBig=null, tableContent=
Assay123456RSD (n = 6)
Relative potency/%A1988410810612110811.8%
A2101122123868110117.1%
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重组人源中期因子生物活性测定方法的建立与验证
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张影 1, 2 , 贺婷 2 , 邱明丰 1 , 彭生斌 2, *
药学学报 | 研究论文 2024,59(1): 198-201
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药学学报 | 研究论文 2024, 59(1): 198-201
重组人源中期因子生物活性测定方法的建立与验证
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张影1, 2, 贺婷2, 邱明丰1, 彭生斌2, *
作者信息
  • 1.上海交通大学药学院, 上海 200240
  • 2.祐森健恒生物医药 (上海) 有限公司, 上海 201203

通讯作者:

*彭生斌, Tel: 86-21-53308801, E-mail:
Establishment and validation of bioactivity measurement method for recombinant human midkine
Ying ZHANG1, 2, Ting HE2, Ming-feng QIU1, Sheng-bin PENG2, *
Affiliations
  • 1. School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China
  • 2. Usynova Pharmaceuticals Ltd., Shanghai 201203, China
出版时间: 2024-01-12 doi: 10.16438/j.0513-4870.2023-0338
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摘要
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建立和优化重组人源中期因子(recombinant human midkine, rhMK) 活性检测方法, 并对其方法进行验证。本文采用细胞计数试剂盒-8 (cell counting kit-8, cck-8) 法检测rhMK作用大鼠膝软骨细胞的促增殖活性, 并对方法进行专属性、准确性、精密性、线性、耐用性验证。RhMK的缓冲液及重组人源白介素-1受体拮抗剂无明显活性效力, 该方法专属性良好; 相对活性为50%、75%、100%、125%、150%的样品效价回收率(n = 6) 统计在80.0%~124.0%之间, 各相对效价的相对标准偏差均在20%以内, 线性相关系数R2 ≥ 0.98, 表明方法的准确性、精密性、线性均良好; 耐用性相关系数R2 ≥ 0.92, 量效曲线的最大值与最小值的比值≥ 1.5, 方法的耐用性良好。研究建立的rhMK的生物学活性检测方法, 其方法验证良好, 为rhMK开发过程中常规样品的生物学活性分析提供了可靠的检测方法。本研究已获得上海南方模式生物科技股份有限公司动物看护与使用委员会批准(批准号: 2019-0008-06)。

重组人源中期因子  /  cck-8法  /  生物学活性  /  增殖  /  方法验证

To establish and optimize a method for the detection of recombinant human midkine (rhMK) activity and verify its methodology, cell counting kit-8 (cck-8) method was used to measure the proliferation activity of rat knee chondrocytes. The specificity, accuracy, precision, linearity and robustness of the method were also verified in this study. The established method was proven to have good specificity because the buffer of rhMK and recombinant human interleukin-1 receptor antagonist have no obvious active effect; the recoveries of the samples with relative activities of 50%, 75%, 100%, 125%, 150% were in the range of 80.0% to 124.0% by statistical analysis, the relative standard deviations (RSD) of relative potency were all within 20%, the linear correlation coefficient, R2 ≥ 0.98, suggesting that the accuracy, precision and linearity of the method were good; the robustness correlation coefficient, R2 ≥ 0.92 and the ratio of maximum to minimum of sigmoidal dose-response were no less than 1.5, indicating that robustness of the methods was good. In conclusion, a bioactivity measurement method for rhMK was established and fully validated in this study and it provides a reliable method for the bioactivity analysis of rhMK routine samples during the development. This study was approved by the Animal Care and Use Committee of Shanghai Model Organisms Center, Inc. (approval number: 2019-0008-06).

recombinant human midkine  /  cck-8  /  bioactivity  /  proliferation  /  method validation
张影, 贺婷, 邱明丰, 彭生斌. 重组人源中期因子生物活性测定方法的建立与验证. 药学学报, 2024 , 59 (1) : 198 -201 . DOI: 10.16438/j.0513-4870.2023-0338
Ying ZHANG, Ting HE, Ming-feng QIU, Sheng-bin PENG. Establishment and validation of bioactivity measurement method for recombinant human midkine[J]. Acta Pharmaceutica Sinica, 2024 , 59 (1) : 198 -201 . DOI: 10.16438/j.0513-4870.2023-0338
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骨关节炎(osteoarthritis, OA) 是一种常见的软骨退行性疾病, 其根源在于软骨损伤自修复很弱[1, 2]。传统药物治疗以非甾体类抗炎药为主, 只能缓解症状, 不能根治[3]。目前在全球市场中, 还没有能够抑制OA疾病进展的药物, 存在较多尚未满足的临床需求[4-6]。
中期因子是肝素结合生长因子, 是多营养蛋白/中期因子家族的成员之一, 其促进胚层重塑, 脂肪细胞存活及软骨形成发挥重要作用, 在炎症诱导下其表达量增加[7, 8]。重组人源中期因子(recombinant human midkine, rhMK) 通过软骨细胞表面低密度脂蛋白受体相关蛋白-1 (low density lipoprotein-related protein-1, LRP-1) 进入细胞, 与核仁蛋白结合, 激活细胞外信号调节激酶信号通路, 促进软骨细胞增殖[3]。研究表明, rhMK可能作为潜在的治疗OA的药物。
为检测rhMK对软骨的修复作用, 通常将rhMK对软骨细胞的促增殖能力作为检测指标[9, 10]。以往的研究中, 常采用较为复杂的饥饿刺激法检测, 需要在细胞培养的过程中去除血清, 对细胞的生长影响较大, 难以形成稳定的检测方法, 不利于药物开发过程对样品的生物活性的评价。建立良好的rhMK临床前生物活性检测方法, 推动抑制OA疾病治疗药物的早期开发和应用显得尤为重要和迫切。基于之前的研究, 经过系列的试验, 开发了一套稳定的检测方法, 并进行了方法验证, 有效地解决了临床前rhMK样品的检测。
本实验依据不同剂量的rhMK促进单层大鼠膝软骨细胞增殖数量的不同, 利用活细胞内脱氢酶可将试剂盒中水溶性四唑盐还原为可溶性橙色甲瓒物质的原理[11], 在450 nm波长处可检测出不同的吸光度, 其与活细胞数及活力呈正比, 得到rhMK促进细胞增殖的S型量效曲线, 建立活性检测方法并进行方法验证。
材料与方法
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实验动物  正常雄性SD大鼠(SPF级), 体质量为250~300 g, 购自浙江维通利华实验动物技术有限公司[生产许可证号: SCXK (浙) 2019-0001], 饲养于上海南方模式生物科技股份有限公司, 使用许可证号: SCXK (沪) 20170012。饲养条件: 温度22~24 ℃, 湿度45%~65%。本研究已获得上海南方模式生物科技股份有限公司动物看护与使用委员会批准(批准号: 2019-0008-06)。
药物和试剂  RhMK原液样品(由毕赤酵母发酵生产的分子质量为13.2 kDa的重组蛋白, 批号P20190301)、rhMK缓冲液(成分为20 mmol·L-1柠檬酸缓冲液, pH为5.5, 批号P20190301-B)、重组人源白介素-1受体拮抗剂(recombinant human interleukin-1 receptor antagonist, rhIL-1Ra) 成品(批号20180706)、软骨细胞取自4~6周龄SD大鼠膝关节软骨组织(批号C20190603、C20190625、C20190720、C20190815) 均由祐森健恒生物医药(上海) 有限公司提供; Ⅱ型胶原酶(批号2195526)、培养基(Dulbecco's modified eagle medium, DMEM, 批号8119233)、胎牛血清(fetal bovine serum, FBS, 批号1907082) 及0.25%乙二胺四乙酸-胰蛋白酶(批号2053591) 均购买自美国Gibco公司; 细胞计数试剂盒-8 (cell counting kit-8, cck-8, 批号SC760) 购买自日本同仁化工有限公司。
仪器  多功能酶标仪SynergyTM H1购买自美国BioTek公司。
方法建立  将大鼠软骨组织用0.2%的Ⅱ型胶原酶溶液在37 ℃、5% CO2培养箱中消化4 h, 用200目过滤器过滤收集软骨细胞, 用10%FBS-DMEM培养[12]。0.25%乙二胺四乙酸-胰蛋白酶消化后离心, 10% FBS-DMEM重悬细胞, 调整细胞密度为4×104 cells·mL-1, 接种于96孔板, 每孔50 μL; 用10% FBS-DMEM培养基将rhMK稀释至200 μg·mL-1, 再2倍稀释至100 μg·mL-1, 后3倍稀释, 共9个浓度, 每个浓度3复孔, 每孔50 μL, 37 ℃、5% CO2共孵育120 h; 移除细胞上清液, 加入新鲜cck-8, 每孔100 μL, 静置2 h; 使用多功能酶标仪测定OD450值, 采用GraphPad Prism v 9.2拟合四参数进行数据分析。
方法验证  首先, 根据活性法测定rhMK、rhMK缓冲液及rhIL-1Ra的活性, 评价方法的专属性; 其次, 将待测样品分别稀释至50、75、100、125、150 μg·mL-1, 另取稀释至100 μg·mL-1的样品作为参考品, 方法验证采用终浓度50 μg·mL-1起, 3倍稀释, 共9个浓度。其余均采用上述活性法对5个待测样品进行不同水平的测定, 由两名分析人员分别进行3次测定, 共3天, 以评估50%、75%、100%、125%、150%效价水平样品的准确性、精密性和线性; 精密性中的重复性验证采用每名分析人员对100%效价水平样品同一次实验内分别测定6次。计算公式为: 试验样品的相对增殖活性(%) = 100%参考品EC50 / 试验样品EC50×100%。
结果
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1 方法建立及优化
RhMK促大鼠软骨细胞48、72、120及144 h最大增殖率分别为68.8%、111.5%、141%及53.3%, 如图 1A所示; 作用120 h时, 效应窗口最宽, 如图 1B所示, 剂量效应曲线趋于最优。
2 方法验证
2.1 专属性
RhMK样品缓冲液及rhIL-1Ra对大鼠软骨细胞无明显促增殖效应, 而rhMK对大鼠软骨细胞有良好的量效关系, 方法专属性良好, 如图 2所示。
2.2 准确性
两名分析员分别对50%、75%、100%、125%、150%效价样品各重复3次, 回收率(n = 6) 分别为80.0%~90.0%、84.0%~106.7%、89.0%~119.0%、89.6%~108.0%、94.0%~124.0%, 回收率范围为±30%之内, 相应的相对标准偏差(relative standard deviations, RSD) (n = 3) 分别为2.4%、11.9%、12.3%、6.9%、6.9% (分析员1), 6.2%、2.7%、7.6%、7.6%、5.6% (分析员2), RSD值均小于20%, 方法准确性良好, 如表 1所示。
2.3 精密性
2.3.1 重复性
两名分析员对100%效价样品进行6次检测的结果显示, RSD (n = 6) 分别为11.8%、17.1%, RSD值均小于20%, 方法重复性良好, 如表 2所示。
2.3.2 中间精密性
两名分析员在不同的时间对50%、75%、100%、125%、150%效价样品的3次重复试验, 相应的RSD (n = 3) 与准确性中RSD相同, RSD (n = 6) 分别为5.0%、9.6%、9.9%、6.8%、9.3%, RSD值均小于20%, 方法的中间精密性良好。
2.4 线性
5个效价样品的相对活性和理论活性的线性回归方程为y = 1.256x-24.2 (R2 = 0.980 5), 斜率在0.8~1.3之间, R2 ≥ 0.98, 在50%~150%效价内线性良好。
2.5 耐用性
3个不同批次的原代大鼠膝软骨细胞的耐用性结果显示(图 3), OD450的最大值/OD450最小值的比值分别为2.54、2.42和2.21倍, 均不小于1.5, R2分别为0.990、0.998和0.991, 均不小于0.92, 耐用性良好。
讨论
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rhMK作为新药, 在没有标准品对比的情况下, 需建立纯度、理化和生物学活性良好表征的过渡期参考品, 满足开发需求[13]。
本文采用非饥饿法测定大鼠膝软骨细胞活性, 整个流程未改变细胞培养条件, 步骤简单, 稳定性好, 可较好地反映药物的生物学活性。50 μg·mL-1 rhMK作用不同传代次数的大鼠膝软骨细胞, 如原代(passage 0, P0)、P1、P2、P3及P4, 其促增殖率分别为122.7%、107.7%、63.0%、67.4%及52.5%, 结果表明, 原代软骨细胞最佳, 是此方法验证的关键因素; rhMK经70 ℃加热处理48 h, 其活性下降至55.5%, 说明此方法能反映出热破坏样品的活性; 不同批次的软骨细胞存在一定的差异, 需使用4~6周龄的SD大鼠提取的软骨细胞, 若大鼠太老, 软骨细胞提取困难, 进而影响实验。综合以上结果, 本研究完成了方法开发与验证。
此外, 考虑到使用大鼠的原代细胞的不灵活性和周期成本, 本文研究了rhMK作用猿猴空泡病毒40永生化人正常软骨细胞C28/I2的增殖活性, 发现在10%血清存在时, 与对照组相比, rhMK促C28/I2细胞增殖效应不明显, 但在微量血清存在的情况下, 随着rhMK剂量的增加, 有明显的剂量依赖性促增殖效应。因此, 可根据rhMK对不同细胞的作用情况, 进行其方法验证。
随着技术的不断更新, 以及国家对生物制品的生物活性检测方法验证的新的要求, 需要进一步开发并完善更为稳定的活性测定方法, 应依照2020版《中华人民共和国药典》四部通则9401[14]指导原则完成。
值得一提的是, 近年来, 在作用机制明确的生物类药物活性检测中, 报告基因法因其具有较高的准确性以及较小的变异性, 被广泛地应用于细胞活性检测[15, 16]。因此, 可以利用包含荧光素酶的质粒以及包含过表达LRP-1基因与核仁蛋白基因的慢病毒, 分别转染永生化大鼠软骨细胞或永生化人软骨细胞C28/I2, 在细胞的表型(如细胞增殖, 凋亡等) 发生之前, 与荧光素底物反应, 缩短实验周期, 并可以提高方法的稳定性, 此方法有待开发。而本文阐述的方法验证确实保证了目前药物开发的进展。
作者贡献: 张影负责实验思路的设计、实验主体部分数据的完成及分析, 以及文章撰写; 贺婷负责实验的主体部分数据的完成及分析; 实验思路的设计和论文的润色由邱明丰及彭生斌导师负责指导; 彭生斌负责文章的项目支持。
利益冲突: 所有作者声明不存在利益冲突, 本研究内容无任何利益冲突。
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参考文献 引证文献
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doi: 10.16438/j.0513-4870.2023-0338
  • 接收时间：2023-03-20
  • 首发时间：2025-11-28
  • 出版时间：2024-01-12
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  • 收稿日期：2023-03-20
  • 修回日期：2023-05-27
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    1.上海交通大学药学院, 上海 200240
    2.祐森健恒生物医药 (上海) 有限公司, 上海 201203

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*彭生斌, Tel: 86-21-53308801, E-mail:
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鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
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