中药鉴定创新方法体系的建立与推广课件

1、中药鉴定创新方法体系的建立与推广中药鉴定创新方法体系的建立与推广中药鉴定技术瓶颈 一万余种中药材物种鉴定 品种混乱传统四大鉴定方法体系的局限 路在何方？基原鉴定形态鉴定显微鉴定理化鉴定中药鉴定技术瓶颈 一万余种中药材物种鉴定 品种混乱DNA条形码 中药鉴定领域的变革与突破经典中药鉴定方法难以满足现代需求鉴定方法缺失导致严重安全性事件 - 关木通误当木通服用，导致急性肾功能衰竭（马兜铃酸肾病事件） -土三七误当三七服用，导致严重肝毒性Hoang et al. Science Translational Medicine, 2013, 5: 197ra102.Nortier J L, Martin

2、ez M C M, Schmeiser H H, et al. New England Journal of Medicine, 2000, 342(23): 1686-1692.关木通百度百科木通中药材天地网DNA条形码 中药鉴定领域的变革与突破经典中药鉴定方法难以DNA barcoding can achieve a rapid and accurate identification for herbsA powerful tool for identifying medicinal plants and traditional Chinese medicinal materialsAbr

3、us cantoniensis Abrus precatorius Medicinal plantsChinese herbal medicinesDNA barcoding technology uses a short and standardized gene region to identify speciesOne sequence One speciesDNA barcoding can achieve a ra课题组首次提出并验证ITS2条形码，引起国内外广泛关注 国际期刊PLoS ONE关注药用植物DNA条形码。该文提出ITS2序列为药用植物通用的条形码序列，为陆地植物DNA条

4、形码序列筛选提出了新的视角，由此引起同行的关注和讨论。被德国专业网站选为近25年来在ITS2领域最受关注的16篇文章之一http:/its2.bioapps.biozentrum.uni-wuerzburg.de/该文发表后3年引文288次课题组首次提出并验证ITS2条形码，引起国内外广泛关注 首次验证 ITS2为药用植物鉴定通用条形码通过研究比较psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS2 和 ITS等七个候选DNA条形码，发现ITS2是最佳条形码序列ITS2长度约230bp，易于扩增和测序，物种鉴定能力强，适合中药材等存在DNA降解材料的鉴定人参、西洋参

5、、羌活、宽叶羌活DNA条形码鉴定方法正在纳入国家药典首次验证 ITS2为药用植物鉴定通用条形码通过研究比较psb Relative distribution of inter-specific divergence between congeneric species and intra-specific variation for four loci. The two bars in each box represent inter-specific (above) and intra-specific (below) genetic distances.Identification of

6、the family RosaceaeW+W-Relative Ranks, n, P valueResultITS2matKW+ = 703, W- = 0, n = 37, P 1.139 10-7ITS2 matKITS2rbcLW+ = 703, W- = 0, n = 37, P 1.137 10-7ITS2 rbcLITS2rpoC1W+ = 703, W- = 0, n = 37, P 1.133 10-7ITS2 rpoC1matKrbcLW+ = 620, W- = 46, n = 36, P 6.479 10-6matK rbcLmatKrpoC1W+ = 655, W-

7、= 11, n = 36, P 4.174 10-7matK rpoC1rbcLrpoC1W+ = 359, W- = 106, n = 30, P 0.009rbcL rpoC1Wilcoxon signed-rank tests of inter-specific divergence among loci.ITS2 and rbcL, matK, rpoC1 were compared.For 54 samples from 43 species in 19 diverse genera, the successful identification rate with ITS2 was

8、100%, followed by matK (94%) and rbcL(65%). For 1410 samples from 893 species in 96 genera No. of genera (2 species): 47 (1351 samples) No. of species ( 2 samples ): 297 (814 samples) Identification rate with ITS2 was 100% and 78% at genus and species level, respectively.X Pang, Shilin Chen*. Cladis

9、tics, 2011, 27 Relative distribution of inteComparative analysis of a large dataset indicates that ITS should be incorporated into the core barcode for seed plantsChina Plant BOL Group, Li Dezhu et al., PNAS, 2011 We therefore propose that ITS/ITS2 should be incorporated into the core barcode for se

10、ed plants. (建议ITS/ITS2应成为种子植物的核心条形码)In cases where it is difficult to amplify and directly sequence ITS in its entirety, just using ITS2 is a useful back-up as it is easier to amplify and sequence this subset of the marker. (ITS难以扩增和测序时，ITS2可以有效地弥补该缺陷)Comparative analysis of a larg创建ITS2为主体条形码的中草药鉴定

11、体系研究一万余份药材样本（涉及数千个基原物种）70余篇相关文章发表于Cladistics、PLoS ONE、PNAS、BMC Evol Biol、 中国科学等杂志出版中药DNA条形码分子鉴定 人民卫生出版社起草中国药典中药材DNA条形码分子鉴定指导原则，已获准纳入国家药典建立中药DNA条形码分子鉴定标准流程创建ITS2为主体条形码的中草药鉴定体系研究一万余份药材样冬虫夏草混伪品及近缘种人造伪品：滇紫菀冬虫夏草及其混伪品/近缘种背景：分布范围狭窄，生态环境脆弱，资源蕴藏量有限（产量每年80-100吨）； 市场产不足销，价格已超黄金 (Guo, 2012) 混伪品泛滥，如古尼虫草，蛹虫草，人造伪品

12、滇紫菀等 样品：冬虫夏草与13种混伪品及近缘种，冬虫夏草采自四川、青海、西藏等主产区 185 条ITS/ITS2序列 (冬虫夏草样品53份，伪品45份，GenBank序列87 条)A：正品：冬虫夏草B：伪品： 亚香棒虫草/古尼虫草C：人造伪品： 滇紫菀D：伪品 凉山虫草E：伪品：蝉花F：伪品：下垂虫草G：伪品： Cordyceps robertsii（新西兰） 结果：基于BLAST/DISTANCE的鉴定效率：100% 基于NJ tree 的鉴定结果：所有的冬虫夏草序列单独聚为一支， 并能与所有的混伪品及近缘种区分开 Xiang L, Chen SL, et al. FEMS microbio

13、logy letters, 2013, 347(2): 156-162.DNA条形码鉴定冬虫夏草及其伪品冬虫夏草混伪品及近缘种人造伪品：滇紫菀冬虫夏草及其混伪品/近中国药典中药材DNA条形码分子鉴定指导原则中国药典中药材DNA条形码分子鉴定指导原则Morphological characteristics of the powder, decoction pieces, seed ,seedling, and fibre among the P.ginseng , P. quinquefolius, P notoginseng and its closely related species a

14、re similar.It is difficult to identify so many different panax species and ginseng produce in medicinal uses by traditional classification methods.Using DNA barcode to identify panax medicinal species 人参类及其混伪品的鉴定Morphological characteristics13 1 5 10 15 20 25 30 35 40 45 50SN01: p.ginseng;SN03: p.qu

15、inquefolius 显示人参、西洋参ITS2序列的SNPs位点信息。Position 32 and 43为区别人参和西洋参的SNPs位点为验证该方法准确性，我们下载了GenBank所有人参、西洋参ITS2序列，表明两个SNPs位点稳定存在样品来源：采集了主产区吉林，辽宁，黑龙江，北京，山东,加拿大及上海和香港药市人参西洋参样品共计72份应用ITS2序列两个SNPs可以快速准确鉴别人参西洋参，所得组合为C-T，则确定为人参，所得组合为T-C，则确定为西洋参ITS2 sequence position 1-50 of p.ginseng and p. quinquefolius 方 法结 果结

16、 论基于ITS2条形码鉴定人参和西洋参Gene, 2013, 530(1): 39-43.A Fast SNP Identification of Medicinal Panax Species Based on DNA Barcoding Analysis, 15 1 5 14金银花药材及其混伪品的ITS2条形码鉴定金银花（忍冬）山银花（红腺忍冬）山银花（黄褐毛忍冬）山银花（灰毡毛忍冬）研究对象：包括24份金银花药材。M: marker1、26: 阴性对照(CK) 2-25: 金银花27-47：混伪品DNA提取：取金银花药材约25 mg，利用植物基因组DNA提取试剂盒(Tiangen Bio

17、tech Co., China)提取总DNA。ITS2序列PCR扩增程序及电泳图94 变性5 min94 变性30 sec58 退火30 sec72 延伸45 sec72 延伸10 min40个循环BLAST鉴定16金银花药材及其混伪品的ITS2条形码鉴定金银花（忍冬）山 Identification of Cortex HerbsSun ZY, Chen SL*. J Nat Med. 2012.The identification efficiency of 51 samples belonging to 19 kinds of cortex herbs in Chinese Pharma

18、copoeia examined was 100% using ITS2 sequence as DNA barcode.Apply the ITS2 barcode to authenticate medicinal materials 皮类药材的条形码鉴定 Identification of Cortex Herb条羌N. franchetii大头羌N. franchetii蚕羌N. incisumPhylogenetic tree constructed with the ITS/ITS2 sequencesadulterantsITSITS231 medicinal materials

19、 samplesStability and Accuracy of the Identification using ITS/ITS2 barcodes (Notopterygii )羌活药材ITS/ITS2条形码鉴定及其稳定性与准确性研究The NJ trees showed that Qianghuo and its adulterants can be easily differentiated according to their monophyly.ITSITS2HaplotypeVariable sitesHaplotypeVariable sitesN. incisum4433N

20、. franchetii61045Xin T Chen S *, et al. Acta Pharm Sin. 2012, 8条羌N. franchetii大头羌N. franchetiUse of the potential DNA barcode ITS2 to identify herbal materials 全草类DNA条形码鉴定 The ITS2 barcode could distinguish successfully all the 34 herbs from their 111 adulterants.Figure 1. The presence/absence of ba

21、rcode gaps between herbs and their adulterants. Pang XH, Chen SL* et al. J Nat Med. 2012Use of the potential DNA barco建立中药材DNA条形码鉴定系统网站建立中药材DNA条形码鉴定系统网站http:/www.tc DNA barcode system shows strong abilities in identifying herb cryptic species BLAST PROCESS打开网址，进入中药材DNA条形码鉴定系统主页点击物种鉴定，选择植物类药材鉴定（ITS2/

22、ITS）进入右图界面。 DNA barcode system shows 在空白处粘贴入需要校对的序列（Query）点击左下角的提交比对序列相似性信息100.0%为人参，说明该序列鉴定结果为人参Quick-and-easy DNA identification of herbs is under way在空白处粘贴入需要校对的序列（Query）点击左下角的提交比华润三九中药材DNA条形码鉴定专用软件华润三九中药材DNA条形码鉴定专用软件2022/9/92022/9/5 七叶皂苷注射剂娑罗子药材的基原植物为七叶树Aesculus chinensis、浙江七叶树Aesculus chinensis

23、 var. chekiangensis和天师栗Aesculus wilsonii娑罗子药材DNA条形码鉴定1. 材料 七叶树属10个物种42份样本采自云南、四川、陕西、湖北、浙江和北京的各个分布区域结 论七叶树属候列扩增效率与测序成功率均为100%，并且物种鉴定效率最高，为95.4%，更适用于七叶树属的鉴定分析。psbA-trnH序列的种间差异最大，在区分物种上具有优势。基于psbA-trnH序列构建的NJ树，可准确鉴别其各个基原物种。七叶树属植物psbA-trnH序列比对变异位点图正品 七叶皂苷注射剂娑罗子药材的基原植物为七叶树Aesc柴胡 Chaihu正品：为伞形科植物柴胡Bupleuru

24、m chinense DC.、狭叶柴胡B. scorzonerifolium Willd.的干燥根。近缘种：同属植物丽江柴胡B. rockii，小叶黑柴胡B. smithii var. parvifolium ，抱茎柴胡B. longicaule var. amplexicaule，细茎有柄柴胡B. petiolulatum var. tenerum 和窄竹叶柴胡B. marginatum var. stenophyllum。（三）柴胡与其近缘种的NJ树图所示：柴胡正品基原柴胡与狭叶柴胡均独立聚为一支，与其混伪品可以区别开。结果表明：基于ITS2序列能很好的区分柴胡及其近缘种（一）柴胡及其及其

25、近缘物种ITS2序列的二级结构：（二）柴胡与其混伪品ITS2序列间存在较多变异位点柴胡不同基原ITS2 条形码consensus 序列柴胡ITS2序列峰图柴胡 Chaihu正品：为伞形科植物柴胡Bupleurum 常用中药材进行了DNA条形码研究课题组已经进行DNA条形码研究的常用中药材包括：人参，西洋参，三七，大黄，石斛，重楼，金银花，丹参，天南星，红豆蔻，肉苁蓉，京大戟，麻黄，高良姜 ，何首乌，吴茱萸，连翘，甘草，玉竹，冬虫夏草，羌活等常用中药材进行了DNA条形码研究课题组已经进行DNA条形码研 Published papers on DNA barcode Cladistics, 2011, 27:BMC Evol. Biol., 2010, 10:324 PLoS ONE, 2010,5: e13102.China Plant BOL Group. PNAS, 2011,108(49) FEMS Microbiology Letters, 2013 PLoS ONE, 2012, 7(5):e35146. Jingyuan Song, Shilin Chen*. PLoS ONE, 201