- 學位論文
台灣金線連真偽藥材之分子鑑定系統探討
Molecular assay System for Authentication Study for Anoectochilusformosanus Hayata
摘要
台灣金線連為台灣原生之珍貴中藥， 此藥材基原為蘭科 （Orchidaceae） 開唇蘭屬（Anoectochilus Bl.）之台灣金線連（A. formosanus Hayata）。目前台灣市售的台灣金線連疑被高雄金線連（A. Koshunensis Hayata）、越南金線連（A. spp.）、美國金線連（Ludisia discolor (Ker-Gawl.) A. Rich）或銀線連（Goodyera matsumurana Schltr.） 替代，由於此5 種植物的外觀形態相似，不易區別，因此本研究利用 5 種分生技術，建立台灣金線連基原之分子鑑定系統。經由ITS1-5.8S rRNA-ITS2 序列分析顯示，台灣金線連與偽品之相似度介於90-99% 之間，利用此等序列之差異處，建立專一性之multiplex PCR 及生物 晶片之檢測方法；同時利用5 種基原ITS1-5.8S rRNA-ITS2 序列，於 ApaL I、BssS I、BstB I 及Ssp I 限制酵素切位之差異，建立PCR-RFLP 之檢測方法。利用RAPD 及ISSR 分子標誌，分別篩選之OPC-R2、 OPC-R4、810 及836 等引子，亦可明顯鑑別台灣金線連、高雄金線連、 越南金線連、美國金線連及銀線連等5 種基原。此台灣金線連基原之 分子鑑定技術，不僅可落實於台灣重要原生植物之保護，亦可配合中 草藥於優良栽培制度(GAP)以及生產履歷制度的執行，提供正確而優 質的藥材基原，協助中藥材品質控管機制之建立。
關鍵字
分子鑑定系統並列摘要
Taiwan jewel orchid (Anoectochilus formosanus Hayata) is an indigenous Orchidaceae found in Taiwan. Extract of the whole plant had been applied for liver protection, anti-inflammation, pain relief, blood sugar reduction and blood pressure reduction. Taiwan jewel orchid had been confused and possibly be replaced by Ludisia discolor, A. koshunensis or Goodyera matsumurana, and Indonesian species in Taiwanese herbal markets. Because the whole plant morphology and outlooks of the mentioned herbs are not easily distinguished. In this study, molecular identification system for authentication of Taiwan jewel orchid had been established using 5.8S rDNA-ITS sequential difference analysis with PCR-RFLP based analysis, multiplex PCR techniques and bio-chip, ISSR (inter simple sequence repeats) and RAPD (Random amplified polymorphic DNA) molecular targeting. The results from 5.8S rDNA-ITS sequential difference analysis showed that the similarity between Taiwan jewel orchid and the “flaws” were between 90-99 %. However, Taiwan jewel orchid and the flaw species could also be distinguished with PCR-RFLP analysis by ApaL I、BssS I、BstB I and Ssp I restriction enzymes. By using specific designed primers and probes and comparing the differences of 5.8S rDNA-ITS between Taiwan jewel orchid and the flaws species with multiplex PCR and bio-chip. The result of ISSR primers and RAPD, 2 of them showed polymorphous bands patterns that were useful for distinguishing Taiwan jewel orchid from other “flaw” species after PCR amplification. This result indicated that the methods are more rapid, accurate and applicable in identification of Taiwan jewel orchid at the molecular level.