利用Nested_PCR-DNA定序方法鑑定大黃藥材及其製劑成分

Identification of Rhei Radix et Rhizoma and Its Preparations by Nested PCR and DNA Sequencing Methods

大黃藥材歷來使用基原混亂，不乏Rheum同屬植物入藥情形，本研究即在建立大黃藥材及製劑的Nested PCR與DNA定序鑑別方法。本局中藥標本室存有購自大陸原產地且經鑑定及GPS定位之三種大黃標準藥材：掌葉大黃(Rheum palmatum L.)、唐古特大黃(Rheumtanguticum Maxim. ex Balf.)及藥用大黃(Rheum officinale Baill.)，抽取這些標準藥材之DNA，經PCR擴增作為DNA標記的ITS (internal transcribed spacer)，並加以定序後，作為標準序列，用以比對鑑定檢體大黃基原，並依標準序列設計引子，以偵測檢體中之大黃成份。20件大黃製劑檢體中可檢出12件含R. officinale、2件R. tanguticum與4件Rheum palmatum 與2件非R. officinale、R. tanguticum、Rheum palmatum之大黃品種，12件以外觀性狀及組織鏡檢不易鑑別的藥材檢體，以本研究方法鑑定為10件R. officinale及2件R. tanguticum。

Many Rheum species are used as Rhei Radix et Rhizoma. This study applied Nested PCR and DNA sequencing methods to identify which Rheum specie is present in the preparations of Chinese medicine. Three kinds of standard raw materials including Rheum palmatum L., Rheum tanguticum Maxim. ex Balf. and Rheum officinale Baill. were deposited in our specimen room. The standard raw materials were authenticated and purchased from source area orientated by global positioning system. ITS (internal transcribed spacer) of the extracted DNA from standard raw materials was amplified by PCR, and then analyzed by auto DNA sequencer to obtain the standard sequences. One set of primers based on the standard sequences, was thus designed to identify Rhei Radix et Rhizoma components. Sequence data of samples were compared with the standard sequences to confirm the Rheum species in the preparation. The results showed that 12 in 20 samples of preparation contained R. officinale, two contained R. tanguticum and four contained R. palmatum. Two contained Rheum species which are not R. palmatum, R. tanguticum or R. officinale. Twelve raw material samples could not be authenticated with the morphology and microscopy. The proposed method could be applied to identify these samples of raw material as that 10 samples are R. officinale and two are R. tanguticum.