利用Nested_PCR-DNA定序方法鑑定秦艽藥材及其製劑成分

Identification of Gentianae Macrophyllae Radix and Its Preparations by Nested PCR and DNA Sequencing Method

秦艽Gentianae Macrophyllae Radix為秦艽Gentiana macrophylla Pall.、麻花秦艽Gentiana straminea Maxim.、粗莖秦艽Gentiana crassicaulis Duthie ex Burk. 或小秦艽Gentiana dahurica Fisch.的乾燥根，各種秦艽藥材外觀、鏡檢等物理方法上常不容易區分，在這次的研究中，將應用Nested PCR-DNA定序方法做藥材基原上的鑑定，同時對秦艽製劑中所使用的藥材作鑑別。以本局秦艽標準品internal transcribed spacer (ITS) DNA序列作為比對標準，向中藥廠及藥房價購秦艽藥材及製劑檢體，DNA萃取是以修正後的傳統有機萃取方法，配合純化套組，以取得純化之DNA，再應用Nested PCR擴增藥材成分的ITS片段後定序，比對秦艽標準品及GenBank的序列，以取得鑑定結果。結果顯示20件藥材檢體中有6件外觀、組織鏡檢方法鑑別的結果不同，而11件製劑檢體中，7件使用小秦艽、2件使用粗莖秦艽、2件使用麻花秦艽及1件使用Gentiana triflora。本研究方法可協助外觀、鏡檢方法鑑別秦艽藥材，同時可鑑定秦艽製劑中所使用之藥材基原。

Gentianae Macrophyllae Radix (秦艽) is the dried root of Gentiana macrophylla Pall., G. straminea Maxim., G. crassicaulis Duthie ex Burk. or G. dahurica Fisch. It is difficult to differentiate these raw materials by morphological examination and micrography. In this study, we applied a Nested PCR and DNA sequencing method to discriminate among these raw materials of Gentianae Macrophyllae Radix and to identify these herbs in Chinese medicine preparations. The internal transcribed spacer (ITS) sequences of 18S-26S ribosomal DNA from the authentic Gentianae Macrophyllae Radix were used as standards. Samples were purchased from Chinese medicine pharmaceutical factories and local traditional Chinese herbal pharmacies. Samples of raw materials were previously identified by morphological examination and micrography. The total DNA were extracted by organic solutions and purified using a commercial kit. The ITS sequence data deduced from Nested PCR following by DNA sequencing were compared with standards and GenBank database for further identification of the origin of Gentianae Macrophyllae Radix in samples. The results obtained from morphological examination and micrography were different those from Nested PCR-DNA sequencing method in six samples of raw materials. The origin of Gentianae Macrophyllae Radix were identified as G. dahurica component present in seven samples, G. crassicaulis in two, G. straminea in two and G. triflora in one. Our methods can be applied both for the identification of raw materials of Gentianae Macrophyllae Radix and its presence in Chinese medicine preparations.