應用巢式 PCR-DNA 定序方法鑑別中藥製劑中的梔子藥材   全文下載

Authentication of Gardeniae Fructus in Chinese Medicine Preparations by Nested PCR and DNA Sequencing Methods

中藥製劑依方劑種類的不同，由多種的中藥材製成，因所含藥材種類複雜，不易鑑定製劑中藥材成分，本篇研究中將應用Nested PCR與DNA定序方法，建立中藥製劑中的梔子藥材的鑑別方法。梔子藥材的基原是Gardenia jasminoides Ellis.植物的乾燥成熟果實，其與Gardenia jasminoides var. grandiflora (Lour.) Nakai的乾燥成熟果實外觀相似，而常有誤用情形。收集28件梔子藥材及22件梔子製劑檢體，進行DNA的抽取與純化，利用Nested PCR擴增ITS（internal transcribed spacer）片段，並以自動定序儀分析DNA序列。所有檢體的DNA序列，除了位置153的鹽基外都一致，3件依外觀鑑定為G. jasminoides var. grandiflora的藥材檢體，在153的位置鹽基皆為G，而G. jasminoides.則為G或A。所有製劑檢體均能夠用研究中所建立之Nested PCR與DNA定序方法鑑定其中所含之梔子的藥材成分。

Chinese medicinal preparations are usually composed of various raw material components, and the complexity of herbal ingredients therein complicates the identification of constituent species. In this study, nested PCR coupled with DNA sequencing methods were established for Gardeniae Fructus in Chinese medicinal preparation. The botanical origin of Gardeniae Fructus is the dried mature fruits of Gardenia jasminoides Ells. The morphology of the dried mature fruits of G. jasminoides var. grandiflora (Lour.) Nakai is similar to that of G. jasminoides, which results in misuse. In this study, 28 raw materials and 22 preparation samples were collected. The total DNA of all samples were extracted by CTAB method and purified. Authentication was executed based on the sequence of internal transcribed spacer (ITS) which was amplified by nested PCR followed by sequencing. The ITS sequences of all samples were the same, with the exception of position 153. The nucleotide base of 3 raw material samples, identified as G. jasminoides var. grandiflora by morhpology, were base “G”. The raw material samples, identified as G. jasminoides by morphology, had the base “G” or “A” on this position. Gardeniae Fructus could be identified in all preparation samples by nested PCR and DNA sequencing.