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

應用巢式PCR-DNA定序方法鑑別中藥製劑中的梔子藥材

呂康祖(Kang-Tsu Lu) ； 謝詠筌(Yung-Chuan Hsieh) ； 李蕙君(Hui-Chun Lee) ； 劉宜祝(Yi-Chu Liu) ； 羅吉方(Chi-Fang Lo)

《藥物食品分析》 20卷4期 (2012/12) Pp. 974-977+987

https://doi.org/10.6227/jfda.2012200429

摘要

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。

關鍵字

梔子 ； 巢式PCR ； DNA定序 ； ITS

並列摘要

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.

並列關鍵字

Gardeniae Fructus ； Gardenia jasminoides ； internal transcribed spacer (ITS) ； nested PCR ； DNA sequencing

參考文獻

Payne-James, J. J., De Gara, C. J., Grimble, G. K. and Silk, D. B. A. 1995. Artificial nutrition support in the United Kingdom - 1994: 3rd National Survey. Clin. Nutr. 14: 329-335.

Anderton, A. 1993. Bacterial contamination of enteral feeds and feeding systems. Clin. Nutr. 12: 16.

Scrimshaw, N. S. 1991. Effect of infection on nutrient requirements. J. Parenter Enterol. Nutr. 15: 589.

Gikas, A., Kritsotakis, E. and Maraki, S. et al. 2007. A nosocomial, foodborne outbreak of Salmonella Enterica serovar Enteritidis in a University hospital in Greece: the importance of establishing HACCP systems in hospital catering. J. Hosp. Infect. 66: 194-196.

Beattie, A. and Anderton, A. 1998. Bracterial contamination of enteral feeding systems due to faulty handling procedures - a comparison of a new system with two established systems. J. Hum. Nutr. Diet. 11: 313-321.