A thin-layer chromatography-bioautographic method for detecting dipeptidyl peptidase IV inhibitors in plants

doi:10.1016/j.chroma.2015.07.123

	A thin-layer chromatography-bioautographic method for detecting dipeptidyl peptidase IV inhibitors in plants
	Gu, L H 2,4; Liao, L P 2,4; Hu, H J 2,4; Annie Bligh, S W 1; Wang, C H 2,4; Chou, G X 3,4; Wang, Z T 3,4
刊名	Journal of chromatography. A
	2015-09-11
卷号	1411 页码:116-22
ISSN号	1873-3778
DOI	10.1016/j.chroma.2015.07.123
文献子类	Article
英文摘要	A thin-layer chromatography (TLC)-bioautographic method was developed with the aim to detect dipeptidyl peptidase IV (DPP IV) inhibitors from plant extracts. The basic principle of the method is that the enzyme (DPP IV) hydrolyzes substrate (Gly-Pro-p-nitroaniline) into p-nitroaniline (pNA), which diazotizes with sodium nitrite, and then reacts with N-(1-naphthyl) ethylenediamine dihydrochloride in turn to form a rose-red azo dye which provides a rose-red background on the TLC plates. The DPP IV inhibitors showed white spots on the background as they blocked enzymolysis of the substrate to produce pNA. The method was validated with respect to selectivity, sensitivity, linearity, precision, recovery, and stability after optimizing key parameters including plate type, time and temperature of incubation, concentration of substrate, enzyme and derivatization reagents, and absorption wavelength. The results showed good lineary within amounts over 0.01-0.1μg range for the positive control, diprotin A, with the coefficient of determination (r(2))=0.9668. The limits of detection (LOD) and quantification (LOQ) were 5 and 10ng, respectively. The recoveries ranged from 98.9% to 107.5%. The averages of the intra- and inter-plate reproducibility were in the range of 4.1-9.7% and 7.6-14.7%, respectively. Among the nine methanolic extracts of medicinal herbs screened for DPP IV inhibitors by the newly developed method, Peganum nigellastrum Bunge was found to have one white active spot, which was then isolated and identified as harmine. By spectrophotometric method, harmine hydrochloride was found to have DPP-IV inhibitory activity of 32.4% at 10mM comparing to that of 54.8% at 50μM for diprotin A.
语种	英语
内容类型	期刊论文
源URL	[http://119.78.100.183/handle/2S10ELR8/266479]
专题	中国科学院上海药物研究所
通讯作者	Chou, G X; Wang, Z T
作者单位	1.Department of Life Sciences, Faculty of Science and Technology, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK; 2.Shanghai R&D Centre for Standardization of Chinese Medicines, Shanghai 201210, China; 3.Shanghai R&D Centre for Standardization of Chinese Medicines, Shanghai 201210, China 4.Key Laboratory of Standardization of Chinese Medicines, Ministry of Education, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, China;
推荐引用方式 GB/T 7714	Gu, L H,Liao, L P,Hu, H J,et al. A thin-layer chromatography-bioautographic method for detecting dipeptidyl peptidase IV inhibitors in plants[J]. Journal of chromatography. A,2015,1411:116-22.
APA	Gu, L H.,Liao, L P.,Hu, H J.,Annie Bligh, S W.,Wang, C H.,...&Wang, Z T.(2015).A thin-layer chromatography-bioautographic method for detecting dipeptidyl peptidase IV inhibitors in plants.Journal of chromatography. A,1411,116-22.
MLA	Gu, L H,et al."A thin-layer chromatography-bioautographic method for detecting dipeptidyl peptidase IV inhibitors in plants".Journal of chromatography. A 1411(2015):116-22.