A thin-layer chromatography-bioautographic method for detecting dipeptidyl peptidase IV inhibitors in plants.
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.
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; Shanghai R&D Centre for Standardization of Chinese Medicines, Shanghai 201210, China.
L H
LH
Gu
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; Shanghai R&D Centre for Standardization of Chinese Medicines, Shanghai 201210, China.
L P
LP
Liao
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; Shanghai R&D Centre for Standardization of Chinese Medicines, Shanghai 201210, China.
H J
HJ
Hu
Department of Life Sciences, Faculty of Science and Technology, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK.
S W
SW
Annie Bligh
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; Shanghai R&D Centre for Standardization of Chinese Medicines, Shanghai 201210, China.
C H
CH
Wang
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; Shanghai R&D Centre for Standardization of Chinese Medicines, Shanghai 201210, China. Electronic address: chouguixinzyb@126.com.
G X
GX
Chou
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; Shanghai R&D Centre for Standardization of Chinese Medicines, Shanghai 201210, China. Electronic address: ztwang@shutcm.edu.cn.
Z T
ZT
Wang
D002855:Chromatography, Thin Layer / Q000379:methods; D054873:Dipeptidyl-Peptidase IV Inhibitors / Q000032:analysis*; D006247:Harmine / Q000032:analysis; D000432:Methanol; D032443:Peganum / Q000737:chemistry; D010936:Plant Extracts / Q000737:chemistry*; D010946:Plants, Medicinal / Q000737:chemistry*; D015203:Reproducibility of Results; D012997:Solvents
D054873:Dipeptidyl-Peptidase IV Inhibitors; D010936:Plant Extracts; D012997:Solvents; D006247:Harmine; D000432:Methanol
DPP IV inhibitor; Method validation; Plant; TLC-bioautography