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 0.01–0.1 μg range for the positive control, diprotin A, with the coefficient of determination (r2) = 0.9668. The limits of detection (LOD) and quantification (LOQ) were 5 and 10 ng, 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 10 mM comparing to that of 54.8% at 50 μM for diprotin A.

Next generation e-learning platform: virtual laboratory simulations

Bioscience subjects require a significant amount of training in laboratory techniques to produce highly skilled science graduates. Many techniques which are currently used in diagnostic, research and industrial laboratories require expensive equipment for single users; examples of which include next generation sequencing, quantitative PCR, mass spectrometry and other analytical techniques. The cost of the machines, reagents and limited access frequently preclude undergraduate students from using such cutting edge techniques. In addition to cost and availability, the time taken for analytical runs on equipment such as High Performance Liquid Chromatography (HPLC) does not necessarily fit with the limitations of timetabling. Understanding the theory underlying these techniques without the accompanying practical classes can be unexciting for students. One alternative from wet laboratory provision is to use virtual simulations of such practical which enable students to see the machines and interact with them to generate data. The Faculty of Science and Technology at the University of Westminster has provided all second and third year undergraduate students with iPads so that these students all have access to a mobile device to assist with learning. We have purchased licences from Labster to access a range of virtual laboratory simulations. These virtual laboratories are fully equipped and require student responses to multiple answer questions in order to progress through the experiment. In a pilot study to look at the feasibility of the Labster virtual laboratory simulations with the iPad devices; second year Biological Science students (n=36) worked through the Labster HPLC simulation on iPads. The virtual HPLC simulation enabled students to optimise the conditions for the separation of drugs. Answers to Multiple choice questions were necessary to progress through the simulation, these focussed on the underlying principles of the HPLC technique. Following the virtual laboratory simulation students went to a real HPLC in the analytical suite in order to separate of asprin, caffeine and paracetamol. In a survey 100% of students (n=36) in this cohort agreed that the Labster virtual simulation had helped them to understand HPLC. In free text responses one student commented that "The terminology is very clear and I enjoyed using Labster very much”. One member of staff commented that “there was a very good knowledge interaction with the virtual practical”.