Effect of Perilla frutescens Extracts on Porcine Jejunal Epithelial Cells.

Green-leaved Perilla frutescens extracts were investigated on their effect on cell proliferation of the porcine jejunal epithelial cell line, IPEC-J2, as well as on the gene expression of cell cycle or cancer-related genes. Some extracted compounds were, however, susceptible to degradation in cell culture medium, whereas others were found to be stable during the entire experimental time. Control experiments also included the assessment of H2 O2 generation in cell culture medium caused by oxidation of natural extract compounds, which was proved to be absent at low extract concentrations. A fast and significant inhibition of cell growth at low physiological extract concentrations could be observed. This finding, along with an immediate downregulation of 67 kDa laminin receptor and cyclin D1 expression, can be accounted to the presence of Perilla frutescens extract. Copyright © 2016 John Wiley & Sons, Ltd.

HPLC method development for the online-coupling of chromatographic Perilla frutescens extract separation with xanthine oxidase enzymatic assay.

Enzyme-regulatory effects of compounds contained in complex mixtures can be unveiled by coupling a continuous-flow enzyme assay to a chromatographic separation. A temperature-elevated separation was developed and the performance was tested using Perilla frutescens plant extracts of various polarity (water, methanol, ethanol/water). Owning to the need of maintaining sufficient enzymatic activity, only low organic solvent concentrations can be added to the mobile phase. Hence, to broaden the spectrum of eluting compounds, two different organic solvents and various contents were tested. The chromatographic performance and elution was further improved by the application of a moderate temperature gradient to the column. By taking the effect of eluent composition as well as calculated logD values and molecular structure of known extract compounds into account, unknown features were tentatively assigned. The method used allowed the successful observation of an enzymatic inhibition caused by P. frutescens extract.

Enzymatic Assays Coupled with Mass Spectrometry with or without Embedded Liquid Chromatography.

This article reviews monitoring strategies for enzymatic assays coupled with mass spectrometric detection. This coupling has already been shown to be helpful in providing versatile and detailed knowledge about enzyme kinetics. Various available publications address two general approaches. 1) The continuous-flow setup allows real-time determination of substrate degradation. Simultaneously, resulting product or potential intermediates can be detected. 2) The online coupled continuous-flow mixing assay allows the direct coupling of an enzymatic assay to chromatographic separation of complex mixtures. The latest efforts in improving the methodology have been made with regard to miniaturization. This is especially advantageous with regard to reducing costly consumption of chemicals. Finally, these developments are applicable for diverse bioanalytical purposes in the realms of pharmaceutical, biotechnological, food, and environmental research.

Utilization of real-time electrospray ionization mass spectrometry to gain further insight into the course of nucleotide degradation by intestinal alkaline phosphatase.

RATIONALE: Related with its ability to degrade nucleotides, intestinal alkaline phosphatase (iAP) is an important participant in intestinal pH regulation and inflammatory processes. However, its activity has been investigated mainly by using artificial non-nucleotide substrates to enable the utilization of conventional colorimetric methods. To capture the degradation of the physiological nucleotide substrate of the enzyme along with arising intermediates and the final product, the enzymatic assay was adapted to mass spectrometric detection. Therewith, the drawbacks associated with colorimetric methods could be overcome. METHODS: Enzymatic activity was comparatively investigated with a conventional colorimetric malachite green method and a single quadrupole mass spectrometer with an electrospray ionization source using the physiological nucleotide substrates ATP, ADP or AMP and three different pH-values in either methodological approach. By this means the enzymatic activity was assessed on the one hand by detecting the phosphate release spectrometrically at defined time points of enzymatic reaction or on the other by continuous monitoring with mass spectrometric detection. RESULTS: Adaption of the enzymatic assay to mass spectrometric detection disclosed the entire course of all reaction components--substrate, intermediates and product--resulting from the degradation of substrate, thereby pointing out a stepwise removal of phosphate groups. By calculating enzymatic substrate conversion rates a distinctively slower degradation of AMP compared to ADP or ATP was revealed together with the finding of a substrate competition between ATP and ADP at alkaline pH. CONCLUSIONS: The comparison of colorimetric and mass spectrometric methods to elucidate enzyme kinetics and specificity clearly underlines the advantages of mass spectrometric detection for the investigation of complex multi-component enzymatic assays. The entire course of enzymatic substrate degradation was revealed with different nucleotide substrates, thus allowing a specific monitoring of intestinal alkaline phosphatase activity.