At‐line LC‐QTOF‐MS micro‐fractionation of Derris scandens (Roxb.) Benth, coupled to radioassay for the early identification of PDE5A1 inhibitors

Abstract

Introduction Chromatographic techniques coupled with bioassays are popularly used for the detection of bioactive compounds in natural products. In this study phytochemicals responsible for showing Phosphodiesterase type 5 (PDE5) inhibitory activity in Derris scandens were studied using at‐line method. Objective The objective of this study was to develop an at‐line liquid chromatography quadrupole time‐of‐flight mass spectrometry (LC‐QTOF‐MS) micro‐fractionation method for rapid separation and identification of PDE5A1 inhibitors in 95% ethanolic extract of D. scandens. Methodology Initially, the correlation between LC‐MS and PDE5A1 inhibitory activity was studied using three concentrations of 1:1 mixture of sildenafil and derrisisoflavone A; PDE5A1 inhibitors. The mixture was separated by high‐performance liquid chromatography (HPLC) column and the eluent was split into two flows in the ratio of 1:9. The major part was collected in a 96‐well plate, in each well consecutively every 30 s. The minor part was fed into an electrospray ionisation (ESI)‐QTOF‐MS system. After subsequent solvent removal, the collected micro‐fractions were subjected to radioassay to determine PDE5A1 inhibition. Results The result showed, PDE5A1 inhibitory activities of the micro‐fractions were observed in a dose response manner and found to be in agreement with an off‐line study. Similarly, 95% ethanolic extract of D. scandens was subjected to the at‐line LC‐QTOF‐MS micro‐fractionation developed, resulting in separation and tentative identification of 25 compounds with PDE5A1 inhibitory activity. Most of the compounds contained prenylated isoflavone skeleton. Additionally, the active micro‐fractions also showed selectivity on PDE5A1 over PDE6 and PDE1B. Conclusion Our results demonstrated that the at‐line coupled LC‐QTOF‐MS micro‐fractionation with PDE5A1 inhibitory assay is a valuable tool for identifying PDE5A1 inhibitors from complex extracts.

Phosphodiesterase‐5 Inhibitory Activity of Canthin‐6‐One Alkaloids and the Roots ofEurycoma longifoliaandEurycoma harmandiana

“…Crude PDE‐5 was obtained from a previous study using the cell transfection method [24] . Crude snake venom ( Crotalus atrox ), cGMP sodium salt, and ethylene glycol‐bis (2‐aminoethylether)‐N,N,N,N‐tetraacetic acid (EGTA) were purchased from Sigma‐Aldrich.…”

“…The PED‐5 assay procedure was modified from a previous study [24] . Briefly, the samples for preliminary screening were prepared by dissolving in 5 % DMSO to obtain a final concentration of 10 μM (reference compounds) and 50 μg crude extract/mL (plant extracts).…”

Identification of Antioxidants and Acetylcholinesterase Inhibitors from Hapaline benthamiana Schott. Using at Line LC-ESI-QTOF MS/MS

“…Liquid chromatography (LC) coupled with mass spectrometry (LC-MS) is one of the techniques widely used in natural product research to identify compounds in complex mixtures. Several researchers have successfully combined this technique with online bioassays for the analyses of biologically active compounds in natural products [11][12][13][14][15]. However, the method is not suitable for some assays, such as enzymatic assays, that require a longer reaction time.…”

“…In addition, the molecular structure can be obtained from fragmentation data of the compounds of interest. Therefore, this technique is convenient, uses a small amount of sample, and is useful for bioassays that need a long reaction time [14,16,17].…”

Estrogenic Activity of Derris scandens Stem Extract and its Major Compounds Using MCF-7 Cell Proliferation Assay and Estrogen-Related Gene Expression

“…Fig 4. The expression levels of the following involved estrogenic activity genes were assessed: ERα gene (A), ERβ gene (B), AR gene (C) and TMPRSS2 gene (D).…”