Withania somnifera is a traditional Indian herb described under the ‘Rasayana’ class in Ayurveda, which gained immense popularity as a dietary supplement in the USA, Europe, Asia, and the Indian domestic market. Despite enormous research on the pharmacological effect of withanosides and withanolides, bioanalytical method development and pharmacokinetics remained challenging and unexplored for these constituents due to isomeric and isobaric characteristics. In current research work, molecular descriptors, pharmacokinetic, and toxicity prediction (ADMET) of these constituents were performed using Molinspiration and admetSAR tools. A rapid, selective, and reproducible bioanalytical method was developed and validated for seven withanosides and withanolides as per USFDA/EMA guidelines, further applied to determine pharmacokinetic parameters of Withania somnifera root extract (WSE) constituents in male Sprague Dawley rats at a dose of 500 mg/kg. Additionally, an ex vivo permeability study was carried out to explore the absorption pattern of withanosides and withanolides from the intestinal lumen. In silico, ADMET revealed oral bioavailability of withanosides and withanolides following Lipinski’s rules of five with significant absorption from the gastrointestinal tract and the ability to cross the blood-brain barrier. Upon oral administration of WSE, C_max was found to be 13.833 ± 3.727, 124.415 ± 64.932, 57.536 ± 7.523, and 7.283 ± 3.341 ng/mL for withanoside IV, withaferin A, 12-Deoxy-withastramonolide, and withanolide A, respectively, with T_max of 0.750 ± 0.000, 0.250 ± 0.000, 0.291 ± 0.102, and 0.333 ± 0.129 h. Moreover, at a given dose, withanoside V, withanolide B, and withanone were detected in plasma; however, the concentration of these constituents was found below LLOQ. Thus, these four major withanoside and withanolides were quantified in plasma supported by ex vivo permeation data exhibiting a time-dependent absorption of withanosides and withanolides across the intestinal barrier. These composite findings provide insights to design a clinical trial of WSE as a potent nutraceutical.

The conserved three-dimensional structure of receptor tyrosine kinases (RTKs) has been varyingly observed in prokaryotes to humans that actively participate in the phosphorylation process of tyrosine residues in the protein, which results in the alteration of protein’s function. Mutation and transcriptional or post-translational modifications lead to a deregulation of kinases, which ultimately fallout into the development of pathological conditions like cancer. The human genome encodes two kinds of tyrosine kinases: non-receptor tyrosine kinases (NRTKs) and receptor tyrosine kinases (RTKs). Among these kinases, VEGF/VEGFR-2 signaling cascade is an important target to develop novel small-molecule inhibitors for the therapy of abnormal angiogenesis incorporated with cancer. Due to advances in the knowledge of the catalytic domain and ‘DFG-motif’ region, selective ‘DFG-in’ (type I) and ‘DFG-out’ (type II) VEGFR-2/KDR inhibitors were successfully developed, and some are in different phases of a clinical trial. ‘DFG-out’ (inactive) confirmation has significant advantages over ‘DFG-in’ (active) confirmation concerning the affinity of the ATP at the catalytic domain. Further, in the catalytic domain, between front and back cleft, smaller gatekeeper residue (Val916) present; therefore, selectivity against VEGFR-2 could be precisely achieved. In this review, small molecule type II/‘DFG-out’ inhibitors, their conformation, interaction at receptor binding pocket, and structural requirements to inhibit VEGFR-2 at the molecular level are discussed. Graphical Abstract Communicated by Ramaswamy H. Sarma