Purified Stigmasterol Related Novel Phytosterol from Chrysopogon zizanioides (L.) Roberty Root Extract Exhibits Strong Cholesterol Esterase (CEase) and Diabetic Foot Ulcer (DFU) Causing Bacterial Pathogens Inhibitory Properties

Background: Chrysopogon zizanioides Roberty root extracts have documented amylase and α-glucosidase inhibition, hepatoprotection, antioxidant, antimicrobial, antihyperglycemic, antidepressant, and antitubercular properties but lack ample reports on health care applications of purified phytoconstituents. Objective: To study the inhibitory properties of phytosterol eluate against cholesterol esterase (CEase) and diabetic foot ulcer (DFU) causing pathogens (clinical isolates). Methods: The shake flask method was adopted to extract the phytoconstituents and standard spectrophotometric assays were carried out to quantify phytosterol constituent and CEase inhibitory activity. Central composite design (CCD) based response surface methodology (RSM) was adopted to observe the improvement of phytosterol constituent and CEase inhibitory activity. Two dimensional preparative thin layer chromatography (2D PTLC) technique was executed to purify the phytosterol content and HRLC-based APCI – Q-TOF/ MS² analysis was done to predict the phytosterol structures. Standard disc assay was used to explore the inhibitory effect of extract and purified phytosterol content against DFU-causing pathogens. Results: The crude extracts exhibited a moderate CEase inhibitory activity (Ethylacetate: 67 ± 3.2%) and appreciable phytosterol content (Acetone: 11.24 ± 0.52 mg/g tissue). CCD based RSM has improved the CEase inhibitory activity (Ethylacetate: 96.56 ± 1.1%) as well as the phytosterol content (6.45 ± 0.5 mg/g tissue). The purified phytosterol fractionate (PTLC eluate) was found to contain stigmasterol related novel phytosterol and revealed a significant CEase inhibitory activity (81 ± 2.5 %; Ki: 54.89 μg) with mixed type inhibition. Conclusion: Both the extract and the PTLC eluate have recorded a substantial control over DFU causing bacterial pathogens.