Background: Blepharis integrifolia is in the preparation of traditional formulation for the treatment of various diseases, including digestive disorders such as flatulence and dysentery. Although a very few studies have assessed the pharmacological properties, nothing is known about the phytochemicals present and the effect of extracts on its pharmacological activities. Objectives: Therefore, the objectives of the present study were to assess the role of methanol extract of B. integrifolia (MBI) for its anti-inflammatory, antioxidant, and antibacterial activities. Materials and Methods: The phytochemicals present in the extract were evaluated using high-resolution liquid chromatography and mass spectrometry (HR-LCMS). The extract and the isolated compound were subjected to antioxidant, antibacterial, and anti-inflammatory studies. Results: MBI exhibited an effective antibacterial, antioxidant, and anti-inflammatory activities at a more significant level than the standards. HR-LCMS analysis of MBI revealed the presence of kaempferol, rutin, and several phytochemicals. The activity-guided repeated fractionation of the methanol extract by silica gel column chromatography yielded a compound that exhibited strong antioxidant activity. Based on various physicochemical and spectroscopic analyses (ultraviolet, infrared, proton nuclear magnetic resonance [NMR], carbon-13 NMR, and MS), the bioactive compound isolated was elucidated as 3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one (kaempferol). Furthermore, the molecular docking studies for kaempferol revealed that it binds β-lactamase at MEG binding site and was found to interact with binding site residues PHE91, TRP94, ASN109, MET148, GLU147, ASN36, and ILE201. Conclusion: The results from the present study suggest that the potent antioxidant, antibacterial, and anti-inflammatory activities observed are a result of the presence of these bioactive compounds within the extract. In addition, these results also demonstrate the antioxidant potency of kaempferol which could be the basis for its alleged health-promoting potential.
