Characterization of crude β-glucuronidase from the pod and callus of Oroxylum indicum and its application in a directly integrated biocatalytic conversion of flavonoid glycosides in NADES extracts

The growing demand for nutraceuticals promoting healthy aging has heightened interest in bioactive flavonoids from Oroxylum indicum (OI) and related species, including baicalein, scutellarein, and chrysin. These plants primarily accumulate glycosylated derivatives—baicalin, scutellarin, oroxin A, and chrysin-7-O-glucuronide—rather than the more pharmacologically potent aglycones. In this study, β-glucuronidase crude enzymes were extracted and characterized from OI pods and calli. The enzyme exhibited high specific activity (9.8 U/mg protein at 30 min), a Km of 10 mM toward baicalin, and an optimum pH of 4.0. The crude extract displayed dual hydrolytic activity: β-glucuronidase efficiently cleaved β-glucuronide bonds in baicalin, scutellarin, and chrysin-7-O-glucuronide, reaching 97% conversion, while β-glucosidase hydrolyzed the β-glucosidic bond of oroxin A, both targeting sugar moieties at the C-7 hydroxyl of the flavone ring. Enzymatic hydrolysis of ethanolic-derived crude extracts from OI and Scutellaria baicalensis (SB), as well as direct post-extraction biocatalysis in natural deep eutectic solvent (NADES) extracts, enabled substantial conversion of flavonoid glycosides to aglycones, resulting in up to a 68-fold increase in aglycone yield. Bioactivity assays using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages showed that hydrolyzed extracts exhibited stronger nitric oxide (NO) inhibition than untreated samples and surpassed the positive control (L-NAME). Gene expression analysis confirmed downregulation of pro-inflammatory markers. These findings demonstrate that OI pod-derived crude enzymes effectively convert flavonoid glycosides to bioactive aglycones, enhancing both yield and anti-inflammatory activity. Furthermore, plant-based enzyme production is sustainable and environmentally friendly, with OI callus offering a scalable, high-yield alternative for future pharmaceutical and nutraceutical applications.