One-pot NADES-UAE extraction and in situ hydrolysis of Pueraria candollei isoflavones using crude enzyme extract from Luffa cylindrica

The rising demand for nutraceuticals, coupled with a rapidly aging population, has increased consumption of bioactive compounds. Pueraria candollei (PC) is recognized for its estrogenic properties due to its high phytoestrogen content. Previous studies on PC extraction and callus cultures report high levels of isoflavone glycosides (daidzin and genistin), which have lower bioavailability than their aglycones and require β -glucosidase conversion to daidzein and genistein. A crude enzyme extract from Luffa cylindrica (LC) fruits, which exhibited β -glucosidase-like activity, was employed as a biocatalyst to enhance aglycone production. The crude enzyme showed broad substrate selectivity, including a low K m for daidzin (0.013 mM), preferential hydrolysis of genistin, and efficient cleavage of oroxin B and cucurbitacin B glycosides, indicating strong affinity for β -D-glycosidic bonds across a broad spectrum of glycosylated phytochemicals. The enzyme was integrated with natural deep eutectic solvents (NADES) in a one-pot ultrasound-assisted extraction (UAE) system, providing a sustainable green platform for simultaneous co-extraction and enzymatic hydrolysis. HPLC analysis demonstrated a significant conversion of glycosides to aglycones, with aglycone levels increasing up to twofold following enzymatic hydrolysis. MCF-7 and LPS-stimulated RAW 264.7 cells were used to assess estrogenic and anti-inflammatory activities, confirming the enhanced bioactivity of the hydrolyzed extracts. The findings demonstrated that hydrolyzed extracts exhibited significantly greater estrogenic and anti-inflammatory activities than non-hydrolyzed extracts in both ethanolic and NADES-based preparations. This approach highlights the efficiency of the crude enzyme and its broad applicability across diverse plant matrices, offering a practical strategy to enhance bioactivity and bioavailability for large-scale production.