Enrichment of Vitexin and Isovitexin From <i>Santalum album</i> L. Leaves and Evaluation of Their In Vitro and In Vivo Biological Activities

ABSTRACT Vitexin and isovitexin are flavonoids with diverse pharmacological properties; however, their enrichment usually relies on chromatographic techniques, which are labor‐intensive and costly. This study reports, for the first time, a simple and chromatography‐free method to prepare a vitexin‐ and isovitexin‐enriched extract (VIEE) from Santalum album L. leaves. The method involved (i) biphasic partitioning between n‐hexane and water, followed by (ii) cold‐water recrystallization. UHPLC analysis confirmed that VIEE contained 456.038 mg/g vitexin and 221.422 mg/g isovitexin (67.75% total), corresponding to a 6.1‐fold increase compared with the hydroethanolic extract. Toxicological assessment demonstrated high safety, with LD 50 &gt; 2.5 g/kg and no adverse effects in a 28‐day sub‐acute study at doses up to 900 mg/kg. Biologically, VIEE exhibited moderate inhibition of α‐glucosidase and α‐amylase , potent antioxidant activity in DPPH and ABTS assays, and acetylcholinesterase inhibition comparable to berberine. In streptozotocin‐induced diabetic mice, VIEE (300 mg/kg) lowered blood glucose by 43.2% after 14 days, prevented weight loss, and showed efficacy comparable to glibenclamide (47.9% reduction), while not affecting normoglycemic mice. Overall, this study introduces a cost‐effective, scalable enrichment strategy that adds value to S. album leaves and supports their development as a safe, multi‐target phytopharmaceutical for diabetes management.