Psidium guajava L. leaves are a valuable source of phenolic compounds, particularly glycosylated flavonoids, with possible applications as standardised nutraceutical ingredients. However, the intrinsic variability of botanical matrices requires reproducible production processes integrating extraction optimisation, phytochemical characterisation, and biological validation. This study aimed to develop an integrated workflow for obtaining a standardised Psidium guajava leaf extract from a pilot cultivation in south-eastern Sicily. A Design of Experiments (DoE) approach was used to optimise the aqueous extraction process. The optimal extraction conditions were a drug-to-solvent ratio of 1:30 (w/v) and an extraction temperature and time of 95 °C and 24 min, respectively. The optimised crude extract yielded a dry residue of 0.81% w/v, with a total polyphenol content of 0.253% w/v and a total flavonoid content of 0.052% w/v. Comparative HPLC-DAD screening of two macroporous resins identified Resin B as the most effective material for flavonoid enrichment, mainly targeting avicularin, guaijaverin, and hyperoside, three biomarker compounds highly characteristic of this plant species. The resulting enriched extract showed a dry residue of 3.22% w/w and a total flavonoid content of 11.071% w/v, including 3.019% w/v avicularin, 2.845% w/v guaijaverin, and 1.196% w/v hyperoside. The enriched extract was subsequently formulated on a maltodextrin carrier to obtain a standardised ingredient with a total flavonoid content of 7.75% w/v. The final formulation exhibited strong antioxidant activity in cell-free assays and significantly reduced nitric oxide production in LPS-stimulated RAW264.7 macrophages, supporting its possible use as a reproducible botanical ingredient for nutraceutical applications.

Psidium guajava L. leaves are a valuable source of phenolic compounds, particularly glycosylated flavonoids, with possible applications as standardized nutraceutical ingredients. However, the intrinsic variability of botanical matrices requires reproducible production processes integrating extraction optimization, phytochemical characterization, and biological validation. This study aimed to develop an integrated workflow for obtaining a standardized P. guajava leaf extract from a pilot cultivation in southeastern Sicily. A Design of Experiments (DoE) approach, implemented by MODDE software, was used to optimize the aqueous extraction process. The optimal extraction conditions were a drug-to-solvent ratio of 1:30 (w/v) and an extraction temperature and time of 95 °C and 24 min., respectively. The optimized crude extract yielded a dry residue of 0.81% w/v, with total polyphenol contents of 0.253% w/v and a total flavonoid content of 0.052% w/v. Comparative HPLC-DAD screening of two macroporous resins identified Resin B as the most effective material for flavonoid enrichment, mainly targeting avicularin, guaijaverin, and hyperoside, three biomarker compounds highly characteristic of this plant species. The resulting enriched extract showed a dry residue of 3.22% w/w and a total flavonoid content of 11.071% w/v, including 3.019% w/v avicularin, 2.845% w/v guaijaverin, and 1.196% w/v hyperoside. The enriched extract was subsequently formulated on a maltodextrin carrier to obtain a standardized ingredient with a total flavonoid content of 7.75% w/v. The final formulation exhibited strong antioxidant activity in cell-free assays and significantly reduced nitric oxide production in LPS-stimulated RAW264.7 macrophages, supporting its possible use as a reproducible botanical ingredient for nutraceutical applications.