BIOACTIVE

This study aims to optimize the extraction of bioactive compounds from guava leaves using the Soxhlet extraction method to investigate the impact of key variables such as mass of the solvent, temperature and extraction time (hours) on the bioactive extraction yield, to characterize the extracted bioactive compounds to identify key functional groups, and to optimize the bioactive yield. The study employed a central composite design (CCD), with 19 experimental runs where Response Surface Method (RSM) was utilized to optimize extraction conditions, evaluating the effects of mass (1.00-10.00 g), extraction time (30-300 min), and temperature (50-90°C). ANOVA and quadratic regression models assessed the influence of these variables on the yields of terpenoids and flavonoids. The qualitative and quantitative analysis of extracted compounds was conducted using colorimetric chemical tests and FTIR spectroscopy. Statistical validation included model significance testing (p-values), R², adjusted R², predicted R², and adequate precision. The qualitative analysis of guava leaf extract identified flavonoids (yellow), terpenoids (reddish- brown), saponins (froth), alkaloids (reddish-brown precipitate), and tannins (greenish-black). Quantitative results showed the highest percentages in flavonoids (15%) and terpenoids (16%), followed by saponins (2%), alkaloids (1.75%), and tannins (0.183%). Extraction efficiency was highest at intermediate conditions, with significant quadratic effects observed for all three independent variables. The regression models yields for the two major extract, terpenoid and flavonoid, demonstrated high accuracy with R2 is 0.7915 for terpenoid and R² = 0.8957 for flavonoid, with ANOVA confirming model significance (F-value = 0.17, p = 0.9585) and (F- value = 8.59, p = 0.0019) for terpenoid and flavonoid respectively. Also, the extraction yield was significantly affected by mass, time, and temperature. Terpenoid yield declined beyond 55 g and 165 min due to solvent saturation, while flavonoids degraded above 70°C. Optimal conditions enhanced solubilization and diffusion, but excessive parameters caused thermal degradation, volatilization, poor solvent penetration, and reduced extraction efficiency. These findings support guava leaves as a rich source of bioactive compounds with antioxidant, anti- v | P a g e inflammatory, and antimicrobial properties, valuable for pharmaceutical and nutraceutical applications.