Optimization of HPLC method for simultaneous detection of isomeric flavonoids from guava leaf extracts.

Guava (Psidium guajava L.) leaves are a rich source of polyphenolic compounds, particularly the flavonoids including morin, quercetin, etc which are reported to exhibit numerous pharmacological effects. In the present study, young guava leaves were subjected to maceration extraction using aqueous and 70% ethanol followed by characterization by qualitative phytochemical screening, total phenolic content, total flavonoid content, total antioxidant activity, and high-performance liquid chromatography analysis. The extraction yields were found to be higher for the ethanolic extract (14.9 %) compared with the aqueous extract (6.8 %), indicating greater solubilization of leaf constituents in hydroalcoholic medium. Interestingly, the aqueous extract showed higher total phenolic content per gram of extract (204.8 ± 10.04 mg gallic acid equivalents (GAE)/g) than the ethanolic extract (171.1 ± 25.3 mg GAE/g), whereas on a dry leaf basis the ethanolic extract yielded more phenolics overall (25.49 vs. 13.92 mg GAE/g dry powder). Total flavonoid content was marginally higher in the ethanolic extract (121.65 ± 0.91 mg catechin equivalents (CE)/g extract; 18.12 mg CE/g dry powder) compared to the aqueous extract (112.1 ± 9.61 mg CE/g extract; 7.61 mg CE/g dry powder). Both the extracts demonstrated strong DPPH radical scavenging activity, with the aqueous extract exhibiting the IC50 at around 9.7 μg/mL compared with the ethanolic extract at 9.4 μg/mL in comparison to the standard, ascorbic acid at 4.4 μg/mL, suggesting slightly higher antioxidant potency of the aqueous extract. Further, HPLC analysis successfully resolved morin and quercetin with retention time of approximately 3.7 ± 0.5 min and 4.1 ± 0.5 min, respectively, confirming the presence of isomeric flavonoids in the guava
leaf extracts. Overall, this study demonstrated that guava leaves are a promising source of morin and quercetin, and that maceration with aqueous and ethanol yields extracts with substantial phenolic content and strong antioxidant activity. The optimized HPLC method provides a basis for future quantitative and qualitative studies targeting isomeric flavonoids from guava leaves and other medicinal plant matrices.