ANTIOXIDANT ACTIVITY, PHYTOCHEMICAL AND PROXIMATE ANALYSIS OF LEAVES OF Psidium guajava, Garcinia quaesita, AND Eryngium foetidum.

Sri Lanka is home to a diverse range of plant species, including endemic species that have  long been used in herbal medicine. Scientific data on the phytochemical compositions, proximate compositions, and antioxidant capacity of locally grown Psidium guajava, Garcinia quaesita, and Eryngium foetidum plants are not adequately reported. The objective of this study was to investigate those factors to close the gap in scientific data on these plants. Even though Sri Lanka has many guavas and garcinia varieties, seven guava (Common guava, Apple-guava, Getta-pera, Embul-pera, Kanthi, Pubudu, Costorican) and two garcinia varieties (G. quaesita and G. zeylanica), along with E. foetidum, were chosen to compare  the above parameters. Macerated methanolic extracts of all selected plants were subjected to phytochemical  quantification and antioxidant analysis. Methanol was used as a solvent as it can extract a  high number of phytochemicals; this statement was validated by HPLC chemical profiling of different solvent extracts of E. foetidum. The maximum quantity of quantified phytochemicals such as polyphenolics, flavonoids, tannins, terpenoids, saponins, and  alkaloids varied by species. Total antioxidant power measured by FRAP assay and radical scavenging capacity measured by DPPH assay were found to be higher in common guava (722.44 ± 6.58 mg Trolox Eq/ g, and IC₅₀ value: 192.89 ± 0.07 ppm, respectively) than other  guava varieties. G. quaesita outperformed the other garcinia varieties in terms of antioxidant potential. When the antioxidant potency of the selected plants was evaluated, guava was found to be the most potent, followed by garcinia and finally E. foetidum. The essential oil was also extracted from the leaves of all the plants described above, and the chemical composition of the oil was evaluated using GC-MS. Chemical compositions in  isolated essential oils, only compounds with a higher than 90% matching value were considered. Sixty eight compounds were discovered in guavas essential oils, twenty-eight  known compounds of which were discovered for the first time. It is worth mentioning that this is the first study to isolate essential oil from the leaves of G. quaesita and G. zeylanica, and thirty three compounds have been identified. All isolated essential oils shown to have good antioxidant potential, with P. guineense having the highest antioxidant capacity (329.56 ± 2.01 μL Trolox Eq/L), followed by common-guava. When comparing both garcinia varieties, G. quaesita has the highest antioxidant capacity (274.74 ± 1.32 μL Trolox Eq/L). Antioxidant potent of essential oil of E. foetidum, it was found to be 118.16 ± 1.32 μL Trolox Eq/L.  Based on these analyses, common-guava, G. quaesita, and E. foetidum were chosen to  proceed further. This study was well developed with four different extraction techniques (Sonication: EN03, Maceration with agitation: EN04, Maceration with agitation upon heating: EN05, and Soxhlet: EN06). Interestingly, the total antioxidant capacity of all three plants, common guava, G. quaesita, and E. foetidum, was found to be higher in extracts obtained using the EN05 (432.57 ± 0.51, 72.08 ± 0.00, and 47.17 ± 0.20 mg Trolox Eq/g, respectively). It should also be highlighted that several of the key phytochemicals were  measured, and which changed with extraction techniques. According to the results, the EN05 is the best extraction method for extracting antioxidant-rich fraction from the leaves of  common-guava, G. quaesita, and E. foetidum, and this statement is supported by the HPLC  analysis of all four different aqueous extracts of E. foetidum. As next part of the study, gallic acid, catechin, and quercetin, well-known antioxidants, were isolated for the first time from the leaves of G. quaesita. Catechin, gallic acid, and quercetin yielded 0.06%, 0.09%, and 0.19%, respectively, based on dried powdered leaves.  To  confirm the structures of these compounds, melting point, λ_max analyses, and HPLC techniques & spectroscopy such as ¹H NMR, ¹³C NMR, FTIR were performed. In the FRAP and DPPH assays, isolated gallic acid outperforms the other two such as catechin and quercetin (FRAP value; 4,112.98 ± 42.21 mg Trolox Eq/ g and IC₅₀ value; 32.64 ± 0.05 ppm). To the best of our knowledge, no saponins have been isolated from guava or garcinia leaves.  As a result, this research was expanded to extract saponins.  Solvent extraction techniques were established and fourteen distinct saponin fractions were extracted from the leaves of common-guava and G. quaesita. These saponins were evaluated by analytical HPLC and  validated by further investigation at Kanagawa University in Japan. The isolated saponins demonstrated higher antioxidant ability, with some outperforming the standards that were  utilized, such as Trolox and ascorbic acid. When FRAP and DPPH assays on fourteen saponins were performed, the compound GuSBF3 isolated from common-guava displayed  the highest antioxidant capacity (1753.86 ± 1.52 mg Trolox Eq/g and the lowest IC50 value, 78.47 ± 0.02 ppm).  In conclusion, this is the first systematic and detailed study of phytochemicals, proximate  compositions, and antioxidant capacity variation with varieties and extraction types. The overall study yielded many active compounds, with three pure natural antioxidants from G. quaesita and fourteen saponins with high antioxidant capacity from common-guava and G.  quaesita. The findings of the study will be immensely beneficial for improving the value chain of selected plants and the development of value addition to enhance the market and be  used in the discovery of novel drug candidates.