Phytochemical characterization and antioxidant potential of Moringa oleifera leaf extracts under different solvent systems

Introduction

Moringa oleifera, commonly known as the drumstick tree or miracle tree, has emerged as one of the most promising medicinal plants of the 21st century [1] . Native to the sub-Himalayan regions, this fast-growing species has spread throughout tropical and subtropical regions, gaining recognition for its exceptional nutritional density and diverse pharmacological activities [2] . The leaves, in particular, contain a remarkable concentration of proteins, vitamins, minerals, and bioactive phytochemicals that underpin its traditional use in treating over 300 ailments [3] . The therapeutic potential of M. oleifera is attributed primarily to its rich content of phenolic compounds, flavonoids, and other secondary metabolites possessing antioxidant, antiinflammatory, and antimicrobial properties [4] . These bioactive constituents combat oxidative stress implicated in chronic diseases including diabetes, cardiovascular disorders, and cancer [5] . However, the efficiency of extracting these compounds depends critically on the solvent system employed, as solvent polarity determines which compound classes are preferentially dissolved [6] . Solvent selection represents a crucial decision in phytochemical research, influencing both extraction yield and bioactivity of the resulting extract [7] . Polar solvents like methanol and ethanol effectively extract phenolics and flavonoids, while non-polar solvents like hexane preferentially dissolve lipophilic compounds including terpenoids and fatty acids [8] . Systematic comparison of extraction solvents provides essential guidance for optimizing bioactive compound recovery and standardizing herbal preparations [9] . This research aimed to characterize phytochemical profiles of M. oleifera leaf extracts prepared using different solvent systems, quantify total phenolic and flavonoid contents, evaluate antioxidant activities through multiple assay systems, and identify the optimal solvent for maximizing antioxidant potential. Material and Methods Material: Fresh M. oleifera leaves were collected from mature trees (3-5 years old) at the Botanical Garden of Monterrey Institute of Biotechnology, Monterrey, Mexico (25.6866°N, 100.3161°W) during September 2022. Plant specimens were authenticated by the institutional herbarium (Voucher No. MIB-2022-147). Leaves were washed, shadedried at room temperature for seven days, and ground to fine powder using an electric grinder (particle size <0.5 mm) [10] .

Reagents and Chemicals

Analytical grade solvents including methanol, ethanol, hexane, and chloroform were purchased from Sigma-Aldrich (St. Louis, MO, USA). DPPH (2,2-diphenyl-1picrylhydrazyl), ABTS (2,2'-azino-bis(3ethylbenzothiazoline-6-sulfonic acid)), Folin-Ciocalteu reagent, gallic acid, quercetin, ascorbic acid, aluminum chloride, ferric chloride, and sodium carbonate were obtained from Merck (Darmstadt, Germany). All reagents were of analytical grade with purity >98% [11] .

Methods

Extraction Protocol: Powdered leaf material (50 g) was extracted separately with 500 mL each of methanol, ethanol, distilled water, hexane, and chloroform using maceration technique at room temperature (25±2°C) for 72 hours with intermittent shaking [12] . Extracts were filtered through Whatman No. 1 filter paper and concentrated under reduced pressure using rotary evaporator (Büchi R-210) at 40°C. Aqueous extract was lyophilized using freeze dryer. Dried extracts were weighed to calculate percentage yield and stored at -20°C until analysis [13] .

Phytochemical Screening

Qualitative phytochemical analysis was performed using standard colorimetric and precipitation methods. Alkaloids were detected using Dragendorff's and Mayer's reagents. Flavonoids were identified by alkaline reagent and lead acetate tests. Phenolics were detected using ferric chloride test. Tannins were confirmed by gelatin and ferric chloride tests. Saponins were detected by frothing test. Terpenoids were identified using Salkowski test. Glycosides were detected using Keller-Killiani test [14] . Total phenolic content was determined using Folin-Ciocalteu method with gallic acid as standard, expressed as mg gallic acid equivalents per gram (mg GAE/g). Total flavonoid content was measured using aluminum chloride colorimetric method with quercetin standard, expressed as mg quercetin equivalents per gram (mg QE/g) [15] . Antioxidant activity was evaluated using DPPH radical scavenging assay, ABTS assay, ferric reducing antioxidant power (FRAP), and total antioxidant capacity (TAC) assay. IC₅₀ values were calculated from dose-response curves [16] .

Statistical analyses used one-way ANOVA with Tukey's post-hoc test. Pearson correlation assessed relationships between variables (p<0.05) [17] .

Results

Extraction yields varied substantially across solvent systems (Table 1). Aqueous extraction provided highest yield (18.6±1.2%), followed by methanol (14.7±0.8%) and ethanol (12.3±0.6%). Non-polar solvents showed lower yields, with hexane producing only 4.2±0.3%. Extract colors ranged from dark green (methanol) to yellowish (hexane), reflecting differential compound extraction. Qualitative phytochemical screening revealed distinct profiles across solvents (Table 2). Flavonoids and phenolics were most abundant in methanol and ethanol extracts, while tannins and saponins showed highest intensity in aqueous extract. Non-polar hexane extract contained primarily terpenoids with minimal polar compound content. 3).

Comprehensive Interpretation

Correlation analysis revealed strong positive relationships between total phenolic content and antioxidant activity (DPPH: r = -0.94, p<0.001; ABTS: r = -0.92, p<0.001), indicating phenolics as primary contributors to antioxidant potential. Flavonoid content also correlated strongly with FRAP values (r = 0.91, p<0.001). Solvent polarity showed positive correlation with phenolic extraction efficiency (r = 0.78, p<0.05).

Discussion

This research demonstrates that solvent selection profoundly influences both phytochemical composition and antioxidant activity of M. oleifera leaf extracts. Methanol emerged as the optimal solvent, yielding extracts with highest phenolic and flavonoid contents alongside strongest antioxidant activity [18] . This superiority likely reflects methanol's intermediate polarity, enabling efficient dissolution of diverse phenolic compound classes while minimizing extraction of inactive polar macromolecules that dilute bioactivity in aqueous extracts [6,9] . The strong correlation between phenolic content and antioxidant activity (r = 0.94) confirms phenolics as the primary antioxidant constituents in M. oleifera leaves [4,19] . These compounds donate hydrogen atoms or electrons to neutralize free radicals, breaking oxidative chain reactions that damage cellular components. The observed IC₅₀ values compare favorably with previous reports and support the traditional use of M. oleifera as an antioxidant-rich functional food [20] . Interestingly, aqueous extraction provided highest crude yield but intermediate antioxidant activity, suggesting that water extracts substantial inactive material alongside bioactive compounds [8] . For applications prioritizing antioxidant potency over mass yield, methanol or ethanol extraction appears preferable, while aqueous extraction may suit applications requiring food-grade solvents [21] .

Conclusion

Moringa oleifera leaf extracts exhibit solvent-dependent variations in phytochemical profiles and antioxidant activities. Methanol extraction yields highest total phenolic content (127.4 mg GAE/g), flavonoid content (84.6 mg QE/g), and strongest DPPH scavenging activity (IC₅₀ 87.4 µg/mL). Strong correlations between phenolic content and antioxidant activity confirm phenolics as primary bioactive constituents. These findings establish methanol as the optimal solvent for preparing antioxidant-rich M. oleifera extracts for pharmaceutical and nutraceutical applications.

Acknowledgement

Acknowledgements Funding SourcesThis research was supported by Monterrey Institute of Biotechnology internal research grant. The funding body had no involvement in research design or manuscript preparation. Institutional SupportWe acknowledge the Department of Phytochemistry and the institutional herbarium for plant authentication and laboratory facilities. Contributions Not Qualifying for AuthorshipWe thank Dr. Elena Torres for spectroscopic analysis assistance and laboratory technicians for sample preparation support.

References

1. Anwar F, Latif S, Ashraf M, Gilani AH. Moringa oleifera: a food plant with multiple medicinal uses. Phytother Res. 2007;21(1):17-25.
2. Gopalakrishnan L, Doriya K, Kumar DS. Moringa oleifera: a review on nutritive importance and its medicinal application. Food Sci Hum Wellness. 2016;5(2):49-56.
3. Fahey JW. Moringa oleifera: a review of the medical evidence for its nutritional, therapeutic and prophylactic properties. Trees Life J. 2005;1(5):1-15.
4. Sreelatha S, Padma PR. Antioxidant activity and total phenolic content of Moringa oleifera leaves. J Med Food. 2009;12(2):431-434.
5. Verma AR, Vijayakumar M, Mathela CS, Rao CV. In vitro and in vivo antioxidant properties of different fractions of Moringa oleifera leaves. Food Chem Toxicol. 2009;47(9):2196-2201.
6. Do QD, Angkawijaya AE, Tran-Nguyen PL, et al. Effect of extraction solvent on total phenol content and antioxidant activity. Food Chem. 2014;145:1007-1012.
7. Sultana B, Anwar F, Ashraf M. Effect of extraction solvent/technique on the antioxidant activity of selected medicinal plant extracts. Molecules. 2009;14(6):21672180.
8. Vongsak B, Sithisarn P, Mangmool S, et al. Maximizing total phenolics, total flavonoids contents and antioxidant activity of Moringa oleifera leaf extract. Ind Crops Prod. 2013;44:566-571.
9. Zhang L, Tu ZC, Wang H, et al. Comparison of different methods for extracting polyphenols from Ipomoea batatas leaves. Int J Food Sci Technol. 2015;50(3):731-738.
10. Amaglo NK, Bennett RN, Lo Curto RB, et al. Profiling selected phytochemicals and nutrients in different tissues of Moringa. Food Chem. 2010;122(4):10471054.
11. Singleton VL, Orthofer R, Lamuela-Raventós RM. Analysis of total phenols and other oxidation substrates by means of Folin-Ciocalteu reagent. Methods Enzymol. 1999;299:152-178.
12. Azwanida NN. A review on the extraction methods used in medicinal plants. J Chem Pharm Res. 2015;7(1):1006-1010.
13. Sasidharan S, Chen Y, Saravanan D, et al. Extraction, isolation and characterization of bioactive compounds from plant extracts. Afr J Tradit Complement Altern Med. 2011;8(1):1-10.
14. Trease GE, Evans WC. Pharmacognosy. 16th ed. London: Saunders Elsevier; 2009.
15. Chang CC, Yang MH, Wen HM, Chern JC. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J Food Drug Anal. 2002;10(3):178-182.
16. Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to evaluate antioxidant activity. LWT Food Sci Technol. 1995;28(1):25-30.
17. Field A. Discovering Statistics Using IBM SPSS Statistics. 5th ed. London: SAGE Publications; 2018.
18. Siddhuraju P, Becker K. Antioxidant properties of various solvent extracts of total phenolic constituents from three different agroclimatic origins of drumstick tree. J Agric Food Chem. 2003;51(8):2144-2155.
19. Leone A, Spada A, Battezzati A, et al. Cultivation, genetic, ethnopharmacology, phytochemistry and pharmacology of Moringa oleifera leaves. Int J Mol Sci. 2015;16(6):12791-12835.
20. Moyo B, Masika PJ, Hugo A, Muchenje V. Nutritional characterization of Moringa oleifera Lam. leaves. Afr J Biotechnol. 2011;10(60):12925-12933.
21. Chumark P, Khunawat P, Sanvarinda Y, et al. The in vitro and ex vivo antioxidant properties, hypolipidaemic and antiatherosclerotic activities of water extract of Moringa oleifera. J Ethnopharmacol. 2008;116(3):439446.