Comparative Determination of Saponins in Nerium Oleander Using Foam-Forming, Optical Activity, and Spectrophotometric Techniques

Abstract

Majorobela Motaung, Fanyana Mtunzi, Imelda Ledwaba, Gcobisa Valencia Manzane, Rosemary Montle and Michael Klink

Nerium oleander L is a medicinal plant of significant ethnopharmacological importance, yet its well-documented toxicity necessitates rigorous phytochemical characterization and standardization. This study comprehensively evaluated the influence of eight extraction solvents of varying polarity (water, methanol, ethanol, acetone, dichloromethane, chloroform, ethyl acetate, and hexane) and three analytical methods (foam-forming, optical activity, and spectrophotometric vanillin- acetic acid) on saponin quantification in N. oleander leaf extracts. Plant material was collected, dried, pulverized, and extracted via maceration, with saponin content determined using the three analytical approaches. Data were analyzed using two-way ANOVA with replication and Tukey's HSD post-hoc testing. Results demonstrated statistically significant variation in saponin yield attributable to both extraction solvent (F = 143.44, p < 0.001) and analytical method (F = 90.53, p < 0.001), with a significant interaction effect (F = 6.35, p < 0.001). Polar solvents, particularly methanol (18.22 ± 0.57%) and ethanol (16.32 ± 3.94%), exhibited superior extraction efficiency, consistent with the glycosidic nature of saponins. Hexane yielded anomalously elevated content (18.00 ± 2.57%), potentially due to unique hydrogen-bonding interactions. The spectrophotometric method consistently produced the highest saponin values due to enhanced sensitivity, while the foam-forming method proved suitable only for preliminary screening, and optical activity lacked specificity due to interference from other chiral constituents. This study conclusively demonstrates that methodological selection critically influences saponin quantification, with significant implications for ethnopharmacological standardization and quality control. Based on these findings, methanol extraction followed by spectrophotometric vanillin-acetic acid analysis is recommended as the optimal protocol for routine quantitative analysis, providing the highest sensitivity, reproducibility, and practical feasibility. These findings provide an evidence-based framework for method selection and underscore the necessity of harmonized analytical protocols to ensure accuracy, reproducibility, and comparability in natural product research, thereby supporting the safe and effective development of N. oleander-based therapeutics and cosmetic formulations.

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