Optimization of the Synergistic Antioxidant Activity of Clove (Syzygium aromaticum) Flower Bud Extract, Turmeric (Curcuma longa) Rhizome Extract, Vitamin C, and Vitamin E Using a Non-Factorial Central Composite Design

Abstract

Oxidative stress plays a central role in the development of metabolic disorders, prompting increased interest in synergistic antioxidant formulations derived from natural and synthetic sources. This study aimed to evaluate and optimise the antioxidant potential of vitamin C, vitamin E, turmeric (Curcuma longa) extract, and clove (Syzygium aromaticum) extract using a non-factorial central composite design (CCD), and to investigate their synergistic interactions and statistical associations in multicomponent blends. Ethanolic extracts of turmeric and clove were prepared using maceration and rotary evaporation techniques. Antioxidant activities were determined using DPPH radical-scavenging, ferric reducing antioxidant power (FRAP), and ABTS radical-scavenging assays, together with analyses of total phenolic and flavonoid contents. In addition, a composite antioxidant response was developed to evaluate formulation efficiency. Synergistic interactions and statistical associations were assessed using a synergy index (SI), chi-square (χ²) deviation analysis, and Pearson correlation coefficients to quantify the strength of interactions among the blend components across 16 CCD experimental runs. The results showed significant variability in antioxidant performance according to assay type. Turmeric exhibited the highest FRAP (8.42 ± 2.20%) and ABTS (0.69 ± 0.02 mmol TE/L) activities, whereas vitamin C showed the strongest DPPH radical-scavenging activity (IC₅₀ = 534.816 µg/mL). Clove extract recorded the highest total phenolic content (653.46 ± 7.58 mg GAE/g), whereas turmeric had a higher flavonoid content (0.21 ± 0.01 mg QE/g). Synergy analysis revealed that formulations containing balanced proportions of the vitamin C/E blend and turmeric and clove extracts exhibited strong synergistic interactions (SI > 1.6), whereas imbalanced formulations showed antagonistic effects (SI < 1.0). Chi-square analysis confirmed significant nonlinear interaction effects (χ² up to 6.84), and correlation analysis indicated strong positive associations (r = 0.52–0.88) between the optimised blends and antioxidant responses. In conclusion, the study demonstrates that antioxidant efficacy is significantly enhanced through synergistic interactions between vitamin-based and plant-derived antioxidants. Integrating response surface methodology with synergy and statistical interaction analyses provides a robust framework for optimising multicomponent antioxidant systems. The optimised blends may be potential candidates for developing functional foods and nutraceuticals targeting oxidative-stress-related disorders.