Antioxidant Synergy of <i>Crocodylus siamensis</i> Blood and <i>Kaempferia parviflora</i> Extract in Protecting RAW 264.7 Macrophages from Oxidative Stress

Natural substances offer alternative antioxidant properties with fewer side effects than synthetic compounds. <i>Crocodylus siamensis</i> blood (CB) and <i>Kaempferia parviflora</i> extract (KP) are valued in traditional Asian medicines for their antioxidant properties, yet their synergistic effects have not previously been explored. This study is the first to investigate the synergistic antioxidant effects of CB blood and KP in RAW 264.7 macrophages under H₂O₂-induced oxidative stress. Initial radical-scavenging activity was measured using the ABTS assay, yielding IC₅₀ values of 109.00 μg/mL for CB and 41.72 μg/mL for KP. Using these values as references, combinations at multiple concentrations were prepared and analyzed for synergy. Three formulations, at CB:KP ratios of 1:1 (F1), 1:3 (F2), and 1:0.5 (F3), demonstrated significant synergistic antioxidant activity (combination index, CI < 1), effectively scavenging ABTS radicals and protecting RAW 264.7 cells from oxidative damage. At optimal concentrations, F1, F2, and F3 restored cell viability to 95.65%, 76.49%, and 96.22%, respectively, compared to H₂O₂-treated controls (∼45% viability) while reducing apoptosis to 12.93%, 32.13%, and 6.33%. Mechanistic studies revealed modulation of antioxidant responses with increased mRNA levels of <i>SOD1</i>, <i>SOD2</i>, <i>CAT</i>, <i>GPX1</i>, <i>Nrf2</i>, and <i>GCLM</i> and decreased <i>Keap1</i>. Label-free proteomics combined with bioinformatic analysis revealed the involvement of Notch and NF-κB signaling pathways and suggested a possible interaction between Nrf2 and Notch1. These combined extracts show potential as natural antioxidant agents for mitigating oxidative stress and merit further investigation for application in functional foods.