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

Natural substances offer alternative antioxidant properties
with
fewer side effects than synthetic compounds. Crocodylus
siamensis blood (CB) and Kaempferia
parviflora 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 SOD1, SOD2, CAT, GPX1, Nrf2, and GCLM and
decreased Keap1. 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.