Optimal formulation of bitter gourd and black galingale extract: Evaluation of effects on inflammation and oxidative stress-related genes

Reactive nitrogen and oxygen species (RNOS) are defined as highly reactive molecules that can be generated endogenously and exogenously. The overproduction of RNOS leads to inflammation, neurodegenerative diseases, diabetes, obesity, and cancer. Natural antioxidants derived from plants are molecules that can neutralize those RNOS. In this study, to increase the antioxidant capacity in neutralizing RNOS, an in vitro evaluation of a binary combination of bitter gourd ( Momordica charantia L.) and black galingale ( Kaempferia parviflora) ) extracts as potential natural antioxidant sources at different ratios was conducted. A combination at the ratio of 1:6 showed the highest total phenolic content (TPC) (336.48 f 0.04 mg GAE/100g DW), while the ratios 1:6 (1511.73 f 0.20 mg QE/100g DW) and 1:8 (1398.91 f 0.28 mg QE/100g DW) exhibited the highest total flavonoid content (TFC) compared to the bitter gourd extract and other mixed ratios. The highest scavenging capacities in the DPPH, ABTS, and FRAP assays were detected at the ratios of 1:3 (IC50 50 9.1 f 0.07 mg/mL; moderate synergism), 1:6 (IC50 50 1.07 f 0.07 mg/mL; synergism), and 1:4 (IC50 50 2.44 f 0.04 mg/mL; strong synergism), respectively. Based on the overall cumulative ability to scavenge free radicals with synergistic or additive antioxidant interactions and the presence of functional groups analyzed using FT-IR, the 1:6 ratio was determined as the best combination ratio to neutralize RNOS where 15 proposed beneficial bioactive compounds were detected. Additionally, in vivo studies showed that the increase of CAT mRNA expression and the suppression of NF-kB and CPT-1 mRNA expression at 250 mg/kg BW of dosage confirms that this methanolic mixed extract potentially can effectively act as both an anti-inflammation and antioxidant agent. This study demonstrates that a specific binary combination ratio of plant extracts can synergistically and additively increase their antioxidant capacity, even when utilized in small quantities.