RF is well known to have various pharmacological effects, including anticancer and anti-inflammatory [
2‐
5,
16] activities, which are attributed to the phenolic compound anthocyanin and EA content [
6,
9,
18]. However, the effect of different harvest times and extraction solvents on the anti-inflammatory effects of RF in LPS-stimulated RAW264.7 cells has not yet been examined. This is the first study to compare the anti-inflammatory effects of different solvent extracts of MRF and IRF in LPS-stimulated RAW264.7 cells. Total phenol and flavonoid contents were higher in 75% ethanol extract of MRF than in 25, 50, and 100% ethanol extract of MRF [
22]. The anti-oxidant effect of 75% ethanol extracts of MRF and IRF was better than that of 25, 50, and 100% ethanol extracts in liver cells and DPPH radical scavenging activity [
22,
23]. In addition, 30% ethanol extract of IRF has beneficial effect osteoporotic by pro-inflammation [
24]. Based on these findings, water, 30% ethanol, and 70% ethanol were selected as extraction solvents in this study.
Injuries due to inflammation are mediated by oxygen-derived free radicals and high-energy oxidation, which cause toxic oxidation reactions in the cells [
14]. In this study, the IC
50 values in both ABTS and DPPH free radical scavenging assays were lower for IRF than for MRF. The 30% ethanol extract of IRF showed better antioxidant activity than MRF. RF contains anthocyanins and phenolic compounds in abundance [
6], and their composition changes during maturation process. In particular, the contents of organic acids, amino acids, and phenolic compounds are reduced [
25], while anthocyanin components formed that are glycosides attach to the phenolics [
9]. Phenolic compounds are found in many fruits and vegetables, and they contribute to protective effects against oxidation in organisms [
26]. EA is known to be a standard phenolic compound in RF and therefore, EA was selected as a standard component for the analysis. EA exerts potential activities such as anti-inflammatory and anticoagulatory effects in cardiac tissues of diabetic mice and UV–B irradiation-induced inflammation model [
20,
21]. In the present study, IRF contained a higher quantity of EA than MRF did, indicating that the EA component in IRF inhibited the production of pro-inflammatory cytokines and inflammation-related factors in LPS-stimulated RAW264.7 cells. Raw 264.7 macrophages perform major functions such as initiation, maintenance, and resolution of inflammatory precess [
27]. Our findings showed that IRF efficaciously inhibited p65 NF-κB inflammatory response via cytokine genes in LPS-stimulated RAW264.7 cells. Treatment of RAW264.7 cells with LPS stimulates toll-like receptor 4 and induces the release of pro-inflammatory cytokines necessary to activate immune response [
28]. We demonstrated the inhibition of pro-inflammatory factors TNF-α, IL-1β, and IL-6 by IRF. In a previous study, 100% ethanol extract of IRF reduced pro-inflammatory cytokines more effectively than MRF extracts did [
16]. However, 0 and 30% ethanol extracts of IRF reduced pro-inflammatory cytokines to a lesser extent than 100% ethanol extract did. Pro-inflammatory cytokine actions involve the activation of NF-κB. Cytokines may be responsible for the induction of COX-2 and iNOS. Furthermore, iNOS produces NO to cause inflammation [
14]. In the present study, treatment of LPS-stimulated RAW264.7 cells with 0 and 30% ethanol extracts of IRF reduced NO and inflammatory-related proteins, and these effects were less potent than those induced by the 100% ethanol extract (similar to effects on pro-inflammatory cytokines) [
16]. The extract with the highest polyphenol content exhibited the highest antioxidant capacity [
6]. A previous study reported that the ethyl acetate fraction of RF, which had the highest polyphenol content, showed the highest NO reduction rate [
8]. These results indicate that the ethanol extract of IRF, which had antioxidant ability, showed better anti-inflammatory effects than MRF.
Our results confirmed that the anti-inflammatory effects of 0 and 30% ethanol extracts of IRF, which were mediated by reduction of NF-κB pathway activity via cytokine inhibition in LPS-stimulated RAW264.7 cells, were higher than that of the 70% ethanol extracts of IRF and MRF. This effect may have been mediated by the anti-inflammatory effects of EA in IRF. Therefore, 0 and 30% ethanol extracts of IRF have the potential to be used as therapeutic agents for inflammation-related diseases.