Anti-inflammatory effect of isoegomaketone isolated from radiation mutant Perilla frutescens var.crispa

Abstract

About 165 lines of radiation-induced mutant P. frutescens var. crispa were screened for their anti-inflammatory activities. Among those screened, the one mutant with the highest inhibitory activity on NO production in lipopolysaccharide (LPS)-treated RAW264.7 cells was selected. The enhanced anti-inflammatory activity of the mutant seemed to be due to the increase in isoegomaketone (IK) content. IK has been shown to exhibit several biological activities including anti-inflammatory, anti-cancer, and anti-obesity effects. The induction of heme oxygenase-1 (HO-1) seemed to contribute to anti-inflammatory activity of IK in RAW264.7 cells. However, there were no studies which investigated the mechanism of HO-1 induction by IK. In the present study, anti-inflammatory effect of IK isolated from radiation mutant P. frutescens var. crispa (Antisperill) was investigated using in vitro and in vivo models. In addition, in an effort to investigate any potentiality of radiation mutant P. frutescens var. crispa as functional food, optimal extraction method was chosen, as well as anti-arthritic properties of the extracts was investigated in CAIA model. In Study 1, the mechanism of IK-induced HO-1 expression was investigated using RAW264.7 cells. RAW264.7 cells were treated with IK (5, 10, 15 M) to study the mechanism of IK-induced HO-1 expression. IK upregulated HO-1 mRNA and protein expression in a dose dependent manner. IK-induced HO-1 mRNA expression was suppressed only by SB203580, a specific inhibitor of p38 MAPK. ROS scavengers (N-acetyl-L-cysteine, NAC, and glutathione, GSH) also blocked the IK-induced ROS production and HO-1 expression. Both NAC and SB203580 suppressed the IK-induced Nrf2 activation. In Study 2, whether IK has an anti-arthritic activity in collagen antibody-induced arthritis (CAIA) animal model was investigated. Rheumatoid arthritis was induced in male Balb/c mice by collagen-antibody injection. Experimental animals were randomly divided into five groups: normal, CAIA, CAIA + IK (5 mg/kg/day), CAIA + IK (10 mg/kg/day), and CAIA + apigenin (16 mg/kg/day) and respective treatments were administered via oral gavage once per day for 4 days. Mice treated with IK (10 mg/kg/day) showed improvement in disease outcome. Arthritic score, paw volume, and paw thickness were significantly lower compared to the control CAIA mice at day 7 (73%, 15%, and 14% lower, respectively). Furthermore, histopathological examination of ankle for inflammation showed that infiltration of inflammatory cells and edema formation were reduced by IK treatment. Similarly, neutrophil to lymphocyte ratio (NLR) in whole blood was lower in mice treated with IK (10 mg/kg/day) by 51.9% compared to the control CAIA mice. In Study 3, to determine the optimal extraction method for developing radiation mutant P. frutescens var. crispa as functional food, extracts were obtained by two methods: extract obtained by supercritical carbon dioxide extraction (SFE) and extract obtained by ethanol extraction (EE). SFE contained 5-fold higher levels of IK compared with EE. When LPS-induced RAW264.7 cells were treated with extracts at 25 g/mL, the SFE inhibited the expression of inflammatory mediators such as nitric oxide (NO), monocyte chemoattractant protein-1 (MCP-1), interleutkin-6 (IL-6), interferon- (IFN-), and inducible nitric oxide synthase (iNOS) to a much greater extent compared with EE. In Study 4, whether SFE (in Study 3) has an anti-arthritic activity in collagen antibody-induced arthritis (CAIA) animal model was investigated. Extracts were obtained by supercritical carbon dioxide extraction method from radiation mutant P. frutescens var. crispa leaf (SFE-M) and from wild type species leaf (SFE-W). Experimental animals were randomly divided into four groups: normal, CAIA, CAIA + SFE-M (100 mg/kg/day), and CAIA + SFE-W (100 mg/kg/day) and respective treatments were administered via oral gavage once per day for 4 days. Mice treated with SFE-M showed improvement in disease outcome. Arthritic score, paw volume, and paw thickness were significantly lower compared to the control CAIA mice from day 3 to day 7. Furthermore, histopathological examination of ankle for inflammation showed that infiltration of inflammatory cells and edema formation were reduced by SFE-M treatment. Similarly, NLR in whole blood was lower in mice treated with SFE-M by 37% compared to the control CAIA mice. However, SFE-W didnt show any significant effect compared to the control CAIA group. IK showed anti-inflammatory properties by HO-1 expression via ROS/p38 MAPK/Nrf2 pathway in RAW264.7 cells, as well as real, actual, and palpable anti-arthritic effect in CAIA animal model. Furthermore, supercritical carbon dioxide extraction was found to be the better method compared with the ethanol extraction method for the presentation of extract from leaves of radiation mutant P. frutescens var. crispa to be used as functional food because of higher IK content. Efficacy of the extract from radiation mutant P. frutescens var. crispa by SFE was confirmed as the treatment with the extract reduced the incidence of clinically evident signs and symptoms in CAIA animal model. Taken together, the results of this study encourage the commercial use of the extract of radiation mutant P. frutescens var. crispa as a functional food in the chronic inflammatory situation like RA.