Simultaneous HPLC quantification and comparative anti-inflammatory activity of Ixeris dentata, Ixeris dentata var. albiflora and Ixeris sonchifolia in LPS-stimulated RAW 264.7 cells

Abstract

Species of the genus Ixeris belonging to Compositae family are used as food garnish and traditional herbal medicines for strengthening of the stomach, sedatives, and diuretic agents in Korea. Extensive phytochemical investigations of the Ixeris genus have been carried out, and components including sesquiterpenes lactones, triterpenes, phenylpropanoids, phenols, amino acids, fatty alcohols, and fatty acids have been isolated and shown to exhibit diverse significant bioactivities such as antioxidant, antitumor, hepatoprotective, cardiovascular, and neuroprotective effects. However, Ixeris are known to have a high content of flavonoids, which have been established as the main bioactive constituents. Among these plants, Ixeris dentata Nakai (ID), Ixeris dentata var. albiflora Nakai (IDA) and Ixeris sonchifolia Bge Hance (IS) are herbaceous plants used in Korea. Therefore, the aim of the present study was to evaluate the comparative anti-inflammatory activities of Ixeris dentata (ID), Ixeris dentata var. albiflora (IDA) and Ixeris sonchifolia (IS) belonging to Compositae family along with simultaneous HPLC quantification of the main compounds present in extracts. Anti-inflammatory activity was evaluated via lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 murine macrophages. Five main compounds consisting of chlorogenic acid, caffeic acid, luteolin 7-O-glucoside, luteolin 7-O-glucuronide, and luteolin were used for simultaneous HPLC quantification. The total phenolic content present in ID (30 mg/g GAE), IDA (35.33 mg/g GAE) and IS (43.79 mg/g GAE) is correlated to the corresponding LPS-induced NO production inhibitory effect in RAW 264.7 cells as expressed with IC50 values 26.19, 21.43 and 7.59 µg/ml, respectively. Luteolin 7-O-glucoside was found as the major compound in ID (8.76 mg/g dry weight) and IDA (10.35 mg/g dry weight) and it was luteolin 7-O-glucuronide in case of IS (34.66 mg/g dry weight). The LPS-induced NO production IC50 value for luteolin 7-O-glucoside and luteolin 7-O-glucuronide was 30 and 4.5 µM, respectively. Furthermore, these two compounds exhibited potent inhibitory activities of IC50 30 and 4.5 µM with regard to LPS-induced NO production in RAW 264.7 cells. Further, luteolin, luteolin 7-O-glucoside and luteolin 7-O-glucuronide suppressed the expression of iNOS and COX-2, and t-BHP-induced ROS generation in LPS-stimulated RAW 264.7 cells. These results clearly showed that the anti-inflammatory potential of ID, IDA and IS extract are primarily due to their higher content of luteolin 7-O-glucoside and luteolin 7-O-glucuronide, respectively.