Isolation and characterization of anti-oxidant and anti-inflammatory constituents from Echinacea purpurea (L.) Moench

A variety of anecdotal claims have been attributed to Echinacea purpurea (L.) Moench, including immune systems enhancement and pain and fever relieving properties. However, questions still remain as to the identity and mode of action of the compounds responsible for these biological effects. Research to identify specific constituents responsible for reported anecdotal claims has established several compounds with significant biological potential. Alkamides (undeca-2E,4Z-dien-8,10-diynoic acid isobutylamide, undeca-2Z,4E-dien-8,10-diynoic acid isobutylamide, dodeca-2E,4Z-dien-8,10-diynoic acid isobutylamide, undeca-2E,4Z-dien-8,10-diynoic acid 2-methylbutylamide, dodeca-2E,4Z-dien-8,10-diynoic acid 2-methylbutylamide, and a mixture of dodeca-2E,4 E,8Z,10E-tetraenoic acid isobutylamide and dodeca-2E,4E,8Z,10 Z-tetraenoic acid isobutylamide) were isolated from E. purpurea roots and examined in in vitro model systems. Significant cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) inhibitory activities, ranging from of 36–60% and 15–46%, respectively, were observed for these compounds at a concentration of 100 μg/mL. The alkamides from E. purpurea were mosquitocidal against Aedes aegyptii L. larvae at 10 and 100 μg/mL. Flavonoids (quercetin-3-O-glucoside, quercetin-3-O-rutinoside, and kaempferol-3-O-robinobioside) and anthocyanins (cyanidin-3-O-β-D glucopyranoside and cyanidin-3-O-malonyl-(1 → 6)-β-D glucopyranoside), were isolated from methanolic extracts of lyophilized E. purpurea flowers and examined for antioxidant and COX-1 and COX-2 inhibitory activities. Four additional reported constituents of E. purpurea, caffeic acid and the caffeoyl derivatives caftaric acid, chlorogenic acid, and cichoric acid were also tested. The anthocyanins demonstrated the greatest cyclooxygenase inhibitory activities at 100 μg/mL, with inhibitions of COX-1 and COX-2 ranging from 23–28%. Co-treatment of RAW 264.7 macrophages with several of the isolated compounds (at 1, 10, 50, and 100 μg/mL) and 100 or 1000 μg/mL lipopolysaccharide (LPS) suppressed induction of the pro-inflammatory cytokines IL-6 and TNF-α. Cytotoxicity was noted at 100 μg/mL for all four alkamides, based upon results obtained from a concurrent MTT assay. Cells treated with compounds from E. purpurea did not induce IL-6 or TNF-α production; however, co-treatment of macrophages with caffeoyl derivatives (50 and 100 μg/mL) and LPS (100 and 1000 μg/mL) resulted in an increase in IL-6 and TNF-α levels, suggesting potential immune-enhancing activity. The alkamides demonstrated the greatest ability to reduce LPS-induced IL-6 and TNF-α production. The anti-inflammatory activity of the alkamides reported here supports the claims of pain and fever relief associated with Echinacea use. The opposing activities noted for compounds from E. purpurea demonstrate the importance of testing individual compounds rather than crude extracts.