New Compounds from Evolvulus linarioides and Their Anti-inflammatory Activity

The genus Evolvulus (Convolvulaceae) comprises approximately seventy species widely distributed in Brazil. Evolvulus species are used in traditional medicine for the treatment of inflammation-related diseases such as edema, dysentery, asthma, bronchitis and other respiratory problems. E. linarioides is an erect sub-shrub with small blue flowers. Previous phytochemical studies have resulted in the identification of chromones and sesquiterpenoids. This study aimed to reports the isolation, structure elucidation, and evaluate the immunomodulatory properties of new compounds. Fresh aerial parts of E. linarioides were collected at Matureia, Paraíba, Brazil in June 2009 and identified by M. de F. A. Access registration in the National Management System of Genetic Patrimony and Associated Traditional Knowledge was obtained under number A18E40E. A voucher specimen (AGRA et al. 6970) was deposited at the herbarium Lauro Pires Xavier, Federal University of Paraíba, Brazil. Powdered E. linarioides aerial parts (1.5 kg) were extracted using methanol (three times for 72 h) at room temperature. The resulting extract was concentrated under reduced pressure, yielded 300 g, which 100 g was suspended in MeOH−H2O (7:3) and successively partitioned with hexane, CH2Cl2, EtOAc and n-butanol. The hexane phase was subjected to normal silica gel column chromatography followed by repeated HPLC separation and gave two new compounds, 1 and 2. The n-butanol phase was chromatographed on a Sephadex LH-20 column followed by repeated HPLC separation to afford new compounds 3 and 4. The structures of the new compounds were elucidated by HR-ESIMS, ESIMS/MS, 1D and 2D NMR data. Thus, compounds 1−4 was characterized and named as linaritol A, linaritol B, linarioside A and linarioside B. Compounds 1, 2 and 4 were studied for their immunomodulatory properties. Initially, the cytotoxicity of 1, 2 and 4 was determined in J774 macrophage lineage. Immunomodulatory activity of 1, 2 and 4 was next evaluated in activation-induced macrophage cytokine and nitric oxide (NO) production. Cytokines (IL-1β) concentrations in supernatants from macrophage cultures were determined by enzyme-linked immunosorbent assay (ELISA). Nitric oxide production was estimated by measuring nitrite content in supernatants using the Griess method. Addition of 1, 2 and 4 (6.25–100 μM) to macrophage cultures stimulated with lipopolysaccaride (LPS) and interferon-γ (INF-γ) caused a concentration-dependent inhibition in NO production, as indicated by the nitrite concentrations in supernatants (p < 0.05). The effects of 1, 2 and 4 on NO production by activated macrophages were not due to toxicity, as J774 macrophage cultured in the presence of 1, 2 and 4 at 100 μM showed no cytotoxicity. The effects of 1, 2 and 4 on cytokine production by activated macrophages was next evaluated. Addition of 1, 2 and 4 (100 μM) to macrophage cultures stimulated with LPS and INF-γ significantly (p < 0.05) decreased the IL-1β production. These results indicate that these compounds, in non-cytotoxic concentrations, inhibit the production of inflammatory mediators by stimulated macrophages, thus exhibiting potential anti-inflammatory action.