Anti-Inflammatory Activity and Chemical Analysis of Different Fractions from Solidago chilensis Inflorescence

Abstract

Solidago chilensis Meyen (Compositae) is a species native to South America (Brazil) popularly known as arnica. In Brazilian popular medicine, inflorescences and rhizomes of this plant have been used since the end of the 19th century to replace the exogenous and hepatotoxic Arnica montana L. in the treatment of edema and inflammatory pathologies. Although the anti-inflammatory activity of S. chilensis is evidenced in the literature, there is a lack of studies with enriched fractions or compounds isolated from it. The objective of the current study was to characterize phytochemically and to evaluate the pharmacological action in vivo and in vitro of the crude extract and the different fractions (hexane, dichloromethane, acetal, butanolic, and aqueous) isolated from the inflorescence of S. chilensis. The inflorescence crude extract (ScIE) and fractions were administered by intraperitoneal route to mice at different doses. In an LPS-induced pleurisy model, inhibition of leukocyte influx was observed for the ScIE and all fractions tested, as compared to controls. Dichloromethane (ScDicF), butanolic (ScButF), and aqueous (ScAquF) were selected for further analysis as they showed the best inhibitory effects in leukocyte migration and inflammatory cytokine and chemokine production: TNF-α, CXCL1/KC, CXCL2/MIP-2, and CCL11/eotaxin-1. In LPS-stimulated J774A.1 cell line, ScIE and the ScDicF exhibited an inhibitory effect on nitric oxide (NO) production and downmodulated the COX-2 expression; ScAquF failed to modulate NO production and COX-2 expression. In phytochemical analysis, HPLC-UV-DAD chromatograms of ScDicF and ScAquF showed the main peaks with UV spectrum characteristics of flavonoids; chlorogenic acid and isoquercetin were the most present phytochemicals identified in the ScAquF, and a high number of n-alkanes was found in ScHexF. Our study was the first to address biological effects and correlate them to phytochemically characterized fractions from inflorescences of S. chilensis.

Figure 1

Effects of S. chilensis crude extract pretreatment (0.1, 1, 10, and 100 mg/kg) on pleural accumulation of total leukocytes (a), mononuclear cells (b), neutrophil (c), and eosinophil (d) populations in the pleural cavity of mice (n = 6) challenged with LPS (12.5 ng/animal). Dexamethasone was used as the reference drug. Mice were treated 1 hour before the challenge. Data represent mean ± standard error of the mean (SEM) (n = 6). Representative of two independent experiments performed. ^#p < 0.05 stimulated group (LPS) vs. nonstimulated group (saline); ^∗p < 0.05 treated vs. untreated group.

Figure 2

In vivo effect of the crude extract and different fractions of S. chilensis (10 mg/kg) on the recruitment of total leukocytes (a) and neutrophil (b) and eosinophil (c) populations in the pleural cavity of Swiss Webster mice stimulated (i.t.) with LPS (12.5 ng/per cavity). Dexamethasone (5 mg/kg, i.p.) and diclofenac sodium (10 mg/kg) were used as reference drugs. Mice were treated 1 hour before the challenge. Data represent mean ± standard error of the mean (SEM) (n = 6). Representative of three independent experiments performed. ^#p < 0.05 stimulated group (LPS) vs. nonstimulated group (saline); ^∗p < 0.05 treated vs. untreated group.

Figure 3

In vivo effect of crude extract and different fractions of S. chilensis (10 mg/kg) on the production of TNF-α (a), CXCL1/KC (b), CXCL2/MIP-2 (c), and CCL11/eotaxin-1 (d) mediators in the pleural lavage of Swiss Webster mice (n = 6) challenged with LPS (12.5 ng/animal, i.t.). Dexamethasone (5 mg/kg, i.p.) and diclofenac sodium (10 mg/kg, i.p.) were used as reference drugs. Data represent mean ± standard error of the mean (SEM) (n = 6). One representative ELISA from two independent experiments performed. ^#p < 0.05 stimulated group (LPS) vs. nonstimulated group (saline); ^∗p < 0.05 treated vs. untreated stimulated group (LPS).

Figure 4

(a) The NO production was determined by the Griess reagent through the supernatant collected 24 h after the LPS simulation in treated cells with S. chilensis fractions (10 μg/mL). Dexamethasone (20 pg/mL) was used as the reference drug. (b, c) Effect of S. chilensis fractions in COX-2 expression in LPS-induced J774A.1 cells. Data represent mean ± standard error of the mean (SEM) (n = 4). Representative of two independent experiments performed. ^#p < 0.05 stimulated group (LPS) vs. nonstimulated group (medium); ^∗p < 0.05 treated vs. untreated stimulated group (LPS).

Figure 5

HPLC-UV-DAD of the ScDicF (a) and ScAquF (b) for flavonoid characterization. The chromatogram A showed the main peaks with UV spectrum characteristics of chlorogenic acid (1), isoquercetin (4), quercitrin (8), afzelin (1), and quercetin (15). And the chromatogram B showed the main peaks with UV spectrum characteristics of chlorogenic acid (1) and isoquercetin (5). Chromatographic conditions: temperature = 250°C; flow rate = 1.0 mL min⁻¹; ʎ = 360 nm; Inj.V. = 10 μL. Mobile phase : acetonitrile (eluent A) and acetic acid : water (1 : 40) (eluent B). In the first 15 min, 14% of the A eluent and 84% of the B eluent were used. 35% of the eluent A in the following 30 min and 100% of eluent A in the last 2 minutes.

Figure 6

HPLC-UV-DAD of the ScDicF for the characterization of terpenes. The presence of the labdanic diterpene solidagenone was not found (retention time 71-72 min), but a great diversity of signs suggestive of terpenes can be observed, as well as a great sign (1) of a substance still not identified (26% of the area, retention time = 53.71 min). Chromatographic conditions: temperature = 300°C; flow rate = 1.0 mL min⁻¹; ʎ = 254 nm; Inj.V. = 10 μL. Isocratic elution system; 0.05% trifluoroacetic acid (54.94 mL) and acetonitrile (27.06 mL).