Anti-Inflammatory and Anti-Oxidant Potential of the Root Extract and Constituents of Doronicum austriacum

Abstract

Doronicum austriacum Jacq., Asteraceae, is a plant which is used in traditional alpine medicine. Historical sources describe the medical use of the root, but up until now only a few studies evaluated its pharmacological properties. The evaluation of the dichloromethane extract, and its major compounds for their anti-inflammatory and anti-oxidant potential was performed in macrophages J774A.1 and C6 astrocytes. Nitric oxide (NO) and reactive oxygen species (ROS) release, as well as nitrotyrosine formation, were evaluated. Moreover, in order to evaluate the potential anti-proliferative activity, under the same experimental conditions, 3-(4,5-dimethyltiazol-2yl)-2,5-phenyl-2H-tetrazolium bromide (MTT) assay was also performed. Our results indicate that Doronicum austriacum has a significant effect in inhibiting both pro-inflammatory and pro-oxidative mediators. All isolated compounds were able to significantly inhibit NO and ROS release both in macrophage and in astrocytes cells, even if the effect was more pronounced in macrophage. In particular, among the tested compounds, 6,12-dihydroxy-(-)-2S-tremetone exerted stronger activity. Both extract and single compounds did not affect cellular viability. This study provides evidence for the pharmacological anti-inflammatory and anti-oxidant potential of Doronicum austriacum extract. These effects could be due to the activity of its major constituents and subsequent identification of benzofurans as a promising compound class to combat inflammation and related diseases.

Keywords: Doronicum austriacum; NO; ROS; astrocytes; benzofurans; inflammation; macrophages; nitrotyrosine; oxidative stress.

Figure 1

Chromatogramm of the HPLC-DAD analysis of the investigated dichloromethane (DCM) extract of the roots of Doronicum austriacum (2.5 mg/mL MeOH) at 205 nm. Analytical conditions: stationary phase: Phenomenex Synergi Max-RP 80 Å, 4 µm (4.6 mm × 150 mm); temperature: 35 °C; mobile phase: A = water + 0.025% TFA, B = acetonitrile; flow rate: 1.00 mL/min; detection: 205 nm; injection volume: 10 µL; solvent composition during analysis: 0′: 75% A; 10′: 60% A; 30′: 30% A; 35′: 2% A; 45′: stop; post time: 10′.

Figure 2

Chemical structure of compounds 1 to 3.

Figure 3

Measured (acetonitrile) and calculated ECD-spectra of compound 1.

Figure 4

Effect of Doronicum austriacum DCM root extract on nitrite release, index of NO production (A,D), measured by Griess reaction, iNOS expression, evaluated by cytofluorimetry (B,E), and on ROS production (C,F), measured by DCHF assay, in J774A.1 macrophages (A–C) and C6 cells (D–F). Data are expressed as mean ± S.E.M.; *** denotes p < 0.001 vs. LPS (1 μg/mL) or LPS + IFN-γ (1 μg/mL; 100 U/mL). L-NAME (1 μM) was used as positive control for inhibition of nitrite release and iNOS expression and inhibited nitrite production with a percentage of 49.00 ± 2.08% and with a percentage of 59.00 ± 0.74% iNOS expression in J774A.1 macrophages. In C6 cells, L-NAME inhibited nitrite release with a percentage of 39.33 ± 4.37% and iNOS expression with a percentage of 46.21 ± 2.65%. N-acetyl cysteine (NAC; 10 mM) was used as positive control for ROS release inhibition, and gave a percentage of inhibition of ROS production of 34.50 ± 0.50% in J774A.1 and of 43.33 ± 1.45% in C6 cells, respectively.

Figure 5

Effect of Doronicum austriacum DCM root extract (25 and 50 µg/mL) on nitrotyrosine formation evaluated by confocal microscopy in J774A.1 macrophages (A) and C6 astrocytes (B) alone, or in combination with LPS (1 μg/mL) or LPS + IFN-γ (1 μg/mL; 100 U/mL) after 24 h.

Figure 6

Effect of compounds 1–3 (5–50 µM) on nitrite release (A,B), iNOS expression (C,D), and on ROS production (E,F), in J774A.1 macrophages (A,C,E) and C6 astrocytes (B,D,F) after 24 h. Data are expressed as mean ± S.E.M.; *** denotes p < 0.001 vs. LPS (1 μg/mL) or LPS + IFN-γ (1 μg/mL; 100 U/mL). °°°, °°, ° denote p < 0.001, p < 0.01, p < 0.05 vs. 2. òòò, òò, ò denote p < 0.001, p < 0.01, p < 0.05 vs. 3. L-NAME (1 μM), used as positive control for NO and iNOS inhibition gave a percentage of inhibition of 49.00 ± 2.08% for nitrite production of and a percentage of inhibition of 59.00 ± 0.74% of iNOS expression in J774A.1 macrophages. In C6 astrocytes, L-NAME gave a percentage of inhibition of 39.33 ± 4.37% and 46.21 ± 2.65% of nitrite production and iNOS expression, respectively. NAC (10 mM) was used as positive control for ROS release and gave a percentage of inhibition of ROS production of 34.50 ± 0.50% in J774A.1 macrophages and 43.33 ± 1.45% in C6 astrocytes.