Unusual Secondary Metabolites of the Aerial Parts of Dionysia diapensifolia Bioss. (Primulaceae) and Their Anti-Inflammatory Activity

Abstract

The genus Dionysia, belonging to the Primulaceae family, encompasses more than 50 species worldwide with a center of diversity located in the arid Irano-Turanian mountains. In this study, a phytochemical investigation of the aerial parts of D. diapensifolia Bioss. led to the isolation of 24 phenolic compounds 1-7 and 9-25, and one sesquiterpenoid 8. Compound 1 was identified as new natural product, while isolation of 2 and 3, already known as synthetic products, from a natural source is reported for the first time in the present study. Isolation of compound 8 from a Dionysia species and indeed the whole Primulaceae family is reported for the first time too. Structure elucidation was performed by extensive spectroscopic analyses (1D-, 2D-NMR, and MS), and by comparison with reported literature data. Furthermore, DP4+ chemical shift probability calculations were performed to establish the relative configuration of compound 1. Additionally, subfractions obtained by liquid-liquid extraction of the methanolic extract of the plant, and subsequently the isolated new and selected known compounds 1-4, 6, 8-11 obtained from the diethyl ether subfraction were investigated for their inhibitory effect on NO release and iNOS and COX-2 expression in J774A.1 murine macrophages. The results showed a potential anti-inflammatory activity of the obtained subfractions, of which the diethyl ether subfraction was the most active one in inhibiting NO release and COX-2 expression (p < 0.001). Among the investigated isolated compounds, compound 4 significantly (p < 0.001) inhibited NO release and iNOS and COX-2 expression in a comparable manner like the used positive controls (L-NAME and indomethacin, respectively). Moreover, other isolated substances displayed moderate to high inhibitory activities, illustrating the potential anti-inflammatory activity of Dionysia diapensifolia.

Keywords: Dionysia diapensifolia Bioss.; anti-inflammatory; flavone; macrophages; phenolic compounds.

Figure 1

Structures of the isolated compounds from the aerial parts of Dionysia diapensifolia.

Figure 2

Key COSY and HMBC correlations of the two new phenolic compounds, 1 and 2, from aerial part of D. diapensifolia.

Figure 3

Chromatogram of the HPLC-DAD analysis of the diethyl ether subfraction (A) and an enlarged view (B) at λ 254 nm obtained from the methanolic extract of the aerial parts of D. diapensifolia. Analysis conditions: stationary phase: Phenomenex Aqua C18 5 µm, 250 × 4.6 mm; mobile phase: A = H₂O + 0.02% TFA, B = acetonitrile; gradient: 0 min: B = 2%; 20 min: B = 50%; 40 min: B = 98%; 50 min B = 98%; T: 35 °C; flow: 1 mL/min; sample concentration and injection volume: 8 mg/mL, 10 µL.

Figure 4

Effect of isolated compounds from aerial parts of Dionysia diapensifolia on NO release inhibition in J774A.1 macrophages (n = 3). Data are expressed as mean ± S.E.M.; *** denotes p < 0.001 vs. LPS (1 µg/mL). L-NAME (5, 10, and 50 µM) was used as a positive control.

Figure 5

Effect of isolated compounds from aerial parts of Dionysia diapensifolia on iNOS expression in J774A.1 macrophages (n = 3). Data are expressed as mean ± S.E.M.; *** denotes p < 0.001 vs. LPS (1 µg/mL). L-NAME (5, 10, and 50 µM) was used as a positive control.

Figure 6

Effect of isolated compounds from aerial parts of Dionysia diapensifolia on COX-2 expression in J774A.1 macrophages (n = 3). Data are expressed as mean ± S.E.M.; *** denotes p < 0.001 vs. LPS (1 µg/mL). Indomethacin (5, 10, and 50 µM) was used as a positive control.

Figure 7

HPLC-DAD chromatogram of methanolic extract of the aerial parts of D. diapensifolia. Analysis condition: stationary phase: Phenomenex Synergi Max-RP C18 4 µm, 150 × 4.6 mm; mobile Phase: A = H₂O + 0.02% TFA, B = acetonitrile; gradient: 0 min: B = 2%; 20 min: B = 98%; 35 min: B = 98%; T: 35 °C; flow: 1 mL/min; sample concentration and injection volume: 2 mg/mL, 10 µL; detection = 254 nm.