Anti-Inflammatory Effects of Mitrephora sirikitiae Leaf Extract and Isolated Lignans in RAW 264.7 Cells

Abstract

Mitrephora sirikitiae Weeras., Chalermglin & R.M.K. Saunders has been reported as a rich source of lignans that contribute to biological activities and health benefits. However, cellular anti-inflammatory effects of M. sirikitiae leaves and their lignan compounds have not been fully elucidated. Therefore, this study aimed to investigate the anti-inflammatory activities of methanol extract of M. sirikitiae leaves and their lignan constituents on lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 mouse macrophage cells. Treatment of RAW 264.7 cells with the methanol extract of M. sirikitiae leaves and its isolated lignans, including (-)-phylligenin (2) and 3',4-O-dimethylcedrusin (6) significantly decreased LPS-induced prostaglandin E₂ (PGE₂) and nitric oxide (NO) productions. These inhibitory effects of the extract and isolated lignans on LPS-induced upregulation of PGE₂ and NO productions were derived from the suppression of cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) production, respectively. In addition, treatment with 2-(3,4-dimethoxyphenyl)-6-(3,5-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (3) and mitrephoran (5) was able to suppress LPS-induced tumor necrosis factor alpha (TNF-α) secretion and synthesis in RAW 264.7 cells. These results demonstrated that M. sirikitiae leaves and some isolated lignans exhibited potent anti-inflammatory activity through the inhibition of secretion and synthesis of PGE₂, NO, and TNF-α.

Keywords: Mitrephora sirikitiae; anti-inflammation; cytokines; lignans; mRNA expression; polyphenols.

Figure 1

Isolated lignans 1–6 from the methanol extract of M. sirikitiae leaves.

Figure 2

Cytotoxic effects of the methanol extract and isolated lignans 1–6 from M. sirikitiae leaves in RAW 264.7 cells; The percentage of cell viability of RAW 264.7 cells exposed to 0.05–50 µg/mL of methanol extract and 0.1–250 µg/mL of isolated lignans 1–6. Data are shown as the mean ± SEM (n = 5).

Figure 3

Effects of the methanol extract of M. sirikitiae leaves on LPS-induced PGE₂ and TNF-α secretion in RAW 264.7 cells; Serum-starved cells were pretreated with various concentrations of 0, 1, and 10 µg/mL of crude methanol extract for 3 h, and then stimulated with LPS (5 µg/mL) for 24 h at 37 °C. The levels of PGE₂ and TNF-α secreted into the medium were assessed by ELISA assay. The PGE₂ (A) and TNF-α (B) levels were quantified using a standard curve and expressed as the mean ± SEM (n = 3). *, p < 0.05 vs. vehicle; #, p < 0.05 vs. LPS.

Figure 4

Effects of the methanol extract and isolated lignans 1–6 from M. sirikitiae leaves on LPS-induced PGE₂ and TNF-α secretion in RAW 264.7 cells; Serum-starved cells were pretreated with crude methanol extract (C) (10 µg/mL), lignans (5 or 10 µg/mL), or indomethacin (10 µM) for 3 h, and then stimulated with LPS (5 µg/mL) for 24 h at 37 °C. The levels of PGE₂ and TNF-α secreted into the medium were assessed by ELISA assay. The relative PGE₂ (A) and TNF-α (B) levels were quantified using a standard curve and expressed as the mean ± SEM (n = 3). *, p < 0.05 vs. vehicle; #, p < 0.05 vs. LPS.

Figure 5

Effects of the methanol extract and isolated lignans 1–6 from M. sirikitiae leaves on LPS-induced nitrate and nitrite productions in RAW 264.7 cells; Serum-starved cells were pretreated with crude methanol extract (C) (10 µg/mL) or lignans (5 or 10 µg/mL) for 3 h, and then stimulated with LPS (5 µg/mL) for 24 h at 37 °C. The levels of nitrate and nitrite in the medium were assessed by a total nitric oxide assay. The relative nitrate (A) and nitrite (B) productions were quantified using a standard curve and expressed as the mean ± SEM (n = 3). *, p < 0.05 vs. vehicle; #, p < 0.05 vs. LPS.

Figure 6

Effects of the methanol extract and isolated lignans 1–6 from M. sirikitiae leaves on LPS-induced mRNA expressions of inflammatory biomarkers in RAW 264.7 cells; Serum-starved cells were pretreated with crude methanol extract (C) (10 µg/mL) or lignans (5 or 10 µg/mL) for 3 h, and then stimulated with LPS for 6 h at 37 °C. After treatment, the total RNA was extracted from the cells and the mRNA expressions of TNF-α (A), IL-6 (B), IL-10 (C), NF-κB (D), COX-2 (E), and iNOS (F) were analyzed by RT-qPCR with gene-specific primers. The relative mRNA levels were quantified and shown as the mean ± SEM (n = 3). *, p < 0.05 vs. vehicle; #, p < 0.05 vs. LPS.