Anti-Inflammatory Effect of Charantadiol A, Isolated from Wild Bitter Melon Leaf, on Heat-Inactivated Porphyromonas gingivalis-Stimulated THP-1 Monocytes and a Periodontitis Mouse Model

Abstract

Porphyromonas gingivalis has been identified as one of the major periodontal pathogens. Activity-directed fractionation and purification processes were employed to identify bioactive compounds from bitter melon leaf. Ethanolic extract of bitter melon leaf was separated into five subfractions by open column chromatography. Subfraction-5-3 significantly inhibited P. gingivalis-induced interleukin (IL)-8 and IL-6 productions in human monocytic THP-1 cells and then was subjected to separation and purification by using different chromatographic methods. Consequently, 5β,19-epoxycucurbita-6,23(E),25(26)-triene-3β,19(R)-diol (charantadiol A) was identified and isolated from the subfraction-5-3. Charantadiol A effectively reduced P. gingivalis-induced IL-6 and IL-8 productions and triggered receptors expressed on myeloid cells (TREM)-1 mRNA level of THP-1 cells. In a separate study, charantadiol A significantly suppressed P. gingivalis-stimulated IL-6 and tumor necrosis factor-α mRNA levels in gingival tissues of mice, confirming the inhibitory effect against P. gingivalis-induced periodontal inflammation. Thus, charantadiol A is a potential anti-inflammatory agent for modulating P. gingivalis-induced inflammation.

Keywords: Porphyromonas gingivalis; anti-inflammation; bitter melon; charantadiol A.

Figure 1

Schemes of the extraction and isolation of charantadiol A. Due to the C-19 hemiacetal carbon in charantadiol A (1), its 19(S) epimer (2) was also found as impurity in the NMR spectra.

Figure 2

Effects of sub-fraction (Fra. 5-3) isolated from wild bitter melon leaf extract on P. gingivalis-induced pro-inflammatory cytokine productions in human monocytic THP-1 cells. Cells were incubated with 0.1% (v/v) DMSO (as a vehicle control), or co-cultured with P. gingivalis (M.O.I. = 10) and different concentrations of sub-fraction 5-3 (2.5, 5 or 10 μg/mL) for 24 h. The cell-free culture supernatants were subsequently collected and analyzed for the content of IL-8 and IL-6. Experiments were performed three times in triplicate. Each value represents the mean ± SD. Values with different letters are significantly different at p < 0.05.

Figure 3

Effects of charantadiol A on P. gingivalis-induced pro-inflammatory cytokine production in human monocytic THP-1 cells. Cells were incubated with 0.1% (v/v) DMSO (as a vehicle control), or co-cultured with P. gingivalis (M.O.I. = 10) and different concentrations of charantadiol A (5, 10 or 20 μM) for 24 h. The cell-free culture supernatants were subsequently collected and analyzed for the content of IL-8 and IL-6. Experiments were performed three times in triplicate. Each value represents the mean ± SD. Values with different letters are significantly different at p < 0.05.

Figure 4

Effect of charantadiol A on P. gingivalis-induced triggering receptors expressed on myeloid cells-1 (TREM-1) mRNA expression. THP-1 cells were cultured with 0.1% (v/v) DMSO (vehicle control), or co-incubated with P. gingivalis (M.O.I. = 10) and different concentrations of charantadiol A (5, 10 or 20 μM) for 4 h to determine the TREM-1 mRNA levels. Experiments were performed three times in triplicate. Each value shows the mean ± SD. Values with different letters are significantly different at p < 0.05.

Figure 5

Charantadiol A suppressed pro-inflammatory cytokine mRNA expressions in P. gingivalis-stimulated gingival tissue of mice. Each value shows the mean ± SD. Values with different letters are significantly different at p < 0.05.