Use of ethanol extracts of Terminalia chebula to prevent periodontal disease induced by dental plaque bacteria

Abstract

Background: The fruit of the Terminalia chebula tree has been widely used for the treatment of various disorders. Its anti-diabetic, anti-mutagenic, anti-oxidant, anti-bacterial, anti-fungal, and anti-viral effects have been studied. Dental plaque bacteria (DPB) are intimately associated with gingivitis and periodontitis. In the quest for materials that will prove useful in the treatment and prevention of periodontal disease, we investigated the preventive effects of an ethanol extract of Terminalia chebula (EETC) on DPB-induced inflammation and bone resorption.

Methods: The anti-bacterial effect of EETC was analyzed using the disc diffusion method. The anti-inflammatory effect of EETC was determined by molecular biological analysis of the DPB-mediated culture cells. Prevention of osteoclastic bone resorption by EETC was explored using osteoclast formation and pit formation assays.

Results: EETC suppressed the growth of oral bacteria and reduced the induction of inflammatory cytokines and proteases, abolishing the expression of PGE2 and COX-2 and inhibiting matrix damage. By stimulating the DPB-derived lipopolysaccharides, EETC inhibited both osteoclast formation in osteoclast precursors and RANKL expression in osteoblasts, thereby contributing to the prevention of bone resorption.

Conclusions: EETC may be a beneficial supplement to help prevent DPB-mediated periodontal disease.

Keywords: Dental plaque bacteria (DPB); Ethanol extract of Terminalia chebula (EETC); Gingivitis; Inflammation; Lipopolysaccharide (LPS); Osteoclast; Periodontitis.

Fig. 1

EETC suppresses bacterial growth. Inhibitory effect of EETC on the growth of Streptococcus mutans (S. mutans), Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), and different types of dental plaque bacteria (DPB^#1, DPB^#2, and DPB^#3) compared with ampicillin (Amp). The data shown are representative of three independent experiments

Fig. 2

Effect of the EETC on DPB-induced PGE2 and COX-2. a The effect of DPB on PGE2 levels in various cell types. Data are expressed as mean ± SE of three independent experiments. *P < 0.01 vs. bacteria-free control medium from each cell line. b The effect of LPS extracted from DPBs (DPB^#1-LPS, DPB^#2-LPS, and DPB^#3-LPS) on PGE2 levels. PBS was used for control. *P < 0.01, ^# P < 0.001 vs. PBS control medium from each cell line. c The effect of DPBs-LPS on COX-2 expression. mRNA expression were detected by RT-PCR. The inhibitory effects of EETC on PGE2 (d) and COX-2 (e) levels in DPB-LPS-treated cells. DMSO was used for control. Data represent the mean ± SE of three independent experiments performed by triplicate. ^# P < 0.001 vs. DMSO control (Ctl) medium, *P < 0.01 vs. DPB^#1 or DPB^#1-LPS medium. f The effect of EETC on the cell viability

Fig. 3

Effect of EETC on DPB-induced molecules involve in inflammation and tissue damage. a Inflammatory cytokine modulated by DPB-LPS and EETC in gingival epithelial cell. Analysis of culture medium (CM) collected from DMSO-treated cells, which served as the control. Altered factors are indicated with rectangles and circled numbers. The list altered factor is shown as fold-change in the graph. The data shown are representative of three independent experiments. *P < 0.01 vs. DPB1^#-LPS-treated medium, ^# P < 0.001 vs. DMSO control medium. b Protease modulated by DPB-LPS and EETC in gingival epithelial cell. Altered factors are indicated with rectangles and circled numbers. The list altered factor is depicted in the graph and is shown as fold-change. The data shown are representative of three independent experiments. *P < 0.01 vs. DPB1^#-LPS-treated medium, ^# P < 0.001 vs. DMSO control medium. c Effect of tissue damage by DPB-LPS and/or EETC on FITC-conjugated gelatin matrix-coated coverslips. The data shown are representative of five independent experiments

Fig. 4

Effect of EETC on DPB-induced osteoclastic bone resorption. a For osteoclast formation, mouse BMMs were treated with M-CSF and RANKL and were stained to detect expression of TRAP. Pit formation was also performed in M-CSF and RANKL on calcium phosphate apatite-coated plates, and light microscopy indicated resorptive pitting. To observe the stimulating effect of DPB-LPS on osteoclast formation and pit formation, both assays were performed at a low concentration of RANKL (10 ng/ml). The graph shows the total number of TRAP-positive multinucleated (≥3 nuclei) osteoclasts (MNC) per well. Data are expressed as mean ± SE of three independent experiments. *P < 0.01 vs. only 10 ng/ml RANKL condition, ^# P < 0.001 vs. DPB1^#-LPS plus 10 ng/ml RANKL condition. b RANKL and OPG mRNA expression was analyzed in hFOB1.19 human osteoblastic cells. Eco-LPS (LPS from Escherichia coli) was the positive control. The data shown are representative of three independent experiments. c Effect of EETC on DPB^#1-LPS-induced RANKL and OPG mRNA expression were analyzed by RT-PCR. The ratio of RANKL to OPG was determined after normalization to the intensity of GAPDH. The data shown are representative of three independent experiments. *P < 0.001 vs. control (Ctl), ^# P < 0.001 vs. DPB^#1-LPS condition