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

Inflammation is the biological response of tissues to infection with pathogens and cell damage [15]. When harmful stimuli cause an inflammatory response, the body’s innate immune system involves the phagocytic activation of leukocytes and macrophages, and the increased release of inflammatory cytokines. Uncontrolled inflammation leads to disease progression because of the excessive production of inflammatory cytokines [16]. DPB are a leading cause of inflammation in periodontal disease [17]. Untreated gingivitis readily progresses to periodontitis and leads to tissue destruction. Bacterial plaque elaborate various compounds that elicit an inflammatory response and lead to destruction of the gingival tissue, which may progress to destruction of the periodontal ligaments. Plaque control is required to prevent further progression of inflammation. In this study, we tried to elucidate the critical factors involved in plaque bacteria-induced inflammation and effective natural anti-inflammatory substances for plaque bacteria control.

The in vitro anti-bacterial activity of EETC against DPB was evaluated. Disc diffusion assay results showed that EETC effectively suppressed the DPB growth and the anti-bacterial activity of EETC were increased with increasing concentration of extract. This concentration of EETC was not cytotoxic to gingival epithelial cells, gingival fibroblasts and macrophage. The induction of inflammatory factors and proteases that contributed to the inflammatory reaction were also abolished by EETC. These results indicate that EETC could effectively suppress the inflammatory responses induced by plaque bacteria. The collective data indicate that EETC is useful in reducing bacterial plaque accumulation and gingival inflammation, thereby preventing periodontal disease. Since the efficacy and safety of EETC has been established, it can be developed into a mouthwash without added alcohol. Many commercially available mouthwashes contain alcohol, which increases the risk of oral cancers and dry mouth.

LPS is also known as lipoglycans and endotoxins. The molecules is a component of the outer membrane of the cell wall of gram-negative bacteria, and is one of the most powerful bacterial virulence factors in the pro-inflammatory response [18]. Initiation of the inflammatory response by LPS stimulates a strong immune response. LPS acts as the prototypical endotoxin through binding to the CD14/TLR4/MD2 receptor complex in many cell types, but especially in monocytes, dendritic cells, macrophages, and B cells, which promotes the secretion of pro-inflammatory cytokines, nitric oxide, and eicosanoids [19]. Therefore, LPS is a central molecule in the pathogenesis of certain bacterial infections [20]. In this study, in vitro co-culture with gingival epithelial cells and plaque bacteria caused pH changes of the culture medium during 24 h of incubation. Therefore, LPS was extracted from DPBs and its capacity to induce inflammation was verified. DPB-LPS significantly increased the levels of inflammatory factors in gingival epithelial cells, gingival fibroblasts and macrophages. EETC also effectively suppressed the levels of DPB-LPS-induced inflammatory factors.

EETC significantly suppressed growth of DPB and DPB-mediated periodontal disease. Antibiotics are used to both treat and prevent bacterial infections [21]. Antibiotics like penicillin and erythromycin used to be highly effective against many bacterial species and strains. They are now less effective because these organisms have developed increased resistance to the drugs [22]. Dental plaque, which adheres to the gingival sulcus, consists mainly of bacterial cells, salivary polymers, and bacterial extracellular products. Bacterial LPS stimulates the host cells, including macrophages, fibroblasts, and epithelial cells, which abundantly produce cytokines, thereby triggering a direct or an indirect immune response through inflammatory processes [23]. The growth of bacterial biofilms increases antibiotic resistance and has been implicated in the etiopathogenesis of gingivitis and periodontal disease by inducing inflammation.

An effective substance is needed that will prevent and treat bacterial plaque-mediated disease by controlling bacterial growth and the inflammatory reaction. The fruits of the T. chebula, which has been amply verified as being medicinally valuable, has been used to treat cancer, cardiovascular diseases, paralysis, leprosy, ulcers, gout, arthritis, epilepsy, cough, fever, diarrhea, gastroenteritis, skin disorders, urinary tract infection, and wound infections [10, 24]. T. chebula fruit is also effective in the treatment of bacterial infections [25, 26]. Clinical trials of T. chebula fruit extract as a mouthwash preparation have reported reduced plaque accumulation and gingival inflammation [27, 28]. The present findings provide mechanistic details for these beneficial effects.

Most dental care products exert their antibacterial effects by eliminating oral bacteria. Instead, LPS from other types of bacterial cells, such as E. coli and Pseudomonas aeruginosa, have been used to induce the inflammation. Inflammatory events related to DPB-derived endotoxins have not been evaluated as intensively [29, 30], and the value of anti-microbial dental care products to treat periodontal disease has not been sufficiently verified. Thus, there was a need to simultaneously investigate inflammatory events due to DPB-derived endotoxin and elucidate an effective anti-microbial component for periodontal disease. Here we demonstrate that DPB and DPB- significantly increase inflammation-associated elements such as COX-2 and PGE2. Many inflammatory molecules and proteases are also upregulated by DPB-LPS and lead to matrix damage. In addition, DPB-LPS stimulation promotes osteoclast formation, causing bone resorption. The risk of bone loss tends to increase in patients with inflammatory conditions [31]. Inflammatory cytokines modulate osteoblast and osteoclast activity [32]. Interleukins (IL), tumor necrosis factor (TNF), and tumor necrosis factor receptor superfamily members (TNFRSF) perturb bone homeostasis, leading to increased cartilage degradation and bone resorption by osteoclasts and inhibiting bone formation by osteoblasts [33].

In the present study, IL-3, IL-16, CXCL10, TNFRSF1A, TNFRSF1B, M-CSF 1, and CCL4 were increased in DPB-LPS-stimulated gingival epithelial cells. Proteases including MMP-3, MMP-7, KLK 10, CTSC, and PC9, which were also increased by DPB-LPS stimulation, might also contribute to the inflammatory reaction, thereby leading to matrix degradation and bone destruction. However, the levels of inflammation-related molecules and osteoclastic bone resorption were significantly reduced by treatment with EETC. EETC also exhibited anti-bacterial properties by arresting the growth of DPB. These findings suggest that the EETC could be used to treat DPB-mediated periodontal disease.

Osteoclast differentiation and bone resorption activity require stimulation by the receptor activator of nuclear factor-kappaB (RANKL) expressed on osteoblasts [34]. In the present study, increased RANKL mRNA expression was observed in DPB-LPS-stimulated hFOB1.19 human osteoblast cells. In addition, DPB-LPS stimulated osteoclast formation and bone resorption under RANKL-limited conditions. These results are consistent with the results of previous studies showing that LPS stimulated osteoclastogenesis [35–37]. These results indicate that DPB-LPS promotes functional osteoclast formation through osteoblast-dependent and osteoblast-independent pathways. DPB-LPS-induced osteoclast formation and bone resorption were also significantly abolished by the EETC treatment.