The effect of ellagic acid on the repair process of periodontal defects related to experimental periodontitis in rats

Abstract Objective This study aims to evaluate the effect of ellagic acid (EA) by measuring the levels of alveolar bone resorption and inflammatory and oxidative stress markers in the periodontal tissues and serum on the periodontal repair process related to experimental periodontitis in rats. Methodology Forty Wistar rats were divided into four study groups as follows: Group 1=healthy control (n=10); Group 2=EA control (15 mg/kg)(n=10); Group 3=periodontitis (n=10); Group 4=periodontitis+EA (15 mg/kg) (n=10). The periodontitis model was established by ligating bilateral mandibular first molars for 14 days. Then, rats were given normal saline or EA for another 14 days by gavage administration. Serum and gingiva myeloperoxidase (MPO) activity, 8-hydroxydeoxyguanosine(8-OHdG), and glutathione (GSH) levels were analyzed by ELISA. İmmunohistochemical analysis was used to detect Interleukin (IL)-6, IL-10, and tumor necrosis factor-alpha (TNF-α) immunoreactivities in the periodontal tissues. Alveolar bone loss (ABL) and attachment loss (AL) was evaluated by histomorphometry analysis. Results ABL and AL were statistically higher in group 3 than in groups 1, 2 and 4 and in group 4 than in groups 1 and 2 (p<0.05). MPO activities in gingival tissue and serum were significantly increased in group 3 compared to groups 1 and 2 (p<0.05). Significantly higher serum GSH levels, lower gingiva, and serum 8-OHdG levels, and MPO activity were observed in group 4 compared to group 3 (p<0.05). Rats with periodontitis (group 3) expressed significantly higher immunoreactivities of IL-6 and TNF-α and lower IL-10 immunoreactivity compared to those other groups (p<0.05). IL-6 and TNF-α immunoreactivities significantly decreased and IL-10 immunoreactivity increased in group 4 after the use of EA compared to group 3 (p<0.001). Conclusions Our findings showed that EA provides significant improvements on gingival oxidative stress and inflammatory markers and alveolar bone resorption in the repair process associated with experimental periodontitis. Therefore, EA may have a therapeutic potential on periodontitis.


Introduction
Periodontitis is one of the most widespread infectious inflammatory diseases. It occurs due to an imbalance between dental plaque bacteria and the host's inflammatory and immune responses. 1 During periodontitis, host cells produce excessive amounts of reactive oxygen species (ROS), such as those produced by myeloperoxidase (MPO), 8-hydroxydeoxyguanosine  and inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6, in response to bacterial infiltration. The release of these markers leads to the destruction of periodontal tissues. [2][3][4] The elimination of microbial dental biofilms is essential to the treatment of periodontal diseases. 1,5 Mechanical removal (scaling and root planning) is not sufficient for the complete elimination of periodontal microflora. 1,5 Therefore, pharmacologic agents (antiseptics, nonsteroidal anti-inflammatory drugs, and antibiotics) are used as adjunctive therapy to ensure the destruction of microorganisms. 6 When these drugs are used systemically with high doses, they can lead to complications such as antibiotic resistance and other side effects. 1 This is the reason why alternative natural products are needed for therapeutic use. 1,6,7 For such purpose, polyphenolic compounds, which have antioxidant, anti-cancer, and anti-inflammatory effects have been suggested as potential candidates. 8,9 Polyphenols have been reported to have several biological activities; they prevent oral disease, inactivate bacterial toxins, promote the antioxidant activity of oral fluids, exhibit antibacterial activity against periodontal pathogens, and can inhibit the proteolytic activity of Porphyromonas gingivalis. 3,9,10 Ellagic acid (EA), a polyphenol, is found in many fruits, including strawberries, walnuts, pomegranates, and grapes, and many medicinal plants. 11,12 EA has been reported to exhibit antioxidant, anti-cancer, anti-allergic, antiproliferative, and anti-inflammatory activities, as well as radical scavenging activity and inhibition of lipid peroxidation. 11-14 Ogawa, et al. 15 (2002) reported that EA eliminated superoxide and hydroxy anions. They stated that EA's effect was stronger than that of α-tocopherol and it was as potent as superoxide dismutase (SOD). EA has been suggested to suppress the production of various cytokines, such as IL-1β, IL-8, and TNF-α. 15 A recent review concluded that EA could be a promising agent for the treatment of various chronic diseases, especially Alzheimer's disease, ulcerative colitis, Crohn's disease, and diabetes. 16 Moreover, Promsong, et al. 17 (2015) showed that EA protects human gingival epithelial cells by reducing IL-2 and IL-8 levels and suppressing defense factors, such as human betadefensin 2 (hBD2) and secretory leukocyte protease inhibitor (SLPI). Bakkiyaraj, et al. 7 (2013)

Methodology Animals
Forty systemically and periodontally healthy adult (8 weeks old) male Wistar albino rats weighing an average of 220 to 250 kg were used in our study.
Rats were placed in separate plastic cages, provided food and water being ad libitum, housed at a room temperature of 22±1°C within 50% humidity conditions in a 12-hour light/dark cycle. All animal care and experimental protocols were approved by The sample size was estimated based on the results of biochemical biomarkers levels in gingival tissue between ellagic acid application groups and their control groups. A sample size of 10 per group was required for detection of a significant difference (80% power, two-sided 5% significant level).
The animals were randomly divided into four groups of ten rats each. 1) Group 1=periodontally healthy control (n=10), in which each rat was gavaged daily with 2 mL of saline by gastric intubation for 14 days; 2) Group 2=periodontally healthy rats+ EA control (15 mg/kg)(n=10), in which rat was gavaged daily with 2 mL of saline containing EA by gastric intubation for 14 days; 3) Group 3=experimental periodontitis group (n=10), in which each rat was gavaged daily with 2 mL of saline by gastric intubation for 14 days; 4) Group 4=experimental periodontitis group + EA (15 mg/kg) (n=10), in which rat was gavaged daily with 2 mL of saline containing EA by gastric intubation for 14 days. 19 The dosage and administration form of drugs were determined based on the literature. 19 Drug treatment began after periodontitis was induced.

Results
Histomorphometric findings Table 1   IL-10 immunoreactivity was significantly greater in group 2 than group 1 (p<0.01), and its level was lower in group 3 than in groups 1, 2, and 4 (p<0.001).          near-normal levels. 26 The study showed that the higher dose of EA (15 mg/kg) was more effective in restoring biochemical parameters to normal than the lower dose (7.5 mg/kg). 26 In another study of Favarin,et al. 11 (2013), they evaluated the anti-inflammatory effect of EA on acute lung injury and reported that 10mg\kg EA application had positive results on inflammatory cytokine levels. Since an effective dose has not yet been investigated in the treatment of periodontal diseases, in our study, we applied a 15 mg/kg dose of EA, 14,26,27 reporting both its anti-inflammatory and anti-oxidant effects.
In our study, we induced experimental periodontitis periodontitis. Therefore, consistent with previous study findings, 6,11,[30][31][32] our results indicate that anti-inflammatory activity of EA in periodontium is evidenced by reducing proinflammatory cytokines and increasing anti-inflammatory cytokine activities in gingival tissue.
Several studies have proved that periodontal tissue loss is associated with increased reactive oxygen species and decreased antioxidant levels. 35

Conclusions
Our study showed that EA provides significant improvements on gingival oxidative stress and inflammatory markers and alveolar bone resorption in the periodontal repair process after experimental periodontitis within the limits of the study. These effects could be attributed to the antioxidative and anti-inflammatory nature of ellagic acid. Thus, EA may have therapeutic potential on periodontitis.
However, studies should further explore and show the therapeutic mechanism of ellagic acid in periodontal inflammation and to examine its clinical effectiveness in human periodontal diseases, thus eliminating the limitations.

Conflict of interest and source of funding
The authors declare that they have no financial relationships related to any products involved in this study. The study was self-funded by the authors. The effect of ellagic acid on the repair process of periodontal defects related to experimental periodontitis in rats