Effect of Punica granatum L. (pomegranate) on the oral pathogens and the identification of metabolites responsible for beneficial effects

Abstract

Dental caries and periodontal diseases are infections caused by accumulation and multiplication of bacteria in the oral cavity. Dental caries is caused by demineralisation of the tooth due to the acids produced by Streptococcus mutans from fermentable carbohydrates. Pathogenic characteristics of S. mutans include biofilm formation, production of extracellular polysaccharides, acidogenicity and aciduricity. Periodontal diseases which are caused by many anaerobic oral bacteria are diseases of the tooth supporting tissues. Among these anaerobes, Porphyromonas gingivalis (Pg) has been identified as a keystone pathogen which also produces cysteine proteases, also called gingipains that cause tissue destruction and inactivate the host immune system. Both these oral infections can be prevented by controlling bacterial biofilm also called plaque with antimicrobial oral hygiene products. Medicinal plants have shown antimicrobial activity against these oral bacteria. Various parts of the Punica granatum L. fruit, commonly known as pomegranate are known to have antimicrobial effect against many bacteria. Its use in the oral cavity to control plaque is also highlighted. However, the effect of the peel extracts on the virulence factors of cariogenic bacteria and the periodontal pathogen, and the responsible beneficial compound has not been studied. The aim of this study was to evaluate the effect of pomegranate fruit peel on the oral pathogens and identify the metabolites responsible for the beneficial effects. Methods Pomegranate peel and seed extracts were prepared using methanol, DCM:methanol, hexane, water and acetone. Minimum bactericidal concentrations (MBC) were obtained against S. mutans and lactobacilli using double dilution technique. Based on MBC, three different subinhibitory concentrations of the methanol peel extract were evaluated for their effect on biofilm formation, acid and extracellular polysaccharides production in S. mutans. The MBC against P. gingivalis, Fusobacterium, Capnocytophaga sp. and P. intermedia were also determined. The DCM: methanol crude peel extract was fractionated using solvent-solvent extraction and chromatography. The MBC’s of fractions and subfractions against S. mutans were determined. Bioactive subfractions with the best activity were examined for the inhibition effect on biofilm formation and acid production. The results were analysed using Kruskal-Wallis and Wilcoxon Rank Sum Tests. The effect of the crude extract and subfractions on the proteinase production in P. gingivalis was also studied. Purification and structural elucidation were performed by means of preparative HPLC, high resolution mass spectrometry (HRMS), nuclear magnetic resonance (NMR) and Fourier transform infra-red spectroscopy (FTIR). In addition, the cytotoxicity of the crude plant extract and the bioactive subfractions on HEK 293 embryonic kidney cell was also established. Results The MBCs for the crude peel and seed extract against S. mutans and Lactobacilli ranged between 6.25 and 25 mg/ml whereas for the periodontal pathogens P. gingivalis, P. intermedia, Capnocytophaga and Fusobacterium they ranged between 0.025 – 3.125 mg/ml. MBC values for the seed extracts were much higher than the peel extracts. After 6 and 24 h, at subinhibitory concentrations, the methanol crude peel extract significantly reduced biofilm-formation by 91 % and 65 % respectively (p < 0.01). It also significantly inhibited acid production in S. mutans (p < 0.01). The crude peel extract did not inhibit the production of soluble extracellular polysaccharides (EPS) in either the biofilm or the planktonic growth. However, it significantly reduced the insoluble EPS in the biofilm and the plantktonic (p < 0.01) form of S. mutans. Chemical analysis resulted in 4 fractions and 10 subfractions. The subfractions F 3.2 and F 4.1 displayed the best MBC on S. mutans of 6.25 and 12.5 mg/ml respectively. Subfractions F 3.2 and F 4.1 significantly reduced the biofilm formation (p <0.01) and acid production compared to the controls. In some tests the reduction in the acid production was due to the antibacterial effect of the subfractions. The crude peel extract, subfractions F 3.2 and F 4.1 reduced proteolytic activity in P. gingivalis where Arg- gingipain was inhibited up to 54 % and Lys- gingipain up to 93 %. The UHPLC-MS showed presence of hydrolysable tannins, major bioactive components in the peel. Structural elucidation by NMR identified 2 compounds to be present, -sitosterol in the subfration F 3.2 while galloyl-HHDP-hexoside was found in both subfractions. At 1.6 mg/ml concentration crude peel extract, subfrations F 3.2 and, F 4.1 showed cell viability of 62, 24 and 58 % respectively. Conclusions Antibacterial activity of the peel extracts of P. granatum proved to be better than the seed extracts therefore the peel extract was further explored. Subfractions, which were identified as -sitosterol and galloyl-HHDP-hexoside showed anti-biofilm and anti-acidogenic activity which were present in the crude extract. However, the purified compounds did not show improved activity. The crude extract and the purified compounds also inhibited the growth of periodontal pathogens and proteolytic activity in P. gingivalis. At high concentrations the crude extract and the compounds will kill the cariogenic bacteria and periodontal pathogens. At low concentrations they will reduce the virulence of these two major oral pathogens. The crude extract and identified compounds proved to be safe. This suggests that P. granatum peel has the potential to control and prevent dental caries and periodontal diseases.