Periodontal implications of Salvadora persica L

University dissertation from Stockholm : Karolinska Institutet, Dept of Dental Medicine

Abstract: Periodontal disease is a major public health problem throughout the world and is the most common cause of tooth loss in adults. For periodontitis to develop, specific gram-negative, anaerobic bacteria must predominate in the subgingival microflora, but simple the presence of these bacteria is insufficient to cause periodontal disease as disease progression involves a complex, sequential relationship between infection, inflammation and tissue destruction. Treatment of periodontal disease targets inflammation through reduction of pathogenic bacteria, eliminating of pathogenic pockets, and use of mechanical debridement techniques along with chemically effective plaque-control agents. To treatment efficacy, disease management should be a holistic approach with medications that have proven antibacterial, and anti-inflammatory properties. Popular concern about the side effects of synthetic drugs and the increasing antibiotic resistance is significant and growing. In the periodontal field, efforts to find natural antimicrobial agents for preventing and treating periodontal disease have been stepped up. Many communities use the Miswak, a chewing stick made from the roots, twigs, or stems of Salvadora persica L., as an oral health tool. Studies have indicated that the Miswak has both an antibacterial and an anti-inflammatory effect, conferred by benzyl isothiocyanate (BITC), the major multi biologically active component. Hence, the overall aim of this thesis was to investigate the possible chemical effects of chewing sticks made from S. persica L., Miswak, in order to create optimum-use guidelines for the Miswak as a way of improving the treatment efficacy of periodontal disease. Study I, a double-blind, cross-over trial evaluated the efficacy of active and inactive S. persica L. on dental plaque, subgingival microbiota, and gingival inflammation in 24 patients with mild-to-moderate periodontitis. Clinical parameters were evaluated before and immediately after all experimental periods. Samples of subgingival plaque and gingival crevicular fluid were also taken. Plaque samples were analyzed with a DNA-DNA hybridization technique. Compared with pre-treatment values, populations of 16 bacterial species increases significantly in the group using a placebo inactive Miswak, (p<0.05), while no species showed a similar change in the group using active Miswak. The plaque and gingival indices on all tooth surfaces, however, differed non-significantly between the active and inactive Miswak groups. Study II measured the amount of BITC released into the mouth after brushing with Miswak and assessed its retention time in saliva. The study also tested the antibacterial and cytotoxic efficacy of the salivary BITC. Salivary BITC and BITC on used brushes were quantified using solid phase microextraction and gas chromatography-mass spectrometry. The antibacterial effects of BITC and Miswak Essential oil (EO) against Aggregatibacter actinomycetemcomitans, Haemophilus influenzae, and Porphyromonas gingivalis; and cytotoxic effects on human gingival fibroblasts and oral keratinocytes were investigated. The highest concentrations of the active compounds were detected in saliva after using a Miswak tip for one time and immediately. Concentrations were significantly decreased when the Miswak tip was used more than once and thus after 10 minutes. Miswak EO and BITC inhibited the growth of the tested bacteria in a dose dependent manner, with P.gingivalis being the most sensitive. A methyl tetrazolium-based MTT assay found BITC and Miswak EO to be cytotoxic to gingival fibroblasts while oral keratinocytes exhibiting resistance, which suggests that, to ensure maximum effect, the Miswak tip should be cut before each use. Study III investigated the anti-inflammatory activities of Miswak EO from S. persica and the main antimicrobial compound BITC by evaluating their effect on the secretion of pro-inflammatory mediators from human gingival fibroblasts and oral keratinocytes: interleukin (IL)-6, IL-8, and matrix metalloproteinase (MMP)-1 from IL-1β (300 pg/ml) stimulated and non-stimulated gingival fibroblasts and oral keratinocytes. An ELISA assessment found a significant decrease in the levels of IL-1β-induced IL-6 and IL-8 in gingival fibroblasts and oral keratinocytes treated with Miswak EO and BITC. In both cell types, levels if secreted MMP-1 were unaltered. Viewing these results, we concluded that Miswak has a weak effect on the subginival microbiota of patients with periodontitis. Cutting a fresh Miswak tip each brushing time is necessary in order to attain a sufficient antibacterial effect. Claimed brushing frequencies with Miswak of five times a day should be considered for maximal effect. Results also showed that Miswak has an anti-inflammatory effect on the pro-inflammatory mediators secreted by gingival fibroblasts and oral keratinocytes. Since periodontitis is a hyper-inflammatory reaction to periodontal pathogens, a treatment that combines an anti-bacterial and an anti-inflammatory effect, like Miswak could be potentially new path of treating periodontitis in a non-synthetic, natural manner.

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