Perillyl alcohol has antibacterial effects and reduces ROS production in macrophages

Abstract Natural products have emerged as a rich source of bioactive compounds for adjunctive treatments of many infectious and inflammatory conditions, including periodontitis. Among the monoterpenes with significant biological properties, there is the perillyl alcohol (POH), which can be found in several essential oils and has shown immunomodulatory properties in recent studies, which may be interesting in the treatment of non-neoplastic inflammatory disorders. Objective To determine the antibacterial and immune modulatory activities of the POH. Methodology The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the POH for two significant Gram-negative periodontal pathogens were determined by macrodilution and subculture, respectively. Cell proliferation and cytotoxicity in RAW 264.7 macrophages were determined by Trypan Blue and mitochondrial enzymatic activity assay. The modulation of reactive oxygen species (ROS) was analyzed by flow cytometry and expression of TNF and arginase-1 by real-time PCR. Results The POH was effective against P. gingivalis (ATCC 33277) and F. nucleatum (ATCC 25586) with MIC= MBC=1600 μM. No cytotoxicity up to 100 µM was observed on macrophages. The cell proliferation was inhibited from 48 hours at 100 μM (p<0.05) and 250 μM (p<0.01). The POH increased ROS production at both 10 μM and 100 μM (p<0.05) in unstimulated cells. The PMA-induced ROS production was not affected by POH, whereas 100 μM significantly reduced lipopolysaccharide-induced (LPS-induced) ROS. The expression of TNF was not affected by POH in unstimulated cells or in cells polarized to M1 phenotype, whereas both concentrations of POH reduced (p<0.05) the expression of arginase-1 in M2-polarized macrophages. Conclusion The POH has antibacterial activity against periodontal pathogens and reduced proliferation of murine macrophages without significant cytotoxicity at concentrations up to 100 μM. In addition, the POH reduced the LPS-induced ROS and the expression of arginase-1 in M2-polarized macrophages.

Salivary arginase activity after mechanical-chemical therapy / Atividade de arginase salivar após tratamento mecânico-químico

Introduction: Salivary components can be used as biomarkers for diagnosing and monitoring oral diseases. There is evidence that one potential biomarker, arginase, is associated with the inflammatory processes of periodontal disease, and its enzymatic activity is reduced according to the improvement in the clinical parameters after treatment. Objective: The present study aimed to evaluate the salivary arginase activity in gingivitis and periodontitis patients treated with full-mouth mechanical procedures combined with the adjunctive use of essential oils or chlorhexidine mouthwash, respectively. Material and method: Twenty-six gingivitis and 16 periodontitis patients received complete periodontal examinations at the baseline and 3 months after therapy, in which the periodontal probing depth, clinical attachment loss, plaque index, and gingival index measurements were taken. At these same appointments, the salivary total protein level and salivary arginase activity were also established via spectrophotometry. Result: There were improvements in all of the clinical parameters (p < 0.05) evaluated from the baseline to 3 months in both groups. In addition, the salivary arginase activity and total protein levels were reduced after the gingivitis treatment. Conclusion: Similar to the clinical results, both therapeutic protocols positively affected the salivary arginase activity; however, further studies are necessary to clarify its potential as a salivary biomarker for periodontal monitoring.

Introdução: Componentes salivares podem ser usados como biomarcadores para diagnóstico e monitoramento de doenças orais. Há evidências de que um potencial biomarcador, arginase, está associado com os processos inflamatórios da doença periodontal, e após o tratamento sua atividade enzimática é reduzida em concordância com a melhora nos parâmetros clínicos. Objetivo: O presente estudo objetivou avaliar a atividade da arginase salivar em pacientes com gengivite e periodontite tratados com procedimentos mecânicos em estágio único combinados ao uso coadjuvante de enxaguatórios com óleos essenciais ou clorexidina, respectivamente. Material e método: Vinte e seis pacientes com gengivite e 16 pacientes com periodontite receberam exame periodontal completo antes e 3 meses após a terapia, em que mensurações de profundidade de sondagem, perda de inserção clínica, índice de placa e índice gengival foram realizadas. Nestas mesmas consultas os níveis de proteína total e a atividade de arginase salivar foram estabelecidos via espectrofotometria. Resultado: Todos os parâmetros clínicos melhoraram, em ambos os grupos, do exame inicial para o de 3 meses (p < 0,05). Adicionalmente, após tratamento para gengivite houve redução da atividade de arginase salivar e do nível de proteína total. Conclusão: Semelhante aos resultados clínicos, ambos os protocolos terapêuticos afetaram positivamente a atividade da arginase salivar; entretanto, estudos futuros são necessários para clarificar seu potencial como biomarcador salivar para o monitoramento periodontal.

Prevalência microbiana e quantificação da arginase na saúde e doença periodontal / Microbial prevalence and arginase quantification in health and periodontal disease

A arginase salivar está aumentada em processos inflamatórios e infecciosos da cavidade bucal. O objetivo do presente estudo foi avaliar a atividade de arginase salivar, correlacionando-a a parâmetros clínicos e microbiológicos em diferentes condições periodontais. Foram avaliados os seguintes parâmetros clínicos: índice de placa, índice de sangramento, profundidade de sondagem e nível de inserção clínica. Foi avaliada ainda a presença dos periodontopatógenos Campylobacter rectus, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tanerella forsythia, Treponema denticola e Prevotella intermedia por meio da reação em cadeia da polimerase (PCR). Finalmente, a presença salivar de arginase — por meio de espectrofotometria —também foi considerada. O tratamento estatístico dos dados foi realizado pelos testes Qui-quadrado e Kruskal-Wallis, adotando-se significância estatística quando p<0,05. Foram alocados no presente estudo 78 indivíduos assim caracterizados: 26 periodontalmente saudáveis (S), 26 com gengivite(G) e 26 com periodontite crônica (P). P. gingivalis, P. intermedia e T. denticola estavam mais prevalentes significativamente em P do que em G e S (p<0,05). A. actinomycetemcomitans foi significativamente mais prevalente (p<0,05) em P e G do que em S. C. rectus apresentou distribuição similar nos três grupos (p>0,05). Com relação aos níveis de atividade de arginase, os indivíduos portadores de periodontite crônica apresentaram maiores níveis (p<0,05) em comparação aos com gengivite ou periodontalmente saudáveis. A expressão da arginase salivar parece acompanhar a mudança dos sinais clínicos e a alteração do perfil microbiológico das diferentes condições periodontais

The salivary arginase is increased in inflammatory and infectious processes of the oral cavity. The aim of this study was to evaluate the salivary arginase activity, correlating it to clinical and microbiological parameters in different periodontal conditions. We evaluated the following parameters: plaque index, bleeding index, probing depth and clinical attachment level. It also assessed the presence of periodontopathogens Campylobacter rectus, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tanerellaforsythia, Treponema denticola, and Prevotella intermedia by polymerase chain reaction (PCR). Finally, presence of salivary arginase - by spectrophotometry - was also considered. Statistical treatment of data was performed by chi-square and Kruskal-Wallis, adopting statistical significance at p <0.05. Eighty-nine individuals were allocated in this study:26 periodontally healthy (S), 26 with gingivitis (G) and 26 with chronic periodontitis (P). P. gingivalis, P. intermedia and T. denticola were significantly more prevalent in P-group than G- and S-group (p <0.05). A. actinomycetemcomitans was significantly more prevalent (p <0.05) in P- and G-group than in S-group. C. rectus showed a similar distribution in the three groups (p> 0.05). With respect to the levels of arginase activity, individuals with chronic periodontitis had higher levels (p <0.05) than those with gingivitis or periodontally healthy. Expression of the salivary arginase seems to followthe changes of clinical signs and microbiological profile in different periodontal conditions.