The Potentiation of Anti-inflammatory Effect and INOS and COX-2 Gene Expression Inhibition by Rut in When Complexed with Cooper.

Aims: The aim of this study was to evaluate both the antioxidant effect and antiinflammatory activity of a new transition metal coordinated rut in compound, Rutin-Cu2 complex. Study Design: Flavonoids have proven antioxidant and anti-inflammatory activities. Moreover, recent researches demonstrate that the antioxidant activity of flavonoids is believed to increase when they are coordinated with transition metal ions. Our group has recently synthesized new compounds by the reaction of rut in (a flavonoid) with divalent metal salts (copper acetate, nickel acetate or iron sulfate), rendering new transitional metal coordinated rut in compounds, named R-Fe1 [(FeC27H32O21S)2], R-Cu2 [C31H32O18Cu.2H20] and R-Ni2 (C31H42O23Ni). In order to investigate the ability of these new compounds in modulating biological activity and to compare if metal coordinated rut in could increase anti-inflammatory activity of rut in alone, we used murine experimental model of peritonitis to measured cell migration and In vitro models of antioxidant activity to evaluate radical superoxide scavenging activity and of macrophage cell line culture to quantify nitric oxide production and iNOS and COX-2 gene expression. Methodology: To characterize physical-chemical the new generated compounds we used elemental analysis, FT-IR and UV/Vis. The antioxidant effect was evaluate by radical superoxide scavenging assay, using NBT methodology. The anti-inflammatory activity of the new compounds were investigated by peritonitis models induced by carrageenan (1%, 4h), bradikynin (10nmol/cavity, 1h), histamine (100μg/cavity, 1h), substance P (20nmol/cavity, 1,5h) and PGE2 (10nmol/cavity, 1h). Total and leucocytes subtypes numbers were evaluated in harvest cells from mice peritoneum after phlogistic agents administration, in controls groups (not treated or treated with dexamethasone or rut in alone) or tested groups (treated with metal coordinated compounds). RAW 264.7 cells were stimulated with LPS on the absence or presence of rut in alone (0.01–90mMr), or Rutin-Cu2 complex (0.01–90mM). The production of NO was measured in culture supernatant after 24h of cell incubation, by Griess assay. And iNOS and COX-2 transcripts were quantified by real time PCR with SYBR GREEN, on cDNAs obtained after 24h of cell incubation, in a step one instrument (Applied bio systems). Results: Complex formation was also verified by 1H RNM, using DMSO-d6 as solvent. The proton signals from Hydroxyl groups 5-OH, 7-OH, 3’-OH and 4’-OH shifted to lower and broader frequencies in coordinated complex R-Fe1, R-Cu2 and R-Ni2, compared with signals from free rut in. The results showed that R-Cu2 complex presented a higher superoxide scavenging effect when compared with rut in alone (6.95% and 46.42%, at 10μM; and 51.80% and 71.32%, at 100μM, respectively). The results also showed that R-Cu2 inhibited significantly (P<0.05; ANOVA) cell migration (neutrophils, lymphocytes and monocytes) in peritonitis induced by carrageenan, bradikynin and PGE2, in mice, when compared to controls ones (without treatment or Ru alone treatment). Furthermore, rutin and R-Cu2 significantly (P<0.05, paired t test) inhibited iNOS and COX-2 gene expression in LPS-induced macrophage cells. Conclusions: Taken together, our results show for the first time that the R-Cu2 complex, a metal coordinated rut in compound, produces anti-inflammatory effects in mice, at least in part, by means of increasing the antioxidant activity and inhibition of iNOS and COX-2 gene expression. We suggest that cooper coordinated rut in compound can potentiates some biological properties of this flavonoid and could be more effective for therapeutic treatment of diseases related to oxidative stress.

Randomized, double-blind, placebo-controlled clinical trial on the effects of propolis and chlorhexidine mouthrinses on gingivitis / Ensaio clínico duplo-cego randomizado e placebo-controlado dos efeitos de enxaguatórios a base de própolis e clorexidina na saúde gengival, para análise

Objective: The aim of this study was to compare the effects of typified propolis and chlorhexidine mouthrinses on gingival health in a randomized double-blind placebo-controlled clinical trial. Material and Methods: Sixty participants were randomized to 3 mouthrinse study groups: 1) 2% typified propolis (n = 20); 2) 0.12% chlorhexidine (n = 20), and 3) placebo (n = 20). Participants rinsed unsupervised twice a day for 28 days. The Papillary Bleeding Score (PBS) was measured on the mesio-buccal surfaces of all teeth at baseline and 28 days thereafter. Co-variance analysis was employed to compare PBS average values and the number of sites with PBS ≥ 2 among study groups. Sub-group analysis was further applied to participants who were < 40 years-old. Results: The results show efficacy of propolis mouthrinse when comparing before and after treatment protocols significantly for a reduction of mean PBS scores. For younger participants propolis mouthrinse was superior to all groups in reducing mean PBS scores and significant when compared to 0.12% chlorhexidine mouthrinse. Conclusion: The efficacy of 2% typified propolis mouthrinse was demonstrated in reducing the levels of gingival inflammation. These results need to be duplicated by other investigators under similar study protocols.

Objetivos: O propósito deste estudo foi o de comparar os efeitos de enxaguatórios a base de própolis tipificada e clorexidina na saúde gengival, empregando-se um ensaio clínico duplo cego, randomizado, e placebo-controlado. Material e Métodos: Sessenta participantes foram randomizados em 3 grupos de enxaguatórios, a saber: própolis tipificada 2% (n = 20), clorexidina 0,12% (n = 20) e placebo (n = 20). Os participantes bochecharam duas vezes ao dia por 28 dias os respectivos enxaguatórios. Medidas do índice de sangramento papilar (PBS) foram feitas na superfície mésio-bucal de todos os dentes dos participantes no tempo basal e após 28 dias. Análise de co-variância foi empregada permitindo comparações entre os grupos das médias do PBS e do número de sítios ≥ 2. Análise de sub-grupo foi efetuada em participantes com idade < 40 anos. Resultados: Constataram-se os efeitos positivos do enxaguatório de própolis a 2% na reducão da inflamação gingival após uso não supervisionado por 28 dias. Análise no sub-grupo de parcipantes com idade < 40 anos constatou superioridade do enxaguatório de própolis quando comparado com o enxaguatório de clorexidina a 0,12%. Conclusão: Este ensaio clínico demonstrou a eficácia de enxaguatório de própolis tipificada a 2% na redução da inflamação gengival. Estes resultados necessitam ser duplicados por outros investigadores.

Effects of typified propolis on mutans streptococci and lactobacilli: a randomized clinical trial / Efeitos da própolis tipificada nos estreptococcos do grupo mutans e lactobacilos: ensaio clínico randomizado

Objective: The aim of this study was to determine in a randomized, double-blind, placebo-controlled clinical trial the effects of typified propolis and chlorhexidine rinses on salivary levels of mutans streptococci (MS) and lactobacilli (LACT). Methods: One hundred patients were screened for salivary levels of MS >100,000 CFUs/mL of saliva. All patients presented with at least one cavitated decayed surface. Sixty patients met entry criteria. Subjects were adults 18-55 years old. After restoration of cavitated lesions patients were randomized to 3 experimental groups:1) PROP-alcohol-free 2% typified propolis rinse (n= 20); 2) CHX- 0.12% chlorhexidine rinse; 3) PLplacebo mouthrinse. Patients rinsed unsupervised 15 mL of respective rinses twice a day for 1 min for 28 days. Patients were assessed for the salivary levels of MS (Dentocult SM) and LACT (Dentocult LB) at baseline, 7-day, 14-day, and at 28-day visits (experimental effects) and at 45-day visit (residual effects). General linear models were employed to analyze the data. Results: PROP was superior to CHX at 14-day and 28-day visits in suppressing the salivary levels of MS (p < .05). PROP was superior to PL at all visits (p < .01). The residual effects of PROP in suppressing the salivary levels of MS could still be observed at the 45-day visit, where significant differences between PROP and CHX (p < .05), were demonstrated. PROP was significantly superior than CHX in suppressing the levels of salivary LACT at the 28-day visit (p < .05). Conclusion: Typified propolis rinse was effective in suppressing cariogenic infections in caries-active patients when compared to existing and placebo therapies.

Objetivo: O objetivo deste estudo foi determinar, em um estudo randomizado, duplo-cego, placebo-controlado os efeitos da própolis tipificada e clorexidina sobre os níveis salivares de estreptococos do grupo mutans (EM) e lactobacilos (LACT). Métodos: Cem pacientes foram selecionados para níveis salivares de MS > 100.000 UFC/mL de saliva. Todos os pacientes apresentaram pelo menos uma superfície cariada com cavitação. Sessenta pacientes preencheram os critérios de inclusão. Os indivíduos eram adultos com 18-55 anos de idade. Após a restauração das lesões cavitadas, os pacientes foram randomizados para três grupos experimentais: 1) PROP- bochecho livre de álcool de 2% de própolis tipificada (n = 20), 2) CHX- bochecho de clorexidina 0,12%, 3) PL- bochecho placebo. Os participantes bochecharam sem supervisão 15 mL dos enxaguatórios duas vezes por dia, durante 1 minuto, durante 28 dias. Os pacientes foram avaliados para os níveis salivares de MS (Dentocult SM) e LACT (Dentocult LB) na linha de base, e após 7 dias, 14 dias, 28 dias (efeitos experimentais) e 45 dias (efeitos residuais). Modelos lineares foram utilizados para analisar os dados. Resultados: PROP foi superior ao CHX nas visitas de 14 dias e de 28 dias na supressão dos níveis salivares de SM (p < 0,05). PROP foi superior ao PL em todas as visitas (p < 0,01). Os efeitos residuais de PROP na supressão dos níveis salivares de MS ainda foi observado na visita de 45 dias, onde diferenças significativas entre PROP e CHX (p < 0,05) foram demonstradas. PROP foi significativamente superior a CHX na supressão dos níveis salivares de LACT na visita de 28 dias (p < 0,05). Conclusão: O enxaguatório de própolis tipificada foi eficaz na supressão de infecções cariogênicas em pacientes com atividade de cárie quando comparado a terapias existentes e ao placebo

Histopathological analysis of corticosteroid-antibiotic preparation and propolis paste formulation as intracanal medication after pulpectomy: an in vivo study

Intracanal medication in pulpectomy therapy is used between appointments with the objective of reducing pain and inflammatory processes in pulp and periapical tissues. Propolis has been known as a natural antibiotic and has been subject of medical and dental research due to its therapeutic properties such as antibiotic, analgesic and anti-inflammatory effects. OBJECTIVE: The aim was to carry out an in vivo evaluation of the periapical tissue response to propolis paste when used as an intracanal medication in the teeth of dogs after pulpectomy. MATERIAL AND METHODS: 72 dog's incisors were selected for the experiment. After biomechanical preparation the root canal was filled with a corticosteroid-antibiotic preparation, experimental propolis paste, non-medicament (negative control) or non-pulpectomy at all (positive control). The medications were left inside the root canal for 7, 14 or 28 days. At the end of the experimental period histological sections were prepared and all laboratories processes for Harris hematoxylin and eosin staining was proceeded followed by the analysis using an optical microscope. Sections were classified according to a score representing the inflammatory events observed: the presence of polymorphonuclear neutrophils, polymorphonuclear eosinophils, lymphocytes and plasma cells, macrophages and/or giant cells, fibrous condensation and abscesses. RESULTS: There were statistically significant differences between the tissue reactions caused by the two substances being tested, after different experimental periods, with the periapical tissue that was in contact with propolis paste exhibiting fewer inflammatory reactions in comparison to corticosteroid-antibiotic preparation. CONCLUSIONS: The low tissue responses from propolis paste suggest that this material could be considered as an option for root canal medication after pulpectomy.

Green propolis phenolic compounds act as vaccine adjuvants, improving humoral and cellular responses in mice inoculated with inactivated vaccines

Adjuvants play an important role in vaccine formulations by increasing their immunogenicity. In this study, the phenolic compound-rich J fraction (JFR) of a Brazilian green propolis methanolic extract stimulated cellular and humoral immune responses when co-administered with an inactivated vaccine against swine herpesvirus type 1 (SuHV-1). When compared to control vaccines that used aluminium hydroxide as an adjuvant, the use of 10 mg/dose of JFR significantly increased (p < 0.05) neutralizing antibody titres against SuHV-1, as well as the percentage of protected animals following SuHV-1 challenge (p < 0.01). Furthermore, addition of phenolic compounds potentiated the performance of the control vaccine, leading to increased cellular and humoral immune responses and enhanced protection of animals after SuHV-1 challenge (p < 0.05). Prenylated compounds such as Artepillin C that are found in large quantities in JFR are likely to be the substances that are responsible for the adjuvant activity.

Spectrophotometric and HPLC determination of deflazacort in pharmaceutical dosage forms

Deflazacort (DFZ) is a glucocorticoid used as an anti-inflammatory and immunosuppressant drug. No official methods are available for DFZ determination in pharmaceutical formulations. The objective of this study was to develop, validate and compare spectrophotometric (UV and colorimetric) and high-performance liquid chromatography (HPLC) methods, for the quantitative determination of DFZ in tablets and oral suspension. For the UV method, ethanol was used as the solvent, with detection at 244 nm. The colorimetric method was based on the redox reaction with blue tetrazolium in alkaline medium, with detection at 524 nm. The method by HPLC was carried out using a C18 column, mobile phase consisting of acetonitrile:water (80:20, v/v) with a flow rate of 1.0 mL min-1 and detection at 244 nm. The methods proved linear (r > 0.999), precise (RSD < 5 percent) and accurate (recovery > 97 percent). Statistical analysis of the results indicated that the UV and HPLC methods were statistically equivalent, while the values obtained for the colorimetric method differed significantly from the other methods.

O deflazacorte (DFZ) é um fármaco glicocorticóide usado como antiinflamatório e imunossupressor. Métodos oficiais não estão disponíveis para a determinação de DFZ em formas farmacêuticas. Este estudo teve como objetivo desenvolver, validar e comparar métodos por espectrofotometria (UV e colorimetria) e cromatografia líquida de alta eficiência (CLAE), na determinação quantitativa de DFZ em comprimidos e suspensão oral. O método por UV utilizou etanol como solvente, com detecção em 244 nm. O método colorimétrico foi baseado na reação de redução com azul de tetrazólio em meio alcalino, com detecção em 524 nm. O método por CLAE utilizou coluna C18; fase móvel constituída de acetonitrila:água (80:20, v/v), com fluxo de 1,0 mL min-1 e detecção em 244 nm. Os métodos foram lineares (r > 0,999); precisos (RSD < 5 por cento), e exatos (recuperação > 97 por cento). As análises estatísticas dos resultados obtidos indicaram que os métodos por UV e por CLAE foram estatisticamente equivalentes, enquanto os valores obtidos para o método colorimétrico diferiram significativamente dos demais métodos.

Análise - microscópia e MEV - da superficie do canal radicular após utilizacao do extrato de própolis a 0, 25 porcento, como irrigante / Analysis - macroscopic and SEM - of root canal surface after irrigation with 0, 25 propolis solution

O objetivo deste trabalho foi verificar a superficie do canal radicular em relacão a coloracão e a limpeza (smear layer), após irrigacão com extrato de própolis a 0,25 porcento (Epl). Quarenta dentes foram seccionados na juncão amelo-cemetária. Vinte dentes foram utilizados para análise macroscópica. Para o controle, utilizou-se 5mm cervicais de cada dente. Dez raízes receberam irrigacão com NaOCI a 1 porcento (grupo I) e 10 com Epl (grupo II). Após isso, as raízes e controles foram clivados, limpos e fotografados. A estas 20 hemifaces foram adicionados o restante dos 20 dentes para análise em MEV, constituindo outros grupos: NaOCI a 1 porcento + EDTA a 17 porcento (III) e Epl+EDTA a 17 porcento (IV). Quatro examinadores cegos fizeram a análise das fotografias, atribuindo escores 0 (sem diferenca), 1 (controle mas claro) e 2 (controle mais escuro). Para smear layer os escores foram: 0 (ausência), 1 (túbulos abertos + smear) e 2 (smear). Resultados: correlacão - não houve diferenca, com não diferenca de coloracão entre o controle e as raízes. MEV - grupos I e II havendo presenca de smear. No grupo III e IV houve remocão de smear, sem haver diferenca estatística, porém no Rank de posicão o grupo III apresentou-se mais limpo, havendo smear plugs no IV. Conclui-se que não houve diferenca de coloracão entre o controle e a raiz preparada para ambas as solucoes; houve presenca de smear em ambas as solucoes, quando utilizadas isoladas; com associacão ao EDTA apesar de não haver diferenca estatística, o Epl permitiu a presenca de smear plugs, não ocorrendo para o NaOCI a 1 porcento

The plants of the genus Phyllanthus as a potential source of new drugs

The plants of the genus Phyllanthus (Euphorbiaceae) comprise about 550 to 750 species which are widely distributed in most tropical and subtropical countries. About 200 species are believed to occur in the Americas, mainly in the Caribbean and Brazil. The plants of the genus Phyllanthus have long been used in folk medicine to treat, among others, kidney and urinary bladder disorders, intestinal infections, diabetes and hepatitis B. In recent years, substantial progress in chemical and pharmacological studies, and a few clinical studies of some Phyllanthus species, were made. This review discusses the current knowledge gained by the in vitro and in vivo pharmacological and biochemical studies performed with the extracts and the main active constituents isolated from different species of plants of the genus Phyllanthus. Data available in the literature strongly support the idea that the extract and some constituents isolated from these plants, including flavanoids, tannins, alkaloids, coumarins, lignans and terpenes, account for their reported antinociceptive, anti-inflammatory, antiviral, antispasmodic and antiallergic properties. In addition, some of these compounds were found to interact with most key enzymes, such as aldose reductase, angiotensin converting enzyme, mitochondrial ATPase, both cyclo- and lipooxygenases, phospholipase A2, tyrosine kinase, reverse transcriptase, and phosphodiesterases. The complex mechanism of action of such compounds could explain, at least in part, the wide therapeutic use of the plants of the genus Phyllanthus in folk medicine. Thus, the plants of the genus Phyllanthus present potential therapeutic interest as a source of new drugs.