'Green' silver nanoparticles combined with tyrosol as potential oral antimicrobial therapy.

OBJECTIVES: This study aimed to evaluate silver nanoparticles (AgNPs) obtained by a 'green' route associated or not to tyrosol (TYR) against Streptococcus mutans and Candida albicans in planktonic and biofilms states. METHODS: AgNPs were obtained by a 'green' route using pomegranate extract. The minimum inhibitory concentration (MIC) against S. mutans and C. albicans was determined for AgNPs and TYR combined and alone, and fractional inhibitory concentration index (FICI) was calculated. Single biofilms of C. albicans and S. mutans were cultivated for 24 h and then treated with drugs alone or in combination for 24 h. RESULTS: AgNPs and TYR were effective against C. albicans and S. mutans considering planktonic cells alone and combined. The MIC values obtained for C. albicans was 312.5 µg/mL (AgNPs) and 50 mM (TYR) and for S. mutans was 78.1 µg/mL (AgNPs) and 90 mM (TYR). The combination of these antimicrobial agents was also effective against both microorganisms: 2.44 µg/mL/0.08 mM (AgNPs/TYR) for C. albicans and 39.05 µg/mL /1.25 mM (AgNPs/TYR) for S. mutans. However, synergism was observed only for C. albicans (FICI 0.008). When biofilm was evaluated, a reduction of 4.62 log10 was observed for S. mutans biofilm cells treated with AgNPs (p < 0.05, Tukey test). However, the addition of TYR to AgNPs did not improve their action against biofilm cells (p > 0.05). AgNPs combined with TYR demonstrated a synergistic effect against C. albicans biofilms. CONCLUSIONS: These findings suggest the potential use of AgNPs with or without TYR against C. albicans and S. mutans, important oral pathogens. CLINICAL SIGNIFICANCE: AgNPs obtained by a 'green' route combined or not with TYR can be an alternative to develop several types of oral antimicrobial therapies and biomaterials.

Pomegranate extract in polyphosphate-fluoride mouthwash reduces enamel demineralization.

OBJECTIVES: To evaluate the anti-demineralizing effect of a mouthwash comprising pomegranate peel extract (PPE 3%), sodium trimetaphosphate (TMP 0.3%), and fluoride (F 225 ppm) in an in situ study, and to assess its irritation potential in an ex vivo study. METHODS: This double-blind crossover study was conducted in four phases with 7 days each. Twelve volunteers used palatal appliances containing enamel blocks, which were subjected to cariogenic challenges. The ETF formulation (PPE + TMP + F, pH 7.0), TF formulation (TMP + F, pH 7.0), deionized water (W, pH 7.0), and essential oil commercial mouthwash (CM, 220 ppm F, pH 4.3) were dropped onto the enamel twice daily. The percentage of surface hardness loss, integrated loss of subsurface hardness, calcium, phosphorus, and fluoride in enamel and biofilms were determined. In addition, alkali-soluble extracellular polysaccharide concentrations were analyzed in the biofilms. The irritation potential was evaluated using the hen's egg chorioallantoic membrane test through the vascular effect produced during 300-s of exposure. RESULTS: ETF was the most efficacious in preventing demineralization. It also showed the highest concentrations of calcium and phosphorus in the enamel and in the biofilm, as well as the lowest amount of extracellular polysaccharides in the biofilm. In the eggs, ETF produced light reddening, whereas CM led to hyperemia and hemorrhage. CONCLUSIONS: The addition of PPE to formulations containing TMP and F increased its anti-demineralizing property, and this formulation presented a lower irritation potential than the CM. CLINICAL RELEVANCE: ETF can be a promising alternative alcohol-free mouthwash in patients at high risk of caries.

Pomegranate Extract Potentiates the Anti-Demineralizing, Anti-Biofilm, and Anti-Inflammatory Actions of Non-Alcoholic Mouthwash When Associated with Sodium-Fluoride Trimetaphosphate.

This study investigated the anti-caries and anti-inflammatory effects of mouthwash formulations containing Punica granatum (pomegranate) peel extract (PPE), sodium-trimetaphosphate, and low concentrations of fluoride. PPE was characterized using high-performance liquid chromatography (ellagic acid and punicalagin). Total phenolics were quantified among formulations, and their stability was analyzed for 28 days. The formulation effects were evaluated as follows: (1) inorganic component concentration and reduced demineralization on bovine enamel blocks subjected to pH cycling; (2) anti-biofilm effect on dual-biofilms of Streptococcus mutans ATCC 25175 and Candida albicans ATCC 10231 treated for 1 and 10 min, respectively; and (3) cytotoxicity and production of inflammatory mediators (interleukin-6 and tumor necrosis factor-alpha). The formulation containing 3% PPE, 0.3% sodium-trimetaphosphate, and 225 ppm of fluoride resulted in a 34.5% surface hardness loss; a 13% (treated for 1 min) and 36% (treated for 10 min) biofilm reduction in S. mutans; a 26% (1 min) and 36% (10 min) biofilm reduction in C. albicans; absence of cytotoxicity; and anti-inflammatory activity confirmed by decreased interleukin-6 production in mouse macrophages. Thus, our results provide a promising prospect for the development of an alcohol-free commercial dental product with the health benefits of P. granatum that have been recognized for a millennium.

Green and Chemical Silver Nanoparticles and Pomegranate Formulations to Heal Infected Wounds in Diabetic Rats.

Infected cutaneous ulcers from diabetic rats with Candida albicans and Streptococcus aureus were treated with spray formulations containing green silver nanoparticles (GS), chemical silver nanoparticles (CS), or pomegranate peel extract (PS). After wound development and infection, the treatments were performed twice per day for 14 days. The wound healing was analyzed on days 2, 7, and 14 through the determination of CFUs, inflammatory infiltrate, angiogenesis, fibroplasia, myeloperoxidase, and collagen determination. Expressive improvement in wound healing was noted using both silver nanoparticles for 7 days. All the treatments were superior to controls and promoted significant S. aureus reduction after 14 days. CS presented better anti-inflammatory results, and GS and CS the highest number of fibroblasts. Despite the techniques' limitations, GS and CS demonstrated considerable potential for managing infected wounds, especially considering no early strategies prior to the drugs, such as the debridement of these wounds, were included.

Influence of Chlorhexidine Gluconate on the Immediate Bond Strength of a Universal Adhesive System on Dentine Subjected to Different Bonding Protocols: An In Vitro Pilot Study.

PURPOSE: The aim of this in vitro study was to evaluate the influence of preapplication of 2% chlorhexidine gluconate on the immediate microtensile bond strength of a universal adhesive system on dentine subjected to different bonding protocols. MATERIALS AND METHODS: Twenty human molars were used in this study, and the tooth surface was abraded to expose the dentine. The teeth were randomly divided into four groups according to the surface treatment (n = 5): SBU group: Single Bond Universal without acid etching; SBUPA group: 37% phosphoric acid + Single Bond Universal; SBUCG group: 2% chlorhexidine gluconate + Single Bond Universal; and SBUPACG group: 37% phosphoric acid + 2% chlorhexidine gluconate + Single Bond Universal. The microtensile bond strengths were measured using a microtensile tester 24 h after bonding. The bond strength data were subjected to analysis of variance (ANOVA) and Sheffé's least statistically significant difference test (α = 0.05). RESULTS: No statistically significant differences between the analysed groups were observed (p > 0.05). However, conditioning with phosphoric acid without the action of the chlorhexidine gluconate group resulted in higher numerical values of bond strengths than that for the chlorhexidine gluconate without the acid conditioning group. CONCLUSION: The preapplication of 2% chlorhexidine gluconate did not reduce the immediate bond strength of the Single Bond Universal adhesive system under different bonding protocols.

Extrato da casca de Punica granatum potencializa o efeito anti cárie de um enxaguatório bucal contendo trimetafosfato de sódio e flúor / Punica granatum peel extract enhances the anti caries effect of mouthwash containing sodium trimetaphosphate and fluoride

A cárie dentária está entre as principais e mais comuns doenças bucais. É causada por ácidos produzidos pelo biofilme microbiano que levam à desmineralização do esmalte. A prevenção e controle dessa doença crônica consistem na desorganização periódica do biofilme e na promoção da remineralização dentária. Para resolver esse problema, associamos o extrato de casca de Punica granatum (romã) (PPE) ao trimetafosfato de sódio (TMP) e fluoreto (F) em formulações para uso como enxaguatório bucal, e avaliamos sua eficácia na redução do processo de desmineralização do esmalte dental, bem como seu potencial anti biofilme contra importantes patógenos orais presentes na cárie dentária (Streptococcus mutans ATCC 25175 e Candida albicans ATCC 10231). Blocos de esmalte bovino (4 mm × 4 mm) selecionados por dureza superficial inicial (SHi) foram alocados aleatoriamente de acordo com grupos de tratamentos de formulação (n = 12 / grupo): ETF1 (3,0% PPE + 0,2% TMP + 100ppmF), TF1 (0,2% TMP + 100ppmF), ETF2 (3,0% PPE + 0,3% TMP + 225ppmF), TF2 (0,3% TMP + 225ppmF), F1 (100 ppmF), F2 (225 ppmF) e P (formulação sem E / T / F - placebo). Os blocos foram tratados 2x / dia com cada formulação e submetidos a cinco ciclos de pH (soluções desmineralizantes / remineralizantes) a 37° C. A seguir, determinaram-se a dureza superficial final (SHf), a dureza integrada da subsuperfície de perda (ΔKHN) padronizada e as concentrações de fluoreto de esmalte (F) de cálcio (Ca) e fósforo (P). A porcentagem de perda de dureza superficial (% SH) foi calculada (% SH = [(SHf - SHi) / SHi)] x 100), e as formulações que promoveram menores porcentagens de desmineralização do esmalte (% SH) e suas contrapartes foram selecionadas para os ensaios anti biofilme, bem como a formulação contendo apenas PPE (E). Para isso, ETF2 e TF2 (% SH = -34,5% e -53,1%, respectivamente), e a formulação E foram usadas para tratar por 1 ou 10 minutos biofilmes duplos de C. albicans e S. mutans crescidos por 24 horas em discos de hidroxiapatita (HA). A desmineralização da superfície do esmalte foi menor nas amostras tratadas com a formulação ETF2, resultando em uma diminuição de 46% na% SH em comparação com a F2. Novamente, a capacidade de reduzir o corpo da lesão (ΔKHN) foi maior (~ 26%) com ETF2 em relação a F2, e F2 proporcionou a maior concentração de F, Ca e P na superfície do esmalte. Entre as formulações de enxaguatório bucal ETF2, TF2 e E, as maiores taxas de redução de células viáveis foram exibidas tratando o biofilme com ETF2 por 10 minutos, independentemente do microrganismo testado. Em conclusão, a adição de PPE (3%) em enxaguatórios bucais contendo TMP (0,3) e F (225ppm) promoveu uma diminuição considerável no mineral sem perda de esmalte dental, além de reduzir consideravelmente o biofilme cariogênico formado por S. mutans e C albicans. Assim, cria uma perspectiva promissora para o desenvolvimento de um produto comercial dental sem álcool com os benefícios de milênios reconhecidos à saúde do Punica granatum(AU)

Dental caries is among the main and most common oral diseases. It is caused by acids produced by microbial biofilm that lead to enamel demineralization. The prevention and control of this chronic disease consist of periodic disorganization of the biofilm and the promotion of dental remineralization. To address this problem, we associate Punica granatum (pomegranate) peel extract (PPE) with sodium trimemtaphosphate (TMP), and fluoride (F) in formulations for being used as mouthwash, and evaluate its efficacy on reducing dental enamel demineralization process as well as its antibiofilm potential against important oral pathogens present in dental caries (Streptococcus mutans ATCC 25175 and Candida albicans ATCC 10231). Bovine enamel blocks (4 mm × 4 mm) selected by initial surface hardness (SHi) were randomly allocated according to groups of formulation treatments (n= 12/group): ETF1 (3.0%PPE+0.2%TMP+100ppmF), TF1 (0.2%TMP+100ppmF), ETF2 (3.0%PPE+0.3%TMP+225ppmF), TF2 (0.3%TMP+225ppmF), F1 (100 ppmF), F2 (225 ppmF), and P (formulation without E/T/F - placebo). The blocks were treated 2×/day with each formulation and submitted to five pH cycles (demineralizing/remineralizing solutions) at 37°C. Next, final surface hardness (SHf), integrated loss subsurface hardness (ΔKHN), and enamel fluoride (F) calcium (Ca) and phosphorus (P) concentrations were determined. The percentage of surface hardness loss (%SH) was calculated (%SH = [(SHf - SHi)/SHi)] x 100), and the formulations which promoted lower percentages of enamel demineralization (%SH) and its counterparts were selected to the antibiofim assays, as well as formulation containing only PPE (E). For that, ETF2 and TF2 (%SH= -34.5% and -53.1% respectively), and formulation E were used to treat for 1 or 10 minutes dual biofilms of C. albicans and S. mutans grown for 24 hours on hydroxyapatite discs (HA). Demineralization of the enamel surface was lower in samples treated with formulation ETF2, resulting in a 46% decrease in %SH in comparison with F2. Again, the capacity to reduce the lesion body (ΔKHN) was higher (~ 26%) with ETF2 in relation to F2, and F2 provided the highest concentration of F, Ca and P in enamel surface. Amongst the mouthwash formulations ETF2, TF2 and E, the highest rates of viable cells reduction were exhibited by treating biofilm with ETF2 for 10 minutes regardless of the microorganism tested. In conclusion, the addition of PPE (3%) in mouthwashes containing TMP (0.3) and F (225ppm) promoted a considerably decrease in the mineral loss of dental enamel besides considerable reducing cariogenic biofilm formed by S. mutans and C. albicans. It thus creates a promising prospect for the development of an alcohol free dental commercial product with the millennial recognized health benefits of Punica granatum(AU)

Antimicrobial Activity of Compounds Containing Silver Nanoparticles and Calcium Glycerophosphate in Combination with Tyrosol.

Nanocomposites containing antimicrobial agents and calcium phosphates have been developed. Thus, this study assessed the effects of two compounds containing silver nanoparticles (AgNPs) and ß-calcium glycerophosphate (CaGP), associated or not with tyrosol (TYR), against planktonic cells and biofilms of Candida albicans and Streptococcus mutans. The nanocompounds were synthesized through chemical and 'green' processes and characterized by scanning electron microscopy. The minimum and fractional inhibitory concentrations of each compound were determined for planktonic cells. Next, 24-h single biofilms of C. albicans and S. mutans were treated for 24 h with the nanocompounds alone or in combination with TYR, and the antibiofilm effect was assessed through enumeration of colony forming units. Biofilm data were statistically examined using one-way ANOVA and the Kruskal-Wallis test (α = 0.05). The chemically synthesized nanocompound in combination with TYR demonstrated a synergistic effect against planktonic cells of C. albicans and S. mutans. For the nanocompound obtained through the 'green' route associated with TYR, a synergistic effect was observed only against C. albicans. For biofilms, only the combination obtained through the 'green' route + TYR demonstrated a synergistic effect against C. albicans. Our results may contribute to the development of oral care products containing AgNPs-CaGP and TYR to combat oral infections.

Antimicrobial Potential and Cytotoxicity of Silver Nanoparticles Phytosynthesized by Pomegranate Peel Extract.

The phytosynthesis of metal nanoparticles is nowadays attracting the increased attention of researchers and is much needed given the worldwide matter related to environmental contamination. The antimicrobial activity of colloidal and spray formulation of silver nanoparticles (AgNPs) synthesized by pomegranate peel extract against Candida albicans and Staphylococcus aureus, and their cytotoxicity in mammalian cells were tested in the present study. Dry matter, pH, total phenolics, and ellagic acid in the extract were determined. Then, AgNPs were phytosynthesized and characterized by X-ray diffraction, electron transmission microscopy, dynamic light scattering, zeta potential, and Ag⁺ dosage. Spray formulations and respective chemical-AgNP controls were prepared and tested. The peel extract reduced more than 99% of Ag⁺, and produced nanoparticles with irregular forms and an 89-nm mean size. All AgNP presented antimicrobial activity, and the spray formulation of green-AgNP increased by 255 and 4 times the effectiveness against S. aureus and C. albicans, respectively. The cytotoxicity of colloidal and spray green-AgNP was expressively lower than the respective chemical controls. Pomegranate peel extract produced stable AgNP with antimicrobial action and low cytotoxicity, stimulating its use in the biomedical field.

Nanosynthesis of Silver-Calcium Glycerophosphate: Promising Association against Oral Pathogens.

Nanobiomaterials combining remineralization and antimicrobial abilities would bring important benefits to control dental caries. This study aimed to produce nanocompounds containing calcium glycerophosphate (CaGP) and silver nanoparticles (AgNP) by varying the reducing agent of silver nitrate (sodium borohydride (B) or sodium citrate (C)), the concentration of silver (1% or 10%), and the CaGP forms (nano or commercial), and analyze its characterization and antimicrobial activity against ATCC Candida albicans (10231) and Streptococcus mutans (25175) by the microdilution method. Controls of AgNP were produced and silver ions (Ag⁺) were quantified in all of the samples. X-ray diffraction, UV-Vis, and scanning electron microscopy (SEM) analysis demonstrated AgNP associated with CaGP. Ag⁺ ions were considerably higher in AgCaGP/C. C. albicans was susceptible to nanocompounds produced with both reducing agents, regardless of Ag concentration and CaGP form, being Ag10%CaGP-N/C the most effective compound (19.5⁻39.0 µg Ag mL−1). While for S. mutans, the effectiveness was observed only for AgCaGP reduced by citrate, also presenting Ag10%CaGP-N the highest effectiveness (156.2⁻312.5 µg Ag mL−1). Notably, CaGP enhanced the silver antimicrobial potential in about two- and eight-fold against C. albicans and S. mutans when compared with the AgNP controls (from 7.8 to 3.9 and from 250 to 31.2 µg Ag mL−1, respectively). The synthesis that was used in this study promoted the formation of AgNP associated with CaGP, and although the use of sodium borohydride (B) resulted in a pronounced reduction of Ag⁺, the composite AgCaGP/B was less effective against the microorganisms that were tested.

Virulence Factors in Candida albicans and Streptococcus mutans Biofilms Mediated by Farnesol.

The aim of this study was to evaluate the effect of farnesol on the production of acids and hydrolytic enzymes by biofilms of Streptococcus mutans and Candida albicans. The present study also evaluated the time-kill curve and the effect of farnesol on matrix composition and structure of single-species and dual-species biofilms. Farnesol, at subinhibitory concentrations, showed a significant reduction in S. mutans biofilm acid production, but did not alter C. albicans hydrolytic enzyme production. The number of cultivable cells of both microorganisms was significantly reduced after 8 h of contact with farnesol. Extracellular matrix protein content was reduced for biofilms formed in the presence of farnesol. In addition, confocal laser scanning and scanning electron microscopy displayed structural alterations in all biofilms treated with farnesol, which included reduction in viable cells and extracellular matrix. In conclusion, farnesol showed favorable properties controlling some virulence factors of S. mutans and C. albicans biofilms. These findings should stimulate further studies using this quorum-sensing molecule, combined with other drugs, to prevent or treat biofilm-associated oral diseases.

Sodium trimetaphosphate and hexametaphosphate impregnated with silver nanoparticles: characteristics and antimicrobial efficacy.

This study aimed to synthesize and characterize materials containing silver nanoparticles (AgNP) with polyphosphates (sodium trimetaphosphate (TMP) or sodium hexametaphosphate (HMP), and evaluate their effect against Candida albicans and Streptococcus mutans. The minimum inhibitory concentration (MIC) was determined, which was followed by the quantification of the biofilm by counting colony-forming units (CFUs), the amount of metabolic activity, and the total biomass. The MICs revealed greater effectiveness of composites containing 10% Ag (TMP + Ag10% (T10) and HMP + Ag10% (H10)) against both microorganisms. It was observed that T10 and H10 reduced the formation of biofilms by 56-76% for C. albicans and by 52-94% for S. mutans for total biomass and metabolic activity. These composites promoted significant log reductions in the number of CFUs, between 0.45-1.43 log10 for C. albicans and 2.88-3.71 log10 for S. mutans (p < .001). These composites demonstrated significant antimicrobial activity, especially against S. mutans, and may be considered a potential alternative for new dental materials.

Biofilm formation by Candida albicans and Streptococcus mutans in the presence of farnesol: a quantitative evaluation.

The aim of this study was to evaluate the effect of the QS molecule farnesol on single and mixed species biofilms formed by Candida albicans and Streptococcus mutans. The anti-biofilm effect of farnesol was assessed through total biomass quantification, counting of colony forming units (CFUs) and evaluation of metabolic activity. Biofilms were also analyzed by scanning electron microscopy (SEM). It was observed that farnesol reduced the formation of single and mixed biofilms, with significant reductions of 37% to 90% and 64% to 96%, respectively, for total biomass and metabolic activity. Regarding cell viability, farnesol treatment promoted significant log reductions in the number of CFUs, ie 1.3-4.2 log10 and 0.67-5.32 log10, respectively, for single and mixed species biofilms. SEM images confirmed these results, showing decreases in the number of cells in all biofilms. In conclusion, these findings highlight the role of farnesol as an alternative agent with the potential to reduce the formation of pathogenic biofilms.