Propolis in Oral Healthcare: Antibacterial Activity of a Composite Resin Enriched With Brazilian Red Propolis.

The aim of this study was to obtain a Brazilian red propolis (BRP) enriched composite resin and to perform the characterization of its antibacterial activity, mechanical, and physical-chemical properties. Brazilian red propolis ethyl acetate extract (EABRP) was characterized by LC-ESI-Orbitrap-FTMS, UPLC-DAD, antibacterial activity, total flavonoids content, and radical scavenging capacity. BRP was incorporated to a commercial composite resin (RC) to obtain BRP enriched composite at 0.1, 0.15 and 0.25% (RP10, RP15 and RP25, respectively). The antibacterial activity RPs was evaluated against Streptococcus mutans by contact direct test and expressed by antibacterial ratio. The RPs were characterized as its cytotoxicity against 3T3 fibroblasts, flexural strength (FS), Knoop microhardness (KHN), post-cure depth (CD), degree of conversion (DC%), water sorption (Wsp), water solubility (Wsl), average roughness (Ra), and thermal analysis. Were identified 50 chemical compounds from BRP extract by LC-ESI-Orbitrap-FTMS. EABRP was bacteriostatic and bactericide at 125 and 500 µg/ml, respectively. The RP25 exhibited antibacterial ratio of 90.76% after 1 h of direct contact with S. mutans (p < 0.0001) while RC no showed significative antibacterial activity (p = 0.1865), both compared with cell control group. RPs and RC no showed cytotoxicity. RPs exhibited CD from 2.74 to 4.48 mm, DC% from 80.70 to 83.96%, Wsp from 17.15 to 21.67 µg/mm3, Wsl from 3.66 to 4.20 µg/mm3, Ra from 14.48 to 20.76 nm. RPs showed thermal resistance between 448-455°C. The results support that propolis can be used on development of modified composite resins that show antibacterial activity and that have compatible mechanical and physical-chemical properties to the indicate for composite resins.

Polyphenols and Brazilian red propolis incorporated into a total-etching adhesive system help in maintaining bonding durability.

OBJECTIVES: The aim of this study was to evaluate the degree of conversion and bond strength of a commercial dental adhesive modified by the incorporation of quercetin, resveratrol (RES), and Brazilian red propolis (BRP). METHODS: BRP markers were identified using ultra-performance liquid chromatography coupled with a diode array detector, and the antioxidant activity (AAO) of the three substances was analyzed. Single Bond 2 adhesive (3M ESPE) was modified by adding BRP, quercetin, and RES, separately, at 20 µg/mL, 250 µg/mL, and 500 µg/mL, respectively. The degree of conversion (DC) was measured using near-infrared spectroscopy 24 h after photopolymerization. Measurements of the resin-dentin microtensile bond strength (µTBS) were carried out after 1 day and 1 year. Student's t test and ANOVA with Tukey's test were used for data analysis (α = 0.05). RESULTS: The markers daidzein, liquiritigenin, pinobanksin, isoliquiritigenin, formononetin, pinocembrin, and biochanin A were found in the ethanolic extract of BRP. Quercetin, RES, and BRP showed high AAO. The DC of the tested adhesives remained adequate for this category of material, with a slight increase in the DC of adhesives with quercetin and BRP (P > 0.05). Comparisons between µTBS measurements made at 1 day and 1 year showed that, contrary to the control group, µTBS values for all modified adhesives were maintained after 1 year in distilled water (P > 0.05). CONCLUSIONS: These findings suggest that quercetin, RES, or BRP might be useful in adhesive dentistry to help improve hybrid layer resistance. CLINICAL SIGNIFICANCE: Dentin bonding agents with quercetin, RES, and BRP have potential to increase the longevity of composite restorations.

Inhibition of matrix metalloproteinases: a troubleshooting for dentin adhesion.

Matrix metalloproteinases (MMPs) are enzymes that can degrade collagen in hybrid layer and reduce the longevity of adhesive restorations. As scientific understanding of the MMPs has advanced, useful strategies focusing on preventing these enzymes' actions by MMP inhibitors have quickly developed in many medical fields. However, in restorative dentistry, it is still not well established. This paper is an overview of the strategies to inhibit MMPs that can achieve a long-lasting material-tooth adhesion. Literature search was performed comprehensively using the electronic databases: PubMed, ScienceDirect and Scopus including articles from May 2007 to December 2019 and the main search terms were "matrix metalloproteinases", "collagen", and "dentin" and "hybrid layer". MMPs typical structure consists of several distinct domains. MMP inhibitors can be divided into 2 main groups: synthetic (synthetic-peptides, non-peptide molecules and compounds, tetracyclines, metallic ions, and others) and natural bioactive inhibitors mainly flavonoids. Selective inhibitors of MMPs promise to be the future for specific targeting of preventing dentin proteolysis. The knowledge about MMPs functionality should be considered to synthesize drugs capable to efficiently and selectively block MMPs chemical routes targeting their inactivation in order to overcome the current limitations of the therapeutic use of MMPs inhibitors, i.e., easy clinical application and long-lasting effect.

Comprehensive multivariate correlations between climatic effect, metabolite-profile, antioxidant capacity and antibacterial activity of Brazilian red propolis metabolites during seasonal study.

The standardization of apiceutical products like as propolis extracts has been widely debated worldwide and variations in the propolis chemical composition are still very relevant topics for use-standardized of different propolis-type as medication by much of the world's population. The present manuscript discuss important issues related to the climate effect and variations in propolis metabolite-profiling changes, antioxidant capacity and variations of the antibacterial activity of the Brazilian red propolis metabolites using comprehensive multivariate correlations. It was observed the increasing of guttiferones concentrations during the intense drought period and drastic decreasing in rainy period. The climate variation induced the high concentration of flavonoids in rainy period with pronounced dropped in some rainy months. The Pearson´s analysis demonstrated correlation between IC50 from DPPH and guttiferones and flavonoids concentrations. The PCA-X and Hotelling T2 test showed outliers during the months with lowest concentrations of formononetin and isoliquiritigenin was observed in antibacterial tests. The PLS-DA, OPLS-DA and VIP analysis demonstrate guttiferone E, guttiferone B, liquiritigenin, naringenin are considered important substances responsible by anti-staphylococcal activity in red propolis composition during the rainy season and drought period, but a synergistic effect with other flavonoids and isoflavonoids are not ruled out.

Characterization and Stability of the Antimony-Quercetin Complex.

Purpose: Quercetin is a flavonoid known for its therapeutic properties and for forming complexes. Although the antimony-quercetin (SbQ) complex has been produced before, no previous exploration of its characteristics has been published in literature. Thus, this study aimed to characterize this complex, assess its stability and investigate its complexation site through its antibacterial activity. Methods: The SbQ complex was synthetized using Sb(III) potassium tartrate trihydrate and quercetin anhydrous (1:1) (v/v) as a solution and dried using three methods: rotaevaporation, lyophilization and spray drying. The material, in solution, was analyzed by UV-vis and fluorimetry; and, in the powder, by X-ray diffraction (XRD), both scanning electronic and fluorescence microscopy and infrared spectroscopy (FT-IR). Antimicrobial activity was evaluated via broth microdilution. Results: UV-vis exhibited a shoulder peak at 291 nm indicating metal chelation at C-ring of quercetin and confirmed 1:1 stoichiometry. Spectrofluorimetry showed an increase of intensity with the complex formation with an emission band (525 nm). After drying, XRD and SEM indicated loss of crystallinity and a difference in shape and size of the complex compared to its precursors. FT-IR suggested by a shift of frequency of the carbonyl group (1661 cm-1) that the quercetin bond to antimony by the C-3, followed by positions C-5 and C-4 carbonyl, which has been confirmed by MIC through the structure-activity relationship of the antibacterial activity of quercetin. Conclusion: These results provided a characterization of SbQ complex with the confirmation of its binding site, working as a guide for future studies involving this complex.

Chemical and microbiological characterization of tinctures and microcapsules loaded with Brazilian red propolis extract.

The aim of this study was to characterize tinctures and microcapsules loaded with an ethanol extract of red propolis through chemical, physicochemical and microbiological assays in order to establish quality control tools for nutraceutical preparations of red propolis. The markers (isoflavonoids, chalcones, pterocarpans, flavones, phenolic acids, terpenes and guttiferones) present in the tinctures A and B were identified and confirmed using LC/ESI/FTMS/Orbitrap. Four compositions (A, B, C and D) were prepared to contain B tincture of the red propolis with some pharmaceutical excipients and submitted to two drying processes, i. e. spray-drying and freeze-drying to obtain microcapsules loaded with the red propolis extract. The tinctures and microcapsules of the red propolis were submitted to the total flavonoid content and antioxidant activity tests. The antibacterial activity and minimum inhibitory concentration (MIC) were tested using Staphylococcus aureus ATCC 25293 and Pseudomonas aeruginosa ATCC 27853 strains. The tinctures and microcapsules presented high flavonoid quantities from 20.50 to 40.79 mg/100 mg of the microcapsules. The antioxidant activity and IC50 were determined for the tinctures A and B (IC50: 6.95 µg/mL and 7.48 µg/mL), the spray-dried microcapsules (IC50: 8.89-15.63 µg/mL) and the freeze-dried microcapsules (IC50: 11.83-23.36 µg/mL). The tinctures and microcapsules were proved to be bioactive against gram-positive and gram-negative bacteria with inhibition halos superior to 10 mm at concentration of 200 µg/mL and MIC values of 135.87-271.74 µg/mL using gram-positive strain and 271.74-543.48 µg/mL using gram-negative strain. The tinctures and microcapsules of the red propolis have a potential application for nutraceutical products.

Polymeric Nanoparticles of Brazilian Red Propolis Extract: Preparation, Characterization, Antioxidant and Leishmanicidal Activity.

The ever-increasing demand for natural products and biotechnology derived from bees and ultra-modernization of various analytical devices has facilitated the rational and planned development of biotechnology products with a focus on human health to treat chronic and neglected diseases. The aim of the present study was to prepare and characterize polymeric nanoparticles loaded with Brazilian red propolis extract and evaluate the cytotoxic activity of "multiple-constituent extract in co-delivery system" for antileishmanial therapies. The polymeric nanoparticles loaded with red propolis extract were prepared with a combination of poly-ε-caprolactone and pluronic using nanoprecipitation method and characterized by different analytical techniques, antioxidant and leishmanicidal assay. The red propolis nanoparticles in aqueous medium presented particle size (200-280 nm) in nanometric scale and zeta analysis (-20 to -26 mV) revealed stability of the nanoparticles without aggregation phenomenon during 1 month. After freeze-drying method using cryoprotectant (sodium starch glycolate), it was possible to observe particles with smooth and spherical shape and apparent size of 200 to 400 nm. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and thermal analysis revealed the encapsulation of the flavonoids from the red propolis extract into the polymeric matrix. Ultra performance liquid chromatography coupled with diode array detector (UPLC-DAD) identified the flavonoids liquiritigenin, pinobanksin, isoliquiritigenin, formononetin and biochanin A in ethanolic extract of propolis (EEP) and nanoparticles of red propolis extract (NRPE). The efficiency of encapsulation was determinate, and median values (75.0 %) were calculated using UPLC-DAD. 2,2-Diphenyl-1-picryhydrazyl method showed antioxidant activity to EEP and red propolis nanoparticles. Compared to negative control, EEP and NRPE exhibited leishmanicidal activity with an IC50 value of ≅38.0 µg/mL and 31.3 µg/mL, 47.2 µg/mL, 154.2µg/mL and 193.2 µg/mL for NRPE A1, NRPE A2, NRPE A3 and NRPE A4, respectively. Nanoparticles loaded with red propolis extract in co-delivery system and EEP presented cytotoxic activity on Leishmania (V.) braziliensis. Red propolis extract loaded in nanoparticles has shown to be potential candidates as intermediate products for preparation of various pharmaceutical dosage forms containing red propolis extract in the therapy against negligible diseases such as leishmaniasis. Graphical Abstract Some biochemical mechanisms of cellular debridement of Leishmania (V.) braziliensis species by the flavonoids of red propolis extract (EEP) or NRPE loaded with red propolis extract.

Brazilian red propolis: phytochemical screening, antioxidant activity and effect against cancer cells.

BACKGROUND: The implementation of new public healthcare models that stimulate the use of natural products from traditional medicine, as a so-called integrated medicine, refers to an approach that use best of both conventional medicine and traditional medicine. Propolis is a widely used natural product by different ancient cultures and known to exhibit biological activities beneficial for health. The large number of studies conducted with propolis had shown that its chemical composition differs as a function of the climate, plant diversity and bee species and plays an important role on its therapeutic properties. The aim of this study was to analyse the phytochemical profile of the ethanolic extract of red propolis (EEP) and its fractionation, antioxidant action of EEP and its fractions hexane, cloroform and ethyl acetate and cytotoxic activity of EEP on human tumour cell lines SF-295 (glioblastoma), OVCAR-8 (ovary) and HCT-116 (colon). METHODS: EEP was obtained by maceration with absolute ethanol, then it was concentrated in rotaevaporator up to complete evaporation of the solvent. The crude extract was fractionated with hexane, ethyl acetate, chloroform and methanol and they were subjected to phytochemical screening and total phenolic compounds. Antioxidant activity of EEP and fractions was done by means of the 2,2-diphenyl-1-picryhydrazyl (DPPH) method. Biomarkers of red propolis were identified by LC-Orbitrap-FTMS. To assess cytotoxic activity of the extract, cells were exposed to EEP over 72 h. Cell viability was assessed by means of MTT assay. The percentage of cell growth inhibition (IC50) was analysed by means of non-linear regression, and the absorbance values of the various investigated concentrations were subjected to one-factor analysis of variance (ANOVA) followed by Tukey's or Tamhane's tests (α = 0.05). RESULTS: The results obtained using phytochemical screening and LC-Orbitrap-FTMS indicated the presence of phlobaphene tannins, catechins, chalcones, aurones, flavonones, flavonols, xanthones, pentacyclic triterpenoids and guttiferones in Brazilian red propolis. EEP and its hexane, chloroform and ethyl acetate fractions obtained by liquid-liquid partitioning exhibited satisfactory antioxidant percentages. EEP (IC50 < 34.27 µg/mL) exhibited high levels of cytotoxicity on all human tumour cell lines tested when compared to negative control. CONCLUSIONS: C-Orbitrap-FTMS was useful to establish the chemical profile of the red propolis. Brazilian red propolis has antioxidant properties and decreases substantially the percentage of cell survival of human tumour cells; thus, it has potential to serve as an anticancer drug.

Short-term stability studies of ampicillin and cephalexin in aqueous solution and human plasma: Application of least squares method in Arrhenius equation.

A limited number of studies with application of the Arrhenius equation have been reported to drugs and biopharmaceuticals in biological fluids at frozen temperatures. This paper describes stability studies of ampicillin and cephalexin in aqueous solution and human plasma applying the Arrhenius law for determination of adequate temperature and time of storage of these drugs using appropriate statistical analysis. Stability studies of the beta-lactams in human plasma were conducted at temperatures of 20°C, 2°C, -20°C and also during four cycles of freeze-thawing. Chromatographic separation was achieved using a Shimpak C(18) column, acetonitrile as organic modifier and detection at 215nm. LC-UV-MS/MS was used to demonstrate the conversion of ampicillin into two diastereomeric forms of ampicilloic acid. Stability studies demonstrated degradation greater than 10% for ampicillin in human plasma at 20°C, 2°C and -20°C after 15h, 2.7days, 11days and for cephalexin at the same temperatures after 14h, 3.4days and 19days, respectively, and after the fourth cycle of freezing-thawing. The Arrhenius plot showed good prediction for the ideal temperature and time of storage for ampicillin (52days) and cephalexin (151days) at a temperature of -40°C, but statistical analysis (least squares method) must be applied to avoid incorrect extrapolations and estimated values out uncertainty limits.

Validation of a method for determination of ampicillin in human plasma using LC-DAD.

We describe the validation data of a simple but selective chromatographic method for determination of ampicillin in human plasma using liquid chromatography-diode array detector. Blank plasma free of drugs was transferred to eppendorf's tubes and spiked with ampicillin stock solution to obtain quality control samples at 1.00, 2.50, 5.00, and 10.00 microg/mL. Extraction of ampicillin and cephalexin (internal standard) from plasma samples (250 microL) was investigated using three different methods: precipitation with perchloric acid, ultra-filtration and solid-phase extraction. Chromatographic separation was achieved using a Shimpak C(18) column (300 mm x 4.6 mm i.d.; 5 microm), and detection was done at 215 nm with a diode array UV-Vis detector. The mobile phase consisted of dihydrogen phosphate (pH 3.5)-acetonitrile (87.5:12.5, v/v) delivered at a flow rate of 1.00 mL/min. Selectivity was evaluated with different pools of human plasma. Perchloric acid precipitation showed an excellent selectivity for normal plasma. The precipitation method presented recoveries above 84.0 +/- 3.3% and 82.0 +/- 1.6%, (n = 3) for ampicillin and cephalexin, respectively. The method has a limit of detection of 0.15 microg/mL and is linear in the range of 0.30 to 100.00 microg/mL. Standardized residue analysis demonstrated normality and homocedasticity. Inter-day precision was 4.5%, and accuracy was 11.1% (n = 9). Stability studies demonstrated instability of b-lactamics in human plasma at 20 and 2 degrees C after 6 and 360 h of storage, respectively.

Simultaneous determination of ranitidine and metronidazole in human plasma using high performance liquid chromatography with diode array detection.

The development and validation of a simple method for the simultaneous determination of ranitidine and metronidazole in human plasma is described. Plasma samples (250 microL) were deproteinized by precipitation with 60% perchloric acid, centrifuged and the supernatant directly injected into the HPLC. Separation was achieved in isocratic mode with a Shimpak C(18) column and a mobile phase consisting of 10mM potassium dihydrogen phosphate pH 3.5:acetonitrile (90:10, v/v) with UV detection at 315 nm. The method showed good selectivity and sensitivity. Good and consistent recovery for metronidazole and ranitidine was obtained: 96.22+/-3.52 and 95.00+/-4.50% for ranitidine (25-1000 ng/mL) and metronidazole (60-10,000 ng/mL), respectively (n=3). With this one-step sample preparation method, both ranitidine and metronidazole could be quantified simultaneously in human plasma with good precision (R.S.D.<15%) and accuracy (bias values below 15%). The limit of quantification for ranitidine and metronidazole were 20 and 40 ng/mL plasma, respectively.