Augusto César Cropanese Spadaro

A convenient manual trinitrobenzenesulfonic acid method for monitoring amino acids and peptides in chromatographic column effluents

Abstract The trinitrobenzenesulfonic acid (TNBS) method of R. Fields (1971, Biochem. J. , 124 , 581–590) has been modified for the manual detection of amino acids and peptides in chromatographic column effluent by changing the reaction conditions to 1 m m TNBS in 0.4 m potassium borate buffer, pH 9.2, at room temperature for 30 to 50 min. The reaction with amines and the spontaneous hydrolysis of TNBS are stopped by neutralization to pH 6.25 with sodium monobasic phosphate (0.33 m ). Sodium sulfite (3 m m ) is added to increase the absorptivity of the product. The TNBS reagent blank is less than 0.100 A 420 after 50 min of reaction. Since the Δ A 420 of the reagent blank is ∼0.002/min before quenching the reaction, and zero afterward, the time required for reaction and for absorbance measurements need not be controlled precisely. Alkaline hydrolysis of peptides is carried out prior to detection to increase the sensitivity of the method. This procedure is convenient for the manual determination of 5 to 100 nmol of amino acids in the 50–100 samples required to define a chromatographic elution profile.

Development of a colorimetric system for evaluation of the masticatory efficiency

The stomatognathic system is responsible for complex vital functions such as chewing, deglutition, respiration and phonation. Food breakdown is one of the masticatory functions, which enables reducing the attrition against the oral soft tissues and increasing the surface contact of food particles with digestive secretions, thus leading to satisfactory and rapid physiological processes. The methods of evaluation the masticatory efficiency are restricted to electromyography, sieving and clinical observation. However, the validity of these methods has been questioned due to the complexity of the procedures, variances in the material used and inaccuracy of methodologies. This study presents a new method in which a capsule of a synthetic material using fuchsin-containing granules was devised. Such capsules were submitted to several laboratory tests in order to determine their resistance and absorbency. Ten calibrated individuals were instructed to chew the capsules. Masticatory efficiency was determined by analyzing fuchsin concentration in a solution obtained from chewed granules measuring the absorbance at 546 nm. The method was found to be fast, simple, reproducible, inexpensive and efficient and can be used as a complementary evaluation of the masticatory efficiency in different situations.

Liquid crystalline phase nanodispersions enable skin delivery of siRNA.

The ability of small interfering RNAs (siRNAs) to potently but reversibly silence genes in vivo has made them particularly well suited as a new class of drugs that interfere with disease-causing or disease-promoting genes. However, the largest remaining hurdle for the widespread use of this technology in skin is the lack of an effective delivery system. The aim of the present study was to evaluate nanodispersed systems in liquid crystalline phases that deliver siRNA into the skin. The proposed systems present important properties for the delivery of macromolecules in a biological medium, as they are formed by substances that have absorption-enhancing and fusogenic effects; additionally, they facilitate entrapment by cellular membranes due to their nano-scale structure. The cationic polymer polyethylenimine (PEI) or the cationic lipid oleylamine (OAM) were added to monoolein (MO)-based systems in different concentrations, and after dispersion in aqueous medium, liquid crystalline phase nanodispersions were obtained and characterized by their physicochemical properties. Then, in vitro penetration studies using diffusion cell and pig ear skin were carried out to evaluate the effect of the nanodispersions on the skin penetration of siRNA; based on these results, the nanodispersions containing MO/OA/PEI/aqueous phase (8:2:5:85, w/w/w/w) and MO/OA/OAM/aqueous phase (8:2:2:88, w/w/w/w) were selected. These systems were investigated in vivo for skin penetration, skin irritation, and the ability to knockdown glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein levels in animal models. The results showed that the studied nanodispersions may represent a promising new non-viral vehicle and can be considered highly advantageous in the treatment of skin disorders; they were effective in optimizing the skin penetration of siRNA and reducing the levels of the model protein GAPDH without causing skin irritation.

Clinical and biochemical evaluation of the saliva of patients with xerostomia induced by radiotherapy

Clinical aspects and biochemical properties in the saliva of 21 patients prior to and following radiotherapy for head and neck cancer were evaluated (experimental group) and compared with the same properties in a control group of 21 subjects free of cancer. Salivary flow was evaluated by measuring the time necessary, in seconds, for the output of 2 ml of stimulated saliva; and the buffering capacity changes were determined using a simple colorimetric method. Total salivary protein concentration was determined by the Bradford 4 method. Amylase activity was measured by reducing sugars released from a soluble starch substrate, quantified by the dinitrosalicylic method. The electrophoretic profile was evaluated in polyacrylamide gel (12% SDS-PAGE) using samples of 5 mg of salivary protein. A statistically significant reduction (p < 0.01) of the salivary flow was observed, (162.47 s +/- 28.30 before and 568.71 s +/- 79.75 after irradiation), as well as a reduction in the salivary buffering capacity (pH 5.45 +/- 0.14 before and pH 4.40 +/- 0.15 after irradiation). No statistically significant alteration was observed in total salivary protein concentration. A statistically significant reduction (p < 0.01) of salivary alpha-amylase activity (856.6 ng/mg +/- 88.0 before and 567.0 ng/mg +/- 120.6 after irradiation) was observed. Electrophoretic profile differences in salivary protein bands were also observed after radiotherapy, mainly in the range of molecular weight of 72000 to 55000 Daltons. Clinically, patients presenting xerostomia induced by radiotherapy presented an increase in oral tissue injury.

Seasonality Role on the Phenolics from Cultivated Baccharis dracunculifolia

Baccharis dracunculifolia is the source of Brazilian green propolis (BGP). Considering the broad spectrum of biological activities attributed to green proplis, B. dracunculifolia has a great potential for the development of new cosmetic and pharmaceutical products. In this work, the cultivation of 10 different populations of native B. dracunculifolia had been undertaken aiming to determine the role of seasonality on its phenolic compounds. For this purpose, fruits of this plant were collected from populations of 10 different regions, and 100 individuals of each population were cultivated in an experimental area of 1800 m2. With respect to cultivation, the yields of dry plant, essential oil and crude extract were measured monthly resulting in mean values of 399 ± 80 g, 0.6 ± 0.1% and 20 ± 4%, respectively. The HPLC analysis allowed detecting seven phenolic compounds: caffeic acid, ferulic acid, aromadendrin-4′-methyl ether (AME), isosakuranetin, artepillin C, baccharin and 2-dimethyl-6-carboxyethenyl-2H-1-benzopyran acid, which were the major ones throughout the 1-year monthly analysis. Caffeic acid was detected in all cultivated populations with mean of 4.0%. AME displayed the wide variation in relation to other compounds showing means values of 0.65 ± 0.13% at last quarter. Isosakuranetin and artepillin C showed increasing concentrations with values between 0% and 1.4% and 0% and 1.09%, respectively. The obtained results allow suggesting that the best time for harvesting this plant, in order to obtain good qualitative and quantitative results for these phenolic compounds, is between December and April.

Development of lamellar gel phase emulsion containing marigold oil (Calendula officinalis) as a potential modern wound dressing

Appropriate therapeutics for wound treatments can be achieved by studying the pathophysiology of tissue repair. Here we develop formulations of lamellar gel phase (LGP) emulsions containing marigold (Calendula officinalis) oil, evaluating their stability and activity on experimental wound healing in rats. LGP emulsions were developed and evaluated based on a phase ternary diagram to select the best LGP emulsion, having a good amount of anisotropic structure and stability. The selected LGP formulation was analyzed according to the intrinsic and accelerated physical stability at different temperatures. In addition, in vitro and in vivo studies were carried out on wound healing rats as a model. The LGP emulsion (15.0% marigold oil; 10.0% of blend surfactants and 75.0% of purified water [w/w/w]) demonstrated good stability and high viscosity, suggesting longer contact of the formulation with the wound. No cytotoxic activity (50-1000 μg/mL) was observed in marigold oil. In the wound healing rat model, the LGP (15 mg/mL) showed an increase in the leukocyte recruitment to the wound at least on days 2 and 7, but reduced leukocyte recruitment after 14 and 21 days, as compared to the control. Additionally, collagen production was reduced in the LGP emulsion on days 2 and 7 and further accelerated the process of re-epithelialization of the wound itself. The methodology utilized in the present study has produced a potentially useful formulation for a stable LGP emulsion-containing marigold, which was able to improve the wound healing process.

A Simple and Efficient Device for Treatment of Fluoride Samples in Analysis by the Selective Electrode Method.

Abstract A simple, efficient and economic device for fluoride mass transference is presented in this study. Hexamethyldisiloxane (HMDS) is used to volatilize fluoride and, in addition to 0.05M NaOH, 0.3M citrate/phosphate/1 M KCl, pH 7.8 (buffer A) and 0.5 M citrate pH 5.5 (buffer B) are used as the trapping solution with the aim of an additional gain when concentration is a limiting condition in the use of the selective electrode. Using 0.05M NaOH, the fluoride mass transferred was in the range of 40 - 0.2 μg, and with buffer A the mass transferred was around 0.2 μg, with a total recovery in a few hours. The influence of interfering substances on fluoride volatilization was assessed. Fluoride was transferred from standard solutions in either the absence or presence of interfering substances (ethanol, propyleneglycol, glycerol, sorbitol, artificial saliva), and the respective recoveries were determined. Due to its simplicity and low cost several device units can be assembled allowing simultaneous analysis...

Chromatographic determination of angiotensin-converting enzyme and angiotensinase activity

An analytical method utilizing an automatic amino acid analyzer is described for the separation, identification, and measurement of 5 to 50 nmol of angiotensin I, angiotensin II, [Des-Phe8]angiotensin II, Phe-His-Leu, His-Leu, isoleucine, leucine, tyrosine, and phenylalanine. Aminex A-5 cation-exchange resin (0.9 x 15 cm) is sequentially eluted with three sodium citrate buffers: pH 3.25, 0.2 N; pH 4.85, 0.54 N, and pH 6.5, 0.39 N at 60 and 80 degrees C. Reaction with ninhydrin is used for detection. This chromatographic system was used to determine angiotensin-converting enzyme activity and the angiotensinase activity of rabbit brain endopeptidase B. In each assay, the unhydrolyzed substrate and both products were measured simultaneously in one step without pretreatment of the hydrolysate. Products were recovered in 1:1 molar ratios and the overall recovery of an hydrolyzed substrate of products was quantitative.

Método para avaliaçäo clínica da capacidade tamponante salivar

The present study aimed to develop and standardize a colorimetric method for assessing salivary buffer capacity adapted to the features of the Brazilian population. Samples of stimulated saliva were titrated without CO2 elimination. The assessment was carried out to study the influence of the loss of CO2 from the samples during the few minutes necessary for titration, and to standardize the instants for measuring pH. Saliva samples were titrated from 206 individuals from both genders, between 5 and 50 years of age, and in a proportion of 0.77 : 1 : 0.44, respectively of children, youngsters, and adults. Case frequency distribution; determination of average values of initial pH, pKa, µ equivalents related to titration end point and final pH; and estimate of the percentage of saliva titrated at a pH of 5.0 were calculated based on the data collected. The results were used to classify individuals according to their salivary buffer capacity by colorimetric pH determinations using 11,12, and 13 µ equivalents of H+/ml saliva, and were compared to case frequency distribution obtained from saliva titration data. The results indicate that 11 µ equivalents H+/ml of saliva is the most appropriate for clinically evaluating the salivary buffer capacity of our population.

Determination of fluoride with an ion-selective electrode in the presence of water-soluble organic substances

The interference of propylene glycol, ethanol, glycerol and sorbitol in the determination of fluoride by the selective electrode method was investigated. Operational procedures are described which involve both the use of the calibration graph method and the standard additions technique to eliminate possible errors due to the presence of the above water-soluble substances, which are widely used in pharmaceutical products for oral hygiene.