Quantificação sérica de vitamina C por CLAE-UV e estudo de estabilidade

Vitamin C, an exogenous antioxidant, is essential to human health. In this study, a method was validated to serum vitamin C quantification by HPLC-UV. Its stability with and without the use of tris [2-carboxy-ethyl] phosphine hydrochloride (TCEP), at -20 and -80 °C, in serum and supernatant were also evaluated. Analysis showed r² > 0.99, precision CV% < 15% and % bias < 15%, being linear, precise and accurate. The stability test revealed that using TCEP in serum storage at -20 and -80 °C or in supernatant at -80 °C the vitamin C levels remain stable for 30 and 12 days, respectively.

Determinação simultânea de olanzapina e cloridrato de fluoxetina em formulações farmacêuticas por espectrofotometria derivativa

Simple, sensitive and accurate spectrophotometric derivative methods were developed for the simultaneous determination of olanzapine and fluoxetine hydrochloride in pharmaceutical formulations by derivative spectrophotometry. On all orders of derivative studied, the linear response range was 10 to 60 mg L-1, with limit of quantitation (LoQ) ranging from 0.73 to 1.49 mg L-1 for fluoxetine hydrochloride and from 0.18 to 0.96 mg L-1 for olanzapine. The best orders for derivative analyses showed recoveries ranging from 99 to 103% and from 98 to 100%, and inter-day accuracy < 2.1% and < 2.8%, for fluoxetine hydrochloride and olanzapine, respectively.

A general A³: coupling reaction based on functionalized alkynes

A range of hydroxypropargylpiperidones were efficiently obtained by a one-pot three-component coupling reaction of aldehydes, alkynols, and a primary amine equivalent (4-piperidone hydrochloride hydrate) in ethyl acetate using copper(I) chloride as a catalyst. The developed protocol proved to be equally efficient using a range of aliphatic aldehydes, including paraformaldehyde, and using protected and unprotected alkynols.

Flow injection analysis of vancomycin

A flow injection method for the quantitative analysis of vancomycin hydrochloride, C66H75Cl2N9O24.HCl (HVCM), based on the reaction with copper (II) ions, is presented. HVCM forms a lilac-blue complex with copper ions at pH≅4.5 in aqueous solutions, with maximum absorption at 555 nm. The detection limit was estimated to be about 8.5×10-5 mol L-1; the quantitation limit is about 2.5×10-4 mol L-1 and about 30 determinations can be performed in an hour. The accuracy of the method was tested through recovery procedures in presence of four different excipients, in the proportion 1:1 w/w. The results were compared with those obtained with the batch spectrophotometric and with the HPLC methods. Statistical comparison was done using the Student's procedure. Complete agreement was found at a 0.95 significance level between the proposed flow injection and the batch spectrophotometric methods, which present similar precision (RSD: 2.1 % vs. 1.9%).

Eficácia de Chenopodium ambrosioides (erva-de-santa-maria) no controle de endoparasitos de Gallus gallus (galinha caipira)

A pesquisa foi desenvolvida no Laboratório de Zoologia da Universidade Federal Rural do Rio de Janeiro e Setor de Parasitologia Animal da Universidade Estadual do Norte Fluminense Darcy Ribeiro, estado do Rio de Janeiro, no período de 2011 a 2012. O objetivo foi testar in vitro e in vivo a eficácia da planta medicinal Chenopodium ambrosioides Linnaeus, 1786 (erva-de-santa-maria), nas formas fitoterápica e homeopática, como meios alternativos para o controle de endoparasitos de Gallus gallus Linnaeus, 1758 (galinha caipira), um sério problema que afeta a criação e desempenho de aves domésticas, ocasionando morte quando muito intenso, retardo de crescimento, redução do índice de conversão alimentar e aumento na suscetibilidade às doenças infecciosas. As metodologias utilizadas foram preconizadas por Coles et al. (1992), creditada pela World Association for the Advancement of Veterinary Parasitology (WAAVP). O ensaio in vitro demonstrou alta taxa de redução na inibição de eclosão de ovos (97,18%), e o ensaio in vivo, elevada taxa na redução da contagem de ovos nas fezes (91,67%). A pesquisa evidenciou a presença dos gêneros Ascaridia (35,00%), Capillaria (30,00%), Heterakis (25,00%) e Strongyloides (10,00%). C. ambrosioides mostrou em certos momentos superioridade frente ao produto tradicional (Thiabendazole/Mebendazole) e índices superiores aos preconizados pelo Ministério da Agricultura do Brasil e Organização Mundial da Saúde como indicativos de eficácia.

Systematization, distribution and territory of the caudal cerebral artery on the brain's surface of the turkey (Meleagris gallopavo)

Thirty Meleagris gallopavo heads with their neck segments were used. Animals were contained and euthanized with the association of mebezonium iodide, embutramide and tetracaine hydrochloride (T 61, Intervet ) by intravenous injection. The arterial system was rinsed with cold saline solution (15°C), with 5000IU heparin and filled with red-colored latex. The samples were fixed in 20% formaldehyde for seven days. The brains were removed with a segment of cervical spinal cord and after, the dura-mater was removed and the arteries dissected. The cerebral carotid arteries, after the intercarotid anastomosis, were projected around the hypophysis, until they reached the tuber cinereum and divided into their terminal branches, the caudal branch and the rostral branch. The rostral branch was projected rostrolateralwards and gave off, in sequence, two collateral branches, the caudal cerebral and the middle cerebral arteries and the terminal branch was as cerebroethmoidal artery. The caudal cerebral artery of one antimere formed the interhemispheric artery, which gave off dorsal hemispheric branches to the convex surface of both antimeres. Its dorsal tectal mesencephalic branch, of only one antimere, originated the dorsal cerebellar artery. In the interior of the cerebral transverse fissure, after the origin of the dorsal tectal mesencephalic artery, the caudal cerebral artery emitted occipital hemispheric branches, pineal branches and medial hemispheric branches, on both antimeres. The caudal cerebral artery's territory comprehended the entire surface of the dorsal hemioptic lobe, the rostral surface of the cerebellum, the diencephalic structures, the caudal pole and the medial surface of the cerebral hemisphere and in the convex surface, the sagittal eminence except for its most rostral third. Due to the asymmetry found in the caudal cerebral arteries' ramifications, the models were classified into three types and their respective subtypes.

Eficácia de Chenopodium ambrosioides (erva-de-santa-maria) no controle de endoparasitos de Coturnix japônica (codorna japonesa)

Resumo:O objetivo foi testar in vitro e in vivo a eficácia da planta medicinal Chenopodium ambrosioidesLinnaeus, 1786 (erva-de-santa-maria), nas formas fitoterápica e homeopática, como meios alternativos para o controle de endoparasitos de Coturnix japonica Temminck & Schlegel, 1849 (codorna japonesa), um sério problema que afeta a criação e desempenho de aves domésticas, ocasionando morte quando muito intenso, retardo de crescimento, redução de índice de conversão alimentar e aumento na suscetibilidade às doenças infecciosas. As metodologias utilizadas foram preconizadas por Coles et al. (1992), creditada pela World Association for the Advancement of Veterinary Parasitology (WAAVP). A pesquisa evidenciou a presença dos gêneros Ascaridiae Eimeria. O ensaio in vitro demonstrou alta taxa de redução na inibição de eclosão de ovos de Ascaridiasp. (100,00%) e significativa taxa de redução na destruição de oocistos de Eimeriasp. (47,06%). O ensaio in vivodemonstrou alta taxa de redução na contagem de ovos de Ascaridiasp. nas fezes (100,00%) e expressiva taxa de redução na contagem de oocistos de Eimeriasp. nas fezes (60,33%). Chenopodium ambrosioides mostrou em certos momentos superioridade frente ao produto tradicional (Thiabendazole/Mebendazole) e índices superiores aos preconizados pelo Ministério da Agricultura do Brasil e Organização Mundial da Saúde como indicativos de eficácia.

Role of nitric oxide and superoxide in Giardia lamblia killing

Giardia lamblia trophozoites were incubated for 2 h with activated murine macrophages, nitric oxide (NO) donors or a superoxide anion generator (20 mU/ml xanthine oxidase plus 1 mM xanthine). Activated macrophages were cytotoxic to Giardia trophozoites (~60% dead trophozoites). This effect was inhibited (>90%) by an NO synthase inhibitor (200 µM) and unaffected by superoxide dismutase (SOD, 300 U/ml). Giardia trophozoites were killed by the NO donors, S-nitroso-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) in a dose-dependent manner (LD50 300 and 50 µM, respectively). A dual NO-superoxide anion donor, 3-morpholino-sydnonimine hydrochloride (SIN-1), did not have a killing effect in concentrations up to 1 mM. However, when SOD (300 U/ml) was added simultaneously with SIN-1 to Giardia, a significant trophozoite-killing effect was observed (~35% dead trophozoites at 1 mM). The mixture of SNAP or SNP with superoxide anion, which yields peroxynitrite, abolished the trophozoite killing induced by NO donors. Authentic peroxynitrite only killed trophozoites at very high concentrations (3 mM). These results indicate that NO accounts for Giardia trophozoite killing and this effect is not mediated by peroxynitrite

Pharmacokinetics and pharmacodynamics of propranolol in hypertensive patients after sublingual administration: systemic availability

The bioavailability of propranolol depends on the degree of liver metabolism. Orally but not intravenously administered propranolol is heavily metabolized. In the present study we assessed the pharmacokinetics and pharmacodynamics of sublingual propranolol. Fourteen severely hypertensive patients (diastolic blood pressure (DBP) ³115 mmHg), aged 40 to 66 years, were randomly chosen to receive a single dose of 40 mg propranolol hydrochloride by sublingual or peroral administration. Systolic (SBP) and diastolic (DBP) blood pressures, heart rate (HR) for pharmacodynamics and blood samples for noncompartmental pharmacokinetics were obtained at baseline and at 10, 20, 30, 60 and 120 min after the single dose. Significant reductions in BP and HR were obtained, but differences in these parameters were not observed when sublingual and peroral administrations were compared as follows: SBP (17 vs 18%, P = NS), DBP (14 vs 8%, P = NS) and HR (22 vs 28%, P = NS), respectively. The pharmacokinetic parameters obtained after sublingual or peroral drug administration were: peak plasma concentration (CMAX): 147 ± 72 vs 41 ± 12 ng/ml, P<0.05; time to reach CMAX (TMAX): 34 ± 18 vs 52 ± 11 min, P<0.05; biological half-life (t1/2b): 0.91 ± 0.54 vs 2.41 ± 1.16 h, P<0.05; area under the curve (AUCT): 245 ± 134 vs 79 ± 54 ng h-1 ml-1, P<0.05; total body clearance (CLT/F): 44 ± 23 vs 26 ± 12 ml min-1 kg-1, P = NS. Systemic availability measured by the AUCT ratio indicates that extension of bioavailability was increased 3 times by the sublingual route. Mouth paresthesia was the main adverse effect observed after sublingual administration. Sublingual propranolol administration showed a better pharmacokinetic profile and this route of administration may be an alternative for intravenous or oral administration.

Partially folded intermediates during trypsinogen denaturation

The equilibrium unfolding of bovine trypsinogen was studied by circular dichroism, differential spectra and size exclusion HPLC. The change in free energy of denaturation was = 6.99 ± 1.40 kcal/mol for guanidine hydrochloride and = 6.37 ± 0.57 kcal/mol for urea. Satisfactory fits of equilibrium unfolding transitions required a three-state model involving an intermediate in addition to the native and unfolded forms. Size exclusion HPLC allowed the detection of an intermediate population of trypsinogen whose Stokes radii varied from 24.1 ± 0.4 Å to 26.0 ± 0.3 Å for 1.5 M and 2.5 M guanidine hydrochloride, respectively. During urea denaturation, the range of Stokes radii varied from 23.9 ± 0.3 Å to 25.7 ± 0.6 Å for 4.0 M and 6.0 M urea, respectively. Maximal intrinsic fluorescence was observed at about 3.8 M urea with 8-aniline-1-naphthalene sulfonate (ANS) binding. These experimental data indicate that the unfolding of bovine trypsinogen is not a simple transition and suggest that the equilibrium intermediate population comprises one intermediate that may be characterized as a molten globule. To obtain further insight by studying intermediates representing different stages of unfolding, we hope to gain a better understanding of the complex interrelations between protein conformation and energetics.

Characterization of ß-trypsin at acid pH by differential scanning calorimetry

Trypsin is a serino-protease with a polypeptide chain of 223 amino acid residues and contains six disulfide bridges. It is a globular protein with a predominance of antiparallel ß-sheet and helix in its secondary structure and has two domains with similar structures. We assessed the stability of ß-trypsin in the acid pH range using microcalorimetric (differential scanning calorimetry) techniques. Protein concentrations varied in the range of 0.05 to 2.30 mg/ml. Buffer solutions of 50.0 mM ß-alanine and 20.0 mM CaCl2 at different pH values (from 2.0 to 4.2) and concentrations of sorbitol (1.0 and 2.0 M), urea (0.5 M) or guanidinium hydrochloride (0.5 and 1.0 M) were used. The data suggest that we are studying the same conformational transition of the protein in all experimental situations using pH, sorbitol, urea and guanidinium hydrochloride as perturbing agents. The observed van't Hoff ratios (deltaHcal/deltaHvH) of 1.0 to 0.5 in the pH range of 3.2 to 4.2 suggest protein aggregation. In contrast, deltaHcal/deltaHvH ratios equal to one in the pH range of 2.0 to 3.2 suggest that the protein unfolds as a monomer. At pH 3.00, ß-trypsin unfolded with Tm = 54ºC and deltaH = 101.8 kcal/mol, and the change in heat capacity between the native and unfolded forms of the protein (deltaCp) was estimated to be 2.50 ± 0.07 kcal mol-1 K-1. The stability of ß-trypsin calculated at 298 K was deltaG D = 5.7 kcal/mol at pH 3.00 and deltaG D = 15.2 kcal/mol at pH 7.00, values in the range expected for a small globular protein.

Effects of centrally acting antihypertensive drugs on the microcirculation of spontaneously hypertensive rats

We investigated the acute effects of centrally acting antihypertensive drugs on the microcirculation of pentobarbital-anesthetized spontaneously hypertensive rats (SHR). The effects of the sympatho-inhibitory agents clonidine and rilmenidine, known to activate both alpha2-adrenoceptors and nonadrenergic I1-imidazoline binding sites (I1BS) in the central nervous system, were compared to those of dicyclopropylmethyl-(4,5-dimethyl-4,5-dihydro-3H -pyrrol-2-yl)-amine hydrochloride (LNP 509), which selectively binds to the I1BS. Terminal mesenteric arterioles were observed by intravital microscopy. Activation of the central sympathetic system with L-glutamate (125 µg, ic) induced marked vasoconstriction of the mesenteric microcirculation (27 ± 3%; N = 6, P < 0.05). In contrast, the marked hypotensive and bradycardic effects elicited by intracisternal injection of clonidine (1 µg), rilmenidine (7 µg) and LNP 509 (60 µg) were accompanied by significant increases in arteriolar diameter (12 ± 1, 25 ± 10 and 21 ± 4%, respectively; N = 6, P < 0.05). The vasodilating effects of rilmenidine and LNP 509 were two-fold higher than those of clonidine, although they induced an identical hypotensive effect. Central sympathetic inhibition elicited by baclofen (1 µg, ic), a GABA B receptor agonist, also resulted in vasodilation of the SHR microvessels. The acute administration of clonidine, rilmenidine and LNP 509 also induced a significant decrease of cardiac output, whereas a decrease in systemic vascular resistance was observed only after rilmenidine and LNP 509. We conclude that the normalization of blood pressure in SHR induced by centrally acting antihypertensive agents is paralleled by important vasodilation of the mesenteric microcirculation. This effect is more pronounced with substances acting preferentially (rilmenidine) or exclusively (LNP 509) upon I1BS than with those presenting important alpha2-adrenergic activity (clonidine).

Distribution of small proteoglycans and glycosaminoglycans in humerus-related articular cartilage of chickens

The expression of components present in the cartilaginous extracellular matrix is related to development, gender, and genotype, as well as to the biomechanical properties of each type of cartilage. In the present study, we analyzed small proteoglycans and glycosaminoglycans present in different cartilages of the chicken wing after extraction with guanidine hydrochloride or papain. Quantitative analysis of glycosaminoglycans showed a larger amount in humeral cartilage (around 200 mg/g tissue) than in articular cartilage of the radius and ulna, with 138 and 80 mg/g tissue, respectively. Non-collagenous proteins isolated were predominantly from cartilage in the proximal regions of the humerus and radius. D4 fractions obtained by ultracentrifugation were separated by DEAE-Sephacel and Octyl-Sepharose chromatography and analyzed by SDS-PAGE. Two bands of 57 and 70-90 kDa were observed for all samples treated with ß-mercaptoethanol. Immunoblotting of these proteins was positive for the small proteoglycans fibromodulin and decorin, respectively. Apparently, the 57-kDa protein is present in macromolecular complexes of 160 and 200 kDa. Chondroitin sulfate was detected in all regions. HPLC analysis of the products formed by chondroitinase AC and ABC digestion mainly revealed ß-D-glucuronic acid and N-acetyl ß-D-galactosamine residues. The 4-sulfation/6-sulfation ratio was close to 3, except for the proximal cartilage of the radius (2.5). These results suggest functional differences between the scapula-humerus, humerus-ulna, and humerus-radius joints of the chicken wing. This study contributes to the understanding of the physiology of cartilage and joints of birds under different types of mechanical stress.

Comparison of sweat rate during graded exercise and the local rate induced by pilocarpine

Centrally stimulated sweat rate produced by graded exercise until exhaustion was compared to the local sweat rate induced by pilocarpine, often used as a sweating index for healthy individuals. Nine young male volunteers (22 ± 4 years) were studied in temperate environment in two situations: at rest and during progressive exercise with 25 W increases every 2 min until exhaustion, on a cycle ergometer. In both situations, sweating was induced on the right forearm with 5 ml 0.5% pilocarpine hydrochloride applied by iontophoresis (1.5 mA, 5 min), with left forearm used as control. Local sweat rate was measured for 15 min at rest. During exercise, whole-body sweat rate was calculated from the body weight variation. Local sweat rate was measured from the time when heart rate reached 150 bpm until exhaustion and was collected using absorbent filter paper. Pharmacologically induced local sweat rate at rest (0.4 ± 0.2 mg cm-2 min-1) and mean exercise-induced whole-body sweat rate (0.4 ± 0.1 mg cm-2 min-1) were the same (P > 0.05) but were about five times smaller than local exercise-induced sweat rate (control = 2.1 ± 1.4; pilocarpine = 2.7 ± 1.2 mg cm-2 min-1), indicating different sudorific mechanisms. Both exercise-induced whole-body sweat rate (P < 0.05) and local sweat rate (P < 0.05) on control forearm correlated positively with pilocarpine-induced local sweat rate at rest. Assuming that exercise-induced sweating was a result of integrated physiological mechanisms, we suggest that local and whole-body sweat rate measured during graded exercise could be a better sweating index than pilocarpine.

Spectroscopic studies on the interaction of efonidipine with bovine serum albumin

Efonidipine hydrochloride is an antihypertensive and antianginal agent with fewer side effects and is better tolerated in the treatment of hypertension with renal impairment. Its interaction with bovine serum albumin (BSA) is of great use for the understanding of the pharmacokinetic and pharmacodynamic mechanisms of the drug. The binding of efonidipine to BSA was investigated by fluorescence spectroscopy and circular dichroism. BSA fluorescence was quenched by efonidipine, due to the fact that efonidipine quenched the fluorescence of tryptophan residues mainly by the collision mode. The thermodynamic parameters ΔH0 and ΔS0 were 68.04 kJ/mol and 319.42 J·mol-1·K-1, respectively, indicating that the hydrophobic interactions played a major role. The results of circular dichroism and synchronous fluorescence measurements showed that the binding of efonidipine to BSA led to a conformational change of BSA. The fraction of occupied sites (θ) for the 8-anilino-1-naphthalein-sulfonic acid (ANS)-BSA system is 85%, whereas for the NZ-105-BSA system, it is 53%, which suggests that the interaction of ANS with BSA is stronger than that of NZ-105 with BSA. Binding studies in the presence of ANS indicated that efonidipine competed with ANS for hydrophobic sites of BSA. The effects of metal ions on the binding constant of the efonidipine-BSA complex were also investigated. The presence of metal ions Zn2+, Mg2+, Al3+, K+, and Ca2+ increased the binding constant of efonidipine_BSA complex, which may prolong the storage period of NZ-105 in blood plasma and enhance its maximum effects.