Validade e fiabilidade do protocolo de avaliação da qualidade vocal da Universidade de Aveiro

O presente estudo teve como principal objetivo rever a versão original do Protocolo de Avaliação da Qualidade Vocal da Universidade de Aveiro (PAQVUA), criar o respetivo manual, comprovar a validade de conteúdo do PAQVUA assim como a sua fiabilidade inter e intra avaliador. Para rever a versão original do PAQVUA, foi efetuada uma revisão narrativa da literatura, com o objetivo de encontrar informação que revelasse a pertinência, da versão original do protocolo, para assim se efetuarem as alterações necessárias. Para analisar a validade de conteúdo recorreu-se a um painel de peritos, num total de seis, especialistas na área da voz que avaliaram as provas do PAQVUA, folha de registo e manual. Os peritos avaliaram todos os componentes do PAQVUA através do preenchimento de um questionário com o objetivo de se comprovar a relevância, clareza e precisão de cada uma das provas de avaliação e também o conteúdo do seu manual. Para além disso pretendeu-se também comprovar a fiabilidade inter e intra avaliador do PAQVUA. Para isso, o mesmo protocolo foi aplicado duas vezes por duas avaliadoras diferentes e também foi aplicado em dois momentos temporais diferentes por uma só avaliadora. A amostra teve um total de doze participantes. Para analisar estatisticamente os resultados da validade de conteúdo utilizou-se o método gráfico Bland e Altman modificado e o Coeficiente de Correlação Intraclasses (CCI). Para a fiabilidade inter e intra avaliador recorreu-se ao Coeficiente de Correlação de Spearman (ρ) e ao Kappa de Cohen (k). Relativamente aos resultados obtidos com o método Bland e Altman modificado, verificou-se a existência de concordância entre os peritos através da análise dos gráficos, sendo que a maioria dos pontos se encontrou dentro dos limites esperados de concordância. Quanto aos valores do CCI (entre 0.379 e 0.479), estes revelaram uma correlação regular. No que concerne à fiabilidade inter avaliador, os resultados de correlação e concordância apresentaram-se relevantes, com valores de ρ (de Spearman) superiores a 0.700 e valores de k (de Cohen) superiores a 0.600, na maioria dos casos. O mesmo foi constatado para a fiabilidade intra avaliador. Desta forma pode concluir-se que a nova versão do PAQVUA apresenta validade de conteúdo sendo tal comprovado pelo método de Bland-Altman modificado e pelos valores do CCI. Quanto à fiabilidade inter e intra avaliador, pode afirmar-se que o PAQVUA é fiável, porém como a amostra em estudo é pequena, o que a torna pouco representativa, esta conclusão carece de fundamento com base num estudo mais alargado. Apesar da limitação referida, acredita-se que este protocolo é uma mais-valia para o estudo da patologia vocal em ambiente clínico, assim como para a investigação científica nesta área, pois através do PAQVUA podem-se recolher diversas informações relacionadas com a voz, úteis para uma intervenção terapêutica baseada em evidências científicas.

Avaliação do gasto energético em amputados de membro inferior utilizando o Six-Minute Walk Test

Objetivos do estudo - Impacto na marcha de: características individuais, hábitos e fatores comportamentais, quadro clínico, componentes protésicos. Conclusões do estudo - Foi possível observar a influência de vários fatores na capacidade de deambulação e no consumo energético durante a marcha: idade correlaciona-se com a distância percorrida, velocidade de marcha, VO2 máx. e presença de dor; IMC correlaciona-se com o VO2 máx.; nível de atividade correlaciona-se com a distância percorrida, velocidade de marcha, VO2 máx. e perceção de esforço. Vários fatores, relacionados com a amputação ou não mostram influenciar a capacidade de deambulação pelo que não devem ser tidos em conta separadamente. Aquisição de conhecimentos na área de investigação em Ortoprotesia, sendo que este trabalho pode servir como base para a realização de um trabalho futuro.

Avaliação da dor

Embora com tendência para melhorias, a prática dos cuidados continua a demonstrar que a avaliação da dor é uma actividade ignorada, esquecida ou realizada de forma pouco fidedigna (precisa).3 Esta prática compromete seriamente a qualidade dos cuidados, na medida em que a eficaz prevenção e o tratamento da dor carece de uma avaliação segura e exacta. A não satisfação desta condição é como conduzir um carro em dia de nevoeiro onde o perigo espreita a qualquer momento. Avaliar um fenómeno complexo e subjectivo como é a dor não é tarefa fácil. Contudo, o conhecimento adquirido nesta área permite aos profissionais de saúde o reconhecimento das dificuldades inerentes a esta actividade e oferece soluções viáveis para a sua resolução. Desde 1989 que a avaliação da intensidade da dor como 5º sinal vital é recomendada.3 Os esforços desenvolvidos para a sua implementação na prática dos cuidados em Portugal não se têm revelado tarefa fácil. O desenvolvimento de mais uma tarefa (avaliação e registo da intensidade da dor), as dificuldades na sua execução com especial relevo no grupo pediátrico, geriátrico, com multideficiência e em Unidades de Cuidados Intensivos (UCI) têm ditado o insucesso. As razões que justificam esta Unidade Curricular resultam do facto de se reconhecer que a avaliação da dor é das actividades mais complexas que os Enfermeiros desempenham e a falta de formação existente nesta área. Com este manual pretendemos que os formandos possam desenvolver as suas capacidades de: argumentar criticamente mitos e crenças relacionadas com a avaliação da dor; elaborar uma história de dor; avaliar a intensidade da dor na pessoa de acordo com o seu contexto clínico; analisar a utilidade da aplicação de escalas de avaliação de dor e o registo da sua intensidade; interpretar as propriedades psicométricas e utilidade clínica de alguns instrumentos de dor; elaborar protocolos de implementação da avaliação da intensidade da dor como 5º sinal vital.

Síndrome Takotsubo após procedimento anestésico em idade pediátrica - um caso clínico

A síndrome Takotsubo (STT) é uma forma adquirida e transitória de disfunção sistólica, cuja apresentação clínica e eletrocardiográfica mimetiza um enfarte agudo do miocárdio. A STT é também conhecida como miocardiopatia de stress, síndrome do «coração partido», balonamento apical, insuficiência cardíaca aguda reversível, miocárdio «atordoado» (forma neurogénica) ou miocardiopatia aguda das catecolaminas. Os autores descrevem uma apresentação rara de STT após procedimento anestésico. Adolescente de 14 anos, sexo feminino, com antecedentes pessoais de enxaqueca hemiplé- gica e quisto pineal, submetida a ressonância magnética (RM) cranioencefálica de controlo. Durante a indução anestésica com propofol verificou-se bradicardia, revertida com atropina, seguida de taquidisritmia ventricular, revertida com lidocaína e murro pré-cordial. Nas primeiras horas de internamento evoluiu para edema pulmonar associado a insuficiência respiratória global por disfunção ventricular esquerda aguda. O ecocardiograma transtorácico mostrou dilatação do ventrículo esquerdo com hipocinesia global e fração de ejeção reduzida (< 30%). O eletrocardiograma revelou taquicardia sinusal persistente e alterações inespecíficas do segmento ST. Os biomarcadores cardíacos encontravam-se elevados (troponina 2,42 ng/ml, proBNP 8248 pg/ml). Foi medicada com diuréticos, IECA, digitálico e dopamina, com melhoria clínica, bioquímica e ecocardiográfica ao quarto dia. Os ecocardiogramas subsequentes mostraram normalização da função ventricular. A doente teve alta medicada com carvedilol, que suspendeu após normalização da função cardíaca e RM cardíaca não ter revelado alterações. Estão descritos poucos casos de STT em idade pediátrica. Alguns são desencadeados por patologia aguda do sistema nervoso central, mas nem todos cumprem os critérios de diagnóstico clássicos. Neste caso, o procedimento anestésico poderá ter desencadeado a STT.

Saúde mental: o modelo clínico-comunitário numa unidade socio-ocupacional

Trabalho de projeto apresentado à Escola Superior de Educação de Paula Frassinetti, para obtenção do grau de mestre em Intervenção Comunitária

Avaliação do efeito citotóxico da lectina da esponja marinha Cliona varians contra células de leucemia mielóide crônica

In this study, a BCR-ABL expressing human chronic myelogenous leukaemia cell line (K562) was used to investigate the antitumoral potential of a novel lectin (CvL) purified from the marine sponge Cliona varians. CvL inhibited the growth of K562 cells with an IC50 value of 70 g/ml, but was ineffective to normal human peripheral blood lymphocytes in the same range of concentrations tested (180 g/ml). Cell death occurred after 72 h of exposure to the lectin and with sign of apoptosis as analysed by DAPI staining. Investigation of the possible effectors of this process showed that cell death occurred in the presence of Bcl-2 and Bax expression, and involved a caspase-independent pathway. Confocal fluorescence microscopy indicated a major role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) abolished the cytotoxic effect of CvL. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NFB) expression in CvL-treated cells. These effects were accompanied by increased levels of p21 and downmodulation of pRb, suggesting that CvL is capable of cell cycle arrest. Collectively, these findings suggest that cathepsin B acts as death mediator in CvL-induced cytotoxicity possibly in a still uncharacterized connection with the membrane death receptor pathway

Avaliação do extrato do fungo caripia montagnei e de agonistas de PPAR - α no processo inflamatório

The mushrooms have been object of intense research in view of its potential raising of application in different sectors of the pharmacology and alimentary industry. Among diverse bioactive composites of polyssacharides nature that exist in the fungus the glucans are much searched. These are polymers of glucose and classified as the type of glicosidic linking [α, β]. Peroxisome proliferator-activated receptors (PPARs), ranscription factors belonging to the family of nuclear receptors that bind themselves o specific agonists, have shown their importance in controlling the inflammatory process. The aim of this study was to perform a chemical characterization of extract rom the mushroom Caripia montagnei, assess its antiinflammatory and antibacterial effect and determine if this effect occurs via PPAR. This mushroom is composed of carbohydrates (63.3±4.1%), lipids (21.4l±0.9%) and proteins (2.2± 0.3%). The aqueous solution resulting from the fractionation contained carbohydrates (98.7±3.3%) and protein (1.3±0.25%). Analyses of infrared spectrophotometry and of nuclear magnetic esonance demonstrated that the extract of mushroom C. montagnei is rich in β-glucans. In hioglycolate-induced peritonitis, the C. montagnei glucans (50 mg/kg) educed the inflammatory process in 65.5±5.2% and agonists, pharmacological igands, for PPAR: Wy-14643 (49.3±6.1%), PFOA (48.9±3.8%) and clofibrate in 45.2±3.2%. Sodium diclofenac showed a reduction of 81.65±0.6%. In the plantar edema, the glucans from C. montagnei (50 mg/kg) and L-NAME reduced the edema to a similar degree 91.4±0.3% and 92.8±0,5 %, respectively. In all the groups tested, nitric oxide (NO), an inflammation mediator, showed a significant reduction in the nitrate/nitrite levels when compared to the positive control (P<0.001). The C. montagnei glucans did not show cytotoxicity in the concentrations tested (2.5, 5.0, 10.0, 20.0 and 40.0 µg/100 µL). Antibacterial activity demonstrated that, unlike total extract, there was no inhibition of bacterial growth. The C. montagnei glucans show great potential for antiinflammatory applications. This effect suggests that it is mediated by PPAR activation and by COX and iNOS inhibition

Avaliação das concentrações hepáticas e séricas de retinol em bovinos e do consumo habitual de fígado por gestantes

The vitamin A is essential to animals because of its participation in a great number of biological functions. The investigation of this vitamin s concentrations is important to serve as reference to normality parameters. This study had as aim to analyse the serics and hepatics concentrations of vitamin A in two groups of bovines and to compare the hepatics concentrations to the present requeriments of vitamin A for pregnant women. It was also appraised the consume habit of bovine liver by pregnant women through of the alimentary frequency quest. Two groups of bovine were studied and the first was formad by Nelore bovine breed and the second by bovine without defined breed (WDB). It was analysed 120 samples: 60 of liver and 60 of serum. The method used to dose retinol was High Performance Liquid Cromatography (HPLC). The average (+ sd) of retinol concentrations in Nelore breed bovine and WDB liver were 16947,8 + 6866,9 and 5213,1 + 2517,2 µg of retinol/100g and at serum 39,6 + 17,9 e 28,6 + 9,4 µg of retinol/dL, respectively. No statistically significant correlation was found between hepatic and the serum retinol. The bovines in this study had adequate vitamin A levels. Independently of animal breed, the daily ingestion of bovine liver is not advised for pregnant women who show adequate support of vitamin A. The consume of bovine liver by pregnant women consulted on school maternity hospital Januário Cicco, UFRN, Natal RN, was considered high

Avaliação da concentração de retinol em fígados frescos e congelados de frangos das linhagens Cobb e Ross

The intake of adequate quantities of food, including those rich in vitamins, is necessary for a healthy life. The lack of vitamin A has been characterized as a public health problem in developing countries, however, a high intake of vitamin A can result in toxic and teratogenics effects. High concentrations of vitamin A have been observed in the livers of animals. The objective of this study was to assess the levels of retinol in chicken livers and verify the effect of frozen storage on these levels. 64 livers from two chicken strains, Cobb and Ross, were used, came from four different farms. We examined 32 livers from each strain, 8 samples from each farm. Liver sample were homogenized individually, then 4 aliquots were taken from each sample. One of aliquots was analyzed immediately after slaughter (T0), the others were analyzed after 30, 60 and 90 days of storage at -18oC (T30, T60 and T90, respectively). Retinol dosage in the liver was determined by High Performance Liquid Chromatography (HPLC). The levels of retinol varied significantly according to the strain. The mean retinol value in the fresh samples was 6678.0 ± 1337.7 and 8324.1 ± 1158.5 µg/100g in the Cobb and Ross strain, respectively. Values of 4258 ± 918.7 ± 1391.7 and 4650.5 ± 1391.7 μg/100g were found after 90 days of storage for Cobb and Ross strain, respectively. The liver freezing caused a significant reduction in their levels of retinol, causing a loss of up to 44% with respect to fresh livers. The reduction in retinol levels occurred from 30 days of storage. Even with the losses from the frozen, the ingestion of a typical portion of 100 g of liver, regardless the chicken strain analyzed, surpass all recommendations of consumption and the maximum tolerable intake of vitamin A (3000 μg/day) for adults

Avaliação de megadoses de retinol palmitato no pós-parto imediato sobre o retinol do leite de puérperas atendidas no Hospital José Pedro Bezerra, Natal-RN

Vitamin A deficiency (VAD) is a serious public health problem in developing countries, and as a therapeutic and prophylactic measure retinil palmitate is being supplemented. Nevertheless its efficacy has been questioned. The objective of the study was to evaluate the supplementation of two retinil palmitate megadosis on the serum retinol levels of post partum healthy mothers from Dr. José Pedro Bezerra (Hospital Santa Catarina) hospital, Natal - RN. The enrolled women (n=199) were randomly distributed into three studied groups and supplemented with retinil palmitate immediately after delivery with a single 200,000 IU dose (group S1), two 200,000 IU dose (group S2) with 24h difference between the doses, or no supplementation (group C). Among women selected, 143 remained until the end of the study. The influence of vitamin A dietary intake was evaluated during pregnancy and after 30 days of delivery. The average intake of the population was reasonable, but a high prevalence of inadequate intake was found. Retinol in colostrums and mature milk was determined by high performance liquid chromatography (HPLC). The retinol average in colostrums and mature milk in the supplemented and control groups were adequate according to the reference values. In colostrums, women from groups C, S1 and S2 presented retinol averages by milk volume of 94.8 ± 40.2 µg/dL, 92.2 ± 50.0 µg/dL and 91.8 ± 53.7 µg/dL, respectively. No difference was found between these averages (p=0.965), this was also seen when the values where expressed as µg/g of fat (p=0.905). After 30 days of delivery, retinol per milk volume differed between the control group (36.6 ± 17.5 µg/dL) and groups supplemented with 200,000 IU (51.0 ± 28.8 µg/dL) or 400,000 IU (55.2 ± 31.6 µg/dL) of retinil palmitate (p<0,05). Nevertheless, when S1 and S2 groups where compared, no significant difference was found (p=0.97). Considering retinol/g of fat, the means were 12.7 ± 6.7 µg/g, 15.6 ± 8.3 µg/g and 17.2 ± 8.9 µg/g for groups C, S1 and S2, respectively, with significant difference between groups S2 and C (p=0,01). Subclinical VAD prevalence showed a serious public health problem in the study population (32% in colostrums and 31.5% in mature milk). When analyzing the groups separately, the group which received two doses (200,000 IU + 200,000 IU) presented the lowest VAD prevalence (20.7%). Retinil palmitate supplementations of 200,000 IU and 400,000 IU (divided in two doses) in the immediate post partum showed no significant difference. Nevertheless, the 400,000 IU (divided in two doses) supplementation showed a reduction in VAD

Avaliação da suplementação materna com megadose de vitamina A sobre os níveis de retinol e alfa-tocoferol no colostro

The vitamins A and E are recognizably important in the initial stages of life and the newborn depends on nutritional adequacy of breast milk to meet their needs. These vitamins share routes of transport to the tissues and antagonistic effects have been observed in animals after supplementation with vitamin A. This study aimed to verify the effect of maternal supplementation with vitamin A megadose (200,000 UI) in the immediate post-partum on the concentration of alpha-tocopherol in colostrum. Healthy parturient women attended at a public maternity natalensis were recruited for the study and divided into two groups: control (n = 37) and supplemented (n = 36). Blood samples of colostrum and milk were collected until 12 hours after delivery. The women of the supplemented group was administered a retynil palmitate capsule and 24 hours after the first collection was obtained the 2nd sample of colostrum in two groups for analysis of retinol and alpha-tocopherol in milk. The mean retinol concentration of 50,7 ± 14,4 μg/dL (Mean ± standard deviation) and alpha-tocopherol of 1217.4 ± 959 mg/dL in the serum indicate the nutritional status biochemical appropriate. Supplementation with retynil palmitate resulted in increase not only retinol levels in the colostrum of the supplemented group (p = 0.002), but also the concentration of alpha-tocopherol (p = 0.04), changing from 1456.6 ± 1095.8 mg/dL to 1804.3 ± 1432.0 mg/dL (milk 0 and 24 respectively) compared to values in the control group, 984.6 ± 750.0 mg/dL and 1175.0 ± 730.8 mg/dL. The women had different responses to supplementation, influenced by baseline levels of retinol in colostrum. Those with previous by low levels of retinol in colostrum (<60 mg/dL) had increased the concentration of alpha-tocopherol in milk, whereas those with adequate levels (> 60 mg/dL), showed a reduction after supplementation. Supplementation with retinol palmitate is an important intervention in situations of high risk for vitamin A deficiency, when considering the need to maternal supplementation, since the excess vitamin can offer unfavorable interactions between nutrients essential for the mother-child group

Avaliação do potencial anti-inflamatório, anticoagulante e hemorrágico de heparinóides presentes em "Artemia franciscana"

Heparan sulfate (HS) and Heparin (Hep) glycosaminoglycans (GAGs) are heterogeneous and highly charged polysaccharides. HS is structurally related to Hep but is much less substituted with sulfo groups than heparin and has a more varied structure (or sequence). Because of structural similiarities between these two polymers, they have been described together as heparinoids . Both chains bind a variety of proteins and mediate various physiologically important processes including, blood coagulation, cell adhesion and growth factor regulation. Heparinoids with structural characteristics similar to these described from HS and/or Hep from mammalian tissues have been isolated from different species of invertebrates, although only a few heparinoids from unusual sources have been characterized. The present study describes the presence of unusual heparinoids population from Artemia franciscana, isolated after proteolysis and fractionation by ion exchange resin and named, F-3.0M. The study model in vivo were hemostasis (rat tail scarification) and inflamatoty activity. The tests in vitro were used for coagulations assays (PT and APTT). The analyse of the heparinoids eluted with 3,0M NaCl showed electrophoretic migration in different buffer systems a single band with a behaviour intermediate between those of mammalian HEP and HS. The main products obtained from Artemia heparinoids after enzymatic degradation with heparitinases I and II from F. heparinum were N-sulphated disaccharides (∆U-GlcNS,6S/ ∆U,2S-GlcNS and ∆U-GlcNS) and N-acetylated disaccharides (∆U, GlcNAc). This heparinoid had a lower hemorrhagic effect (400μg/ml) when compared to unfractiionated heparins(25μg/ml).The results also suggest a negligible APTT activity of this heparinoid (62.2s). No action was observed on PT indicating that F-3.0M haven t action on the extrinsic pathway. The results showed that the fraction F- 3.0M have inhibitory effect on migration of leukocytes, 64.5% in the concentration of 10 μg/ml (P<0.001). The search for new heparin and/or heparan sulphates analogs devoid of anticoagulant activity is an atractive alternative and may open up a wide variety of new therapeutic applications

Avaliação das propriedades farmacológicas de polissacarídeos do fungo Scleroderma nitidum

Several pharmacological properties have been attributed to isolated compounds from mushroom. Recently, have these compounds, especially the polysaccharides derived from mushrooms, modulate the immune system, and its antitumor, antiviral, antibiotic and antiinflammatory activities. This study assesses the possible pharmacological properties of the polysaccharides from Scleroderma nitidum mushroom. The centesimal composition of the tissue showed that this fungus is composed mainly of fibers (35.61%), ash (33.69%) and carbohydrates (25.31%). The chemical analysis of the polysaccharide fraction showed high levels of carbohydrates (94.71%) and low content of protein (5.29%). These polysaccharides are composed of glucose, galactose, mannose and fucose in the following molar ratios 0.156, 0.044, 0.025, 0.066 and the infrared analysis showed a possible polysaccharide-protein complex. The polysaccharides from Scleroderma nitidum showed antioxidant potential with concentration-dependent antioxidant activity compared to ascorbic acid. The analysis scavenging of superoxide radical and inhibition of lipid peroxidation showed that the polysaccharides from S. nitidum have an IC50 of 12.70 mg/ml and EC50 10.4 μg/ml, respectively. The antioxidant activity was confirmed by the presence of reducing potential of these polysaccharides. The effect of these polymers on the inflammatory process was tested using the carrageenan or histamine-induced paw edema model and the sodium thioglycolate or zymosan-induced model. The polysaccharides were effective in reducing edema (73% at 50 mg/kg) and cell infiltrate (37% at 10 mg/kg) in both inflammation models tested. Nitric oxide, a mediator in the inflammatory process, showed a reduction of around 26% at 10 mg/kg of body weight. Analysis of pro- and anti-inflammatory cytokines showed that in the groups treated with polysaccharides from S. nitidum there was an increase in cytokines such as IL-1ra, IL-10, and MIP-1β concomitant with the decrease in INF-γ (75%) and IL-2 (22%). We observed the influence of polysaccharides on the modulation of the expression of nuclear factor κB. Thus, polysaccharides from S. nitidum reduced the expression of NF-κB by up to 64%. The results obtained suggest that NF-κB modulation is one of the possible mechanisms that explain the anti-inflammatory effect of polysaccharides from the fungus S. nitidum.

Avaliação do potencial antioxidante e antiinflamatório de galactomanana do fungo Tyllopiillus ballllouiii.

Polymers of mushroom cellular wall are recognized for presenting a lot of biological activities such as anti-inflammatory, antioxidant and anti-tumoral action. Polysaccharides from mushrooms of different molecular mass obtained mushrooms can activate leucocytes, stimulate fagocitic, citotoxic and antimicrobial activity including oxygen reactive species production. In this study were investigated chemical characteristics, in vitro antioxidant activity and anti-inflammatory action in an acute inflammation model of the polysaccharides extracted from Tylopilus ballouii. Results showed that were mainly extracted polysaccharides and that it primarily consisted of mannose and galactose with variable amounts of xylose and fucose. Infrared analysis showed a possible interation between this polysaccharides and proteins. In addition, molecular mass was about 140KDa. Antioxidant activity was tested by superoxide and hydroxyl radical scavenging assay, total antioxidant activity and lipid peroxidation assay. For superoxide and hydroxyl radical generation inhibition, polysaccharides have an IC50 of 2.36 and 0.36 mg/mL, respectively. Lipid peroxidation assay results showed that polysaccharides from Tylopilus ballouii present an IC50 of 3.42 mg/mL. Futhermore, anti-inflammatory assay showed that polysaccharides cause an paw edema decreasing in 32.8, 42 and 56% in 30, 50 and 70 mg/Kg dose, respectively. Thus, these results can indicate a possible use for these polysaccharides from Tylopilus ballouii as an anti-inflammatory and antioxidant.

Purificação, caracterização e avaliação da atividade antiproliferativa de um inibidor de quimotripsina tipo kunitz de semente de Eryhrina velutina

A chymotrypsin inhibitor was purified from Erythrina velutina seeds by ammonium sulphate fractionation, affinities chromatographies on Trypsin-Sepharose, Quimotrypsin-Sepharose and reversed phase C-18 FPLC/AKTA system. The inhibitor, named EvCI, shown molecular mass of 17 kDa, as determined by SDSPAGE. 2D-PAGE showed four isoinhibitors with pI values of 4,42, 4,63, 4,83 and 5,06, with molecular mass of 17 kDa each. The aminoacid sequence of EvCI was determined by MALDI-TOF-MS and showed a high similarity with other Kunitz-type inhibitor of Erythrina variegata. EvCI competitively inhibited chymotrypsin, with Ki of 4 x10-8 M, but did not inhibited trypsin, pancreatic elastase, bromelain and papain. The inhibitory activity of EvCI was stable over wide pH and temperature ranges. In the presence of DTT 100 mM for 120 min, EvCI lost 50 % of activity. Cytotoxicity was studied in HeLa, MDA, HepG2, K562 and PC3 cells after 72-h incubation period. EvCl inhibited HeLa cells growth with an IC50 value of 50 μg/ml. Subsequent studies in HeLa cells analysis of cell death by annexin V/PI double-staining and cell cycle, using flow cytometry. The results provide evidence for a cytostatic activity of EvCl and support further studies on potential application of this inhibitors as an antiproliferative agent in combined therapy against cervical cancer