Aplicación del método bioquímico-colorimétrico para identificar agentes trypanocidas / Application of the biochemical-colorimetric method to identify trypanocidal agents

La tripanosomiasis americana es una enfermedad infecciosa desatendida, causada por el parásito protozoo Trypanosoma cruzi, que no cuenta con tratamiento en la fase crónica de esta enfermedad mortal, uno de los desafíos es encontrar terapias efectivas para esta compleja enfermedad, dado que no presenta síntomas asociables a la parasitosis por lo que es desconocida entre los médicos tradicionales. Nuestra Facultad está evaluando la medicina tradicional tacana como fuente de agentes antiparasitarios potenciales. El objetivo de este trabajo fue identificar productos naturales trypanocidas utilizando el método colorimétrico XTT-PMS. Para ello, se realizaron curvas de crecimiento de epimastigotes de T. cruzi y determinamos el tiempo óptimo de realización de los ensayos. Se seleccionó la población inicial de trabajo (3x106 parásitos/mL), las condiciones de incubación (medio LIT, 27ºC, 72 horas) y revelado (XTT-PMS, 4 horas). Con el protocolo optimizado, se realizaron evaluaciones de actividad de drogas control, controles naturales y 20 extractos crudos de plantas medicinales de la amazonía. La actividad se basó en cálculos de concentración inhibitoria media y se consideraron activos las sustancias con CI50<50µg/mL. De los 20 extractos evaluados, el 40% fueron activos. Las plantas más interesantes fueron Sipu sipu (CI50=8.9±1.7µg/mL), Ejije bid'u (CI50=9.1±1.5µg/mL) e Id'ene eidhue (CI50=10.8±1.1µg/mL) con valores de CI50 cercanos a los controles, confirmando la utilidad y potencial del protocolo desarrollado

American trypanosomiasis is listed among the unattended infectious disease, is caused by the protozoan parasite Trypanosoma cruzi, and has no treatment in the chronic phase of this deadly disease. One of the challenges is finding effective therapies for this complex disease, given that it does not present any associated symptoms to the parasitism and is unknown among traditional doctors. Our Faculty is evaluating tacana traditional medicine as a source of potential antiparasitic agents. The objective of this work was to identify trypanocidal natural products using the XTTPMS colorimetric method. For this, growth curves of T. cruzi epimastigotes were made to determine the optimal time to carry out the tests. The initial work population (3x106 parasites/mL), the incubation conditions (medium LIT, 27ºC, 72 hours) and revealed process (XTT-PMS, 4 hours) were selected. With the optimized protocol, activity evaluations of control drugs, natural controls and 20 crude extracts of medicinal plants of the Amazon were carried out. The activity was based on calculations of mean inhibitory concentration and substances with IC50 <50µg/mL were considered active. Of the 20 extracts evaluated, 40% were active. The most interesting plants were Sipu sipu (IC50=8.9±1.7µg/mL), Ejije bid'u (IC50=9.1±1.5µg/mL) and Id'ene eidhue (IC50=10.8±1.1µg/mL) with values of IC50 close to the controls, confirming the usefulness and potential of the developed protocol.

Comparação entre três métodos analíticos para determinação de soro em leite cru refrigerado / Comparison of three analytical methods to determine serum in refrigerated raw milk

O objetivo deste experimento foi comparar três métodos analíticos para determinação de soro em leite cru refrigerado: cromatografia líquida de alta eficiência, ninidrina ácida e colorimétrico adaptado. Foram coletadas 100 amostras de leite cru refrigerado de tanques de expansão. Estas, quando submetidas à análise pelo método da ninidrina ácida, apresentaram 10 (14,7%) amostras negativas e 58 (85,3%) positivas. O teor médio de ácido siálico encontrado na técnica da ninidrina foi de 5,58(g/mL, com valor mais frequente de 2,70(g/mL. Das 68 amostras negativas pela cromatografia líquida de alta eficiência, duas foram positivas (2,94%) e 66 (97,06%) negativas, quando analisadas pelo método colorimétrico. A frequência relativa de amostras positivas foi de 32%, com a CLAE apresentando a maior média de soro (14,37%), seguida do método colorimétrico (5,28%) e o da ninidrina ácida (3,12%). A técnica de cromatografia líquida de alta eficiência diferiu dos métodos de ninidrina ácida e colorimétrico, enquanto os métodos da ninidrina e colorimétrico não diferiram entre si, podendo ambos serem utilizados como metodologias de triagem. Entre as três técnicas, a cromatografia líquida de alta eficiência foi a metodologia mais sensível na detecção e quantificação do soro em leite cru refrigerado.(AU)

The objective of this study was to compare three analytical methods to determine serum in refrigerated raw milk. High-performance liquid chromatography (HPLC), acidic and colorimetric ninhydrin methods were applied. A collection of 100 samples of raw milk from cooled expansion tanks took place. The results showed that 10 samples (14.7%) were negative and 58 (85.3%) were positive for the acidic ninhydrin method. The mean sialic acid content found in the ninhydrin technique was 5.58µg/mL, with a more frequent value of 2.70µg/mL. From all 68 HPLC negative samples, two were positive (2.94%) and 66 (97.06%) negative to the colorimetric method. The relative frequency of positive samples was 32%, HPLC had the highest mean serum levels (14.37%), followed by the colorimetric method (5.28%) and acid ninhydrin (3.12%). The high-performance liquid chromatography method was different from the acid and colorimetric ninhydrin methods. The ninhydrin and colorimetric methods were not different from each other, both of which could be used as screening methodologies. Among the three techniques, HPLC was the most sensitive methodology for the detection and quantification of serum in refrigerated raw milk.(AU)