Naringinase

Abstract:

Context: Naringin (NRG) is the major flavonoid (flavanone glycoside) in grapefruit juice. Its biological activity has been only partially characterized and little is known about the mechanism of the negative inotropic action of this flavonoid.

Aims: To evaluate the effects of NRG on the surface electrogram (ECG) and the force of contraction (FC) of mice hearts as well as on the sodium (INa), calcium (ICaL) and Na+ – Ca2+ exchange (INaCaX) currents of enzymatically isolated mouse ventricular cardiomyocytes.

Methods: ECG and FC were recorded on mouse hearts perfused in a Langendorff column. Ventricular cardiomyocytes were enzimatically dissociated and ionic currents recorded with the patch-clamp technique.

Results: NRG increased RR interval and shortened corrected QT only at high concentrations (30-100 µM). However, at a fixed heart rate, it decreased FC with an IC50 of 0.4 µM. NRG reduced INa with an IC50 of 0.07 µM but with a maximal inhibition of 60 %. NRG also depressed ICaL with an IC50 of 0.013 µM and increased its fast inactivation time constant. The effects on ICaL were not voltage-dependent. INaCaX was not affected by NRG.

Conclusions: Our results indicate that NRG exerts a negative inotropic effect in mice hearts that could be explained by a decrease in INa and ICaL. These actions should be taken into account when considering this molecule either as a dietetic supplement or as a template to develop therapeutic agents for human diseases.

******************************************************

Resumen:

Contexto: La naringina (NRG) es el principal flavonoide (glicósido de flavanona) en el jugo de toronja. Su actividad biológica ha sido solo parcialmente caracterizada y poco se conoce acerca del mecanismo de la acción inotrópica negativa de este flavonoide.

Objetivos: Evaluar los efectos de la NRG sobre el electrograma de superficie (ECG) y la fuerza de contracción (FC) de corazones de ratón, así como sobre las corrientes de sodio (INa), calcio (ICaL) y del intercambiador Na+ – Ca2+ (INaCaX) en cardiomiocitos ventriculares de ratón, aislados enzimáticamente.

Métodos: El ECG y la FC se registraron en corazones de ratón perfundidos en una columna de Langendorff. Los cardiomiocitos ventriculares se disociaron enzimáticamente y las corrientes iónicas se registraron con la técnica de patch-clamp.

Resultados: La NRG incrementó el intervalo RR intervalo y acortó el QT solo a altas concentraciones (30-100 µM). No obstante, a frecuencia cardíaca fija, disminuyó la FC con un IC50 de 0.4 µM. La NRG redujo INa con un IC50 de 0.07 µM pero con una máxima inhibición de 60 %. La NRG también redujo ICaL con un IC50 de 0.013 µM e incrementó su constante de inactivación rápida. Los efectos sobre ICaL no fueron dependientes del potencial. La INaCaX no fue afectada por la NRG.

Conclusiones: Nuestros resultados indican que la NRG ejerce un efecto inotrópico negativo en corazones de ratón que puede ser explicado por una reducción en INa e ICaL. Esas acciones deben ser tomadas en cuenta al considerar a esta molécula como suplemento dietético o como plantilla para desarrollar nuevos agentes terapéuticos para tratar las enfermedades en humanos.

The present research has been focused on isolation, identification and characterization of new isolate of fungi isolated from rotten pomelo (Citrus maxima), which was able to produce an extracellular debittering enzyme naringinase. The identification of the isolated fungus was based on 18S rRNA gene sequencing analysis and internal transcribed spacer region. The isolated fungi Aspergillus sp. SK01 was found to be capable of producing naringinase enzyme in liquid medium through submerged fermentation and produced enzyme was partially purified, identified, and characterized. The optimum conditions for enzyme production were pH 5.0, temperature 28±2°C, naringin (0.5%), and time 10 days. The activity of the crude enzyme was found 1.63 IU/ml at optimum value of pH 4.0, temperature 50°C in 4 hrs.