Antioxidant Activity of Hydrolyzed Melinjo (Gnetum Gnemon) Seeds Protein Against Neutrophil Superoxide Radical in Vitro
Superoxide radical (O2-) is one type of reactive oxygen species (ROS) that play important role in pathogenesis of various inflammatory diseases. The main source of ROS is inflammatory cell neutrophil, that will produce a large amount of ROS to response against antigen. This study purposed to analyze antioxidant activity of the hydrolyzed melinjo (Gnetum gnemon) seeds protein (Gg-PH) against superoxide radical produced by neutrophil. This in vitro study used neutrophil isolated from human peripheral blood vein using Histopaque Gradient Centrifugation method. Briefly, neutrophil were incubated with antigen and antioxidant for 1 hour and 18 hours. Antioxidant activity were demonstrated by reduction of superoxide production. Experiments were divided into three groups, (1) neutrophil + antigen + Gg-PH, (2) neutrophil + antigen + glutathion (GSH), (3) neutrophil + antigen. The antigen is formyl-methionyl-leucyl-phenylalanine (fMLP). Superoxide production was analyzed by means of Nitro Blue Tetrazolium (NBT) assay. Intracellular superoxide were demonstrated microscopically, whereas extracellular superoxide spectrophotometrically. Result showed that incubation of Gg-PH for 1 hour reduced intracellular superoxide production significantly (p<0,05), but not for extracellular superoxide (p>0.05). Incubation for 18 hours could not prevent superoxide production, both intracellular and extracellular. Inconclusion Gg-PH had antioxidant activity against intracellular superoxide radical for 1 hour incubation but not in extracellular. Effect of dosis and duration of incubation need to be analyzed further.
Keywords— Antioxidants, Gnetum gnemon Protein, Neutrophil, Superoxide Radical, Nitro Blue Tetrazolium (NBT)