1. INTRODUCTIONThe excessive production of reactive oxygen species (ROS) affect cellular signaling pathways, which is associated with pathological and physiological conditions such as cancer, diabetes and neurodegenerative diseases [1] . Cellular defense mechanisms against oxidative damage include enzymatic conversion of ROS (e.g., 
and OH−) into less reactive species, chelation of transition metal catalysts and detoxification of ROS by antioxidants [2] . Thus, application of antioxidants might be an effective therapeutic strategy to cure neurodegenerative disorders initiated by ROS. Because of this, there is growing interest in the scientific community and industry to obtain naturally occurring substances aimed at the prevention and treatment of these pathologies.
It has also reported a number of possible bioactivities that might have, including antimicrobial capacity, hypertensive, antioxidant and anticancer [3]. Accordingly, biotechnological products are among various naturally occurring substances that are receiving growing attention from the viewpoint of antioxidation. The antioxidant peptides can be isolated from different sources such as marine sources [4] and plants [5]. Some of them have been accepted to be one of the important candidates for the development of effective and non-toxic medicines with antioxidant actions. The presence of His (H) within the peptide sequence is characteristic of antioxidant peptides, together with the presence at the N-terminal residues Leu (L) or Pro (P) [6]. According to the findings by Chan and Decker (1994) [7], the structure-activity relationship of antioxidants in their own sequence His, is attributed to the ability of hydrogen donation and/or metal chelation capacity of the imidazole group.
The plants, the subject of a growing number of natural product researches, are now considered as efficient producers of biologically active and/or chemically novel compounds. One source plants used for extraction of bioactive polypeptides is the genus Bauhinia, which has more than 600 species of wide distribution in tropical and subtropical forests [8]. Many proteins have been isolated from its seeds, and in particular bauhinioides species [9] . Thus, functional and biologically active peptides and derivatives obtained from seeds, as well as being a contribution to their nutritional value, have the ability to exert physiological functions that native protein sequenced from a large active, can be obtained by scalable production processes industrial, or by synthesis, by fermentation or by methods of recombinant protein. In this context, our laboratory has utilized the Bios-p peptide analogue, which represents the active 12-amino acid active site, obtained by sequencing from the inhibitor BbKI protease present in the seed of Bauhinia bauhinoides and patented by the working group of Dr. Maria Luiza Vilela Oliva of UNIFESP, Brazil. In this study we investigated the protector effect and antioxidant capacity of Bios-p peptide in the cellular model HEK293T when these were subjected to oxidative stress induced by hydrogen peroxide (H2O2). On the other hand, it is known that the function of the bioactive peptide is dependent on its amino acid sequence and three dimensional structures that these possess [10] and binding with cell membrane components. Because of this, we have obtained a threedimensional structure of Bios-p using modeling basedhomology.
2. MATHERIALS AND METHODS2.1. Cellular Lines and Culture ConditionsHEK293 cells were cultured in D-MEM medium supplemented with 10% FBS (Hyclone) and penicilin-streptomicin 1% (Invitrogen). Cell growth was done at 5% CO2 and 37˚C changing the culture medium every three days.
2.2. Incubation of HEK293T Cells with Bios-p Peptide AnalogueThe cells were grown during 24 h in six-well plates and allowed to reach to 50% confluence. Then, were incubated with Bios-p analogue peptide at different concentrations of 0,1; 1 and 10 µg/ml during 24 h at 37˚C. Additionally cells were treated with H2O2 200 µM for 15 min at 37˚C to induce the oxidative stress.
2.3. Cells-Viability AssessmentThe cells were incubated with the probe SYTOX Green 0.5 µM (Molecular Probes, Eugene, OR) at 37˚C for 15 min in darkness. The cells were analyzed in a confocal microscope at 510 nm emission of SYTOX Green. The percentage of viable cells was calculated by manually counting the number in the x-y reference frame visualized in ten different fields.
2.4. Production of ROS ExtracellularThe production of ROS extracellular were measured by luminescence assay by incubating the cells with luminol 200 µM (5-amino-2, 3 hydro-1, 4-ftalazinediona, Sigma Chemical Co., St. Louis, MO) for 15 min at 37˚C in darkness and immediately quantifying the luminescence in a luminometer Luminoskan mark (Thermos Scientifics, China), expressing the results as relative luminescence units (RLU). In each analysis, a negative control without the addition of luminol and positive control cells treated with H2O2 200 µM, were added.
2.5. Anion Superoxide Intracellular ProductionThe cells were incubated with the probes DHE/SYTOX Green (2 µM and 0.5 µM respectively) (Molecular probes, Eugene, OR) at 37˚C for 15 min in darkness. The results obtained with this probe have been validated as a measure of the ability of cells to generate ROS, specifically definitive identification of the superoxide anion. The cells were analyzed in a confocal microscope at 510 and 670 nm emissions of SYTOX Green and DHE, respectively [11]. The percentage of viable cells producing superoxide anion was calculated by manually counting the number in the x-y reference frame, and dividing by the total number of cells visualized in ten different fields.
2.6. Homology ModelingThe three-dimensional structure of Bios-p peptide analogue was predicted by homology-based modeling using Modeller9v8 [12]. BLAST-P was used to identify the potential template structures for molecular modeling. The templates are in Protein Data Bank (PDB) WEB page, with PDB IDs 2GO2/2GZB. The protein models were validated using prochek [13] and Anolea [14].
2.7. Statistical AnalysisThe data for the different functional parameters evaluated were expressed as mean + SEM. The data were analyzed with GraphPad Prism version 5.02 for Windows (GraphPad Software, San Diego, CA, USA). The differences between the groups were analyzed using the one way analysis of variance (ANOVA) followed by Tukey multiple comparison tests. P values < 0.05 were considered as significant.
3. RESULTS AND DISCUSSION3.1. Bios-p Protects the Cells-ViabilityWas evaluated the protective effect of Bios-p peptide analog against H2O2-induced cytotoxicity in the HEK 293T cellular model (Figure 1). When added to cell culture H2O2 200 µM without pre-treatment with Bios-p, was observed a decreased of cell viability product of H2O2-cytoxicity. However, the pre-treatment with different concentrations of Bios-p (1 - 10 μM) showed a increase of 53.83% ± 3.86% the cellular viability in under oxidative stress compared to control. As shown in the Figure 1(a), the protective effect on the viability of Bios-p is concentration-dependent (P < 0.05). Additionally, we determined the effective concentration of Bios-p to maintain viable cells by 50% (EC50) when these are subjected to H2O2-induced oxidative stress. The EC50 was determined in 7.51 µM ± 0.09 µM.
Furthermore, we directly examined the viability effect when the cells were pretreatment with the EC50 of Bios-p (7.51 µM) during 6-12-18 and 24 h. As shown in the
Figure 1(b) the incubation with Bios-p significantly increased the viability cells (P < 0.05) when the cells are under oxidative stress with H2O2. Moreover, the results show that by incubating the cells at 37˚C during 6, 12, 18 and 24 h with Bios-p (7.51 µM), was not observed a significant effect on the protection of viability respect to control in the absence of Bios-p when these cells are not in conditions of oxidative stress (P > 0.05).