EVALUATION OF THE CYTOTOXIC AND NEUROPROTECTIVE EFFECT OF STANDARDIZED EXTRACT AND RIZONIC ACID from Erythrina velutina

AH Silva1, JT Pires Filho1, AA Lopes1, TM Rocha1, ATA Pimenta2, MAS Lima2, HYF Magalhães3, HV Nobre Júnior1, SMM Vasconcelos3, LKAM Leal1,3. 1Federal University of Ceara, Pharmacy, Brazil, 2Federal University of Ceara, Organic and Inorganic Chemistry, Brazil, 3Federal University of Ceara, Physiology and Pharmacology, Brazil

Introduction and aim: The trunk bark of Erythrina velutina Wild. (Leguminoseae) is largely used in Northeast Brazil for the treatment of central nervous system disorders. Previous chemical studies of this species showed the presence of several bioactive phenols molecules, including flavonoids. The aims of this study were to investigate the citotoxity and neuroprotective effects of extract and rizonic acid (RA) from E. velutina, on neutrophils and SHSY5Y cell line exposure to the neurotoxin 6-hydroxydopamine (6-OHDA). Methods and Results: The spectrophotometric analysis of ethanolic extract from the trunk bark of E. velutina (EEEV) allowed to detect and quantify phenols total (155 ± 3,3 µg/mg), while HPLC analysis establish the fingerprint of the extract detecting several chemical constituents, as hesperidin, abissinin, homoesperidin, rizonic acid (RA) and sigmoidin. The pharmacological tests include the determination of cell viability by the lactate dehydrogenase (LDH) and MTT method and the measurement of nitrite concentration on culture cells. Polymorphonuclear (2.5 x106 cells/mL), predominantly neutrophils (80-90%) with cell viability of 95% (technical Trypan Blue exclusion), were isolated from human blood (Lucisano & Mantovani, 1984) and incubated (37°C) with EEEV (0.1; 1; 10; 100; 200 μg/mL), HBSS (negative control), vehicle (1% DMSO, control) or Triton (0.2%, standard drug). The two highest concentrations of EEEV (100 and 200 µg/mL) promoted a significant (p<0.05; ANOVA, Tukey) increase in LDH activity (97.64 ± 10.86, 108.10 ± 10.41 U/L) as compared to control (44.56 ± 8.31 U/L). On the other hand, the EEEV did not interfere significantly the neutrophil viability evaluated by MTT assay (540 nm) (89.08 ± 8.18; 96.01 ± 4.94; 99.98 ± 4.00; 94.12 ± 3.63 and 98.17 ± 4.41%, respectively) as compared to control (82.16 ± 8.13%). In another set of experiments, the effect of EEEV and AR on neurotoxicity induced by neurotoxin 6-OHDA (25 µM) was investigated through the cell viability (MTT assay) in SHSY5Y cell line (0.7 x 105 cells/mL; medium: DMEM/F12 1, 10% FBS and 1% antibiotics). The addition of EEEV (0.0025; 0.025; 0.25; 0.5; 1 μg/mL) or RA (0.0025; 0.025; 0.25; 0.5 μg/mL) alone to the culture of SHSY5Y cell line did not interfere on cell viability. Furthermore, the EEEV (MTT absorbance: EEEV 1 + 6-OHDA = 0.3854 ± 0.0268) and RA (MTT absorbance: 0.5 + 6-OHDA = 0.3571 ± 0.0401) were effective as a preventive agent, since its addition before 6-OHDA partially reversed (MTT absorbance: control = 0.4241 ± 0.0204, 6-OHDA = 0.2268 ± 0.0172) the neurotoxin’s effect. The EEEV also prevented free radical formation indicated by the reduction of the increased nitrite concentration induced by 6-OHDA. This effect might be explained, at least in part by the presence of phenols compounds in the extract. Conclusion: In the present study the EEEV showed a relative toxicity at high concentration on human neutrophils. However, both EEEV and RA did not present toxic effects on SHSY5Y cell line and showed a neuroprotective effect against 6-OHDA-induced toxicity. These effects may be due, at least in part, to the antioxidant potential of the test drugs. Financial support: CNPq and CAPES.