Química e atividades biológicas de Hancornia speciosa Gomes (Apocynaceae): inibição da enzima conversora de angiotensina (ECA) e efeito na quimioprevenção de câncer

Abstract

Hancornia speciosa Gomes (Apocynaceae) is a plant species found in cerrado, a savannahlike vegetation, popularly used to treat hypertension and inflammatory processes. Its potential anti-hypertensive activity has been previously demonstrated by in vitro inhibition of angiotensin converting enzyme (ACE) and vasodilatation in rat aortic rings. Several studies have evidenced the involvement of inflammatory process in cardiovascular diseases and carcinogenesis. The ability of angiotensin II in modulating the inflammatory process has also been established. Therefore, the goal of the present study was to carry out the bioguidedstudy of H. speciosa, aiming at isolating anti-hypertensive and cancer chemopreventive compounds. The ethanol extract of H. speciosa leaves (EHS), obtained by percolation with EtOH 96%, was initially fractionated by silica gel column chromatography, followed by constituents isolation and purification on different chromatographic supports (C-2, C-18 andSephadex LH-20). The potential anti-hypertensive activity was evaluated in vitro by ACE inhibition colorimetric assay, whereas cancer chemoprevention was tested in several in vitro models. EHS (IC50 = 62.8 ± 39.6 mg/mL) and derived fractions, assayed at 100 mg/mL,produced over 50% ACE inhibition, excepting the n-hexane fraction. Bioguided fractionation of the EtOAc:MeOH (1:1) fraction (IECA = 81.8 ± 32.1%) lead to the isolation of two compounds, identified by spectroscopic methods (U.V., I.R., NMR 1H and 13C) and X-rayanalysis as rutin (IC50 = 453,9 ± 78,4 mM, 1) and L-(+)-bornesitol (IC50 = 41.4 ± 9.6 mM, 2). Those constituents were also isolated or detected in several others ACE inhibiting fractions. L-(+)-bornesitol, its derivative per-O-acetyl-1L-(+)-bornesitol, myo-inositol and elevencarbohydrates (D-manose, D-glucose, D-galactose, D-xylose, D-fructose, L-arabinose, Ddulcitol, melibiose, sucrose, lactose and raffinose) were evaluated in the ACE inhibition assay. Per-O-acetyl-1L-(+)-bornesitol, the disaccharides and rafinose were inactive, whereas 2 (IC50 = 41.4 ± 9.6 mM), D-galactose (IC50 = 35.7 ± 5.6 mM), D-glucose (IC50 = 85.7 ± 23.3mM) and myo-inositol (CI50 = 449.2 ± 39.68 mM) did present ACE inhibiting activity. The common structural feature between L-(+)-bornesitol and D-galactose is an axial located hydroxyl group vicinal to a carbon bearing either a hydroxyl-methylene or a methoxyl groupas substituent. The lack of this group at C6 might explain myo-inositol being tenfold less active than 2, as well as the inactivity of D-xylose. The presence of hydroxyl groups seems to be also essential for ACE inhibition, since peracetylation of 2 resulted in an inactivecompound. Neither EHS nor its fractions were active in the aromatase, ornithine descarboxylase and COX-1 assays. Likewise, EHS did not inhibit COX-2, although the derived fractions EtOAc (ICOX-2 = 58.8 ± 3.0%) and MeOH (ICOX-2 62.8 ± 5.8%) were active. Conversely, 2 and the cyclitols scyllo-inositol, peracetyl-1L-(+)-bornesitol, myo-inositoland b-D-galactoside-myo-inositol were inactive in the COX-1 assay, whereas 2 inhibited COX-2 (ICOX-1= 29.1 ± 3.8%; ICOX-2= 59.8 ± 6.4%) and therefore was disclosed as a selective inhibitor. EHS (CD= 19.7 ± 0.02 mg/mL; IC50= 19.8 ± 0.6 mg/mL; CI = 1.0) was activein the quinone redutase (QR) induction assay, carried out in Hep1c1c7 murine cells, as well as hexane (CD= 21.5 ± 0.7 mg/mL; IC50= 19.6 ± 0.2 mg/mL; CI = 0.9) and EtOAc:MeOH (1:1) fractions (CD= 16.6 ± 0.4 mg/mL; IC50= 48.5 ± 2.9 mg/mL; CI = 2.9); nevertheless, none of thecompounds isolated from them showed activity. EHS (EC50 = 19.7 ± 1.0 mg/mL) and its fractions were active in the ARE induction assay, performed in HepaG2 cell; however, none of the isolated compounds showed activity. Excepting the n-hexane fraction, all fractionsderived from EHS (IC50 = 17.4 ± 5.8 mg/mL) were active in the TPA-induced NF-kB inhibition assay, suggesting both antiinflammatory and cancer chemoprevention effects. Bioguided fractionation of EHS led to the isolation of L-(+)-bornesitol (IC50 = 27.5 ± 3.8 mM), quinic acid(IC50 = 85.0 ± 12.3 mM) and rutin (IC50 = 26.8 ± 6.3 mM), whose exhibited lower activity than the starting fractions, thus suggesting synergic effects. The derivative per-O-acetyl-1L-(+)- bornesitol (IC50 = 38.4 ± 6.2 mM), scyllo-inositol (CI50 = 83.0 ± 13.7 mM) and b-D-galactosidemyo- inositol (IC50 = 52.4 ± 8.4 mM) were active in the TPA-induced NF-kB inhibition assay, while myo-inositol was inactive. None of the active samples showed anti-proliferative activityagainst four cell lines (MCF-7, LNCap, HepG2 and LU-1). Other compounds isolated from H. speciosa active fractions did not show activity in none of the above cited biological assays, including kaempferol-O-3-rutinoside and 5-O-cafeoil-quinic, trans-4-hydroxi-cinamic and cis- 4-hydroxi-cinamic acids. -Amirin, lupeol and a long-chain fatty acid ester derivative from lupeol were not tested in the ACE inhibition assay. With the exception of the 5-O-cafeoilquinicacid, all isolated compounds are reported for the first time in H. speciosa. The results from the biological assays corroborate the traditional use of H.speciosa for treating hypertension and inflammatory processes, and also disclosed L-(+)-bornesitol, rutin and quinic acid as the main constituents responsible for the effects.