Biological activity of 1,2,3,4,6-Pentagalloyl glucose from Astronium graveolens

Introduction. Astronium graveolens Jacq., also commonly known as "Gateado" or "Gateado Galán," is a timber tree often used for making fine furniture, decorative sliced veneer, sawn, keels and long structures such as beams and trusses posts (1-2). The species are used in Brazilian folk medicine to treat allergies, inflammation, diarrhea and ulcers. Previous phytochemical studies report the presence of gallotannins as galloylglucose compounds, trans-ocimene (3) and flavonoids. In this work, we reported the isolation of known compounds as chlorogenic acid, quercitrin, quercetin 3-O-glucoside (4) and 1,2,3,4,6-Pentagalloyl glucose (5). Studies report an interesting anti-angiogenic activity for 1,2,3,4,6-Pentagalloyl glucose (6), we evaluated its ability to bind and inhibit the Human Placental Growth Factor (hPlGF), a key mediator of pathological angiogenesis. Materials and Methods. The leaves of Astronium graveolens collected (July 2010) at the Experimental Station "Caparo,― Barinas State, Venezuela were defatted with n-hexane and successively extracted for 48h with chloroform and and methanol, to give 8.5, 8.0 and 8.3 g of the respective residues. A portion of methanol extract (2.5 g), was chromatographed on Sephadex LH-20 using MeOH as eluent. A total of 106 fractions were collected (8 ml each) and combined by TLC results on silica 60 F254 gel-coated glass sheets with n-BuOH–AcOH–H2O (60:15:25) and CHCl3–MeOH–H2O (40:9:1), to give 11 pooled fractions (A–K). The fractions more interesting were purified by RP-HPLC. The structures of isolated compounds were elucidated by 1D- and 2D-NMR Spectroscopy (1H, 13C, 13C DEPT, DQF-COSY, HSQC, HMBC, ROESY ) and confirmed by mass spectrometry studies. Competitive enzyme-linked immunosorbent assays (ELISA) was used to study the interaction of 1,2,3,4,6-Pentagalloyl glucose and the h-PlGF binding to its receptor (h-VEGF-R1 or h-Flt-1); the concentrations are: 0,5 mg\ml for h-Flt-1, 10 ng\ml for h-PlGF, 0,5 mg\ml for the biotinylated antibody and 200, 100, 50, 25, 12,5, 6,25, 3,12 μM for the natural compound. Finally, we measured the binding affinity between 1,2,3,4,6-Pentagalloyl glucose and h-PlGF with surface plasmon resonance assays, using Biacore technology. Results and Discussion. The phytochemical study of methanol extract led to isolation of phenolic compounds as chlorogenic acid, quercitrin, quercetin 3-O-glucoside (4) and 1,2,3,4,6-Pentagalloyl glucose (5). The competitive enzyme-linked immunosorbent assay (ELISA) showed that 1,2,3,4,6-Pentagalloyl glucose is able to reduce by 58% the PlGF interaction with Flt-1(its specific receptor), at concentration of 50mM. This interaction is further characterized using surface plasmon resonance (SPR); the binding affinity of this compound with hPlGF was KD= 0.114μM.