Avaliação da capacidade antioxidante, antiglicante e de inibição enzimática in vitro e ex vivo do extrato etanólico de Centella asiatica e suas frações orgânicas

Abstract

Centella asiatica (L.) urb., popularly known as Gotu Kola and Pegaga, is a small herbaceous plant belonging to the Apiaceae family. It is found in hot and swampy regions, such as: India, Iran and Pakistan, also found in other locations of Asia, Australia, Central and South America. It is a plant which leaves are widely used in biomedical applications, mainly in traditional Indian medicine (Ayurvedic medicine). Such properties are associated with the presence of triterpenes, asiaticoside and madecassoside compounds. Considering its broad therapeutic potential, the present study aims to evaluate the in vitro and ex vivo antioxidant activity, the antiglycant and enzymatic inhibition potential (α-amylase, α-glycosidase, acetylcholinesterase and butyrylcholinesterase), ability to inhibit oxidation, glycation and peroxidation of LDL (low density lipoprotein) and its antimicrobial activity. Ethanolic extract (ee-Ca) was obtained from static maceration for 7 days, and organic fractions (fh-Ca; fd-Ca; fa-Ca; fb-Ca; fh-Ca) were obtained by liquid-liquid fractionation. Phytochemical prospecting and elucidation of compounds present in fa-Ca and fb-Ca fractions were performed by HPLC-ESI-MS/MS analyses and the antioxidant capacity of ee-Ca and its organic fractions were also studied. Antiglycant activity was evaluated from ee-Ca incubation and fractions with glycation target proteins in the presence of glycation inducers. Inhibition of α-amylase, α-glycosidase, acetylcholinesterase and butyrylcholinesterase activities was determined by colorimetric kinetic assay. The in vitro inhibition of the potential oxidation and peroxidation of LDL in vitro was analyzed through the formation of conjugated dienes and reactive species of thiobarbituric acid (TBARS). Ex vivo assays were also conducted to investigate the properties of lipid peroxidation inhibition in liver tissue. Antimicrobial activity was determined using cariogenical bacterial strains and strains related to skin diseases, and data were expressed as the minimum inhibitory concentration (MIC). In the ORAC assay, it was observed that there were no statistical differences among quercetin (positive control) and all fractions, except for fh-Ca and ee-Ca. In the FRAP and DPPH assays, fa-Ca (978.4 ± 26.05 μmol Trolox eq/g FRAP; 1.601 ± 0.4841 DPPH μg/mL) and fb-Ca (753.2 ± 66.15 μmol Trolox eq/g FRAP; 10.82 ± 2.592 μg/mL) stood out. These fractions also demonstrated the ability to inhibit above 90% the activity of α-amylase at the concentration of 12 μg/mL. No relevant results were observed in view of the inhibition of α-glycosidase, however, studies should be conducted to elucidate the inhibition of acetyl and butyrylcholinesterase enzymes. As for the glycation models, the ee-Ca and the fa-Ca and fb-Ca (25.87 ± 5.59 μg/mL; 4,146 ± 2.52 μg/mL and 6,468 ± 1.10 μg/mL) had suitable activity in the BSA/FRU experiments. Ee-Ca and fa-Ca also expressed antiglycant activity in the ARG/MGO and COL/MGO models. Moreover, ee-Ca, fd-Ca, fa-Ca and fb-Ca demonstrated in vitro inhibition of LDL oxidation and peroxidation at the concentration of 2 μg/mL. However, they were not able to inhibit the glycation. In the ex vivo liver tissue oxidation induction assay, MDA dosages revealed that fa-Ca, fb-Ca and fh-Ca (0.10 ± 0.001; 0.10 ± 0.003; 0.13 ± 0.029 nmol MDA/mg prot, respectively) exhibited tissue protection property, inhibiting lipid peroxidation, when compared with untreated oxidized liver tissue MDA levels (0.36 ± 0.01). In relation to antimicrobial assays, cariogenic bacteria (Streptococcus sobrinus ATCC 33448; Streptococcus mitis ATCC 49456; Streptococcus mutans ATCC 25175) were susceptible to fh-Ca and fd-Ca. Therefore, it was possible to observe great antioxidant potential, antiglycant (BSA/FRU), inhibition of α-amylase, inhibition of LDL oxidation and lipid peroxidation exerted by fa-Ca and fb-Ca, justifying the further studies of these fractions in in vivo models. In relation to fh-Ca and fd-Ca, interesting results were observed in relation to growth inhibition and cariogenic bacteria, indicating possible application in the dentistry area.