Fitoquímica e potencial antidiabético das cascas do fruto do pequi (Caryocar brasiliense)

Abstract

Changes in food habits and lifestyle increased several metabolic diseases, including type 2 diabetes mellitus (T2DM). New therapeutic agents have been demanded to control T2DM. Natural products from plants represent an alternative, including species traditionally used as anti-inflammatory agents like Caryocar brasiliense Camb. (Caryocaraceae), popularly named pequi tree, and the exotic ornamental species Leea rubra Blume ex Spreng. (Vitaceae). This study aimed to evaluate the activity of C. brasiliense and L. rubra extracts in experimental models related to T2DM, in order to identify compounds and/or chemically characterized fractions potentially useful for future therapeutic applications. The ethanolic extracts of leaves (EEFCB) and fruit peels (EECCB) of C. brasiliense were prepared, along with the ethanol extract of L. rubra aerial parts (EEALR). These extracts were fractionated by partition between immiscible solvents and the resulting fractions, along with the original extracts (10, 30 and 90 μg/mL), had their effect on the release of pro-inflammatory cytokines TNF-α, IL-1β and MCP-1 evaluated in LPS-stimulated THP-1 cells, being the cytokines quantified by ELISA. The cell viability of all samples was higher than 90%, tested by the MTT assay, indicating the absence of toxicity for THP-1 cells, except for the hexane and dichloromethane fractions of EEFCB and EECCB. EEALR had no inhibitory effect on the assayed cytokines, while EEFCB inhibited the release of TNF-α, MCP-1 and IL-1β in a concentration-dependent manner, with maximum inhibitions (MI) of 84.9 ± 0.6%, 89. 8 ± 0.8% and 72.2 ± 4.4%, respectively. On the other hand, EECCB inhibited the release of TNF-α, MCP-1 and IL-1β with MI values of 28.5 ± 5.6%, 42.3 ± 9.0% and 74.5 ± 3.7 %, respectively. The ethyl acetate (EtOAc) fraction of EECCB inhibited the release of TNF-α at all tested concentrations (MI of 59.9 ± 2.3%), as well as the release of MCP-1 (MI of 85.7 ± 2.7%) and IL-1β (MI of 71.9 ± 4.9%). Subsequently, the potential antidiabetic activity of the extracts and fractions was tested in vitro by the α-amylase and α-glucosidase enzyme inhibition assay. EECCB and EtOAc inhibited α-glycosidase activity in a concentration-dependent manner (MI of 97.9 ± 1.4% and 97.0 ± 0.7%, respectively) and induced approximately 30% inhibition of α-amylase. EECCB was fractionated by chromatography over a Sephadex-LH20 column to afford tannin-enriched fractions, named FR15C and FR16C. Both fractions inhibited α-glucosidase activity, and FR16C induced a more potent inhibitory effect than EECCB and FR15C, showing the highest pIC50 value (5.3 ± 0.1). Following, EECCB, EtOAc, FR15C and FR16C were assayed in the starch tolerance test in mice. FR16C significantly reduced blood glucose 60 min after starch intake, resulting in a glycemic level of 181 ± 10 mg/dL in comparison to the control group of 238 ± 11 mg/dL. The chemical composition of EECCB, EtOAc and FR16C was investigated by UPLC-ESI-MS/MS. Ions with mass-to-charge ratio compatible with gallic acid, glucogallin, ellagic acid, corilagin and geraniin were identified in EECCB. According to UPLC-ESI-MS/MS analysis, the EtOAc fraction was enriched in the identified constituents of EECCB, whereas FR16C presented a less complex profile, majorly composed by corilagin and geraniin. The ellagitannins corilagin and geraniin were isolated by preparative HPLC and had their structures charaterized unequivocally by 1D (1H and 13C) and 2D (COSY, HMQC and HMBC) NMR experiments. In a chronic model of T2DM induced by the administration of a low dose of streptozotocin with nicotinamide supplementation (LD-STZ-NA), the mice groups treated with EECCB or EtOAc showed a significant reduction in fasting glucose levels between the second and fourth week of treatment, with maximal hypoglycemic effects observed in the EtOAc-treated group. In conclusion, the results obtained from in vitro and in vivo assays herein reported for EECCB and derived fractions indicate that pequi peels contain ellagitannins with hypoglycemic activity, potentially useful for future development of an herbal product or functional food with antidiabetic properties.