Efeito do extrato etanólico da casca de Punica Granatum l. (romã) no cultivo in vitro de tecido ovariano bovino

Abstract

The aim of this study was to investigate the antioxidant potential of the thanolic extractproduced from the peel of pomegranate fruit (Punica granatum L.) on bovine ovarian tissue cultured in vitro. The present study was approved by the ethics committee under protocol number 09326942/2021. For the extract production, fresh fruits from the local market were collected, and the peels were removed from the fruit and immersed in ethanol (99.8% PA) for 5 days. Finally, the obtained extract was lyophilized, diluted in ultrapure water, and stored at - 20°C. The antioxidant capacity of the extract was evaluated (DPPH and ABTS), and its chemical composition was determined through High-Performance Liquid Chromatography with Ultraviolet-Visible Detector (HPLC-UV-Vis). Additionally, ovaries (n=24) from crossbred cows were collected and transported to the laboratory in saline solution with penicillin and streptomycin at 4°C. In the laboratory, ovarian cortices were fragmented and cultured for 6 days in αMEM medium supplemented with ITS (10 µg/mL insulin, 5.5 µg/mL transferrin, and 5 ng/mL selenium), BSA (1.25 mg/mL), Glutamine, and Hypoxanthine (αMEM+), in the absence (cultured control) or presence of different concentrations (10, 50, and 100 µg/mL) of ethanolic extract of Punica granatum L. (EE-cPG). The culture was performed in an incubator at 38.5°C and 5% CO2 humidified atmosphere. After 5 days, the fragments were subjected to morphological (classical histology and ultrastructure) and biochemical nalyses (SOD and CAT enzyme activity, reduced thiol content (GSH), and quantification of malondialdehyde levels). Analysis of variance (ANOVA), with Tukey or Mann-Whitney posthoc tests, respecting the normality test, was used. A P value < 0.05 was considered statistically significant. Results revealed the presence of α-punicalagin in the produced ethanolic extract. Moreover, 50 and 100 µg/mL of EE-cPG showed significantly higher total antioxidant capacity throughout the culture (D2 and D6) compared to the 10 µg/mL EE-cPG treatment. These same concentrations were able to decrease MDA levels and increase Thiol levels. However, only the concentration of 100 µg/mL was able to maintain the percentage of morphologically normal follicles similar to fresh and cultured controls, besides promoting activation and preserving follicular ultrastructural integrity. Therefore, the ethanolic extract from pomegranate fruit peel was able to preserve the morphology and ultrastructure of bovine preantral follicles, in addition to maintaining redox balance. Among the evaluated concentrations, 100 µg/mL stands out for maintaining follicular morphology and preventing oxidative stress.