Extrato metanólico do veneno de Rhinella marina : composição química e atividades antioxidante e imunomoduladora

Abstract

The popular "cane toad", scientifically Rhinella marina (R. marina), arouses interest as it is a fascinating source of bioactive compounds. The objective of this work was to study the chemical composition using methods of ultra high pressure liquid chromatography (UHPLC) coupled to the detector by mass spectrometry (EM). Experimental tests were carried out to verify the immunomodulatory and antioxidant activities of the methanolic extract of R. marina venom. The poison obtained from the collected frogs was dried and extracted with methanol. For biological activity, male Swiss mice were treated daily (100 μL / animal, via gavage) with 8 μg mL-1

, 16 μg mL-1

or 32 μg mL-1

of methanolic extract (0.5% tween solution) of the poison for 7 and 30 days. The control group was treated with water and the vehicle group with 0.5% tween solution. At the end, the animals were euthanized by cervical dislocation. Peritoneal macrophages were obtained for the in vitro quantification of hydrogen peroxide (H2O2) and nitric oxide (NO•). The spleen was removed to obtain the suspension of total splenic cells to perform lymphoproliferation assays using the colorimetric method using MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and the measurement of cytokines IL-4, IL-10, IL-12p70, TNF-α. Part of the liver was used to investigate some parameters of oxidative stress: Thiobarbituric Acid Reactive Substances (TBARS), Reduced Glutathione (GSH), Glutathione-S-transferase (GST) and Catalase (CAT). The organs (heart, liver, kidney and lung) were excised to assess relative weight, absolute weight and histopathological analysis. The identified compounds were: Adipoyl arginine, Pimeloyl

arginine, Diidrobufotenin, Suberoyl arginine, Azelayl arginine, 3-(N- adipoylargininyl)marinobufagin, 3-(N-pimeloylargininyl)marinobufagin, 3-(N- suberoylargininyl)telecinobufagin, 3-suberoylargininyl)marinobufagin, Telecinobufagin,

Marinobufagin and 3-(N-suberoylargininyl)bufalin.. There were no significant differences in the absolute weight of the organs. Only the relative lung weight showed a significant difference between the doses of 8 μg mL-1

and 32 μg mL-1

. Histopathological analyzes showed the presence of edema in all organs in proportion to the increase in doses and treatment time. In addition, the lung had intravascular clots and small peribronchiolar leukocyte infiltrates. The results obtained from the biochemical analyzes demonstrated a biphasic effect of CAT and GSH only between the doses administered in the period of 7 days, not differentiating from the control. TBARS showed an increase in the period of 30 days in the dose 32 μg mL-1

. The immunomodulatory effect was observed only regarding the production capacity of IL-12p70 and TNF-α. The lowest dose showed results that suggest better defense to the treated animal's organism, while the highest dose increased tissue damage. The compounds found in the samples of the methanolic extract of the poison are: Argininyl derivatives, cardioactive steroids and steroids linked to argininyls. In this first study, we cannot verify the antioxidant effect of the extract. We observed that the treatment modulated the production of cytokines in animals and this effect may be related to tissue damage observed microscopically. This study found positive stimuli in the antioxidant and immunomodulatory system, indicating that the molecules found in the extract have biotechnological potential and can be combined in the treatment of diseases that currently generate challenges for the medicinal world.