Investigação etnofarmacológica dos efeitos cardiovasculares e renais de três espécies medicinais utilizadas no Pantanal sul-mato-grossense

Abstract

Cardiovascular diseases are among the leading causes of death worldwide. Medicinal plants can be viable alternatives to the development of new drugs to control these diseases. The aim of this work was to investigate Acanthospermum hispidum, Luehea divaricata and Talisia esculenta, which are medicinal plants popularly used for the treatment of diseases affecting the cardiovascular and renal systems in the Pantanal region of Mato Grosso do Sul state, Brazil. First, we performed a morphological and histochemical evaluation to adequately identify the three species under study. After being properly identified, the aerial parts of A. hispidum and leaves of L. divaricata and T. esculenta were collected. By the infusion method (100 g/1000 mL filtered water) we prepared the crude extract with the aerial parts of A. hispidum and with the leaves of the L. divaricata e T. esculenta. Ethanol (PA) was used to purify the extract and the ethanolic supernatant (ES) was used for the tests. The phytochemical profile of the ESAH, ESLD e ESTE was revealed and all extracts presented phenolic compounds. Male and female Wistar rats were used for the pharmacological and acute toxicity studies. By the acute toxicity test, different groups of male and female rats received 5, 50, 300 e 2000 mg/kg (single dose, v.o.) of the three extracts or filtered water (5 mL/100g). On the 15 th day, blood and tissue samples were obtained for biochemical and histological analyzes. Extracts promoted no signs of toxicity and, therefore, pharmacological studies were conducted. First, we analyzed the diuretic effects in different groups of rats (30, 100 and 300 mg/kg, v.o.). After 4-24 hours (acute treatment) or daily for seven days (prolonged treatment) the urinary volume was determined. Furthermore, sodium, potassium, chloride, pH and density were measured in urine samples. In a second moment, we analyzed the effects of the three extracts on systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP) and heart rate (HR) in normotensive rats. For this, animals were anesthetized with ketamine/xilasin (100/20 mg/kg, i.m.). A polyethylene catheter was introduced into the right carotid artery, and blood pressure (BP) was then measured. The extracts, at the same doses of diuresis, were administered directly into the duodenum of animals. Hydrochlorothiazide (HCTZ 25 mg/kg) and filtered water (5 mL/kg) were used as controls. ESAH did not induce diuresis, however, it presented hypotensive effect (reduced SBP and MAP at the dose of 30 mg/kg). Based on these results, we analyzed the role of nitric oxide (NO), prostacyclin (PGI2) and K-channel involvement in this activity. Different groups of rats were continuously infused through the femoral vein with L-NAME (7 mg/kg/min, NO synthase inhibitor), indomethacin (3 mg/kg/min., a non-selective cyclooxygenase inhibitor), methylene blue (150 nmol/kg/min, glanilate cyclase inhibitor) or TEA (400 μmol/kg/min., a non-selective calcium-sensitive K + channel bloker (KCa)). ESAH (30 mg/kg, i.d.) was then administered. We observed that the ON pathway was involved in the ESAH hypotensive activity, as well as the Ca + sensitive K + channels. ESTE showed no significant diuretic or hypotensive activity. We then evaluated if ESTE presented in vitro antioxidant activity through its ability to eliminate free radical DPPH and to prevent oxidative hemolysis caused in human erythrocytes by AAPH. Although ESTE presented no diuretic and hypotensive activity, it has been shown to have an interesting antioxidant activity. Finally, based on the initial results where ESLD presented diuretic, saluretic and hypotensive effects, and the first two activities were maintained for seven days, we performed the fractionation of this extract with solvents with different polarities. The ESLD (15 g.) was solubilized in water (500 mL) and sequentially fractionated with chloroform, butanol and ethyl acetate resulting in fractions ChloroFr, ButFr and AceFr, respectively. The aqueous residue generated the aqueous fraction (AqueFr). These fractions were evaluated for diuretic and acute hypotensive effects. The fractions Acefr (6 mg/kg), AqueFr (128 mg/kg) and ButFr (65 mg/kg) presented diuretic activity after 24 hours. The last two fractions were also hypotensive, besides the saluretic activity presented by ButFr. From this result, we investigated the involvement of ON and PGI2 in the diuretic activity of ButFr. Different groups of animals received, one hour earlier, L-NAME (60 mg/kg, v.o.) and indomethacin (5 mg/kg, v.o). They were then treated with ButFr (65 mg/kg, v.o.). Urinary volume was measured after 8 and 24 hours. The two pathways were involved with the diuretic activity of this fraction. We also investigated whether the diuretic, saluric and hypotensive effect (ButFr (65 mg/kg)) would be maintained after prolonged treatment (daily for seven days), which was also confirmed. Based on these data, we decided to analyze this fraction in isolated organs. For this, rats were anesthetized and their mesenteric beds and kidneys were removed and perfused with adequate solution and arterial pressure was measure. After stabilization of the arterial pressure, the organs received different concentrations of ButFr. The most effective concentrations of ButFr (0.01-0.1 mg) were analyzed for ON and PGI2 involvement in the diuretic and hypotensive activities presented. We perfused new beds and kidneys with the inhibitors L-NAME (100 μmol) and indomethacin (1 μmol) and then administered different concentrations of ButFr. Two paths were involved. Finally, since AqueFr (128 mg/kg) presented a hypotensive activity in previous studies, we elucidated its phytochemical profile and identified isovitexin as its main metabolite. We investigated AqueFr and isovitexin in mesenteric beds isolated from rats and the mechanisms involved in hypotension. We verified the involvement of ON, PGI2 and K + channels (sensitive to Ca + , voltage and ATP). We isolated the beds, perfused with the inhibitors L-NAME (100 μmol), indomethacin (1 μmol), KCl (40 mM, K + ), TEA (1 mM), 4-aminopyridine (10 μmol- a voltage-dependent (KV) K + channels bloker) and glibenclamide (10 μmol- a seletive Kir6.1 ATP-sensive K + channels bloker) and administered different concentrations of AqueFr (0.01-0.1 mg) and isovitexin (100-1000 nmol). Both treatments presented hypotensive activity via NO and stimulated K + channels sensitive to Ca + and ATP. Thus, we conclude that although A. hispidum was not diuretic, it had hypotensive activity, T. esculenta was neither diuretic nor hypotensive but presented antioxidant activity and L. divaricata presented diuretic, salinetic and hypotensive activity. The diuretic and saluretic effects of L. divaricata were maintained for seven days. Its fractions (AqueFr and ButFr) presented diuretic and hypotensive activity and the mechanisms of action were elucidated in isolated organs, acting through the ON and PGI2 pathways acting on different K + -channels. L. divaricata presented better pharmacological effects than the other two species and its diuretic and hypotensive effects corroborate the popular use in Pantanal. Therefore, it can be considered a potential species for the development of new drugs.