Ethanolic extract of Casearia sylvestris Sw exhibits in vitro antioxidant and antimicrobial activities and in vivo hypolipidemic effect in rats / Extrato etanólico de Casearia sylvestris Sw apresenta atividade antioxidante e antimicrobiana in vitro e efeito hipolipemiante em ratos

ABSTRACT: The Casearia sylvestris Sw (Flacourtiaceae) is a shrub that occurs in forests of Southern Brazil; its leaves are widely used in folk medicine as a depurative, analgesic, anti-inflammatory and antiulcerogenic agent. The objective of this study was to perform the phytochemical description and to evaluate the pharmacological activities (antimicrobial, antifungal, antioxidant and toxicity) of the ethanolic extract (EE) of C. sylvestris Sw. In addition, we also evaluated the effect of the EE of C. sylvestris Sw on the glucose levels and lipid profile in blood serum of rats submitted to a model of streptozotocin-induced diabetes. Material and Methods: In vitro assay: the detection of chemical groups was done through chemical reactions with the development of color or precipitate and by chromatographic profile; the antioxidant activity was measured by the method of reduction of DPPH free radical (2,2-diphenyl-1-picrylhydrazyl); the Minimum Inhibitory Concentration was evaluated by the broth microdilution method, and the Minimum Bactericide Concentration and the Minimum Fungicide Concentration were performed in Petri dishes; the cytotoxic activity was measured by the Artemia salina test. In vivo assay: diabetic and non-diabetic rats were treated with EE of C. sylvestris Sw (300 mg/kg) for 45 days, and the glycaemia and lipid profile were analyzed. Results: The EE showed a Lethal Dose50 of 724.76 μg.mL-1 and important antioxidant, fungicide and fungistatic activities. The EE showed better antimicrobial activity regarding the microorganisms Staphylococcus aureus, Escherichia coli and Salmonella setubal. Conclusion: The EE of C. sylvestris Sw produces a significant decrease in triglycerides, total cholesterol and VLDL levels without any significant alteration in the glycaemia. The EE of C. sylvestris Sw presents antioxidant and antimicrobial activities and it exhibits a potent hypolipidemic effect.

RESUMO: Casearia sylvestris Sw (Flacourtiaceae) é uma planta comumente encontrada em florestas do sul do Brasil; suas folhas são amplamente utilizadas na medicina popular como depurativa, analgésica, anti-inflamatória e anti ulcerogênica. O objetivo deste estudo foi apresentar uma descrição fitoquímica e da atividade farmacológica (antimicrobiana, antifúngica, antioxidante e toxicidade) do extrato etanólico (EE) da C. Sylvestris Sw. Adicionalmente, procurou-se avaliar o efeito do EE da C. Sylvestris Sw sobre os níveis séricos de glicose e perfil lipídico de ratos submetidos a um modelo de diabetes induzida por estreptozotocina. A detecção de grupos químicos foi realizada por reações químicas de coloração ou precipitação, e também por cromatografia; a atividade antioxidante foi mensurada pelo método de redução do DPPH (2,2-difenil-1-picril-hidrazil); a concentração mínima inibitória foi realizada pela técnica de micro-diluição, e concentração mínima bactericida e concentração mínima fungicida foram realizadas em placa de Petri; enquanto a atividade citotóxica foi conduzida pelo teste da Artemia salina. Nos ensaios in vivo, ratos diabéticos e não-diabéticos foram tratado com EE da C. Sylvestris Sw (300mg/kg) por 45 dias, e os níveis glicêmico e perfil lipídico foram medidos. A dose Letal50 do EE foi de 724.76 μg.mL-1; mostrando importante atividades antioxidante, fungicida e fungistática e melhor atividade antimicrobiana contra Staphylococcus aureus, Escherichia coli e Salmonella setubal. O EE da C. Sylvestris Sw promoveu diminuição significativa nos níveis de triglicerídeos, colesterol total e VLDL; porém sem efeito significativo nos níveis glicêmicos. O EE da C. Sylvestris Sw, além de apresentar atividade antioxidante e antimicrobiana; possui também potente efeito hipolipidêmico.

Gadolinium increases the vascular reactivity of rat aortic rings

Gadolinium (Gd) blocks intra- and extracellular ATP hydrolysis. We determined whether Gd affects vascular reactivity to contractile responses to phenylephrine (PHE) by blocking aortic ectonucleoside triphosphate diphosphohydrolase (E-NTPDase). Wistar rats of both sexes (260-300 g, 23 females, 7 males) were used. Experiments were performed before and after incubation of aortic rings with 3 µM Gd. Concentration-response curves to PHE (0.1 nM to 0.1 mM) were obtained in the presence and absence of endothelium, after incubation with 100 µM L-NAME, 10 µM losartan, or 10 µM enalaprilat. Gd significantly increased the maximum response (control: 72.3 ± 3.5; Gd: 101.3 ± 6.4 percent) and sensitivity (control: 6.6 ± 0.1; Gd: 10.5 ± 2.8 percent) to PHE. To investigate the blockade of E-NTDase activity by Gd, we added 1 mM ATP to the bath. ATP reduced smooth muscle tension and Gd increased its relaxing effect (control: -33.5 ± 4.1; Gd: -47.4 ± 4.1 percent). Endothelial damage abolished the effect of Gd on the contractile responses to PHE (control: 132.6 ± 8.6; Gd: 122.4 ± 7.1 percent). L-NAME + Gd in the presence of endothelium reduced PHE contractile responses (control/L-NAME: 151.1 ± 28.8; L-NAME + Gd: 67.9 ± 19 percent AUC). ATP hydrolysis was reduced after Gd administration, which led to ATP accumulation in the nutrient solution and reduced ADP concentration, while adenosine levels remained the same. Incubation with Gd plus losartan and enalaprilat eliminated the pressor effects of Gd. Gd increased vascular reactivity to PHE regardless of the reduction of E-NTPDase activity and adenosine production. Moreover, the increased reactivity to PHE promoted by Gd was endothelium-dependent, reducing NO bioavailability and involving an increased stimulation of angiotensin-converting enzyme and angiotensin II AT1 receptors.

Ectonucleotidase activities in Sertoli cells from immature rats

Sertoli cells have been shown to be targets for extracellular purines such as ATP and adenosine. These purines evoke responses in Sertoli cells through two subtypes of purinoreceptors, P2Y2 and P A1. The signals to purinoreceptors are usually terminated by the action of ectonucleotidases. To demonstrate these enzymatic activities, we cultured rat Sertoli cells for four days and then used them for different assays. ATP, ADP and AMP hydrolysis was estimated by measuring the Pi released using a colorimetric method. Adenosine deaminase activity (EC 3.5.4.4) was determined by HPLC. The cells were not disrupted after 40 min of incubation and the enzymatic activities were considered to be ectocellularly localized. ATP and ADP hydrolysis was markedly increased by the addition of divalent cations to the reaction medium. A competition plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. This result indicates that the enzyme that acts on the degradation of tri- and diphosphate nucleosides on the surface of Sertoli cells is a true ATP diphosphohydrolase (EC 3.6.1.5) (specific activities of 113 + or - 6 and 21 + or - 2 nmol Pi mg-1 min-1 for ATP and ADP, respectively). The ecto-5'-nucleotidase (EC 3.1.3.5) and ectoadenosine deaminase activities (specific activities of 32 + or - 2 nmol Pi mg-1 min-1 for AMP and 1.52 + or - 0.13 nmol adenosine mg-1 min-1, respectively) were shown to be able to terminate the effects of purines and may be relevant for the physiological control of extracellular levels of nucleotides and nucleosides inside the seminiferous tubules