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.

Alterations in the cholinesterase and adenosine deaminase activities and inflammation biomarker levels in patients with multiple sclerosis.

Multiple sclerosis (MS) is one of the main chronic inflammatory diseases of the CNS that cause functional disability in young adults. It has unknown etiology characterized by the infiltration of lymphocytes and macrophages into the brain. The aim of this study was to evaluate the acetylcholinesterase (AChE) activity in lymphocytes and whole blood, as well as butyrylcholinesterase (BChE) and adenosine deaminase (ADA) activities in serum. We also checked the levels of nucleotides, nucleosides, biomarkers of inflammation such as cytokines (interleukin (IL)-1, IL-6, interferon (IFN)-γ, tumor necrosis factor-alpha (TNF-α) and IL-10) and C-reactive protein (CRP) in serum from 29 patients with the relapsing-remitting form of MS (RRMS) and 29 healthy subjects as the control group. Results showed that AChE in lymphocytes and whole blood as well as BChE, and ADA activities in serum were significantly increased in RRMS patients when compared to the control group (P<0.05). In addition, we observed a decrease in ATP levels and a significant increase in the levels of ADP, AMP, adenosine and inosine in serum from RRMS patients in relation to the healthy subjects (P<0.05). Results also demonstrated an increase in the IFN-γ, TNF-α, IL-1, IL-6 and CRP (P<0.05) and a significant decrease in the IL-10 (P<0.0001) in RRMS patients when compared to control. Our results suggest that alterations in the biomarkers of inflammation and hydrolysis of nucleotides and nucleosides may contribute to the understanding of the neurological dysfunction of RRMS patients.

Mild hyperhomocysteinemia alters extracellular adenine metabolism in rat brain.

Since homocysteine (Hcy) is considered a risk factor to cerebral diseases and adenine nucleotides are important molecules to brain normal function, in the present study we investigated the effect of chronic mild hyperhomocysteinemia on ectonucleotidase activities and expression in rat cerebral cortex. The levels of ATP, ADP, AMP and adenosine (Ado) in cerebrospinal fluid (CSF) of adult rats also were evaluated by high-performance liquid chromatography. For the chronic chemically induced mild hyperhomocysteinemia, Hcy (0.03 µmol/g of body weight) was administered subcutaneously from the 30th to the 60th day of life. Control rats received saline solution in the same volumes. Results showed that Hcy significantly decreased nucleotide hydrolysis in the synaptosomal fraction and increased E-NTPDase1 and ecto-5'-nucleotidase transcripts in rat cerebral cortex. ATP levels were significantly increased, while Ado decreased in CSF of Hcy-treated rats. These findings suggest that the unbalance in ATP and Ado levels may be, at last in part, involved in the cerebral toxicity of mild hyperhomocysteinemia.

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.

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%) and sensitivity (control: 6.6 ± 0.1; Gd: 10.5 ± 2.8%) 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%). Endothelial damage abolished the effect of Gd on the contractile responses to PHE (control: 132.6 ± 8.6; Gd: 122.4 ± 7.1%). 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% 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.

NTPDase and 5' ecto-nucleotidase expression profiles and the pattern of extracellular ATP metabolism in the Walker 256 tumor.

In this study, we evaluated the NTPDases and ecto-5'-nucleotidase (CD73) expression profiles and the pattern of adenine nucleotide hydrolysis in rats submitted to the Walker 256 tumor model, 6, 10 and 15 days after the subcutaneous inoculation. Using RT-PCR analysis, we identified mRNA for all of the members of the ecto-nucleoside triphosphate diphosphohydrolase family investigated and a 5'-nucleotidase. By quantitative real-time PCR, Entpd1 (Cd39) and Entpd2 (Cd39L1) and CD73 were identified as the dominant genes expressed by the Walker 256 tumor, at all times studied. Extracellular adenine nucleotide hydrolysis by the Walker 256 tumor was estimated by HPLC analysis. Rapid hydrolysis of extracellular ATP by the tumor cells was observed, leading to the formation of adenosine and inosine in cells obtained from solid tumors at 6 and 10 days after inoculation. Cells obtained from solid tumors at 15 days of growth presented high levels of AMP and presented adenosine as a final product after 90 min of incubation. Results demonstrate that the presence of NTPDases and 5'-nucleotidase enzymes in Walker 256 tumor cells may be important for regulation of the extracellular adenine nucleotides/adenine nucleoside ratio, therefore leading to tumor growth.

Ornithine decarboxylase activity during the development of Anastrepha fraterculus (Diptera, Tephritidae).

Ornithine decarboxylase (ODC) (EC 4.1.1.17) is very important for polyamine biosynthesis, which is required for main biological events. In the present study, ODC activity was measured in samples of Anastrepha fraterculus's egg, larva, pupa body and abdomen, adult body, ovaries, and fat body of young females, and in ovaries of mature flies. The kinetic parameters (Km app and Vmax) for ODC activity were determined for pupa, larva, and young ovary. ODC activity showed fluctuations during A. fraterculus's life development. In its earlier stages, prior to emergence, the egg has high ODC-specific activity probably due to embryogenesis, which is characterized by a high rate of cell division. This enzyme activity is also significantly high in the ovary and fat body of young females possibly related to the increased oogenesis and vitellogenesis. The kinetic parameters (Km app and Vmax) had great variation. Our results using GTP showed that the great variation in kinetic parameters can be accounted for by post-translational modifications.

Thyroid hormone upregulates ecto-5'-nucleotidase/CD73 in C6 rat glioma cells.

Thyroid hormones have profound effects on the central nervous system, such as proliferation, secretion of growth factors and gene expression regulation. Ecto-NTPDases and ecto-5'-nucleotidase can control the extracellular ATP/adenosine levels, which have been described as proliferation factors. Here, we investigated the influence of T(3) on the enzyme cascade which catalyzes interconversion of purine nucleotides in rat C6 glioma cells. Exposure of C6 cells to T(3) caused a dose dependent increase of 30% in the AMP hydrolysis up to 0.25 nM, which was suppressed by actinomycin. No significant alteration was observed on ATP/ADP hydrolysis and T(4) at higher concentrations (10-1000 nM) promoted an increase in AMP hydrolysis that was not dose dependent. T(3) treatment also increased the expression of CD73 mRNA. Besides the importance of the ecto-5'-NT in the cell proliferation and differentiation, its overexpression can enhance extracellular adenosine levels, which could also be an important proliferation signal.

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 +/- 6 and 21 +/- 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 +/- 2 nmol Pi mg(-1) min(-1) for AMP and 1.52 +/- 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.

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

Influence of the biomatrix on the response of Sertoli cells to FSH.

Sertoli cell preparations isolated from 15-day-old Wistar rats were cultured on two different substrates, i.e., plastic and a biomatrix isolated from seminiferous tubules of rat testis. Sertoli cells cultured on a biomatrix acquired a phenotype and morphology more characteristic of in vivo differentiated cells. In order to determine the influence of a biomatrix on the response of Sertoli cells to FSH, on the 7th day of culture, untreated cells, or cells pretreated for 12 h with FSH (1 microgram/ml), were incubated with [U-14C] leucine or [2-3H] mannose. Cells cultured on the biomatrix showed higher [U-14C] leucine and [2-3H] mannose incorporation into proteins and glycoproteins. FSH increased these activities in cells cultured on both substrates, although its stimulating effect was higher on cells cultured on the biomatrix. These results demonstrate that the biomatrix increases protein and glycoprotein synthesis and secretion, and also influences the response of Sertoli cells to FSH.