Evaluation of leucocytes from sputum samples of pulmonary tuberculosis patients using flow cytometry.

SETTING: Information about the sputum cells of pulmonary tuberculosis (PTB) patients is scarce. The analysis of sputum cells using optical microscopy (OM) is a well-established method, but it has some serious limitations. OBJECTIVE: To establish a new flow cytometry (FC) protocol for the leucocyte evaluation of sputum samples from PTB patients. DESIGN: A new FC protocol using 0.1% dithiothreitol and 0.5% paraformaldehyde was developed to fluidise sputum samples and kill Mycobacterium tuberculosis, respectively, to allow the analysis of sputum samples collected from TB patients. The protocol was validated by comparing it with OM, and the cellularity of 30 sputum samples from patients with PTB was evaluated. RESULTS: The comparison between leucocyte subsets analysed using OM and FC showed agreement. Immunophenotyping of leucocytes from sputum samples showed that neutrophils (95.7%) comprised the largest proportion of sputum cells, followed by monocytes/macrophages (2.6%) and lymphocytes (1.6%). Among the total T-lymphocytes (100%), 12.3% were T-helper cells, 24.1% were cytotoxic T-cells and 62.9% were gamma/delta T; none of the T lymphocytes had the CD4+/CD8+ phenotype. CONCLUSION: FC is a useful method for evaluating the different subtypes of leucocytes present in the sputum samples of PTB patients.

Hypotensive and vasorelaxant effects induced by the ethanolic extract of the Mimosa caesalpiniifolia Benth. (Mimosaceae) inflorescences in normotensive rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Caatinga is highly influenced by its seasonality. This species is endemic in the northeastern region, which is rich in plants with pharmacological potential. Many of these plants are used by the population and some of them have confirmed pharmacological properties. Mimosa caesalpiniifolia Benth. (Mimosaceae) is a native plant from northeastern Brazil׳s caatinga, popularly known as sabiá and cascudo. The tea from the inflorescence of this species is used by the population of the semi-arid for the treatment of hypertension, and the utilization of the plant bark for the staunching of bleedings and wound washing in order to prevent inflammation; also, the ingestion of the bark infusion is used in the treatment of bronchitis. However, its pharmacological effects and mechanisms of action have not yet been studied. The aim of the present study was to determine the effect of the ethanolic extract of M. caesalpiniifolia on the cardiovascular system in rats. MATERIAL AND METHODS: In a study for the assessment of the hypotensive effect of the extract, the polyethylene catheters were inserted in the aorta artery and inferior vena cava for the measurement of the arterial pressure and heart rate. When intragastric administration was performed, only one catheter was implanted in the abdominal aorta. In studies for the vasorelaxant activity, mesenteric arterial rings (1-2mm) were used: they were kept in Tyrode׳s solution (95% O2 and 5% CO2) and submitted to tension of 0.75 g/f for 1h. The results were expressed as mean ± S.E.M., significant to the values of p<0.05. RESULTS: The administration of the doses through venous pathway (6.25; 12.5 and 25mg/kg, i.v.) promoted hypotension followed by bradycardia in the higher doses. The pre-treatment with atropine (2mg/kg, i.v.) interrupted both the hypotension and the bradycardia; with hexamethonium, hypotension was reverted and bradycardia was attenuated. While the administration of tea/flowers (25mg/kg i.v.) also promoted a following section of hypotension, a slight increase in heart rate was observed. When administered orally, MC-EtOH/flowers (100mg/kg, v.o.) promoted a decrease in the arterial pressure from 90 min on, without a significant alteration in the heart rate in relation to the control. In the in vitro study, a pharmacological trial was performed with the extracts obtained from parts of the species M. caesalpiifolia (leaves, bark, fruit and inflorescences). Among all extracts tested, the ethanolic extract from the inflorescences (MC-EtOH/flowers) presented higher vasorelaxant potency in relation to the other parts of the plant. Henceforth, MC-EtOH/flowers was used in the sequence. In mesenteric preparations pre-contracted with phenylephrine (10(-5)M), the MC-EtOH/flowers (0.1-750 µg/ml) promoted vasorelaxant effect regardless of the vascular endothelium. MC-EtOH/flowers inhibited the contractions induced by the cumulative addition of phenylephrine (10(-9)-10(-5)mol/l) or CaCl2 (10(-6)-3 × 10(-2)M), in a concentration-dependent way. In contractions induced by S(-)Bay K 8644, a Cav-L activator, the MC-EtOH/flowers promoted concentration-dependent relaxation, corroborating previous results. CONCLUSION: The tea of flowers of M. caesalpiniifolia promotes hypotension and tachycardia, whereas ethanolic extract (MC-EtOH) promotes hypotension and bradycardia involving the participation of the muscarinic and ganglionic pathways, as well as vasorelaxant action involving the Ca(2+) influx inhibition blockade.

Gastroprotective activity of Cenostigma macrophyllum Tul. var. acuminata Teles Freire leaves on experimental ulcer models.

ETHNOPHARMACOLOGICAL RELEVANCE: Cenostigma macrophyllum Tul. var. acuminata Teles Freire (Leguminosae-Caesalpinioideae), popularly known in Brazil as "caneleiro", is widely used in folk medicine against gastrointestinal diseases. In previous studies, the ethanol extract of leaves from Cenostigma macrophyllum Tul. var. acuminata Teles Freire had shown antinociceptive, anti-inflammatory, antibacterial, antioxidant and antiulcerogenic activities. AIM OF THE STUDY: The aim of this study was to assess the gastroprotective effect of the hydroalcoholic fraction of leaves of Cenostigma macrophyllum Tul. var. acuminata Teles Freire (Cm-FHA), as well as to elucidate the possible underlying mechanisms of action. MATERIALS AND METHODS: Mice were used for the evaluation of the acute toxicity, and mice and rats to study the gastroprotective activity. The potential gastroprotective of Cm-FHA was assessed on different gastric ulcer models in rodents, such as absolute ethanol, HCl/ethanol, ischemia-reperfusion, cold restraint stress and indomethacin. The participation of prostaglandins, NO-synthase pathway and ATP-sensitive potassium channels (KATP) in gastroprotective activity of Cm-FHA were evaluated after treatment with a cyclooxygenase inhibitor (indomethacin), a NO-synthase inhibitor (L-NAME) and a KATP channel blocker (glibenclamide 5mg/kg), respectively. Likewise, the catalase activity was determinated in order to assess the possible participation of antioxidant mechanisms. RESULTS: No signs of acute toxicity was observed after oral acute administration of Cm-FHA, considering the analyzed parameters. Likewise, Cm-FHA promoted a protective effect against gastric ulcers induced by absolute ethanol (lesion inhibition by 40% at both 100 and 200mg/kg), HCl/ethanol (lesion inhibition by 50 or 48% at 100 or 200mg/kg, respectively), ischemia-reperfusion (lesion inhibition by 49 or 90% at 100 or 200mg/kg, respectively) and cold restraint stress (lesion inhibition by 63 or 76% at 100 or 200mg/kg, respectively), as well as a increase of catalase activity was observed. Otherwise, Cm-FHA was not able to protect gastric mucosa against indomethacin-induced lesions. Nitric oxide release, the of KATP channels opening and antioxidant activity are the possibly involved in the Cm-FHA-induced gastroprotective activity. CONCLUSION: This study corroborates the folk medicine use of Cenostigma macrophyllum for treatment of gastric ulcers, as well as reinforces this species as a valuable source of promising natural drugs with gastroprotective activity.

Gastroprotective activity of Zanthoxylum rhoifolium Lam. in animal models.

ETHNOPHARMACOLOGICAL RELEVANCE: The stem barks of Zanthoxylum rhoifolium Lam. (Rutaceae), locally known as "mamica de cadela", are popularly used in dyspepsies, stomachic, tonic, antitumoral, antipyretic and are used in treating flatulence and colic. The objective of this study was to evaluate the gastroprotective effect of the ethanolic extract of Zanthoxylum rhoifolium (EEZR) stem barks in acute gastric lesion models, investigating their possible mechanisms. MATERIALS AND METHODS: Mice were used for the evaluation of the acute toxicity, and mice and rats to study the gastroprotective activity. The gastroprotective action of EEZR was analyzed in the absolute ethanol, HCl/ethanol and indomethacin-induced gastric lesion models in mice, hypothermic-restraint stress, and ischemia/reperfusion in rats. In the investigation of the gastroprotective mechanisms of EEZR, the participation of the NO-synthase pathway, ATP-sensitive potassium channels (K(ATP)), the levels of the non-protein sulfhydril groups (NP-SH) and the catalase activity using the ethanol-induced gastric mucosa lesion model and the quantification of the gastric mucus and the antisecretory activity through pylorus ligature model in rats were analyzed. RESULTS: The animals did not present any signs of acute toxicity for the EEZR (up to the 4 g/kg dose, po), and it was not possible to calculate the DL(50). EEZR (125-500 mg/kg) exhibited a significant gastroprotective effect in absolute ethanol, HCl/ethanol, hypothermic-restraint stress, and ischemia/reperfusion-induced gastric lesion models. EEZR (250 and 500 mg/kg) exhibited still a gastroprotective activity in the indomethacin-induced ulcer model. Gastroprotection of EEZR was significantly decreased in pre-treated mice with l-NAME or glibenclamide, the respective nitric oxide synthase and K(ATP) channels inhibitors. Our studies revealed that EEZR (500 mg/kg) prevented the decrease of the non-protein sulfhydril groups (NP-SH) and increased the catalase levels in ethanol-treated animals. Furthermore, the extract (500 mg/kg) significantly increased the mucus production, however, the gastric secretion parameters (volume, [H(+)], pH) did not show any alteration. CONCLUSIONS: Our results indicate that the ethanolic extract of Zanthoxylum rhoifolium exhibits a significant gastroprotection, because it inhibits the formation of gastric lesions using different models. The release of the nitric oxide, the opening of the K(ATP) channels, the participation of the non-protein sulfhydril groups (NP-SH), catalase and the increase of mucous secretion seem to be involved in the gastroprotection activity of the EEZR. Nevertheless, this activity does not seem to be related to antisecretory mechanisms.