Ricardo Bezerra de Oliveira

Chemical composition and antimicrobial activity of the essential oil of Lippia grandis Schauer (Verbenaceae) from the western Amazon.

Lippia grandis Schauer is an aromatic plant that has been used as a spice in Brazilian culinary and in traditional medicine to treat liver disease, disorders of the stomach and throat infections. We determined the chemical composition of the essential oil of L. grandis and evaluated its potential for the treatment of clinically-important pathogenic micro-organisms. The essential oil was obtained by hydrodistillation and analyzed by gas chromatography-mass spectrometry (GC-MS), giving carvacrol (37.12%), ρ-cymene (11.64%), and thymol (7.83%) as the main components. The agar disk diffusion method of the essential oil was effective against 75% of the micro-organisms analyzed, in particular, Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli. The minimum inhibitory concentration was 0.57 mg/ml for E. faecalis and 1.15 mg/ml for all the other strains. The results indicate that the essential oil of L. grandis contains chemical compounds with good potential for the treatment of infections.

Significance of fingernail and toenail mercury concentrations as biomarkers for prenatal methylmercury exposure in relation to segmental hair mercury concentrations.

OBJECTIVE To investigate the appropriateness of mercury (Hg) concentrations in fingernails and toenails at parturition for detecting prenatal exposure to methylmercury (MeHg). METHODS Total Hg concentrations were measured in 54 paired samples of fingernails, toenails, maternal blood, and maternal hair (1cm incremental segments from the scalp toward the tip) collected at 4th weeks of (early) pregnancy, and the same specimens and cord blood collected at parturition. RESULTS Strong correlations were observed between Hg concentrations in fingernails and toenails at early pregnancy (r=0.923, p<0.01) and at parturition (r=0.895, p<0.01). At early pregnancy, Hg concentrations in fingernails and toenails showed the strongest correlations with those in hair 3-4 cm from the scalp (r=0.818 and r=0.747, p<0.01, respectively) among the 1cm incremental hair segments. Mercury concentrations in fingernails and toenails at parturition represented strong correlations with those in cord blood (r=0.803, p<0.01 for fingernails and r=0.792, p<0.01 for toenails, respectively). At parturition, Hg concentrations in fingernails had the highest correlation with those in hair 0-1cm from the scalp (r=0.918, p<0.01), and Hg concentrations in toenails showed the highest correlation with those in hair at 2-3 cm from the scalp (r=0.872, p<0.01). In addition, Hg concentrations in both finger and toe nails at parturition had equally high (p<0.01) correlation coefficients with hair segments at 0-1, 1-2, and 2-3 cm from the scalp. CONCLUSIONS Mercury in fingernails and toenails at early pregnancy reflected the maternal Hg body burden level approximately 5 months retroactively. At parturition, Hg levels in fingernails and toenails also showed strong correlations with those in cord blood. In addition, Hg levels in fingernails and toenails at parturition reflected more recent MeHg exposure, compared with those at early pregnancy. These results suggest that fingernails and toenails at parturition are useful biomarkers for prenatal MeHg exposure for mothers and fetuses, especially during the third-trimester of gestation.

Plants used to treat snakebites in Santarém, western Pará, Brazil: An assessment of their effectiveness in inhibiting hemorrhagic activity induced by Bothrops jararaca venom

ETHNOPHARMACOLOGICAL RELEVANCE The poor distribution and limited availability of antivenoms in Brazil have led to greater use of plants to treat snakebites. Very often such plants are the only alternative available to riverside communities. MATERIALS AND METHODS Direct questionnaire-based interviews were conducted with members of the Cucurunã, São Pedro and Alter do Chão communities in Santarém, Pará, Brazil. For each of the 12 most frequently mentioned species aqueous extracts were prepared and the phytochemical profiles determined by thin layer chromatography. The concentrations of phenolic compounds (tannins and flavonoids) in the aqueous extracts were determined by colorimetric assays. To assess inhibition of the hemorrhagic activity of Bothrops jararaca venom, solutions containing the venom mixed with aqueous extracts in the ratios 1:12 and 1:48 were tested (w/w). SDS-PAGE and Western blot were used to assess the action of the extracts on Bothrops jararaca venom. RESULTS In all, 24 plants belonging to 19 families were mentioned in the survey as being used to treat snakebites. Leaves (84%), seeds (60.9%) and inner bark (53%) were cited as the most frequently used parts in folk remedies, which were usually prepared in the form of a decoction (62.5%), tincture (45%) or maceration (22.5%). Hemorrhage induced by Bothrops jararaca venom was completely inhibited by aqueous extracts of Bellucia dichotoma, Connarus favosus, Plathymenia reticulata and Philodendron megalophyllum, which had a high phenolic content and contained condensed and hydrolyzable tannins. The results of SDS-PAGE showed that some venom protein bands were not visible when the venom was preincubated with the extracts that had completely inhibited hemorrhagic activity of the venom. Western blot showed that the extracts did not have any enzymatic action on the proteins in the venom as it failed to detect low-molecular-weight bands, which are indicative of possible enzymatic cleavage. CONCLUSIONS Traditional use of plants to treat snakebites is a common practice in the western region of Pará, Brazil. Our findings show that some plant extracts were able to inhibit snake venom-induced hemorrhage in vitro. In vivo studies are being carried out to validate the traditional use of these species to treat snakebites.

Methylmercury intoxication and histochemical demonstration of NADPH-diaphorase activity in the striate cortex of adult cats

The effects of methylmercury (MeHg) on histochemical demonstration of the NADPH-diaphorase (NADPH-d) activity in the striate cortex were studied in 4 adult cats. Two animals were used as control. The contaminated animals received 50 ml milk containing 0.42 microgram MeHg and 100 g fish containing 0.03 microgram MeHg daily for 2 months. The level of MeHg in area 17 of intoxicated animals was 3.2 micrograms/g wet weight brain tissue. Two cats were perfused 24 h after the last dose (group 1) and the other animals were perfused 6 months later (group 2). After microtomy, sections were processed for NADPHd histochemistry procedures using the malic enzyme method. Dendritic branch counts were performed from camera lucida drawings for control and intoxicated animals (N = 80). Average, standard deviation and Student t-test were calculated for each data group. The concentrations of mercury (Hg) in milk, fish and brain tissue were measured by acid digestion of samples, followed by reduction of total Hg in the digested sample to metallic Hg using stannous chloride followed by atomic fluorescence analysis. Only group 2 revealed a reduction of the neuropil enzyme activity and morphometric analysis showed a reduction in dendritic field area and in the number of distal dendrite branches of the NADPHd neurons in the white matter (P < 0.05). These results suggest that NADPHd neurons in the white matter are more vulnerable to the long-term effects of MeHg than NADPHd neurons in the gray matter.

Implications of mercury concentrations in umbilical cord tissue in relation to maternal hair segments as biomarkers for prenatal exposure to methylmercury

In this study, we investigated how mercury (Hg) concentrations in umbilical cord tissue are correlated with those in biomarkers for prenatal exposure to methylmercury (MeHg). Total Hg (T-Hg) concentrations were measured in 54 mother-child paired samples of maternal blood, umbilical cord tissue, cord blood, and maternal hair segments (1-cm incremental segments from the scalp) collected at parturition. MeHg concentrations were also measured in the cord tissue. Median T-Hg and MeHg concentrations in cord tissue on a dry-weight basis (d.w.) were 62.2ng/g and 56.7ng/g, respectively. Proportions of MeHg to T-Hg were approximately 95%. Both T-Hg and MeHg in cord tissue (d.w.) showed better correlations with T-Hg in cord blood than did T-Hg in cord tissue on a wet-weight basis (w.w.). Median T-Hg concentrations in maternal blood, cord blood, and maternal hair (0-1cm from the scalp) were 3.79ng/g, 7.26ng/g, and 1.35 μg/g, respectively. Median T-Hg concentration in cord blood was 1.92 times higher than that in maternal blood. T-Hg in cord tissue (d.w.) showed a strong correlation with that in cord blood (r=0.912, p<0.01). Among the hair segments, T-Hg in cord tissue (d.w.) showed the strongest correlation (r=0.854, p<0.01) with that in maternal hair at 0-1cm from the scalp, reflecting growth for approximately 1 month before parturition. Based on the present results, T-Hg and MeHg concentrations in cord tissue may be useful biomarkers for prenatal MeHg exposure of the fetus, especially reflecting the maternal MeHg body burden during late gestation. The conversion factors for T-Hg and MeHg concentrations in cord tissue (d.w.) to T-Hg concentrations in maternal hair (0-1cm from the scalp) were calculated to be 22.37 and 24.09, respectively. This information will be useful for evaluating maternal MeHg exposure levels in retrospective studies using preserved umbilical cord tissue.

Changes in methylmercury accumulation in the brain of rat offspring throughout gestation and during suckling

Higher methylmercury (MeHg) accumulation and susceptibility to toxicity in the fetus than in the mother at parturition is well known. However, the degree of MeHg accumulation in the brain during the late pregnancy period when the human brain is most vulnerable is not clear. In addition, changes in MeHg accumulation in the developing rat tissues with consecutive exposure throughout gestation and lactation periods have not been well studied. The purposes of this study were to evaluate the changes in MeHg accumulation in the brain and other tissues of the offspring, based on constant and consecutive doses of MeHg to mothers throughout gestation and lactation. Adult female rats were given a diet containing 5 ppm Hg (as MeHg) for 8 weeks. Then they were mated and subsequently given the same diet throughout gestation and lactation. On embryonic days 18, 20, 22 and at parturition, the concentrations of Hg in the brains of the offspring were approximately 1.5–2.0 times higher than those in the mothers. On the other hand, during the suckling period Hg concentrations in the brain rapidly declined to about 1/10 of that during late pregnancy. Changes in MeHg accumulation in the blood and liver after parturition were similar to those in the brain. Thus, although mothers are subjected to constant and prolonged MeHg exposure throughout both the gestation and lactating periods, the risk to the offspring may be especially high throughout the late gestation period but rapidly decreases during the suckling period

The potential of aqueous extracts of Bellucia dichotoma Cogn. (Melastomataceae) to inhibit the biological activities of Bothrops atrox venom: A comparison of specimens collected in the states of Pará and Amazonas, Brazil

ETHNOPHARMACOLOGICAL IMPORTANCE The effectiveness of aqueous extract of Bellucia dichotoma Cogn. (Melastomataceae) specimems collected in Santarém, PA, against some biological activities of Bothrops atrox venom (BaV) has been scientifically proven. Here, we analyzed the components and assessed the anti-snakebite potential of aqueous extracts of bark of B. dichotoma collected in Manaus, AM, (AEBd-MAO) and Santarém, PA, (AEBd-STM), both in Brazil. MATERIALS AND METHODS The phytochemical profiles of the aqueous extracts were identified using thin layer chromatography (TLC), and the concentrations of phenolics were determined by colorimetric assay. The inhibitory potential of the extracts was tested against the phospholipase A2, coagulant and gelatinolytic activities of BaV in vitro and its defibrinating and edema-inducing activities in vivo. Interaction between BaV and the extracts was investigated using SDS-Page electrophoresis and Western blotting. Extract cytotoxicity and antioxidant potential were assessed using the human fibroblast cell line MRC-5 and the DPPH assay in cell culture, respectively. RESULTS While there was no difference between the phytochemical profiles of the extracts, AEBd-MAO had higher concentrations of total phenolics, total tannins and hydrolysable tannins. The extracts inhibited 100% of the phospholipase and coagulant activity of BaV when pre-incubated. Without pre-incubation, however, there was no reduction in phospholipase activity, although significant inhibition of coagulant activity was observed. In the doses used in folk medicine, without pre-incubation, both extracts inhibited 100% of the coagulant activity of BaV. In vivo, the extracts were unable to inhibit the defibrinating activity of the venom but were effective in inhibiting its edema-inducing activity. In the profiles of the extracts pre-incubated with BaV, not all the protein bands revealed by SDS-PAGE and Western blot were observed. Both extracts had a high antioxidant potential and neither had a cytotoxic effect. CONCLUSION Although the concentrations of phenolics in each extract were different, the anti-snakebite potential was similar for the concentrations of extract tested. Our findings are of importance for the quality control of this raw material, which, once tested in accordance with Brazilian National Health Surveillance Agency recommendations, may be suitable for use as a phytomedicine to complement treatment of the local effects induced by Bothrops venoms.

The inhibitory potential of the condensed-tannin-rich fraction of Plathymenia reticulata Benth. (Fabaceae) against Bothrops atrox envenomation

ETHNOPHARMACOLOGICAL RELEVANCE Ethnobotanical studies have shown that Plathymenia reticulata Benth. (Fabaceae) has been widely used in cases of snake envenomation, particularly in Northern Brazil. In light of this, the aim of this study was to evaluate the inhibitory potential of the condensed-tannin-rich fraction obtained from the bark of P. reticulata against the main biological activities induced by Bothrops atrox venom (BaV). MATERIALS AND METHODS The chemical composition of the aqueous extract of P. reticulata (AEPr) was first investigated by thin-layer chromatography (TLC) and the extract was then fractionated by column chromatography on Sephadex LH-20. This yielded five main fractions (Pr1, Pr2, Pr3, Pr4 and Pr5), which were analyzed by colorimetry to determine their concentrations of total phenolics, total tannins and condensed tannins and to assess their potential for blocking the phospholipase activity of BaV. The Pr5 fraction was defined as the fraction rich in condensed tannins (CTPr), and its inhibitory potential against the activities of the venom was evaluated. CTPr was evaluated in different in vivo and in vitro experimental protocols. The in vivo protocols consisted of (1) pre-incubation (venom:CTPr, w/w), (2) pre-treatment (orally administered) and (3) post-treatment (orally administered) to evaluate the effect on the hemorrhagic and edematogenic activities of BaV; in the in vitro protocol the effect on phospholipase and coagulant activity using pre-incubation in both tests was evaluated. RESULTS There was statistically significant inhibition (p<0.05) of hemorrhagic activity by CTPr when the pre-incubation protocol was used [55% (1:5, w/w) and 74% (1:10, w/w)] and when pre-treatment with doses of 50 and 100mg/kg was used (19% and 13%, respectively). However, for the concentrations tested, there was no statistically significant inhibition in the group subjected to post-treatment administered orally. CTPr blocked 100% of phospholipase activity and 63.3% (1:10, w/w) of coagulant activity when it was pre-incubated with BaV. There was a statistically significant reduction (p<0.05) in edema induced by BaV in the oral protocols. Maximum inhibition was 95% (pre-treatment). CONCLUSION Our findings indicate that CTPr could be a good source of natural inhibitors of the components of snake venom responsible for inducing local inflammation.

Stable and episodic/bolus patterns of methylmercury exposure on mercury accumulation and histopathologic alterations in the nervous system.

Abstract The main purpose of the present study was to compare the blood and brain mercury (Hg) accumulation and neurological alterations in adult male and pregnant female/fetal rats following stable and episodic/bolus patterns of methylmercury (MeHg) exposure. In addition, MeHg accumulation in the human body was estimated by a one‐compartment model using three different patterns of MeHg exposure. In the adult male rat experiment, doses of 0.3 and 1.5 mg MeHg/kg/day were orally administered to the stable groups for 5 weeks, while 7‐fold higher doses of 2.1 and 10.5 mg MeHg/kg/once a week were administered to the bolus groups. The blood Hg levels increased constantly in the stable groups, but increased with repeated waves in the bolus groups. At completion of the experiment, there were no significant differences in the brain Hg concentrations or neurological alterations between the stable and bolus groups, when the total doses of MeHg were the same. In the pregnant female rat experiment, a dose of 1 mg MeHg/kg/day was administered orally to the stable group for 20 days (until 1 day before expected parturition), while a 5‐fold higher dose of 5 mg MeHg/kg/once every 5 days was administered to the bolus group. In the brains of the maternal/fetal rats, there were no significant differences in the Hg concentrations and neurological alterations between the stable and bolus groups. The mean Hg concentrations in the fetal brains were approximately 2‐fold higher than those in the maternal brains for both stable and bolus groups. Using the one‐compartment model, the Hg accumulation curves in humans at doses of 7 &mgr;g MeHg/day, 48 &mgr;g MeHg/once a week, and 96 &mgr;g MeHg/once every 2 weeks were estimated to be similar, while the bolus groups showed dose‐dependent amplitudes of repeated waves. These results suggest that stable and episodic/bolus patterns of MeHg exposure do not cause differences in Hg accumulation in the blood and brain, or in neurological alterations, when the total doses are the same. HighlightsEffects of episodic/bolus patterns of MeHg exposure on Hg accumulation were examined.Episodic/bolus MeHg exposure patterns did not modify brain Hg and neurological alterations in maternal/fetal rats.In a one‐compartment model, Hg accumulation curves at different MeHg doses were similar.

Three dimensional morphometric analyses of axon terminals early changes induced by methylmercury intoxication in the adult cat striate cortex

The aim of the present report is to investigate in detail morphometric changes of axon terminals of area 17 of adult cat induced by methylmercury intoxication. Six adult male cats (Felix catus), with 12 h day-light cycle and ad libitum water and food regimen, received a single dose of MeHgCl (6.4 mg/kg) dissolved in milk, whereas control subjects (n=6) received only milk. After 30 days, biocytin iontophoretic injections were done into the area 17, (Horsley-Clark coordinates between AP 3.0-6.0) on the crown of the lateral gyrus, near the border with area 18. MeHg and inorganic Hg (Hgi) concentrations were measured in the brain parenchyma of intoxicated cats and corresponded on average to 1.39+/-0.3 and 6.79+/-0.6 ppm (mean+/-s.e.m.) respectively. Twenty four hours after iontophoresis, aldehyde fixed brain sections (200 microm thick), were processed to reveal biocytin labeled terminals. Axonal microscopic 3D reconstructions using Neurolucida software (Microbright Systems Inc.) allowed estimations of boutons, branching points and segment densities for each terminal. Cluster analysis of morphometric axonal features from control and intoxicated group revealed, two distinct axon families (Type I and II) as described elsewhere. Total density values of boutons, branching points and segment densities of intoxicated group, decreased 81, 59 and 91% respectively, as compared to the control group (ANOVA two-way, Bonferroni a priori test p<0.05). Altered axonal morphology associated with MeHg, appeared early in the disease (30 days after contamination), revealing new aspects of the neuronal pathology of the methylmercury intoxication in the visual cortex.