Antibacterial and cytotoxic activity of Brazilian plant extracts - Clusiaceae

Twelve extracts obtained from nine plants belonging to six different genera of Clusiaceae were analyzed against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and Enterococcus faecalis) bacteria using the microdilution broth assay. Tovomita aff. longifolia, T. brasiliensis, Clusia columnaris, Garcinia madruno, Haploclathra paniculata, and Caraipa grandifolia extracts showed significant results against the bacteria. The organic extract obtained from the leaves of T. aff. longifolia showed minimal inhibitory concentration (MIC) = 70 µg/ml and minimal bactericidal concentration (MBC) = 90 µg/ml against E. faecalis and the organic extract made with the stem of C. columnaris showed MIC = 180 µg/ml and MBC = 270 µg/ml against P. aeruginosa. None of the antibacterial extracts showed lethal activity against brine shrimp nauplii. On the other hand, both aqueous and organic extracts obtained from the aerial organs of Vismia guianensis that were cytotoxic to brine shrimp nauplii did not show a significant antibacterial activity in the assay.

Synergism between plant extract and antimicrobial drugs used on Staphylococcus aureus diseases

Searches for substances with antimicrobial activity are frequent, and medicinal plants have been considered interesting by some researchers since they are frequently used in popular medicine as remedies for many infectious diseases. The aim of this study was to verify the synergism between 13 antimicrobial drugs and 8 plant extracts - "guaco" (Mikania glomerata), guava (Psidium guajava), clove (Syzygium aromaticum), garlic (Allium sativum), lemongrass (Cymbopogon citratus), ginger (Zingiber officinale), "carqueja" (Baccharis trimera), and mint (Mentha piperita) - against Staphylococcus aureus strains, and for this purpose, the disk method was the antimicrobial susceptibility test performed. Petri dishes were prepared with or without dilution of plant extracts at sub-inhibitory concentrations in Mueller-Hinton Agar (MHA), and the inhibitory zones were recorded in millimeters. In vitro anti-Staphylococcus aureus activities of the extracts were confirmed, and synergism was verified for all the extracts; clove, guava, and lemongrass presented the highest synergism rate with antimicrobial drugs, while ginger and garlic showed limited synergistic capacity.

In vitro inhibition of acetylcholinesterase by crude plant extracts from Colombian flora

The methanol extracts from five different plant families (Asteraceae, Euphorbiaceae, Melastomataceae, Rubiaceae, and Solanaceae) collected at Regional Natural Park Ucumarí (Colombia), were screened for their acetylcholinesterase inhibitory activity through the modified Ellman's spectrophotometric method. The best inhibitory activities on this study were shown by the extracts of Solanum leucocarpum Dunal (IC50 = 204.59 mg/l) and Witheringia coccoloboides (Damm) (IC50 = 220.68 mg/l), both plants belonging to the Solanaceae family.

Molecular detection of human astrovirus in an urban sewage treatment plant in Rio de Janeiro, Brazil

The objective of this study was to evaluate the prevalence and dissemination of human astroviruses (HAstV) in the environment by analyzing urban sewage samples from a wastewater treatment plant in the city of Rio de Janeiro, Brazil. A one-year study was performed with a total of 48 raw and treated sewage composite samples, which were collected biweekly from an activated sludge plant. Virus particles were concentrated by the adsorption-elution method using negatively charged membranes associated to a Centriprep Concentrator® 50 (Nihon Millipore). HAstV were detected in 16.7% of the samples in raw and treated sewage by using both qualitative and quantitative reverse transcriptase-polymerase chain reactions (RT-PCR and qPCR, respectively). Positive untreated sewage sample exhibited mean values of 1.1 x 10(4) gEq/mL. The qPCR sensitivity was 18 gEq/reaction. Through utilization of qPCR, a HAstV recovery efficiency of 4.2% and 4.3% was demonstrated for raw and treated sewage samples, respectively. The presence of HAstV in both the raw and treated sewage samples demonstrated the dissemination of these viruses in the environment as well as viral permanence after sewage treatment. There was a reduction in the total and faecal coliform levels, indicating efficiency of the wastewater treatment plant.

Biological activity of neosergeolide and isobrucein B (and two semi-synthetic derivatives) isolated from the Amazonian medicinal plant Picrolemma sprucei (Simaroubaceae)

In the present study, in vitro techniques were used to investigate a range of biological activities of known natural quassinoids isobrucein B (1) and neosergeolide (2), known semi-synthetic derivative 1,12-diacetylisobrucein B (3), and a new semi-synthetic derivative, 12-acetylneosergeolide (4). These compounds were evaluated for general toxicity toward the brine shrimp species Artemia franciscana, cytotoxicity toward human tumour cells, larvicidal activity toward the dengue fever mosquito vector Aedes aegypti, haemolytic activity in mouse erythrocytes and antimalarial activity against the human malaria parasite Plasmodium falciparum. Compounds 1 and 2 exhibited the greatest cytotoxicity against all the tumor cells tested (IC50 = 5-27 µg/L) and against multidrug-resistant P. falciparum K1 strain (IC50 = 1.0-4.0 g/L) and 3 was only cytotoxic toward the leukaemia HL-60 strain (IC50 = 11.8 µg/L). Quassinoids 1 and 2 (LC50 = 3.2-4.4 mg/L) displayed greater lethality than derivative 4 (LC50 = 75.0 mg/L) toward A. aegypti larvae, while derivative 3 was inactive. These results suggest a novel application for these natural quassinoids as larvicides. The toxicity toward A. franciscana could be correlated with the activity in several biological models, a finding that is in agreement with the literature. Importantly, none of the studied compounds exhibited in vitro haemolytic activity, suggesting specificity of the observed cytotoxic effects. This study reveals the biological potential of quassinoids 1 and 2 and to a lesser extent their semi-synthetic derivatives for their in vitro antimalarial and cytotoxic activities.

Development of physiological resistance and its stage specificity in Culex quinquefasciatus after selection with deltamethrin in Assam, India

The study investigated the development and stage specificity of physiological resistance to insecticides in a colony of Culex quinquefasciatus Say (Diptera: Culicidae) mosquitoes, which are vectors of bancroftian filariasis in India, after selection with deltamethrin. Resistance was selected by exposing the larvae to the concentration of deltamethrin that caused 50% mortality in the tested population (i.e., LC50). Under continuous selection pressure, the LC50 increased steadily in subsequent generations. The estimated LC50 for the F0 generation was 0.409 μg/L; the LC50 first displayed a substantial increase in the F5 generation (5.616 μg/L) and reached 121.902 μg/L in the F10 generation. The objective of this study was to establish a deltamethrin-resistant colony to develop a research programme that will study the evolution of physiological resistance patterns and stage-specific resistance responses in Cx. quinquefasciatus larvae and adults under laboratory conditions. An approximately 298-fold increase in resistance was recorded after 10 generations, as evidenced by the resistance ratio (RR50). The progress and effect of the selection pressure in the adult stage was monitored with the World Health Organisation (WHO) diagnostic test. The mortality, as observed using the WHO diagnostic test, declined significantly from the F5 generation (85%) onwards and the highest rate of survival (65%) was observed in the F10 generation.

In vitro antiplasmodial activity and toxicity assessment of plant extracts used in traditional malaria therapy in the Lake Victoria Region

As part of our program screening the flora of the Lake Victoria Region, a total of 54 organic extracts from seven plant families (8 species) were individually tested for antiplasmodial activity against chloroquine-sensitive [Sierra Leone (D-6)] and chloroquine-resistant [Vietnam (W-2)] strains. Only 22% of these extracts exhibited very high in vitro antiplasmodial activity. Six methanol (MeOH) extracts and one chloroform extract showed in vitro antiplasmodial activity against the D-6 Plasmodium falciparum strain, while only three MeOH extracts were active against the W-2 strain. All of the ethyl acetate extracts proved to be inactive against both strains of P. falciparum. A brine shrimp cytotoxicity assay was used to predict the potential toxicity of the extracts. The cytotoxicity to antiplasmodial ratios for the MeOH extracts were found to be greater than 100, which could indicate that the extracts are of low toxicity.

The stepwise selection for ketoconazole resistance induces upregulation of C14-demethylase (CYP51) in Leishmania amazonensis

Ketoconazole is a clinically safe antifungal agent that also inhibits the growth of Leishmania spp. A study was undertaken to determine whether Leishmania parasites are prone to becoming resistant to ketoconazole by upregulating C14-demethylase after stepwise pharmacological pressure. Leishmania amazonensis promastigotes [inhibitory concentration (IC)50 = 2 µM] were subjected to stepwise selection with ketoconazole and two resistant lines were obtained, La8 (IC50 = 8 µM) and La10 (IC50 = 10 µM). As a result, we found that the resistance level was directly proportional to the C14-demethylase mRNA expression level; we also observed that expression levels were six and 12 times higher in La8 and La10, respectively. This is the first demonstration that L. amazonensis can up-regulate C14-demethylase in response to drug pressure and this report contributes to the understanding of the mechanisms of parasite resistance.

In vitro and in vivo antimalarial activity and cytotoxicity of extracts, fractions and a substance isolated from the Amazonian plant Tachia grandiflora (Gentianaceae)

Tachia sp. are used as antimalarials in the Amazon Region and in vivo antimalarial activity of a Tachia sp. has been previously reported. Tachia grandiflora Maguire and Weaver is an Amazonian antimalarial plant and herein its cytotoxicity and antimalarial activity were investigated. Spectral analysis of the tetraoxygenated xanthone decussatin and the iridoid aglyone amplexine isolated, respectively, from the chloroform fractions of root methanol and leaf ethanol extracts was performed. In vitro inhibition of the growth of Plasmodium falciparum Welch was evaluated using optical microscopy on blood smears. Crude extracts of leaves and roots were inactive in vitro. However, chloroform fractions of the root and leaf extracts [half-maximal inhibitory concentration (IC50) = 10.5 and 35.8 µg/mL, respectively] and amplexine (IC50= 7.1 µg/mL) were active in vitro. Extracts and fractions were not toxic to type MRC-5 human fibroblasts (IC50> 50 µg/mL). Water extracts of the roots of T. grandiflora administered by mouth were the most active extracts in the Peters 4-day suppression test in Plasmodium berghei-infected mice. At 500 mg/kg/day, these extracts exhibited 45-59% inhibition five to seven days after infection. T. grandiflora infusions, fractions and isolated substance have potential as antimalarials.

Culicidae (Diptera) selection of humans, chickens and rabbits in three different environments in the province of Chaco, Argentina

Studies were conducted to determine the selection of humans, chickens and rabbits by Culicidae in three different environments in the province of Chaco, Argentina. Mosquitoes were collected fortnightly using cylindrical metal traps containing animal bait (chickens and rabbits). The mosquitoes were collected between June 2001-May 2002. During the same period and with the same frequency, mosquitoes biting the human operators of the traps were collected during the first 15 min of exposure within different time intervals: from 09:00 am-11:00 am, 01:00 pm-03:00 pm, 05:00 pm-07:00 pm and 09:00 pm-10:00 pm. A total of 19,430 mosquitoes of 49 species belonging to 10 genera were collected. Culex species mainly selected chicken bait and Wyeomyia species selected rabbit bait. Ochlerotatus and Psorophora species were more abundant in rabbit-baited traps. Anopheles triannulatus, Coquillettidia nigricans, Ochlerotatus scapularis, Mansonia titillans and Psorophora albigenu showed a strong attraction for human bait. The Anopheles, Coquillettidia, Culex and Mansonia species were more active between 05:00 pm-09:00 pm, while Ochlerotatus, Psorophora, Haemagogus and Wyeomyia were most active from 09:00 am-07:00 pm. This study provides additional information about the biology and ecology of arbovirus vectors in Chaco.

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part I: Aspidosperma nitidum (Benth) used as a remedy to treat fever and malaria in the Amazon

Infusions of Aspidosperma nitidum (Apocynaceae) wood bark are used to treat fever and malaria in the Amazon Region. Several species of this family are known to possess indole alkaloids and other classes of secondary metabolites, whereas terpenoids, an inositol and the indole alkaloids harmane-3 acid and braznitidumine have been described in A. nitidum . In the present study, extracts from the wood bark, leaves and branches of this species were prepared for assays against malaria parasites and cytotoxicity testing using human hepatoma and normal monkey kidney cells. The wood bark extracts were active against Plasmodium falciparum and showed a low cytotoxicity in vitro, whereas the leaf and branch extracts and the pure alkaloid braznitidumine were inactive. A crude methanol extract was subjected to acid-base fractionation aimed at obtaining alkaloid-rich fractions, which were active at low concentrations against P. falciparum and in mice infected with and sensitive Plasmodium berghei parasites. Our data validate the antimalarial usefulness of A. nitidum wood bark, a remedy that can most likely help to control malaria. However, the molecules responsible for this antimalarial activity have not yet been identified. Considering their high selectivity index, the alkaloid-rich fractions from the plant bark might be useful in the development of new antimalarials.

Genetic variability in G2 and F2 region between biological clones of human respiratory syncytial virus with or without host immune selection pressure

Human respiratory syncytial virus (HRSV) is an important respiratory pathogens among children between zero-five years old. Host immunity and viral genetic variability are important factors that can make vaccine production difficult. In this work, differences between biological clones of HRSV were detected in clinical samples in the absence and presence of serum collected from children in the convalescent phase of the illness and from their biological mothers. Viral clones were selected by plaque assay in the absence and presence of serum and nucleotide sequences of the G2 and F2 genes of HRSV biological clones were compared. One non-synonymous mutation was found in the F gene (Ile5Asn) in one clone of an HRSV-B sample and one non-synonymous mutation was found in the G gene (Ser291Pro) in four clones of the same HRSV-B sample. Only one of these clones was obtained after treatment with the child's serum. In addition, some synonymous mutations were determined in two clones of the HRSV-A samples. In conclusion, it is possible that minor sequences could be selected by host antibodies contributing to the HRSV evolutionary process, hampering the development of an effective vaccine, since we verify the same codon alteration in absence and presence of human sera in individual clones of BR-85 sample.

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies withAspidosperma ramiflorum in vivo and in vitro

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellowperoba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium bergheiin mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.

Power and selection of contract terms: The case from the Brazilian orange juice sector

ABSTRACT We propose a model to explain how contract terms are selected in the presence of a form of economic power: contract power. The orange juice sector is used to illustrate an analysis that demonstrates the effects of contract power on the economic organization of the sector. We define contract power as the ability to exploit contractual gaps or failures of contractual provisions, which are strategically left incomplete. Empirical evidence from content analysis of antitrust documents supports the logic of contract power in the orange juice sector in three forms: avoiding changes to payment methods from weight to solid contents (quality); using information asymmetries to manipulate indexes that calculate the formula of orange prices; and deliberately harvesting oranges late in order to dehydrate the fruit, which consequently reduces weight and price. The paper contributes to understanding the selection of contract terms and the debate about how antitrust offices can deal with this issue.

Sex allocation and sex-dependent selection for body size in Trypoxylon rogenhoferi Kohl (Hymenoptera, Sphecidae)

Two populations of the wasp Trypoxylon rogenhoferi Kohl, 1884 from São Carlos and Luís Antônio, State of São Paulo, Brazil, were observed and sampled from May 1999 to February 2001 using trap-nests. This mass-provisioning wasp was used to test some aspects of optimal sex allocation theory. Both populations fit all the predictions of the models of Green and Brockmann and Grafen. Maternal provisions determined the size of each offspring, and females allocated well-stocked brood cells to daughters, the sex that benefits most being large. This strategy resulted in a difference in size between the sexes. In São Carlos, female weight at emergence was 1.18 times that of males, in Luís Antônio this value was 1.13. The brood cell volume was correlated with both wing length and weight at emergence in both sexes, and the chance that a given brood cell contained a male offspring decreased with increased brood cell volume. In T. rogenhoferi female body size was related to fitness. Larger females were able to collect more mass of spiders per day, the spiders they captured were heavier, and they provisioned more brood cells per day. They also produced larger daughters. For males, no relationship between body size and fitness was found, but the data were scarce. Since the patterns of provisioning were variable among different females in both study sites, it is possible that the females not follow a unique strategy for sex allocation. The sex ratio and/or investment ratio in the São Carlos population was female-biased and in Luís Antônio, male-biased. In spite of the influence of trap-nests diameters on male production in Luís Antônio, there is some evidence that in São Carlos population the local availability of prey and/or lower rate of parasitism may be major forces in determining the observed sex ratio, but further studies are necessary to verify such hypothesis.