In vitro and in vivo assessment of the anti-malarial activity of Caesalpinia pluviosa.

BACKGROUND: To overcome the problem of increasing drug resistance, traditional medicines are an important source for potential new anti-malarials. Caesalpinia pluviosa, commonly named "sibipiruna", originates from Brazil and possess multiple therapeutic properties, including anti-malarial activity. METHODS: Crude extract (CE) was obtained from stem bark by purification using different solvents, resulting in seven fractions. An MTT assay was performed to evaluate cytotoxicity in MCF-7 cells. The CE and its fractions were tested in vitro against chloroquine-sensitive (3D7) and -resistant (S20) strains of Plasmodium falciparum and in vivo in Plasmodium chabaudi-infected mice. In vitro interaction with artesunate and the active C. pluviosa fractions was assessed, and mass spectrometry analyses were conducted. RESULTS: At non-toxic concentrations, the 100% ethanolic (F4) and 50% methanolic (F5) fractions possessed significant anti-malarial activity against both 3D7 and S20 strains. Drug interaction assays with artesunate showed a synergistic interaction with the F4. Four days of treatment with this fraction significantly inhibited parasitaemia in mice in a dose-dependent manner. Mass spectrometry analyses revealed the presence of an ion corresponding to m/z 303.0450, suggesting the presence of quercetin. However, a second set of analyses, with a quercetin standard, showed distinct ions of m/z 137 and 153. CONCLUSIONS: The findings show that the F4 fraction of C. pluviosa exhibits anti-malarial activity in vitro at non-toxic concentrations, which was potentiated in the presence of artesunate. Moreover, this anti-malarial activity was also sustained in vivo after treatment of infected mice. Finally, mass spectrometry analyses suggest that a new compound, most likely an isomer of quercetin, is responsible for the anti-malarial activity of the F4.

Evaluation of the naturally acquired antibody immune response to the Pv200L N-terminal fragment of Plasmodium vivax merozoite surface protein-1 in four areas of the Amazon Region of Brazil.

Frequency and levels of IgG antibodies to an N-terminal fragment of the Plasmodium vivax MSP-1 (Pv200L) protein, in individuals naturally exposed to malaria in four endemic areas of Brazil, were evaluated by enzyme-linked immunosorbent assay. Plasma samples of 261 P. vivax-infected individuals from communities of Macapá, Novo Repartimento, Porto Velho, and Plácido de Castro in the Amazonian region with different malaria transmission intensities. A high mean number of studied individuals (89.3%) presented with antibodies to the Pv200L that correlated with the number of previous malaria infections; there were significant differences in the frequency of the responders (71.9-98.7) and in the antibody levels (1:200-1:51,200) among the four study areas. Results of this study provide evidence that Pv200L is a naturally immunogenic fragment of the PvMSP-1 and is associated with the degree of exposure to parasites. The fine specificity of antibodies to Pv200L is currently being assessed.

Plasmodium vivax circumsporozoite genotypes: a limited variation or new subspecies with major biological consequences?

BACKGROUND: Plasmodium vivax circumsporozoite variants have been identified in several geographical areas. The real implication of the genetic variation in this region of the P. vivax genome has been questioned for a long time. Although previous studies have observed significant association between VK210 and the Duffy blood group, we present here that evidences of this variation are limited to the CSP central portion. METHODS: The phylogenetic analyses were accomplished starting from the amplification of conserved domains of 18 SSU RNAr and Cyt B. The antibodies responses against the CSP peptides, MSP-1, AMA-1 and DBP were detected by ELISA, in plasma samples of individuals infected with two P. vivax CS genotypes: VK210 and P. vivax-like. RESULTS: These analyses of the two markers demonstrate high similarity among the P. vivax CS genotypes and surprisingly showed diversity equal to zero between VK210 and P. vivax-like, positioning these CS genotypes in the same clade. A high frequency IgG antibody against the N- and C-terminal regions of the P. vivax CSP was found as compared to the immune response to the R- and V- repetitive regions (p = 0.0005, Fisher's Exact test). This difference was more pronounced when the P. vivax-like variant was present in the infection (p = 0.003, Fisher's Exact test). A high frequency of antibody response against MSP-1 and AMA-1 peptides was observed for all P. vivax CS genotypes in comparison to the same frequency for DBP. CONCLUSIONS: This results target that the differences among the P. vivax CS variants are restrict to the central repeated region of the protein, mostly nucleotide variation with important serological consequences.

Plasmodium vivax circumsporozoite variants and Duffy blood group genotypes in the Brazilian Amazon region.

The circumsporozoite protein (CSP) of the Plasmodium vivax infective sporozoite is considered to be a major target for the development of recombinant malaria vaccines. The Duffy blood group molecule acts as the red blood cell receptor for P. vivax. We review the frequency of P. vivax CSP variants and report their association with the Duffy blood group genotypes from Brazilian Amazon patients carrying P. vivax malaria. Peripheral blood samples were collected from 155 P. vivax-infected individuals from five Brazilian malaria-endemic areas. The P. vivax CSP variants and the Duffy blood group genotypes were assessed using PCR/RFLP. In single infections, the VK210 variant was the commonest followed by the P. vivax-like variant. The typing of P. vivax indicated that the frequency of variants among the study areas was significantly different from one to another. This is the first detection of the VK247 and P. vivax-like variant in single infections in endemic areas of Brazil. Association of the CSP P. vivax variants with the heterozygous Duffy blood group system genotype was significant for VK210 single infection. These observations provide additional data on the Plasmodium-host interactions concerning the Duffy blood group and P. vivax capability of causing human malaria.