Francisco Célio Maia Chaves

The anthelmintic effect of plant extracts on Haemonchus contortus and Strongyloides venezuelensis

The indiscriminate use of anthelmintics has resulted in the establishment of parasite resistance. Thus, this study aimed to evaluate the in vitro antiparasitic effect of plant extracts on Haemonchus contortus in sheep and the in vivo effect on Strongyloides venezuelensis in Rattus norvegicus. The plant extracts from Piper tuberculatum, Lippia sidoides, Mentha piperita, Hura crepitans and Carapa guianensis, produced at different research institutions, were chemically analyzed and evaluated through the egg hatch test (EHT) and larval development test (LDT) in H. contortus. P. tuberculatum (150 and 250 mg kg(-1) of body weight) was evaluated for its anthelmintic action on R. norvegicus experimentally infected with S. venezuelensis. In the EHT, the LC(50) and LC(90) of the extracts were respectively as follows: 0.031 and 0.09 mg mL(-1) for P. tuberculatum, 0.04 and 0.13 mg mL(-1) for L. sidoides, 0.037 and 0.10 mg mL(-1) for M. piperita, 2.16 and 17.13 mg mL(-1) for H. crepitans and 2.03 × 10(-6) and 1.22 × 10(-12) mg mL(-1) for C. guianensis. In the LDT, the LC(50) and LC(90) were respectively: 0.02 and 0.031 mg mL(-1) for P. tuberculatum, 0.002 and 0.04 mg mL(-1) for L. sidoides, 0.018 and 0.03 mg mL(-1) for M. piperita, 0.36 and 0.91 mg mL(-1) for H. crepitans and 17.65 and 1890 mg mL(-1) for C. guianensis. The extract of P. tuberculatum showed the following substances: piperamides as (Z)-piplartine, (E)-piplartine, 8,9-dihydropiplartine, piperine, 10,11-dihydropiperine, 5,6 dihydropiperlongumine and pellitorine. The major compounds of the oils were thymol (76.6%) for L. sidoides, menthol (27.5%) for M. piperita and oleic acid (46.8%) for C. guianensis. Regarding the in vivo test, neither dose of P. tuberculatum caused any significant reduction (P>0.05) in worm burden and fecal egg counts compared with the control group. We conclude that the extracts of P. tuberculatum, L. sidoides and M. piperita have effective activity when tested in vitro, but the doses of the extract of P. tuberculatum have no effect when employed in in vivo tests.

In vitro cytocidal effects of the essential oil from Croton cajucara (red sacaca) and its major constituent 7- hydroxycalamenene against Leishmania chagasi

BackgroundVisceral leishmaniasis is the most serious form of leishmaniasis and can be lethal if left untreated. Currently available treatments for these parasitic diseases are frequently associated to severe side effects. The leaves of Croton cajucara are used as an infusion in popular medicine to combat several diseases. Previous studies have demonstrated that the linalool-rich essential oil from C. cajucara (white sacaca) is extremely efficient against the tegumentary specie Leishmania amazonensis. In this study, we investigated the effects of the 7-hydroxycalamenene-rich essential oil from the leaves of C. cajucara (red sacaca) against Leishmania chagasi, as well as on the interaction of these parasites with host cells.MethodsPromastigotes were treated with different concentrations of the essential oil for determination of its minimum inhibitory concentration (MIC). In addition, the effects of the essential oil on parasite ultrastructure were analyzed by transmission electron microscopy. To evaluate its efficacy against infected cells, mouse peritoneal macrophages infected with L. chagasi promastigotes were treated with the inhibitory and sub-inhibitory concentrations of the essential oil.ResultsThe minimum inhibitory concentrations of the essential oil and its purified component 7-hydroxycalamenene against L. chagasi were 250 and 15.6 μg/mL, respectively. Transmission electron microscopy analysis revealed important nuclear and kinetoplastic alterations in L. chagasi promastigotes. Pre-treatment of macrophages and parasites with the essential oil reduced parasite/macrophage interaction by 52.8%, while it increased the production of nitric oxide by L. chagasi-infected macrophages by 80%.ConclusionThese results indicate that the 7-hydroxycalamenene-rich essential oil from C. cajucara is a promising source of leishmanicidal compounds.

Arrabidaea chica Hexanic Extract Induces Mitochondrion Damage and Peptidase Inhibition on Leishmania spp.

Currently available leishmaniasis treatments are limited due to severe side effects. Arrabidaea chica is a medicinal plant used in Brazil against several diseases. In this study, we investigated the effects of 5 fractions obtained from the crude hexanic extract of A. chica against Leishmania amazonensis and L. infantum, as well as on the interaction of these parasites with host cells. Promastigotes were treated with several concentrations of the fractions obtained from A. chica for determination of their minimum inhibitory concentration (MIC). In addition, the effect of the most active fraction (B2) on parasites ultrastructure was analyzed by transmission electron microscopy. To evaluate the inhibitory activity of B2 fraction on Leishmania peptidases, parasites lysates were treated with the inhibitory and subinhibitory concentrations of the B2 fraction. The minimum inhibitory concentration of B2 fraction was 37.2 and 18.6 μg/mL for L. amazonensis and L. infantum, respectively. Important ultrastructural alterations as mitochondrial swelling with loss of matrix content and the presence of vesicles inside this organelle were observed in treated parasites. Moreover, B2 fraction was able to completely inhibit the peptidase activity of promastigotes at pH 5.5. The results presented here further support the use of A. chica as an interesting source of antileishmanial agents.

New antimalarial and cytotoxic 4-nerolidylcatechol derivatives.

4-Nerolidylcatechol (1) was isolated from cultivated Pothomorphe peltata root on a multigram scale using straight-forward solvent extraction-column chromatography. New semi-synthetic derivatives of 1 were prepared and tested in vitro against multidrug-resistant Plasmodium falciparum K1 strain. Mono-O-methyl, mono-O-benzyl, O,O-dibenzyl and O,O-dibenzoyl derivatives 2-8 exhibited IC(50) in the 0.67-22.52 microM range. Mono-O-methyl ethers 6 and 7 inhibited the in vitro growth of human tumor cell lines HCT-8 (colon carcinoma), SF-295 (central nervous system), LH-60 (human myeloblastic leukemia) and MDA/MB-435 (melanoma). In general, derivatives 2-8 are more stable to light, air and pH at ambient temperatures than their labile, natural precursor 1. These derivatives provide leads for the development of a novel class of antimalarial drugs with enhanced chemical and pharmacological properties.

In Vivo and In Vitro Antimalarial Activity of 4‐Nerolidylcatechol

4‐Nerolidylcatechol (4‐NC) isolated from Piper peltatum L. (Piperaceae) was evaluated for in vitro antiplasmodial activity against Plasmodium falciparum (cultures of both standard CQR (K1) and CQS (3D7) strains and two Amazonian field isolates) and for in vivo antimalarial activity using the Plasmodium berghei‐murine model. 4‐NC exhibits significant in vitro and moderate in vivo antiplasmodial activity. 4‐NC administered orally and subcutaneously at doses of 200, 400 and 600 mg/kg/day suppressed the growth of P. berghei by up to 63% after four daily treatments (days 1–4). Also, 4‐NC exhibited important in vitro antiplasmodial activity against both standard and field P. falciparum strains in which 50% inhibition of parasite growth (IC50) was produced at concentrations of 0.05–2.11 μg/mL and depended upon the parasite strain. Interestingly, healthy (non‐infected) mice that received 4‐NC orally presented (denatured) blood plasma which exhibited significant in vitro activity against P. falciparum. This is evidence that mouse metabolism allows 4‐NC or active metabolites to enter the blood. Further chemical and pharmacological studies are necessary to confirm the potential of 4‐NC as a new antimalarial prototype. Copyright

Antioxidant and antimicrobial activities of 7-hydroxy-calamenene-rich essential oils from Croton cajucara Benth.

Croton cajucara is a shrub native to the Amazon region locally known as “sacaca”. Two morphotypes are known: white and red “sacaca”. The essential oils (EO) obtained by hydrodistillation from leaves of the red morphotype were, in general, rich in 7-hydroxycalamenene (28.4%–37.5%). The effectiveness of these EO regarding the antimicrobial activity against pathogenic microorganisms was initially investigated by the drop test method, showing significant inhibition zones. Among the microorganisms tested, the essential oils rich in 7-hydroxycalamenene were more effective against methicillin resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, Mycobacterium tuberculosis, M. smegmatis, Mucor circinelloides and Rhizopus oryzae. The minimum inhibitory concentrations (MIC) of the oils were determined using the broth dilution assay. It was possible to observe that 7-hydroxycalamenene-rich oils presented high antimicrobial activity, with MIC of 4.76 × 10−3 μg/mL for MRSA, 4.88 μg/mL for M. tuberculosis, 39.06 μg/mL for M. smegmatis, and 0.152 μg/mL for R. oryzae and 3.63 × 10−8 μg/mL for M. circinelloides. The antioxidant activity of this EO suggests that 7-hydroxycalamenene provides more antioxidant activity according with EC50 less than 63.59 μg/mL. Considering the bioactive potential of EOs and 7-hydroxycalamenene could be of great interest for development of antimicrobials for therapeutic use in treatment of bacterial and fungal infections in humans and/or veterinary practice.

Stability and Antioxidant Activity of Semi-synthetic Derivatives of 4-Nerolidylcatechol

4-nerolidylcatechol (4-NC) is an unstable natural product that exhibits important antioxidant, anti-inflammatory and other properties. It is readily obtainable on a multi-gram scale through straightforward solvent extraction of the roots of cultivated Piper peltatum or P. umbellatum, followed by column chromatography on the resulting extract. Semi-synthetic derivatives of 4-NC with one or two substituent groups (methyl, acetyl, benzyl, benzoyl) on the O atoms have been introduced that have increased stability compared to 4-NC and significant in vitro inhibitory activity against the human malaria parasite Plasmodium falciparum. Antioxidant and anti-inflammatory properties may be important for the antiplasmodial mode of action of 4-NC derivatives. Thus, we decided to investigate the antioxidant properties, cytotoxicity and stability of 4-NC derivatives as a means to explore the potential utility of these compounds. 4-NC showed high antioxidant activity in the DPPH and ABTS assays and in 3T3-L1 cells (mouse embryonic fibroblast), however 4-NC was more cytotoxic (IC50 = 31.4 µM) and more unstable than its derivatives and lost more than 80% of its antioxidant activity upon storage in solution at −20 °C for 30 days. DMSO solutions of mono-O-substituted derivatives of 4-NC exhibited antioxidant activity and radical scavenging activity in the DPPH and ABTS assays that was comparable to that of BHA and BHT. In the cell-based antioxidant model, most DMSO solutions of derivatives of 4-NC were less active on day 1 than 4-NC, quercetin and BHA and more active antioxidants than BHT. After storage for 30 days at −20 °C, DMSO solutions of most of the derivatives of 4-NC were more stable and exhibited more antioxidant activity than 4-NC, quercetin and BHA and exhibited comparable antioxidant activity to BHT. These findings point to the potential of derivatives of 4-NC as antioxidant compounds.

Isolation and Identification of cis-7- Hydroxycalamenene from the Essential Oil of Croton cajucara Benth.

Abstract During agronomical and chemical studies with Croton cajucara Benth. (Euphorbiaceae), a shrub native to the Amazon area possessing an essential oil rich in linalool, it was observed that some individuals of a germplasm bank produced an oil containing a hydroxylated sesquiterpene as major component (up to 44.3%). Originally misidentified as 5-hydroxycalamenene, the compound was further isolated and properly identified by 1H and 13C NMR as cis-7-hydroxycalamenene. Measured retention indices were 1759 (Ultra-1), 1804 (HP-5), 2212 (FFAP) and 2663 (Carbowax 20M).

Piper peltatum: Biomass and 4-Nerolidylcatechol Production

Piper peltatum L. is used for the treatment of inflammation, malaria, and other ailments. 4-Nerolidylcatechol (4-NC) is a valuable natural product that has important anti-inflammatory, antimalarial, and antioxidant properties. 4-NC is a component of P. peltatum and P. umbellatum extracts, which are used in cosmetics. The aim of this work was to evaluate the production of plant biomass and the production of 4-NC in roots of cultivated P. peltatum over a full life cycle. Seedlings were produced in a greenhouse and then transplanted. The weight of dry plant parts (leaves, stems, roots, and inflorescences); numbers of stems, leaves, and inflorescences; and the leaf-to-stem ratio were evaluated at intervals of 60 days after transplanting (DAT). Extracts were prepared using 1:1 ethanol-chloroform and an ultrasound bath. Roots, leaves, and inflorescences contained 4-NC according to TLC photodensitometry analysis. Quantification of 4-NC in root extracts was performed using HPLC-DAD analysis. Per-hectare production of 4-NC by roots was estimated based on quantitative HPLC analysis and biomass data. Optimal per-hectare yields of 4-NC were obtained by harvesting roots between 350 and 400 DAT. In this period, the average yield was 27 kg 4-NC per hectare. Importantly, at the time of maximal overall production of root biomass (470 DAT), there was a decrease in the production of 4-NC (23.8 kg/ha), probably due to the onset of senescence.

Electrospray ionization mass spectrometry fingerprinting of extracts of the leaves of Arrabidaea chica

Arrabidaea chica (crajiru) e uma importante planta da Amazonia. Seus extratos sao usados como pigmento, agentes antimicrobianos e adstringentes. Tres variedades diferentes desta especie sao cultivadas na regiao Amazonica. Neste trabalho, infusoes diretas de extratos de A. chica destas tres variedade foram analisadas por impressao digital via espectrometria de massas com ionizacao por eletrospray (ESI(+)-MS). Dados derivados destes espectros foram classificados utilizando um metodo de analise multivariada (PLS-DA, analise discriminante com calibracao multivariada por minimos quadrados parciais). O metodo direto aqui apresentado baseia-se na extracao de folhas secas e em po com uma solucao de metanol/agua acidificada sem manipulacao adicional da amostra. A solucao sobrenadante foi analisada atraves de infusao direta por ESI(+)-MS, o que resultou em perfis de composicao para cada variedade. 3-Desoxiantocianidinas sao importantes substâncias presentes na A. chica, seus ions foram usados como marcadores no tratamento de dados utilizando PLS-DA, o que possibilitou a separacao das tres variedades. Impressao digital por ESI(+)-MS funciona como um metodo simples e rapido para diferenciacao de variedades de A. chica. Arrabidaea chica (crajiru) is an important Amazonian plant. Its extracts are used as red pigments, antimicrobial agents and astringents. Three different varieties of this species are cultivated in the Amazon region. In this work, direct infusions of A. chica extracts from these three varieties were analyzed via electrospray ionization mass spectrometry (ESI(+)-MS) fingerprinting. Derived data from the spectra were classified by using a multivariate method (PLS-DA, partial least squares-discriminant analysis). The direct method that is herein presented relies on extraction of dry, powdered leaves with acidified methanol/water solution with no further sample preparation. The resulting supernatants were analyzed by direct infusion ESI(+)-MS, which provides characteristic fingerprints of the sample composition. 3-Deoxyanthocyanidins are important substances in A. chica, their ions were used as markers in the PLS-DA data treatment. PLS-DA was able to differentiate the three varieties. ESI(+)-MS fingerprinting works as a simple and fast method to differentiate varieties of A. chica.