José Luiz P. Ferreira

Nitric oxide production by murine peritoneal macrophages in vitro and in vivo treated with Phyllanthus tenellus extracts.

Phyllanthus spp. are used traditionally for the treatment of viral, bacterial and parasitic infections. Macrophages may play a central role in innate and adaptive response against several infections. Nitric oxide (NO) can be induced during macrophage activation and may exert antimicrobial activity inhibiting the replication of several viruses or parasites. In the present study, we investigated the immunomodulatory role, both in vitro and in vivo, of aqueous extracts of fresh and dried Phyllanthus tenellus as well as an acetone/water extract of the dried plant. NO production by mouse peritoneal macrophages was detected in culture supernatants. Our results demonstrated that: (1) in vitro, a concentration of 100 microg/ml fresh extract stimulated significantly (P< or =0.05) NO production in all assays and the optimal production was achieved at 48-h incubation; (2) 10 and 50 mg/kg fresh extract injected twice intraperitonealy primed macrophages in vivo. Priming was detected by in vitro addition of a second stimulus with 100 microg/ml extract of the fresh plant. Thus, P. tenellus was able to pre-activate macrophages in vivo, and induce full activation in vitro. Further studies should be carried out to better evaluate the optimal dose schedules in terms of time/response for obtaining antiviral or other antimicrobial activity without host damage.

Chemical and biological evaluation of essential oils with economic value from Lauraceae species

This work compares the chemical composition of the essential oils from the leaves of Licaria canella collected in two different seasons. The results of this investigation were compared with the leaf essential oils of other species of the Lauraceae family, Aniba canelilla, collected at the same time. Both essential oils were analyzed by GC-FID and GC-MS. The results demonstrated a larger predominance of benzenoids, being the main constituent benzyl benzoate for L. canella and 1-nitro-2-phenylethane for A. canelilla. The comparison of the biological activities showed that L. canella (IC50 19 µg mL-1) was more active against Leishmania amazonensis strains and less cytotoxic in macrophage cultures than A. canelilla (IC50 40 µg mL-1). On the other hand, the L. canella oil displayed a higher cytotoxicity against Artemia salina with a lethal concentration (LC50), equal to 5.25 µg mL-1.

In vitro evaluation of (-)α-bisabolol as a promising agent against Leishmania amazonensis.

Current treatments for leishmaniasis present some difficulties due to their toxicity, the use of the intravenous route for administration and therapy duration, which may lead to treatment discontinuation. The aim of this study is to investigate new treatment alternatives to improve patients well being. Therefore, we evaluated the inhibitory effect of (-)α-bisabolol, a sesquiterpene alcohol found in various essential oils of different plant species, against the promastigotes and intracellular amastigotes forms of Leishmania amazonensis, as well as the cytotoxic, morphological and ultrastructural alterations of treated cells. Promastigotes forms of L. amazonensis were incubated with (-)α-bisabolol to determine the antileishmanial activity of this compound. The cytotoxicity effect was evaluated by testing against J774.G8 cells. After these tests, the infected and uninfected cells with L. amazonensis were used to determine if the (-)α-bisabolol was able to kill intracellular parasites and to cause some morphological changes in the cells. The (-)α-bisabolol compound showed significant antileishmanial activity against promastigotes with a 50% effective concentration of 8.07 µg/ml (24 h) and 4.26 µg/ml (48 h). Against intracellular amastigotes the IC50 (inhibitory concentration) of (-)α-bisabolol (24 h) was 4.15 µg/ml. The (-)α-bisabolol also showed a cytotoxic effect against the macrophage strain J774.G8. The value of 50% cytotoxic concentration was 14.82 µg/ml showing that (-)α-bisabolol is less toxic to macrophages than to the parasite. Ultrastructural studies of treated promastigotes and amastigotes showed several alterations, such as loss of cytoplasmic organelles, including the nucleus, and the presence of lipid inclusions. This study showed that (-)α-bisabolol has promising antileishmanial properties, as it can act against the promastigote forms and is able to penetrate the cell, and is also active against the amastigote forms. About 69% of the promastigotes forms suffered mitochondrial membrane damage after treatment with IC50 of (-)α-bisabolol, suggesting inhibition of the metabolic activity of parasites. These results open new prospects for research that can contribute to the development of products based on essential oils or isolated compounds from plants for the treatment of cutaneous leishmaniasis.

Chemical composition and insecticidal activity of Cymbopogon citratus essential oil from Cuba and Brazil against housefly

Essential oil of Cymbopogon citratus collected from Brazil and Cuba was tested to a chemical characterization and then was tested on the post-embryonic development of Musca domestica. The chemical composition analysis by GC-MS of the oils from Brazil/Cuba allowed the identification of 13 and 12 major constituents respectively; nine of them common to both. In the both oils, the main components were the isomers geranial and neral, which together form the compound citral. This corresponds to a total of 97.92%/Brazil and 97.69%/Cuba of the compounds identified. The monoterpene myrcene, observed only in the sample of Cuba, presented a large relative abundance (6.52%). The essential oil of C. citratus (Brazil/Cuba) was dissolved in DMSO and tested at concentrations of 5, 10, 25, 50, 75 and 100% and citral was prepared by mixing 16.8 mg with 960 µL DMSO. Both essential oils and monoterpene citral were applied topically to newly-hatched larvae (1µL/larva). The results showed a lethal concentration (LC50) of 4.25 and 3.24% for the Brazilian and Cuban essential oils, respectively. Mortalities of larval and newly-hatched larvae to adult periods were dose-dependent for the two both oils as for monoterpene citral, reaching 90%. Both essential oils and citral caused morphological changes in adult specimens.

Liposomal formulation of turmerone-rich hexane fractions from Curcuma longa enhances their antileishmanial activity.

Promastigote forms of Leishmania amazonensis were treated with different concentrations of two fractions of Curcuma longa cortex rich in turmerones and their respective liposomal formulations in order to evaluate growth inhibition and the minimal inhibitory concentration (MIC). In addition, cellular alterations of treated promastigotes were investigated under transmission and scanning electron microscopies. LipoRHIC and LipoRHIWC presented lower MIC, 5.5 and 12.5 μg/mL, when compared to nonencapsulated fractions (125 and 250 μg/mL), respectively, and to ar-turmerone (50 μg/mL). Parasite growth inhibition was demonstrated to be dose-dependent. Important morphological changes as rounded body and presence of several roles on plasmatic membrane could be seen on L. amazonensis promastigotes after treatment with subinhibitory concentration (2.75 μg/mL) of the most active LipoRHIC. In that sense, the hexane fraction from the turmeric cortex of Curcuma longa incorporated in liposomal formulation (LipoRHIC) could represent good strategy for the development of new antileishmanial agent.

Anatomia Foliar e Caulinar de Picrolemma sprucei Hook (Simaroubaceae)

Anatomical studies of the leaves and stems of Picrolemma sprucei revealed that the epidermes of the leaves are glabrous, with undulating cell walls and anomocytic stomata. The mesophyll is dorsiventral with a layer of cells in palisade and a spongy parenchyma. The vascular bundles are bicollateral and have a dark tannin containing idioblast. The stem possesses a developed cortical parenchyma with schlerenchymatous thick walled cells. These are either isolated or gathered in small groups. Internally the vascular region exhibits xylematic vessels some isolated and some in groups submerged in fibrous tissue. The stem possesses a paratracheal, vasicentric parenchyma with central pith. Histochemical tests (sudan III and ferric chloride) made on the central section of the midrib and on the petiole showed the presence, respectively, of tannin or oleoresin in some cells.

Wound healing properties of Copaifera paupera in diabetic mice

Copaifera oleoresin is one of the most used natural products in popular medicine all over the world. Among other effects (i.e., anti-inflammatory, antinociceptive, microbicidal) one of the most well-known is its wound healing capacity. However, the mechanism by which the oleoresin presents its effect is still not clear. In this study, our aim was to evaluate the wound healing capacity of oleoresin obtained from Copaifera paupera, its mechanism of action and identify its major components. For these purposes, diabetic Swiss Webster mice were topically treated with oleoresin (100, 150 or 200 mg/kg) for 14 consecutive days after an excision was performed in the back of the mice. Cytokines, wound retraction and histological evaluation were conducted at 3, 7 and 10 days (for cytokines); 0, 3, 7, 10 and 14 days (for wound retraction); and 7 and 14 days (for histological evaluation). Our data indicate that oleoresin significantly reduced production of MCP-1 and TNF-α at days 7 and 10 post-excision and increased IL-10 production at both days. All treatments demonstrated an effect similar or higher to that in collagenase-treated mice. Histological evaluations demonstrated that higher dose treatment resulted in better resolution and closure of the wound and higher levels of collagen deposition and indexes of re-epithelialization even when compared with the collagenase-treated group. The treatment with oleoresin from Copaifera paupera demonstrated that it is even better than an ointment routinely used for improvement of wound healing, suggesting this oleoresin as an option for use in diabetic patients.

Chemical Composition of Essential Oils from the Leaves of Endlicheria bracteolata

Abstract This work compares the chemical composition of the essential oils from the leaves of Endlicheria bracteolata (Lauraceae family) collected in two different seasons. These oils obtained by hydrodistillation were analyzed by gas chromatography coupled to mass and FID detectors. Analysis of the chromatograms showed a predominance of sesquiterpene hydrocarbons in the essential oils obtained in the rainy and dry seasons, representing 75.6 % and 86.5 % of these sesquiterpenes, respectively. The main constituents found in the oil obtained during the rainy season were valencene (30.0 %) and (E)-caryophyllene (17.7 %), while for the oil obtained in the dry season, the main constituents were (E)-caryophyllene (21.2 %) and guaia-10(14),11-diene (17.5 %).

Fingerprint by Gas Chromatography-Mass Spectrometry of Two Himatanthus Species of Brazilian North Region

Himatanthus species (Apocynaceae) are popularly used in Brazil to treat inflammation, ulcer, and cancer [1–4]. The genus is chemically characterized by the occurrence of iridoids [2, 5, 6]. Himatanthus sucuuba (Spruce) Woodson grows in the Amazon region, and several chemical research reports on its latex and barks led to the isolation of iridoids such as plumericin and isoplumericin and triterpenes such as -amyrin and lupeol esters [1, 5, 7]. These components are usually associated with the activity of this genus. H. attenuatus (Benth.) Woodson is a tree species distributed in Colombia, Venezuela, Brazil, and Bolivia. Unlike H. sucuuba, there are few studies on H. attenuatus related only to taxonomy, morphology, and screening studies [8–10]. The aim of this work was to contribute to the knowledge of the genus Himatanthus by comparing the hexane and dichloromethane extracts of the scarcely studied species H. attenuatus with the extracts of H. sucuuba using gas chromatography coupled to mass spectrometry (GC-MS) analyses. This procedure also furnished the chromatography fingerprint of both species, popularly known as sucuba in Amazonas and Acre states, among other names. Roots of H. attenuatus and H. sucuuba were collected in the Brazilian Amazon region, and the herbaria vouchers were compared with those previously deposited: 17590 (UFAC Herbarium, Acre State) and 180453 (INPA Herbarium, Amazonas State), respectively. Both plant materials were dried in the oven with air circulation at 35 C for 24 h. Finely triturated roots of H. attenuatus and H. sucuuba (0.5 g each) were successively macerated with agitation five times with hexane (1.2 L/extraction) and CH2Cl2 (1.2 L/extraction). Extracts were concentrated under reduced pressure, yielding (p/p) 1.9% of hexane extract of H. attenuatus (HA-HX), 1.1% of CH2Cl2 extract of H. attenuatus (HA-DC), 1.2% of hexane extract of H. sucuuba (HS-HX), and 2.3% of CH2Cl2 extract of H. sucuuba (HS-DC). Their chemical compositions are shown in Table 1. Extracts were analyzed by GC-MS on an Agilent 6890N GC coupled to a quadripolar mass spectrometer (Agilent 5973N), with ionization by electronic impact (70 eV). Analyses were performed in triplicate, and the injected volume was 1 L, in splitless mode. The apparatus was fitted with a DB-5MS column (30 m 0.25 mm I.D., 0.25 m phase film). The injector temperature was 270 C, ion-source 70 eV, detector 230 C, and scan range 40–700 m/z. The column temperature varied from 70 C to 305 C at a rate of 5 C/min. Helium was the carrier gas at flow rate 1 mL/min. Interpretation and identification of the fragmentation mass spectrum were carried out by comparison with the Wiley NBS mass spectrum database. Results are expressed as relative percentage of peak area in the chromatogram (Table 1). As shown in Table 1, free fatty acids and their esters were detected in both species, and an important content of palmitic acid was found in the CH2Cl2 extract of H. sucuuba (11.5%). The steroid, sitosterol, was present in H. sucuuba extracts.

A General Description of Apocynaceae Iridoids Chromatography

Iridoids are considered atypical monoterpenoid compounds, based on a methylcyclopentan[C]-pyran skeleton, often fused to a six-membered oxygen ring consisting of ten, nine or in rare cases, eight carbon atoms (Figure 1a) [1, 2]. More than 2500 iridoid compounds have been described in nature, with structural differences related mainly to the degree and type of substitution in the cyclopentane ring skeleton [3]. Iridoids can be found in nature as secoiri‐ doids (Figure 1b), a large group characterized by cleavage of the 7,8-bond on the cyclopentane ring, glycosides, mainly 1-O-glucosides, and nor-iridoids, originating from oxidative decar‐ boxylation on C10 or C11 (Figure 1) [3, 4]. A General Description of Apocynaceae Iridoids Chromatography