A new HPLC-DAD-MS/MS method for the simultaneous determination of major compounds in the crude extract of Lychnophora salicifolia Mart. (Brazilian arnicao) leaves: Application to chemical variability evaluation

Lychnophora salicifolia Mart., which occurs in the Brazilian Cerrado in the states of Bahia and Minas Gerais as well as in the southeast of the state of Goias, is the most widely distributed and also the most polymorphic species of the genus. This plant is popularly known to have anti-inflammatory and analgesic activities. In this work, we have studied the variation in terms of polar metabolites of ninety-three Lychnophora salicifolia Mart, specimens collected from different regions of the Brazilian Cerrado. Identification of the constituents of this mixture was carried out by analysis of the UV spectra and MS data after chromatographic separation. Twenty substances were identified, including chlorogenic acid derivatives, a flavonoid C-glucoside, and other sesquiterpenes. The analytical method was validated, and the reliability and credibility of the results was ensured for the purposes of this study. The concentration range required for analysis of content variability within the analyzed group of specimens was covered with appropriate values of limits of detection and quantitation, as well as satisfactory precision and recovery. A quantitative variability was observed among specimens collected from the same location, but on average they were similar from a chemical viewpoint. In relation to the study involving specimens from different locations, there were both qualitative and quantitative differences among plants collected from different regions of Brazil. Statistical analysis revealed that there is a correlation between geographical localization and polar metabolites profile for specimens collected from different locations. This is evidence that the pattern of metabolites concentration depends on the geographical distribution of the specimens. (C) 2012 Elsevier B.V. All rights reserved.

TOWARDS NEW BOTANICAL PESTICIDES: THE TOXIC EFFECT OF Eremanthus goyazensis (Asteraceae) LEAVES ESSENTIAL OIL AGAINST Brevipalpus phoenicis (Acari: Tenuipalpidae)

This work reports the chemical characterization of Eremanthus goyzensis essential oil and its toxic effect over Brevipalpus phoenicis. The essential oil displayed a major composition of sesquiterpenes (61.87%) including trans-caryophillene (26.81%) and germacrene-D (13.31%). The fumigation test indicated a promising bioactivity over adult B. phoenicis individuals at 24 h (2.03 mu L/L of air) and 48 h (1.08 mu L/L of air) of exposition. A brief discussion of essential oils composition and their singular role on the toxic effect over B. phoenicis is provided here. Our results may contribute to a new and profitable use of a species of Brazilian flora on agribusiness.

SEASONAL VARIATION OF THE MAJOR SECONDARY METABOLITES PRESENT IN THE EXTRACT OF Eremanthus mattogrossensis Less (Asteraceae: Vernonieae) LEAVES

The species Eremanthus mattogrossensis, known as "veludo do cerrado" (cerrado velvet), is native to the Brazilian Cerrado. Because the amount of metabolites present in plants may be influenced by biological and environmental factors, here we conducted an HPLC-DAD-MS/MS investigation of the metabolite concentrations found in the MeOH/H2O extract of the leaves of this species. The main compounds were identified and quantified, and the metabolites were grouped by chemical class (caffeoylquinic acids, flavonoids, and sesquiterpene lactone). Statistical analysis indicated a straight correlation between the quantity of metabolites and seasonality, suggesting that environmental properties elicit important metabolic responses.

Leaves of Lolium multiflorum 'Lema' and tropical tree species as biomonitors of polycyclic aromatic hydrocarbons

This study extends the current knowledge regarding the use of plants for the passive accumulation of anthropogenic PAHs that are present in the atmospheric total suspended particles (TSP) in the tropics and sub-tropics. It is of major relevance because the anthropic emissions of TSP containing PAHs are significant in these regions, but their monitoring is still scarce. We compared the biomonitor efficiency of Lolium multiflorum 'Lema' and tropical tree species (Tibouchina pukka and Psidium guajava 'Paluma') that were growing in an intensely TSP-polluted site in Cubatao (SE Brazil), and established the species with the highest potential for alternative monitoring of PAHs. PAHs present in the TSP indicated that the region is impacted by various emission sources. L. multiflorum showed a greater efficiency for the accumulation of PAH compounds on their leaves than the tropical trees. The linear regression between the logBCF and logKoa revealed that L. multiflorum is an efficient biomonitor of the profile of light and heavy PAHs present in the particulate phase of the atmosphere during dry weather and mild temperatures. The grass should be used only for indicating the PAHs with higher molecular weight in warmer and wetter periods. (C) 2012 Elsevier Inc. All rights reserved.

Endothelium-Dependent Vasorelaxant Effect of Butanolic Fraction from Caryocar brasiliense Camb. Leaves in Rat Thoracic Aorta

Caryocar brasiliense Camb. "pequi" is a native plant from the Cerrado region of Brazil that contains bioactive components reported to be antioxidant agents. Previous work has demonstrated that dietary supplementation with pequi decreased the arterial pressure of volunteer athletes. We found that the crude hydroalcoholic extract (CHE) of C. brasiliense leaves relaxed, in a concentration-dependent manner, rat aortic rings precontracted with phenylephrine, and that the butanolic fraction (BF) produced an effect similar to that of the CHE. Aortic relaxation induced by BF was abolished by endothelium removal, by incubation of the nitric oxide synthase inhibitor L-NAME, or the soluble guanylatecyclase inhibitor ODQ. However, incubation with atropine and pyrilamine had no effect on the BF-induced vasorelaxation. Moreover, this effect was not inhibited by indomethacin and tetraethylammonium. The concentration-response curve to calcium in denuded-endothelium rings was not modified after incubation with BF, and the vasorelaxation by BF in endothelium-intact rings precontracted with KCl was abolished after incubation with L-NAME. In addition, administration of BF in anesthetized rats resulted in a reversible hypotension. The results reveal that C. brasiliense possesses both in vivo and in vitro activities and that the vascular effect of BF involves stimulation of the nitric oxide/cyclic GMP pathway.

Electrospray Ionization Mass Spectrometry Fingerprinting of Extracts of the Leaves of Arrabidaea 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.

Anatomical analysis of peach palm (Bactris gasipaes) leaves cultivated in vitro, ex vitro and in vivo

(Anatomical analysis of peach palm (Bactris gasipaes) leaves cultivated in vitro, ex vitro and in vivo). The present work characterized and compared the anatomical structures of the leaves of Bactris gasipaes (Arecaceae) plants grown under different cultivation conditions (in vitro, ex vitro and in vivo) with the goal of identifying the origins of the difficulties encountered in acclimatizing micro-plants. The Quant program was used to determine leaf tissue thicknesses and areas, and histochemical tests were performed on leaf sections and analyzed using light microscopy. Stomatal and trichome densities were determined using the epidermal impression method and by scanning electronic microscopy. Our results indicated that there were no discernible alterations of the anatomical characteristics of the leaves of micro-plants cultivated under differing conditions and that the thickening of the mesophyll and the vascular fibers indicated adaptive responses to ex vitro conditions. As such, the observed difficulties in acclimatizing peach palm micro-plants to ex vitro conditions cannot be attributed to plant anatomical characteristics acquired during in vitro cultivation.

Anti-inflammatory effect of Spiranthera odoratissima A. St.-Hil. leaves involves reduction of TNF-alpha

Spiranthera odoratissima A. St.-Hil., 'manaca', is a medicinal species used in Brazil, especially in central region, for the treatment of several diseases such as pain and inflammation. In this study, the methanol/aqueous phase of the ethanol extract of the leaves of 'manaca' (MAP), at the doses of 50, 150 and 500 mg/kg was used to evaluate the anti-inflammatory and/or antinociceptive effects and the possible anti-inflammatory mechanism. The antinociceptive and anti-inflammatory activities of MAP were assessed using formalin test, carrageenan-induced paw oedema. The myeloperoxidase activity, capillary permeability, leukocyte migration and tumour necrosis factor alpha (TNF-alpha) levels were evaluated in pleural exudate. The MAP reduced the licking time only in the later phase of formalin test, and showed anti-inflammatory activity by reducing the paw oedema, migration cell, myeloperoxidase activity, capillary permeability and TNF-alpha levels. In conclusion, we confirmed the inflammatory activity of MAP and affirm that this effect involves the reduction of TNF-alpha level.

Silicon location through backscattered electron imaging and X-ray microanalysis in leaves of Cyperus ligularis L. and Rhynchospora aberrans C. B. Clarke (Cyperaceae)

(Silicon location through backscattered electron imaging and X-ray microanalysis in leaves of Cyperus ligularis L. and Rhynchospora aberrans C. B. Clarke (Cyperaceae)). The Cyperaceae show the ability to incorporate silicon by depositing colloidal silica, which is recorded by the occurrence of projections in the form of cones, in inner tangential walls of some epidermal cells or "silica cells". Leaves of C. ligularis and R. aberrans were analyzed through the technique of electron backscatter. Cyperus ligularis accumulates silica, in addition to "silica cells", in some stomata, trichomes and the cell walls that surround the cavities of the aerenchyma. The silica in the latter occurs in various forms; however, the cells located near the vascular bundles have conical projections, similar to those of the epidermis. Rhynchospora aberrans presents "silica cells" whose projections have tapered "satellites". In this species, silica also occurs in stomata and certain epidermal cells adjacent to them. It appears that the silicon deposition occurs in combination with the wall (with no apparent structural changes), and structures of secretion, or projections of the wall. These structural changes in the species, and location, are probably related to functional and environmental factors, especially the soil, in addition to relation with taxonomic groups.

Underground leaves of Philcoxia trap and digest nematodes

The recently described genus Philcoxia comprises three species restricted to well lit and low-nutrient soils in the Brazilian Cerrado. The morphological and habitat similarities of Philcoxia to those of some carnivorous plants, along with recent observations of nematodes over its subterranean leaves, prompted the suggestion that the genus is carnivorous. Here we report compelling evidence of carnivory in Philcoxia of the Plantaginaceae, a family in which no carnivorous members are otherwise known. We also document both a unique capturing strategy for carnivorous plants and a case of a plant that traps and digests nematodes with underground adhesive leaves. Our findings illustrate how much can still be discovered about the origin, distribution, and frequency of the carnivorous syndrome in angiosperms and, more generally, about the diversity of nutrient-acquisition mechanisms that have evolved in plants growing in severely nutrient-impoverished environments such as the Brazilian Cerrado, one of the world's 34 biodiversity hotspots.

Exogenous ornithine is an effective precursor and the delta-ornithine amino transferase pathway contributes to proline accumulation under high N recycling in salt-stressed cashew leaves

The role of the delta-ornithine amino transferase (OAT) pathway in proline synthesis is still controversial and was assessed in leaves of cashew plants subjected to salinity. The activities of enzymes and the concentrations of metabolites involved in proline synthesis were examined in parallel with the capacity of exogenous ornithine and glutamate to induce proline accumulation. Proline accumulation was best correlated with OAT activity, which increased 4-fold and was paralleled by NADH oxidation coupled to the activities of OAT and Delta(1)-pyrroline-5-carboxylate reductase (P5CR), demonstrating the potential of proline synthesis via OAT/P5C. Overall, the activities of GS. GOGAT and aminating GDH remained practically unchanged under salinity. The activity of P5CR did not respond to NaCl whereas Delta(1)-pyrroline-5-carboxylate dehydrogenase was sharply repressed by salinity. We suggest that if the export of P5C from the mitochondria to the cytosol is possible, its subsequent conversion to proline by P5CR may be important. In a time-course experiment, proline accumulation was associated with disturbances in amino acid metabolism as indicated by large increases in the concentrations of ammonia, free amino acids, glutamine, arginine and ornithine. Conversely, glutamate concentrations increased moderately and only within the first 24 h. Exogenous feeding of ornithine as a precursor was very effective in inducing proline accumulation in intact plants and leaf discs, in which proline concentrations were several times higher than glutamate-fed or salt-treated plants. Our data suggest that proline accumulation might be a consequence of salt-induced increase in N recycling, resulting in increased levels of ornithine and other metabolites involved with proline synthesis and OAT activity. Under these metabolic circumstances the OAT pathway might contribute significantly to proline accumulation in salt-stressed cashew leaves. (C) 2011 Elsevier GmbH. All rights reserved.

Effect of hydroalcoholic extract from Copaifera langsdorffii leaves on urolithiasis induced in rats

Copaifera langsdorffii Desf. commonly known as "copaiba", produce a commercially valuable oil-resin that is extensively used in folk medicine for anti-inflammatory, antimicrobial and antiseptic purposes. We have found the hydroalcoholic extract of this plant leaf has the potential to treat urolithiasis, a problem affecting similar to 7% of the population. To isolate the functional compounds C. langsdorffii leaves were dried, ground, and macerated in a hydroalcoholic solution 7:3 to produce a 16.8% crude extract after solvent elimination. Urolithiasis was induced by introduction of a calcium oxalate pellet (CaOx) into the bladders of adult male Wistar rats. The treated groups received the crude extract by oral gavage at 20 mg/kg body weight daily for 18 days. Extract treatment started 30 days after CaOx seed implantation. To monitor renal function sodium, potassium and creatinine concentrations were analyzed in urine and plasma, and were found to be in the normal range. Analyses of pH, magnesium, phosphate, calcium, uric acid, oxalate and citrate levels were evaluated to determine whether the C. langsdorffii extract may function as a stone formation prevention agent. The HPLC analysis of the extract identified flavonoids quercitrin and afzelin as the major components. Animals treated with C. langsdorffii have increased levels of magnesium and decreased levels of uric acid in urinary excretions. Treated animals have a significant decrease in the mean number of calculi and a reduction in calculi mass. Calculi taken from extract treated animals were more brittle and fragile than calculi from untreated animals. Moreover, breaking calculi from untreated animals required twice the amount of pressure as calculi from treated animals (6.90 +/- A 3.45 vs. 3.00 +/- A 1.51). The extract is rich in flavonoid heterosides and other phenolic compounds. Therefore, we hypothesize this class of compounds might contribute significantly to the observed activity.

Biochemical, physicochemical and molecular characterization of a genuine 2-Cys-peroxiredoxin purified from cowpea [Vigna unguiculata (L.) Walpers] leaves

Background: Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2 and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx). Methods: Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography. Results: Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56-4.72; its NH2-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% alpha-helix, 39% beta-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys(52) residue and the amino acids Pro(45), Thr(49) and Arg(128) are conserved as in other 2-Cys-Prx. General significance: The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins. (C) 2012 Elsevier B.V. All rights reserved.

Chemopreventive effect of Copaifera langsdorffii leaves hydroalcoholic extract on 1,2-dimethylhydrazine-induced DNA damage and preneoplastic lesions in rat colon

Background Natural antioxidants present in common foods and beverages have drawn great attention to cancer prevention due to its health benefits, remarkable lack of toxicity and side effects. Copaifera langsdorffii, known as “copaiba”, “capaiva”, or “pau-de-óleo“, belongs to the Leguminosae family and occurs in fields and grasslands in the northern and northeastern parts of Brazil. Biological studies of Copaifera corroborate its widespread use by the population. This paper describes the effects of C. langsdorffii leaves hydroalcoholic extract on the 1,2-dimethylhydrazine (DMH)-induced DNA damage and aberrant crypt foci (ACF) in the colon of male Wistar rats. Methods The hydroalcoholic extract of C. langsdorffii was administered to rats by gavage at daily doses of 20, 40 and 80 mg/kg body weight. To evaluate DNA damage by the comet assay, animals received the C. langsdorffii extract for seven days and a single subcutaneous injection (sc) of 1,2-dimethylhydrazine (DMH) at a dose of 40 mg/kg on day 7. Animals were sacrificed 4 h after injection of DMH, to assess DNA damage. For the ACF assay, animals were acclimatized for one week (week 1) and then treated with the C. langsdorffii extract five times a week for four weeks (weeks 2 to 5). The rats received sc injections of DMH (40 mg/kg) on days 2 and 5 of weeks 2 and 3, to induce ACF. Animals were euthanized at week 5; i.e., four weeks after the first DMH treatment. Results Animals treated with different doses of the C. langsdorffii extract combined with DMH had significantly lower frequency of DNA damage as compared with the positive control (animals treated with DMH only). The percentage of reduction in the frequency of DNA damage ranged from 14.30% to 38.8%. The groups treated with 40 and 80 mg/kg C. langsdorffii extract during and after DMH treatment presented significantly lower numbers of ACF and aberrant crypts compared with the control. Conclusion The C. langsdorffii extract significantly reduced the extent of DNA damage and ACF induced by DMH, suggesting that the extract has a protective effect against colon carcinogenesis.

Seasonal variation of the major secondary metabolites present in the extract of Eremanthus mattogrossensis Less (Asteraceae: Vernonieae) leaves

The species Eremanthus mattogrossensis, known as "veludo do cerrado" (cerrado velvet), is native to the Brazilian Cerrado. Because the amount of metabolites present in plants may be influenced by biological and environmental factors, here we conducted an HPLC-DAD-MS/MS investigation of the metabolite concentrations found in the MeOH/H2O extract of the leaves of this species. The main compounds were identified and quantified, and the metabolites were grouped by chemical class (caffeoylquinic acids, flavonoids, and sesquiterpene lactone). Statistical analysis indicated a straight correlation between the quantity of metabolites and seasonality, suggesting that environmental properties elicit important metabolic responses.