Maria Fátima das Graças Fernandes da Silva

Chemosystematics of theRutaceae: Suggestions for a more natural taxonomy and evolutionary interpretation of the family

The chemosystematics ofRutaceae is reviewed on the basis of updated surveys of various secondary metabolites and their biosynthetic derivation. A comparison of these data with the morphological and geographical differentiation clearly shows that the current taxonomic arrangement of the family is to a large extent artificial and needs improvement. Starting from obviously “natural” groups of genera (or single genera) as “basic taxonomic entities” a new system with informal tribal names is suggested. In particular, the subfamilyToddalioideae is broken up altogether and its former members are rearranged among several of the 17 provisional tribes within the subfamilyRutoideae s. lat. Phylogenetic progressions can be recognized from parallel changes of morphological characters and biosynthetic pathways to secondary metabolites. As a general trend, a stepwise replacement of benzylisoquinolines by simple and complex anthranilic acid derived alkaloids, and eventually by coumarins and/or limonoids is confirmed. The available data are summarized in a discussion of the possible evolutionary relationships among theRutaceae, with theZanthoxylum- andEvodia-tribes in a central position.

Simultaneous determination of dextromethorphan, diphenhydramine and phenylephrine in expectorant and decongestant syrups by capillary electrophoresis

The separation of basic nitrogenous compounds commonly used as active ingredients in cold medicine formulations by micellar electrokinetic capillary chromatography and capillary zone electrophoresis with direct absorptiometric detection was investigated. The type and composition of the background electrolyte (BGE) were investigated with respect to separation selectivity and BGE stability. BGE of 10 mM sodium dihydrogenphosphate-sodium tetraborate buffer containing 10 mM SDS and 10% acetonitrile, pH 9.0 was found to be optimal. Dextromethorphan hydrobhromide, diphenhydramine hydrochloride and phenylephrine hydrochloride were baseline-separated in less than 11 min, giving separation efficiencies of up to 494,000 theoretical plates, reproducibility of corrected peaks areas below 3% relative standard deviation and concentration detection limits from 2.5 to 5.5 microg ml(-1). Detection was performed at 196 and 214 nm.

Assessment of trace aluminium content in parenteral solutions by combined cloud point preconcentration—flow injection inductively coupled plasma optical emission spectrometry

A micelle-mediated phase separation without added chelating agents to preconcentrate trace levels of aluminium in parenteral solutions as a prior step to its determination by flow injection inductively coupled plasma optical emission spectrometry has been developed. The enrichment step is based on the cloud point extraction of aluminium with the non-ionic surfactant polyethyleneglycolmono-p-nonylphenylether (PONPE 7.5). The chemical variables affecting the sensitivity of the extractive-spectrometric procedure were studied in detail. After optimization, a preconcentration factor of 200 and a %E higher than 99.9 were achieved. The detection limit (DL) value of aluminium for the preconcentration of 50 ml of parenteral solution was 0.25 microgl(-1). The calibration graph using the preconcentration system for aluminium was linear with a correlation coefficient of 0.9997 at levels near the DLs up to at least 200 microgl(-1). The developed hyphenated assay, which thoroughly satisfies the typical requirements for pharmaceutical control processes, is appropriate to monitor the aluminium concentration in parenteral nutrition.

Trypanocidal activity of Meliaceae and Rutaceae plant extracts.

The in vitro trypanocidal activity of 22 extracts and 43 fractions of plants belonging to the families Meliaceae and Rutaceae was evaluated. The extracts from leaves of Conchocarphus heterophyllus and branches of Trichilia ramalhoi were the most active. The trypanocidal activity seems to be increased by fractionation of the extracts. Fractions from C. heterophyllus and Galipea carinata were the most active and a 100% lysis of the parasites was observed for five fractions. From one of them were isolated two flavonoids: flavone and 7-methoxyflavone, which showed weak trypanocidal activity. The results obtained from the extracts and fractions revealed that the order Rutales is a promising source for the search of new drugs for Chagas disease. Phytochemical studies with the other active fractions are underway in order to isolate compounds, which could be associated with observed activities.

In vitro activity of Rutaceae species against the trypomastigote form of Trypanosoma cruzi.

The activity of crude plant extracts of nine species of Rutaceae against the trypomastigote form of Trypanosoma cruzi was evaluated at 4 mg/ml. Thirty-two crude extracts were tested and eight of them showed significant activity (>80%). The most active extract was obtained from the stems of Pilocarpus spicatus (97.3%). Fractionation of the active crude extracts provided 25 fractions which were tested against the trypomastigote form of T. cruzi at 2 mg/ml. Of these six showed significant activity (>80%). The most active fractions (100%) were obtained from the leaves of Almeidea coerulea (butanol fraction) and Conchocarpus inopinatus (dichloromethane fraction).

Simultaneous quantification of azadirachtin and 3-tigloylazadirachtol in Brazilian seeds and oil of Azadirachta indica: application to quality control and marketing

A rapid, sensitive and selective HPLC-UV method was developed for quantification of azadirachtin (1) and 3-tigloylazadirachtol (2) in Neem kernels and oil, with a good linearity over a range of 5.0–60.0 and 2.5–50.0 μg mL−1 respectively, with r2 > 0.999 for all curves. The limits of detection (LOD) for both limonoids were 25 ng mL−1 for kernels and 0.10 μg mL−1 for oil, and quantification (LOQ) for 1 and 2 were 1.5 μg mL−1 for both matrices. The quantification method was applied to Neem seeds and oil from different areas of Brazil. The low quality of Brazilian oil (concentrations of 1 = 228.5 to 630 mg kg−1 and 2 = 117.1 to 582.9 mg kg−1) could be attributed to mechanical extraction, since the content of azadirachtin and 3-tigloylazadirachtol in seeds was (1 = 2048.8 to 5117.1 mg kg−1, 2 = 224.7 to 1116.5 mg kg−1) similar to values obtained from Indian seeds (1 = 556.9 to 3030.8 mg kg−1, 2 = 43.1 to 590.6 mg kg−1).

Flavonoids as noncompetitive inhibitors of Dengue virus NS2B-NS3 protease: inhibition kinetics and docking studies.

NS2B-NS3 is a serine protease of the Dengue virus considered a key target in the search for new antiviral drugs. In this study flavonoids were found to be inhibitors of NS2B-NS3 proteases of the Dengue virus serotypes 2 and 3 with IC50 values ranging from 15 to 44 μM. Agathisflavone (1) and myricetin (4) turned out to be noncompetitive inhibitors of dengue virus serotype 2 NS2B-NS3 protease with Ki values of 11 and 4.7 μM, respectively. Docking studies propose a binding mode of the flavonoids in a specific allosteric binding site of the enzyme. Analysis of biomolecular interactions of quercetin (5) with NT647-NHS-labeled Dengue virus serotype 3 NS2B-NS3 protease by microscale thermophoresis experiments, yielded a dissociation constant KD of 20 μM. Our results help to understand the mechanism of inhibition of the Dengue virus serine protease by flavonoids, which is essential for the development of improved inhibitors.

Purification and differential biological effects of ginger-derived substances on normal and tumor cell lines

This study describes an optimization of [6]-, [8]- and [10]-gingerol isolation and purification in semi-preparative HPLC scale and their anti-proliferative activity. The gingerols purification was carried out in HPLC system using a Luna-C₁₈ and the best mobile phase evaluated was MeOH/H₂O (75:25, v/v). This new methodology for the gingerols isolation was very effective, since considerable amounts (in the range of milligrams) with a good purity degree (∼98%) were achieved in 30 min of chromatographic run. [6]-, [8]- and [10]-Gingerol purified by this methodology inhibited the proliferation of MDA-MB-231 tumor cell line with IC₅₀ of 666.2±134.6 μM, 135.6±22.6 μM and 12.1±0.3 μM, respectively. These substances also inhibited human fibroblasts (HF) cell proliferation, however in concentrations starting from 500 μM. In conclusion, our results demonstrate an optimization of gingerols isolation and their specific anti-proliferative activities against tumor cells, suggesting their use as important models for drug design in an attempt to develop new compounds with fewer side effects when compared to conventional chemotherapy.

Isolation of xanthyletin, an inhibitor of ants’ symbiotic fungus, by high-speed counter-current chromatography

Xanthyletin, an inhibitor of symbiotic fungus (Leucoagaricus gongylophorus) of leaf-cutting ant (Atta sexdens rubropilosa), as well as suberosin, seselin and xanthoxyletin were isolated from Citrus sinensis grafted on Citrus limonia. A two-phase solvent system composed of hexane/ethanol/acetonitrile/water (10:8:1:1, v/v) was used for the high-speed counter-current chromatographic isolation of xanthyletin with high yield and over 99% purity as determined by liquid and gas chromatography with mass spectrometry detection. Identifications were performed by UV spectra, IR spectra, (1)H NMR and (13)C NMR.

Development of a New Method To Prepare Nano-/microparticles Loaded with Extracts of Azadirachta indica, Their Characterization and Use in Controlling Plutella xylostella

Biodegradable nanoparticles have been widely explored as carriers for controlled delivery of therapeutic molecules; however, studies describing the development of nanoparticles as carriers for biopesticide products are few. In this work, a new method to prepare nanoparticles loaded with neem (Azadirachta indica) extracts is presented. In this study, nanoparticles were formulated as colloidal suspension and (spray-dried) powder and characterized by evaluating pH, particle size, zeta potential, morphology, absolute recovery, and entrapment efficiency. A high-performance liquid chromatography method was used for nanoparticle characterization. The best formulations presented absolute recovery and entrapment efficiencies of approximately 100% and a release profile based on swelling and relaxation of the polymer or polymer erosion. The biological data of the formulated products against Plutella xylostella showed 100% larval mortality. The nanoparticle information improved the stability of neem products against ultraviolet radiation and increased their dispersion in the aqueous phase.