Screening of plants found in Amazonas State for lethality towards brine shrimp

226 methanol and water extracts representing 74 mainly native plant species found in Amazonas State, Brazil, were tested at a standard concentration of 500 μg/mL for lethality towards larvae of the brine shrimp species Artemia franciscana. Several cytotoxic plant species were identified in this work: Aspidosperma marcgravianum, A. nitidum, Croton cajucara, Citrus limetta, Geissospermum argenteum, Minquartia guianensis, Piper aduncum, P. amapense, P. capitarianum, P. tuberculatum and Protium aracouchini. The results were analyzed within the context of the available traditional knowledge and uses for these plants.

Screening of plants found in the State of Amazonas, Brazil for activity against Aedes aegypti larvae

Ethanol, methanol and water extracts representing mostly native plant species found in the Amazon region were prepared, respectively, by maceration, continuous liquid-solid extraction and infusion, followed by evaporation and freeze-drying. The freeze-dried extracts were tested for lethality toward Aedes aegypti larvae at test concentrations of 500 mg / mL. In general, methanol extracts exhibited the greatest larvicidal activity. The following 7 methanol extracts of (the parts of) the indicated plant species were the most active, resulting in 100% mortality in A. aegypti larvae: Tapura amazonica Poepp. (root), Piper aduncum L. (leaf and root), P. tuberculatum Jacq. (leaf, fruit and branch). and Simaba polyphylla (Cavalcante) W.W. Thomas (branch).

Engineering of fatty acid production and secretion in Saccharomyces cerevisiae

Tese de Doutoramento em Ciências - Especialidade em Biologia

Characterization of phenolic compounds and antioxidant properties of Glycyrrhiza glabra L. rhizomes and roots

The present work aims to characterize and quantify the phenolic composition and to evaluate the antioxidant activity of Glycyrrhiza glabra L. (commonly known as licorice) rhizomes and roots. The antioxidant potential of its methanol/water extract could be related with flavones (mainly apigenin derivatives), flavanones (mainly liquirintin derivatives), a methylated isoflavone and a chalcone, identified in the extract. Lipid peroxidation inhibition was the most pronounced antioxidant effect (EC50=0.24±0.01 µg/mL and 22.74±2.42 µg/mL in TBARS and -carotene/linoleate assays, respectively), followed by free radicals scavenging activity (EC50=111.54±6.04 µg/mL) and, finally, reducing power (EC50=128.63±0.21 µg/mL). In this sense, licorice extract could be used as a source of antioxidants for pharmaceutical, cosmetic and/or food industries.

Aminobenzocoumarinylmehtyl esters as photoactive precursors for the release of butyric acid

The evaluation of the photorelease of a carboxylic acid drug, using butyric acid as a representative model, was carried out by using 7-amino-4-chloromethyl-2-oxo-2Hnaphtho[1,2-b] pyran, an aminobenzocoumarin, and its mono- and di-methylated or ethylated derivatives. This study was intended to improve the release of butyric acid from benzocoumarins by the addition of an amino group to the heterocycle by applying the knowledge of second-generation coumarinylmethyl-based photoremovable protecting groups. Photolysis studies were performed on the resultant ester cages by irradiation in a photochemical reactor at 254, 300, 350 and 419 nm, using methanol/HEPES buffer 80:20 solutions as solvent. The data obtained showed that these new fluorescent aminobenzocoumarins are superior to all the previously tested benzocoumarins with the same or different ring fusions. As well as the photolysis, the photophysics of the compounds were characterised by both steady state and time-resolved methods.

Antimicrobial activity of bergenin from Endopleura uchi (Huber) Cuatrec

Endopleura uchi (Huber) Cuatrec. is an Amazon species traditionally used as treatment for inflammations and female disorders. Bergenin was isolated from ethyl acetate fraction of bark of E. uchi by using column chromatography over sephadex LH-20 and then silica gel 60 flash. Its structure was identified on the basis of its NMR spectra. The antimicrobial activity of bergenin and fractions of methanol extract of E. uchi were evaluated against ATCC microorganisms (Escherichia coli, Salmonella enteritidis, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, Candida albicans, C. guilliermondii, Aspergillus flavus, A. nidulans). Clinically isolated strains of all of these microorganisms, along with C. tropicalis, A. niger, Shigella sonnei, Serratia marcenses and Klebsiella pneumoniae were also evaluated. The growth inhibition caused by bergenin, extracts and fractions of E. uchi against ATCC microorganisms were similar to the inhibition to microorganisms clinically isolated. The ethyl acetate fraction and the isolate bergenin inhibit the growth of the yeasts C. albicans, C. tropicalis, and C. guilliermondii, but present lower activity against filamentous fungi Aspergillus flavus, A. nidulans, A. niger, and did not inhibit the Gram positive and Gram negative bacteria. The activity of the ethyl acetate fraction and bergenin are in agreement wit its high concentration found in bark extract of E. uchi. Moreover, the selective activity against three Candida species helps to understand its traditional use against infections that affect women.

Photoactivation of butyric acid from 6-aminobenzocoumarin cages

A new benzocoumarin bearing an amino group is proposed as a photocleavable protecting group for carboxylic acids. The novel heterocycle, 6-amino-4-chloromethyl-2-oxo-2H-naphtho[1,2-b]pyran was used in the preparation of ester conjugates of butyric acid, and of the corresponding mono- and di-methylated or ethylated derivatives. The photolability of the ester conjugates was studied by irradiation at selected wavelengths in methanol/HEPES buffer (80:20) solutions, and the release of butyric acid was followed with HPLC/UV and 1H NMR monitoring. Release of the carboxylic acid was faster for the monoalkylated derivatives (approximately within 20 min), at the longer wavelengths of irradiation (350 and 419 nm). The photophysics of the heterocyclic conjugates was also evaluated by both steady state and time-resolved methods.

In vitro screening of Amazonian plants for hemolytic activity and inhibition of platelet aggregation in human blood

In the present study, different aerial parts from twelve Amazonian plant species found in the National Institute for Amazon Research's (INPA's) Adolpho Ducke Forest Reserve (in Manaus, Amazonas, Brazil) were collected. Separate portions of dried, ground plant materials were extracted with water (by infusion), methanol and chloroform (by continuous liquid-solid extraction) and solvents were removed first by rotary evaporation, and finally by freeze-drying which yielded a total of seventy-one freeze-dried extracts for evaluation. These extracts were evaluated initially at concentrations of 500 and 100 µg/mL for in vitro hemolytic activity and in vitro inhibition of platelet aggregation in human blood, respectively. Sixteen extracts (23 % of all extracts tested, 42 % of all plant species), representing the following plants: Chaunochiton kappleri (Olacaceae), Diclinanona calycina (Annonaceae), Paypayrola grandiflora (Violaceae), Pleurisanthes parviflora (Icacinaceae), Sarcaulus brasiliensis (Sapotaceae), exhibited significant inhibitory activity towards human platelet aggregation. A group of extracts with antiplatelet aggregation activity having no in vitro hemolytic activity has therefore been identified. Three extracts (4 %), all derived from Elaeoluma nuda (Sapotaceae), exhibited hemolytic activity. None of the plant species in this study has known use in traditional medicine. So, these data serve as a baseline or minimum of antiplatelet and hemolytic activities (and potential usefulness) of non-medicinal plants from the Amazon forest. Finally, in general, these are the first data on hemolytic and inhibitory activity on platelet aggregation for the genera which these plant species represent.

Pentacyclic triterpenes and steroids from the stem bark of uchi (Sacoglottis uchi, Humiriaceae)

The ethanol extract from stem bark of Sacoglottis uchi Huber (popularly known as “uchi” in the Amazon Region) was submitted to chromatographic fractionation. The dichloromethane fractions provided the pentacyclic triterpene 3-oxo-friedelin (1). The dichloromethane:methanol fractions provided the pentacyclic triterpenes pseudotaraxasterol (2), lupeol (3), a-amyrin (4), betulin (5), and methyl 2ß,3ß-dihydroxy-urs-12-en-28-oate (6) and a mixture of the steroids sitosterol (7) and stigmasterol (8). Their chemical structures were determined by NMR spectroscopy and comparison with spectroscopic data from the literature. All compounds are described for the first time in this species.

Exploring the properties of genetically engineered silk-elastin-like protein films

Free standing films of a genetically engineered silk-elastin-like protein (SELP) were prepared using water and formic acid as solvents. Exposure to methanol-saturated air promoted the formation of aggregated β-strands rendering aqueous insolubility and improved the mechanical properties leading to a 10-fold increase in strain-to-failure. The films were optically clear with resistivity values similar to natural rubber and thermally stable up to 180 °C. Addition of glycerol showed to enhance the flexibility of SELP/glycerol films by interacting with SELP molecules through hydrogen bonding, interpenetrating between the polymer chains and granting more conformational freedom. This detailed characterization provides cues for future and unique applications using SELP based biopolymers.

Pró-fármacos fotoativáveis do péptido AAPV: síntese e avaliação da libertação controlada por luz

Dissertação de mestrado em Bioquímica Aplicada (área de especialização em Biomedicina)

Triterpenes of leaves from Piranhea trifoliata (Picrodendraceae)

ABSTRACT The Amazon forest is rich in plant species diversity, among them,Piranhea trifoliata stands out, which is popularly known as piranheira, because their fruits are eaten by fish. Their barks are used as bath composition on uterus inflammation and as tea in malaria treatment. This study aimed to fractionate the dichloromethane and dichloromethane phase from methanolic extract of leaves of Piranhea trifoliata. The leaves were dried, grounded and extracted with dichloromethane, methanol and water. The methanol extract was partitioned with dichloromethane and ethyl acetate. The chromatographic fractionation yielded six pentacyclic triterpenoids: friedelan-3-one, 28-hydroxy-friedelan-3-one, 30-hydroxy-friedelan-3-one, lupeol, α- and β-amyrin mixture, besides the mixture of the steroids: β-sitosterol and stigmasterol. The substances structures were identified by 1H- and13C-Nuclear Magnetic Resonance (NMR) analysis and literature data comparison. This is the first report describing the chemical study of P. trifoliata leaves.

Libertação fotocontrolada de moléculas bioativas a partir de conjugados de cumarinas

Dissertação de mestrado em Química Medicinal

Bio-detoxification of mycotoxins by lactic acid bacteria from different food matrices

Lactic acid bacteria (LAB) play a key role in the biopreservation of a wide range of fermented food products, such as yogurt, cheese, fermented milks, meat, fish, vegetables (sauerkraut, olives and pickles), certain beer brands, wines and silage, allowing their safe consumption, which gave to these bacteria a GRAS (Generally Recognised as Safe) status. Besides that, the use of LAB in food and feed is a promising strategy to reduce the exposure to dietary mycotoxins, improving their shelf life and reducing health risks, given the unique mycotoxin decontaminating characteristic of some LAB. Mycotoxins present carcinogenic, mutagenic, teratogenic, neurotoxic and immunosuppressive effects over animals and Humans, being the most important ochratoxin A (OTA), aflatoxins (AFB1), trichothecenes, zearalenone (ZEA), fumonisin (FUM) and patulin. In a previous work of our group it was observed OTA biodegradation by some strains of Pediococcus parvulus isolated from Douro wines. So, the aim of this study was to enlarge the screening of the biodetoxification over more mycotoxins besides OTA, including AFB1, and ZEA. This ability was checked in a collection of LAB isolated from vegetable (wine, olives, fruits and silage) and animal (milk and dairy products, sausages) sources. All LAB strains were characterized phenotypically (Gram, catalase) and genotypically. Molecular characterisation of all LAB strains was performed using genomic fingerprinting by MSP- PCR with (GTG)5 and csM13 primers. The identification of the isolates was confirmed by 16S rDNA sequencing. To study the ability of LAB strains to degrade OTA, AFB1 and ZEA, a MRS broth medium was supplemented with 2.0 g/mL of each mycotoxin. For each strain, 2 mL of MRS supplemented with the mycotoxins was inoculated in triplicate with 109 CFU/mL. The culture media and bacterial cells were extracted by the addition of an equal volume of acetonitrile/methanol/acetic acid (78:20:2 v/v/v) to the culture tubes. A 2 mL sample was then collected and filtered into a clean 2 mL vial using PP filters with 0.45 m pores. The samples were preserved at 4 °C until HPLC analysis. Among LAB tested, 10 strains isolated from milk were able to eliminate AFB1, belonging to Lactobacillus casei (7), Lb. paracasei (1), Lb. plantarum (1) and 1 to Leuconostoc mesenteroides. Two strains of Enterococcus faecium and one of Ec. faecalis from sausage eliminated ZEA. Concerning to strains of vegetal origin, one Lb. plantarum isolated from elderberry fruit, one Lb. buchnerii and one Lb. parafarraginis both isolated from silage eliminated ZEA. Other 2 strains of Lb. plantarum from silage were able to degrade both ZEA and OTA, and 1 Lb. buchnerii showed activity over AFB1. These enzymatic activities were also verified genotypically through specific gene PCR and posteriorly confirmed by sequencing analysis. In conclusion, due the ability of some strains of LAB isolated from different sources to eliminate OTA, AFB1 and ZEA one can recognize their potential biotechnological application to reduce the health hazards associated with these mycotoxins. They may be suitable as silage inoculants or as feed additives or even in food industry.

Food safety in wine: removal of ochratoxin A in contaminated white wine using commercial fining agents

The presence of mycotoxins in foodstuff is a matter of concern for food safety. Mycotoxins are toxic secondary metabolites produced by certain molds, being ochratoxin A (OTA) one of the most relevant. Wines can also be contaminated with these toxicants. Several authors have demonstrated the presence of mycotoxins in wine, especially ochratoxin A (OTA) [1]. Its chemical structure is a dihydro-isocoumarin connected at the 7-carboxy group to a molecule of L--phenylalanine via an amide bond. As these toxicants can never be completely removed from the food chain, many countries have defined levels in food in order to attend health concerns. OTA contamination of wines might be a risk to consumer health, thus requiring treatments to achieve acceptable standards for human consumption [2]. The maximum acceptable level of OTA in wines is 2.0 g/kg according to the Commission regulation No. 1881/2006 [3]. Therefore, the aim of this work was to reduce OTA to safer levels using different fining agents, as well as their impact on white wine physicochemical characteristics. To evaluate their efficiency, 11 commercial fining agents (mineral, synthetic, animal and vegetable proteins) were used to get new approaches on OTA removal from white wine. Trials (including a control without addition of a fining agent) were performed in white wine artificially supplemented with OTA (10 µg/L). OTA analysis were performed after wine fining. Wine was centrifuged at 4000 rpm for 10 min and 1 mL of the supernatant was collected and added of an equal volume of acetonitrile/methanol/acetic acid (78:20:2 v/v/v). Also, the solid fractions obtained after fining, were centrifuged (4000 rpm, 15 min), the resulting supernatant discarded, and the pellet extracted with 1 mL of the above solution and 1 mL of H2O. OTA analysis was performed by HPLC with fluorescence detection according to Abrunhosa and Venâncio [4]. The most effective fining agent in removing OTA (80%) from white wine was a commercial formulation that contains gelatine, bentonite and activated carbon. Removals between 10-30% were obtained with potassium caseinate, yeast cell walls and pea protein. With bentonites, carboxymethylcellulose, polyvinylpolypyrrolidone and chitosan no considerable OTA removal was verified. Following, the effectiveness of seven commercial activated carbons was also evaluated and compared with the commercial formulation that contains gelatine, bentonite and activated carbon. The different activated carbons were applied at the concentration recommended by the manufacturer in order to evaluate their efficiency in reducing OTA levels. Trial and OTA analysis were performed as explained previously. The results showed that in white wine all activated carbons except one reduced 100% of OTA. The commercial formulation that contains gelatine, bentonite and activated carbon (C8) reduced only 73% of OTA concentration. These results may provide useful information for winemakers, namely for the selection of the most appropriate oenological product for OTA removal, reducing wine toxicity and simultaneously enhancing food safety and wine quality.