A new acridone with antifungal properties against Candida spp. and dermatophytes, and antibiofilm activity against C. albicans.

AIM: The increase in the number of fungal infections worldwide, coupled with the limitations of current antifungal chemotherapy, demand the development of safe and effective new antifungals. Here, we presented the synthesis of a novel acridone (M14) and its antifungal properties against Candida and dermatophytes species. METHODS AND RESULTS: A series of 17 acridones was designed, synthesized and tested for its antifungal activity. The minimum inhibitory concentration (MIC) was determined by the broth microdilution method. Only the acridone M14 showed growth-inhibitory activity against reference strains and clinical isolates of Candida and dermatophytes, with MIC range of 7·81-31·25 µg ml-1 . Moreover, M14 exhibited fungicidal activity and prevented biofilm formation by C. albicans as well as reduced the viability of preformed biofilms, even at sub-MICs. The confocal laser scanning microscopy analysis revealed that C. albicans hyphal growth was completely inhibited in the presence of M14. Similarly, there was a severe inhibition on hyphal growth of Trichophyton rubrum. We also found that M14 has relatively low toxicity to human fibroblasts. CONCLUSIONS: The new acridone M14 has antifungal properties against Candida spp. and dermatophytes, and antibiofilm activity against C. albicans. In addition, M14 is relatively selective to fungal cells compared to human normal cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of its in vitro antifungal activity, anti-Candida biofilm effect and moderate cytotoxicity towards normal human cell, M14 may serve as a valuable lead compound to develop a new antifungal agent.

Pinpointing disulfide connectivities in cysteine-rich proteins.

A simple MD-based protocol is presented to accurately predict both the sequence and order of disulfide bond formation in proteins containing multiple cysteine residues. It provides a detailed description of their dynamical and structural features, which can be used to perform ensemble-averaged ECD calculations. Plant cyclotides are used as model compounds.

LC-MS/MS quantitative determination of Tetrapterys mucronata alkaloids, a plant occasionally used in ayahuasca preparation.

INTRODUCTION: Tetrapterys mucronata Cav. (Malpighiaceae) is a plant used in some regions of Brazil in the preparation of ayahuasca. OBJECTIVE: To determine the content of the main tryptamine alkaloids in the stem bark of T. mucronata Cav. and assess their possible toxic and hallucinogenic properties based on the doses found in a water decoction that mimics the ayahuasca preparation. METHODS: Four alkaloids previously described for their toxic and hallucinogenic properties were quantitated by multiple reaction monitoring HPLC combined with electrospray ionisation and tandem MS (HPLC-ESI/MS/MS) in the water decoction and ethanolic extracts from the bark of T. mucronata. RESULTS: Exhaustive extraction of the stem barks with ethanol revealed the following alkaloid levels: bufotenine (1) 3.26 ± 0.31 mg/g, 5-methoxy-N-methyltryptamine (2) 0.88 ± 0.08 mg/g, 5-methoxy-bufotenine (3) 3.07 ± 0.22 mg/g and 2-methyl-6-methoxy-1,2,3,4-tetrahydro-ß-carboline (4) 0.14 ± 0.004 mg/g. The water decoction presented slightly lower levels, ranging between 2.32 ± 0.14, 0.50 ± 0.04, 1.53 ± 0.09 and 0.10 ± 0.01 mg/g for (1), (2), (3) and (4) respectively. CONCLUSIONS: The HPLC-ESI/MS/MS quantitation revealed significant alkaloid levels, in particular for bufotenine and 5-methoxy-bufotenine. As such compounds are known for their toxic and hallucinogenic properties, these results indicate that the consumption of this plant as an ingredient in ayahuasca preparations may present a risk to consumers.

Antibacterial activity of alkyl gallates against Xanthomonas citri subsp. citri.

The plant-pathogenic bacterium Xanthomonas citri subsp. citri is the causal agent of Asiatic citrus canker, a serious disease that affects all the cultivars of citrus in subtropical citrus-producing areas worldwide. There is no curative treatment for citrus canker; thus, the eradication of infected plants constitutes the only effective control of the spread of X. citri subsp. citri. Since the eradication program in the state of São Paulo, Brazil, is under threat, there is a clear risk of X. citri subsp. citri becoming endemic in the main orange-producing area in the world. Here we evaluated the potential use of alkyl gallates to prevent X. citri subsp. citri growth. These esters displayed a potent anti-X. citri subsp. citri activity similar to that of kanamycin (positive control), as evaluated by the resazurin microtiter assay (REMA). The treatment of X. citri subsp. citri cells with these compounds induced altered cell morphology, and investigations of the possible intracellular targets using X. citri subsp. citri strains labeled for the septum and centromere pointed to a common target involved in chromosome segregation and cell division. Finally, the artificial inoculation of citrus with X. citri subsp. citri cells pretreated with alkyl gallates showed that the bacterium loses the ability to colonize its host, which indicates the potential of these esters to protect citrus plants against X. citri subsp. citri infection.

4'-Aminochalcones as novel inhibitors of the chlorinating activity of myeloperoxidase.

The excessive activation of neutrophils generates reactive oxygen species (ROS) and the secretion of primary granular enzymes, such as myeloperoxidase (MPO), which is implicated in numerous inflammatory diseases. The aim of this study was to evaluate chalcones as inhibitors of the chlorinating activity of MPO using in vitro and ex vivo assays. In addition to cytotoxic properties, the inhibition of respiratory burst, the scavenger capacity, and the oxidation potential were measured. 4'-Aminochalcone (1), 4'-amino-4- fluorochalcone (2), and 4'-amino-4-methylchalcone (3) exhibited potent inhibition of the chlorinating activity of MPO, as evaluated in a neutrophil system and a free cell system, to the following degree: (1) IC50 = 0.265 � 0.036 µmol L-1; (2) IC50 = 0.250 � 0.081 µmol L-1; and (3) IC50 = 0.250 � 0.012 µmol L-1. These values were similar to those for 5-fluorotryptamine (IC50 = 0.192 � 0.012 µmol L-1), a compound considered to be a potent MPO inhibitor. These aminochalcones were not toxic to neutrophils at concentrations below 100 µmol L- 1, as determined by the trypan blue exclusion assay. Compounds 1-3 presented a high oxidation potential (Epa1 ≉ 0.80 V), low scavenger capacity against DPPH• and HOCl, and low inhibition of respiratory burst. These data indicated that aminochalcones are potent inhibitors of MPO chlorinating activity, a new property for chalcone derivatives, given that they are neither antioxidant agents nor inhibitors of respiratory burst. In conclusion, the selected aminochalcones have potential as pharmacological agents for inflammatory diseases.

Alkyl hydroxybenzoic acid derivatives that inhibit HIV-1 protease dimerization.

The therapeutic potential of gallic acid and its derivatives as anti-cancer, antimicrobial and antiviral agents is well known. We have examined the mechanism by which natural gallic acid and newly synthesized gallic acid alkyl esters and related protocatechuic acid alkyl esters inhibit HIV-1 protease to compare the influence of the aromatic ring substitutions on inhibition. We used Zhang-Poorman's kinetic analysis and fluorescent probe binding to demonstrate that several gallic and protecatechuic acid alkyl esters inhibited HIV-1 protease by preventing the dimerization of this obligate homodimeric aspartic protease rather than targeting the active site. The tri-hydroxy substituted benzoic moiety in gallates was more favorable than the di-substituted one in protocatechuates. In both series, the type of inhibition, its mechanism and the inhibitory efficiency dramatically depended on the length of the alkyl chain: no inhibition with alkyl chains less than 8 carbon atoms long. Molecular dynamics simulations corroborated the kinetic data and propose that gallic esters are intercalated between the two N- and C-monomer ends. They complete the ß-sheet and disrupt the dimeric enzyme. The best gallic ester (14 carbon atoms, K(id) of 320 nM) also inhibited the multi-mutated protease MDR-HM. These results will aid the rational design of future generations of non-peptide inhibitors of HIV-1 protease dimerization that inhibit multi-mutated proteases. Finally, our work suggests the wide use of gallic and protocatechuic alkyl esters to dissociate intermolecular ß-sheets involved in protein-protein interactions.

Chemopreventive activity of compounds extracted from Casearia sylvestris (Salicaceae) Sw against DNA damage induced by particulate matter emitted by sugarcane burning near Araraquara, Brazil.

Ethanolic extract of Casearia sylvestris is thought to be antimutagenic. In this study, we attempted to determine whether this extract and casearin X (a clerodane diterpene from C. sylvestris) are protective against the harmful effects of airborne pollutants from sugarcane burning. To that end, we used the Tradescantia micronucleus test in meiotic pollen cells of Tradescantia pallida, the micronucleus test in mouse bone marrow cells, and the comet assay in mouse blood cells. The mutagenic compound was total suspended particulate (TSP) from air. For the Tradescantia micronucleus test, T. pallida cuttings were treated with the extract at 0.13, 0.25, or 0.50 mg/ml. Subsequently, TSP was added at 0.3mg/ml, and tetrads from the inflorescences were examined for micronuclei. For the micronucleus test in mouse bone marrow cells and the comet assay in mouse blood cells, Balb/c mice were treated for 15 days with the extract-3.9, 7.5, or 15.0 mg/kg body weight (BW)-or with casearin X-0.3, 0.25, or 1.2 mg/kg BW-after which they received TSP (3.75 mg/kg BW). In T. pallida and mouse bone marrow cells, the extract was antimutagenic at all concentrations tested. In mouse blood cells, the extract was antigenotoxic at all concentrations, whereas casearin X was not antimutagenic but was antigenotoxic at all concentrations. We conclude that C. sylvestris ethanolic extract and casearin X protect DNA from damage induced by airborne pollutants from sugarcane burning.

Avaliação das atividades antioxidante, anti e pró-hemolítica do extrato etanólico das folhas de Pterogyne nitens Tul. (Fabaceae-Caesalpinioideae) / Evaluation of antioxidant, anti- and pro-hemolytic activities of ethanol extract from the leaves of Pterogynenitens Tul. (Fabaceae-Caesalpinioideae)

A pesquisa de produtos naturais permite a descoberta de novos princípios ativos, ou ainda, a descoberta de novas atividades para extratos de plantas (amplamente utilizados pela população brasileira) e princípios ativos naturais já conhecidos. Pterogyne nitens é uma planta cuja descrição das atividades é relativamente recente e, portanto, tem no extrato bruto boa fonte para pesquisas na área de produtos naturais. Desta forma, o objetivo deste trabalho foi estudar o perfil antioxidante do extrato bruto etanólico das folhas de P. nitens e possível interferência sobre a hemólise provocada pelo radical AAPH•. No estudo da ação antioxidante das espécies estudadas, ABTS•+, DPPH•, H2O2 e HOCl, encontrou-se os valores de IC50 de 5,0 µg mL-1, 17 µg mL-1, sem ação e 3,9 µg mL-1, respectivamente, valores relativamente baixos e que indicam bom potencial antioxidante. Foram encontradas atividades pró-hemolítica e anti-hemolítica para o extrato de forma concentração-dependente. O extrato estudado mostro boa fonte de moléculas naturais com potencial de ação biológica.

The search for natural products as a widespread practice enables the discovery of new active principles, or the discovery of new activities for plant extracts (extensively used by the population) and natural active principles already known. Pterogynenitensis is a plant whose descriptions of activities are relatively recent and therefore has in its crude extract a good source for research in the field of natural products. Thus, the aim of this study was to evaluate the antioxidant profile of crude ethanol extract from P. nitens leaves and a possible influence on the hemolysis caused by AAPH• radical. For the studied oxidant species, ABTS•+, DPPH•, HOCl and H2O2, the IC50 values were found of 5.0 µg mL-1, 17 µg mL-1, no action at all, and 3.9 µg mL-1, respectively, relatively low values, indicating a good antioxidant potential. Pro- and anti-hemolytic activities were found for the extract in a concentration-dependent way. The studied extract showed to be a good source of natural molecules with potential biological action.

Anticholinesterasic, nematostatic and anthelmintic activities of pyridinic and pyrazinic compounds.

In the search for acetylcholinesterase inhibitors as a potential target for the discovery of anthelmintic drugs, a series of 27 pyridinic and pyrazinic compounds have been designed on the basis of molecular hybridization of two known AChE inhibitors, namely, tacrine and (-)-3-O-acetylspectaline, and on the concept of isosterism. The synthesized compounds generally presented moderate anticholinesterasic activities when compared with the positive control physostigmine, but one compound (ethyl 2-[(6-chloropyrazin-2-yl)sulfanyl] acetate) exhibited an in vitro ability to immobilize the root-knot nematode Meloidogyne incognita that was highly comparable to that of the positive control Temik. Moreover, in anthelmintic assays against the gastrointestinal parasitic nematode Nippostrongylus brasiliensis (L4), some of the compounds, such as (6-chloropyrazin-2-yl)sulfanyl ethanol (32, EC50 = 33 nM), presented activities that were considerably stronger than that of the positive control albendazole (EC50 = 340 nM). In the light of the positive results obtained in the anthelmintic evaluations, the acute oral toxicity of the representative compound diethyl 2,2'-[(3-nitropyridine-2,6-diyl) bissulfanediyl] diacetate was determined in rats, and the drug was shown to be non-toxic at a dose of 2000 mg/kg. These results, allied with the relatively simple structures of the active compounds and their facile synthesis, highlight their potential use as anthelmintic or nematicidic agents.

Cryptomoscatona D2 de Cryptocarya mandioccana: atividade citotóxica contra linhagem celular de carcinoma cervical humano / Cryptomoschatone D2 from Cryptocarya mandioccana: cytotoxicity against human cervical carcinoma cell lines

Among the substances isolated from Cryptocarya sp, some styrylpyrones, such as goniothalamin, demonstrate antiproliferative activity in a broad range of human cell lines. In the present study, we assessed the cytotoxicity of a styrylpyrone (cryptomoschatone D2), isolated from Cryptocarya mandiocanna, in HPV-infected (HeLa and SiHa) and uninfected (C33A) human cervical carcinoma cell lines and a human lung fibroblast line (MRC-5). The cytotoxicity was tested by the MTT assay. In this assay, cells were treated with cryptomoschatone D2 at 15, 30, 60 or 90 ?M for 6, 24 or 48 hours, as well as for 6 hours followed by a post-treatment recovery period of 24, 48 or 72 hours. High cytotoxicity (dose- and timedependent) was observed in HeLa, SiHa, C33A and MRC-5 cell lines. Although in general the styrylpyrone cytotoxicity was not significantly different among the cell lines tested, it was apparently stronger in HeLa and C33A than in MRC-5 and SiHa in the 24 or 48-hour treatments. Moreover, HeLa and SiHa were able to recover their ability to proliferate, in direct proportion to the post-treatment recovery time. On the other hand, C33A did not demonstrate a similar post-treatment recovery. We can conclude that cryptomoschatone D2 possesses high dose-dependent or time-dependent cytotoxicity.

Dentre as substâncias isoladas de Cryptocarya sp, algumas estirilpironas, como a goniotalamina, apresentam atividade antiproliferativa em diferentes linhagens celulares. No presente estudo, foram avaliadas as atividades citotóxica de uma estirilpirona (criptomoscatona D2) isolada de Cryptocarya mandiocanna, em linhagens celulares de carcinoma cervical humano infectada por HPV (HeLa e SiHa), não infectada (C33A) e fibroblasto pulmonar humano (MRC-5). A atividade citotóxica foi avaliada pelo ensaio do MTT. No ensaio do MTT, as células foram tratadas com criptomoscatona D2 em 15, 30, 60 e 90 ?M por 6, 24 e 48 horas e por 6 horas com período de recuperação de 24, 48 e 72 horas pós-tratamento. O tratamento com a estirilpirona (criptomoscatona D2) ocasionou elevada citotoxicidade dose-resposta e tempo-resposta em HeLa, SiHa, C33A e MRC-5. Embora não haja diferença estatisticamente significativa de citotoxicidade entre as linhagens, aparentemente a citotoxicidade foi maior em HeLa e C33A (tratamento de 24 e 48 horas) que em MRC-5 e SiHa. Ainda, no período de recuperação, HeLa e SiHa aparentemente restabelecem sua capacidade proliferativa, que é diretamente proporcional ao tempo de recuperação, enquanto o mesmo comportamento não é observado em C33A. Estes resultados sugerem que criptomoscatona D2 possui elevada atividade antiproliferativa dose-resposta ou o tempo resposta.

Comparative study of disk diffusion and microdilution methods for evaluation of antifungal activity of natural compounds against medical yeasts Candida spp and Cryptococcus sp / Estudo comparativo entre os métodos de difusão em disco e de microdiluição para avaliação de atividade antifúngica de produtos naturais contra as leveduras de interesse médico Candida spp e Cryptococcus sp

Antifungal activity of natural products has been tested by adapting methods designed for synthetic drugs. Inthis study, two methods for the determination of antifungal activity of natural products, agar diffusionand broth microdilution, the CLSI reference methods for synthetic drugs, are compared and discussed. Themicrodilution method was more sensitive. The minimal inhibitory concentrations (MIC) of crude extracts,fractions and pure substances from different species of the plant families Piperaceae, Rubiaceae, Clusiaceae, Fabaceae and Lauraceae, from the Biota project, were determined. Antifungal activities against Candida albicans, C.krusei, C.parapsilosis and Cryptococcus neoformans were produced by several samples

Atividade antifúngica de produtos naturais foi determinada após algumas adaptações de métodos preconizados para fármacos sintéticos. Neste estudo foram comparados e discutidos os métodos para determinação de atividade antifúngica de produtos naturais por duas metodologias, difusão em ágar e microdiluição em caldo, segundo método preconizado pelo CLSI para fármacos sintéticos. A concentração mínima inibitória foi determinada de extratos brutos, frações e de substâncias puras de diferentes espécies de plantas das famílias Piperaceae, Rubiaceae, Clusiaceae, Fabaceae and Lauraceae do projeto Biota. Vários apresentaram atividade antifúngica para as levedurasCandida albicans, C.krusei, C.parapsilosis and Cryptococcus neoformans.

Antifungal polysulphides from Petiveria alliacea L.

Bioactivity-directed fractionation of the CH(2)Cl(2)/MeOH (2:1, v/v) extract of the roots of Petiveria alliacea, using mutant yeast strains of Saccharomyces cerevisiae and fungi Cladosporium cladosporioides and C. sphaerospermum led to the isolation of dipropyl disulphide (1), dibenzyl sulphide (2), dibenzyl disulphide (3), dibenzyl trisulphide (4), dibenzyl tetrasulphide (5), benzylhydroxymethyl sulphide (6) and di(benzyltrithio) methane (7). Of these, 5-7 are new compounds and this is the first report of the natural occurrence of 2 and 3.

Triterpenes and saponins from Rudgea viburnioides.

A novel triterpene, viburgenin (1), has been isolated from an extract of the ripe fruit rinds of Rudgea viburnioides, together with the known saponins, arjunglucoside I and trachelosperosides B-1 and E-1, and the triterpenes trachelosperogenin B (2) and arjungenin. Compound 2 was previously obtained as a product from enzymatic hydrolysis, and it is reported for the first time as a natural product. The structure of compound 1 was determined as 2alpha,3beta, 19alpha,23,24-pentahydroxyurs-12-ene by extensive use of 1D and 2D NMR spectroscopic methods. Compound 1 exhibited moderate antifungal activity against Cladosporium cladosporioides.