Chromolaena laevigata (Asteraceae) as a source of endophytic non-aflatoxigenic Aspergillus flavus: chemical profile in different culture conditions and biological applications.

Endophytes are microorganisms that form symbiotic relationships with their host. These microorganisms can produce a variety of secondary metabolites, some of which have inhibitory effects on pests and pathogens or even act to promote plant growth. Due to these characteristics, these microorganisms are used as sources of biologically active substances for a wide range of biotechnological applications. Based on that, the aim of this study was to evaluate the production of metabolites of the endophytic Aspergillus flavus CL7 isolated from Chromolaena laevigata, in four different cultivation conditions, and to determine the antimicrobial, cytotoxic, antiviral, and antioxidant potential of these extracts. The multiphasic approach used to identify this strain was based on morphology and ITS gene sequence analysis. The chemical investigation of A. flavus using potato dextrose and minimal medium, using both stationary and agitated methods, resulted in the isolation of kojic acid, α-cyclopiazonic acid, and 20,25-dihydroxyaflavinine. Another 18 compounds in these extracts were identified by UHPLC-HRMS/MS, of which dideacetyl parasiticolide A has been described for the first time from A. flavus. Aflatoxins, important chemomarkers of A. flavus, were not detected in any of the extracts, thus indicating that the CL7 strain is non-aflatoxigenic. The biological potential of all extracts was evaluated, and the best results were observed for the extract obtained using minimal medium against Trichophyton rubrum and Mycobacterium tuberculosis.

Proanthocyanidin Polymer-Rich Fraction of Stryphnodendron adstringens Promotes in Vitro and in Vivo Cancer Cell Death via Oxidative Stress.

Cervical cancer is the fourth most common cancer that affects women, mainly through human papilloma virus (HPV) infection with high-risk HPV16 and HPV18. The present study investigated the in vitro anticancer activity and mechanism of action of a proanthocyanidin polymer-rich fraction of Stryphnodendron adstringens (F2) in cervical cancer cell lines, including HeLa (HPV18-positive), SiHa (HPV16-positive), and C33A (HPV-negative) cells, and also evaluated in vivo anticancer activity. In vitro, cell viability was determined by the MTT assay. Cell migration was determined by the wound healing assay. The mechanism of action was investigated by performing ultrastructural analysis and evaluating reactive oxygen species (ROS) production, mitochondrial metabolism, lipoperoxidation, BCL-2 family expression, caspase expression, and DNA and cell membrane integrity. In vivo activity was evaluated using the murine Ehrlich solid tumor model. F2 time- and dose-dependently reduced cell viability and significantly inhibited the migration of cervical cancer cells. HeLa and SiHa cells treated with F2 (IC50) exhibited intense oxidative stress (i.e., increase in ROS and decrease in antioxidant species) and mitochondrial damage (i.e., mitochondrial membrane potential depolarization and a reduction of intracellular levels of adenosine triphosphate). Increases in the Bax/BCL-2 ratio and caspase 9 and caspase 3 expression, were observed, with DNA damage that was sufficient to trigger mitochondria-dependent apoptosis. Cell membrane disruption was observed in C33A cells (IC50 and IC90) and HeLa and SiHa cells (IC90), indicating progress to late apoptosis/necrosis. The inhibition of ROS production by N-acetylcysteine significantly suppressed oxidative stress in all three cell lines. In vivo, F2 significantly reduced tumor volume and weight of the Ehrlich solid tumor, and significantly increased lipoperoxidation, indicating that F2 also induces oxidative stress in the in vivo model. These findings indicate that the proanthocyanidin polymer-rich fraction of S. adstringens may be a potential chemotherapeutic candidate for cancer treatment.

Induction of Early Autophagic Process on Leishmania amazonensis by Synergistic Effect of Miltefosine and Innovative Semi-synthetic Thiosemicarbazone.

Drug combination therapy is a current trend to treat complex diseases. Many benefits are expected from this strategy, such as cytotoxicity decrease, retardation of resistant strains development, and activity increment. This study evaluated in vitro combination between an innovative thiosemicarbazone molecule - BZTS with miltefosine, a drug already consolidated in the leishmaniasis treatment, against Leishmania amazonensis. Cytotoxicity effects were also evaluated on macrophages and erythrocytes. Synergistic antileishmania effect and antagonist cytotoxicity were revealed from this combination therapy. Mechanisms of action assays were performed in order to investigate the main cell pathways induced by this treatment. Mitochondrial dysfunction generated a significant increase of reactive oxygen and nitrogen species production, causing severe cell injuries and promoting intense autophagy process and consequent apoptosis cell death. However, this phenomenon was not strong enough to promote dead in mammalian cell, providing the potential selective effect of the tested combination for the protozoa. Thus, the results confirmed that drugs involved in distinct metabolic routes are promising agents for drug combination therapy, promoting a synergistic effect.

Anti-biofilm activity against Staphylococcus aureus MRSA and MSSA of neolignans and extract of Piper regnellii

ABSTRACT Many infections worldwide are associated with bacterial biofilms. The effects of isolated neolignans (conocarpan and eupomathenoid-5) and the dichloromethane extract of Piper regnellii (Miq.) C. DC., Piperaceae, were tested against isolates of methicillin-resistant Staphylococcus aureus and methicillin-sensitive S. aureus biofilms and S. aureus planktonic cells. The dichloromethane extract presented better results than isolated neolignans against all of the biofilms tested, with a minimum inhibitory concentration <400 µg/ml for preformed biofilms and minimal biofilm inhibitory concentration of 15.6 µg/ml for biofilm formation. The minimum inhibitory concentration to planktonic cells was <12.5 µg/ml. These results indicate a good effect of the dichloromethane extract against methicillin-resistant S. aureus and methicillin-sensitive S. aureus biofilms and efficient prophylaxis.

Antifungal activity of pomegranate peel extract and isolated compound punicalagin against dermatophytes.

BACKGROUND: Dermatophyte species infect the epidermis and appendages, often with serious social and health-economic consequences. The hydroalcoholic extract of pomegranate fruit peel showed activity against the dermatophyte fungi Trichophyton mentagrophytes, T. rubrum, Microsporum canis and M. gypseum. METHODS: Hydroalcoholic extract was prepared with pomegranate peels. This crude extract was fractionated and submitted to liquid-liquid partition, resulting in an active fraction which was fractionated in a Sephadex LH-20 column, followed by a Lobar column. The structure of the active compound was established with the use of spectroscopic methods. RESULTS: The crude extract of pomegranate fruit peel showed activity against the dermatophytes Trichophyton mentagrophytes, T. rubrum, Microsporum canis, and M. gypseum, with MICs values of 125 µg/ml and 250 µg/ml, respectively for each genus. Punicalagin was isolated and identified by spectroscopic analysis. The crude extract and punicalagin showed activity against the conidial and hyphal stages of the fungi. The cytotoxicity assay showed selectivity for fungal cells than for mammalian cells. CONCLUSIONS: These results indicated that the crude extract and punicalagin had a greater antifungal activity against T. rubrum, indicating that the pomegranate is a good target for study to obtain a new antidermatophyte medicine.

Trypanocidal activity of 1,3,7-trihydroxy-2-(3-methylbut-2-enyl)-xanthone isolated from Kielmeyera coriacea.

This work evaluated the activity and ultrastructural and morphological alterations induced by the xanthone 1,3,7-trihydroxy-2-(3-methylbut-2-enyl)-xanthone (C23) isolated from Kielmeyera coriacea against Trypanosoma cruzi. This xanthone had inhibitory activity against the three forms of this protozoan and did not induce toxicity in mammalian cells. The best activity of this xanthone was against the intracellular amastigote form. Additionally, the mitochondrion was the main target of this compound, reflected by electronic microscopy and rhodamine 123 assays. Our MitoSOX assay results also indicated that C23 increased O2(-) production in mitochondrion. C23 might be a promising chemotherapeutic agent against T. cruzi because its trypanocidal action involves the disruption of mitochondrion, a specific target of Trypanosomatides.

Effect of Brazilian copaiba oils on Leishmania amazonensis.

ETHNOPHARMACOLOGICAL RELEVANCE: Copaiba oil has been used in folk medicine since the 19th century. The use of copaiba oils to treat leishmaniasis is cited in several ethnopharmacological studies. Nevertheless, the potential antileishmania of copaiba oils had not been studied. AIM OF THE STUDY: Eight different kinds of Brazilian copaiba oils were screened for antileishmanial activity. MATERIALS AND METHODS: The antiproliferative effect of copaiba oil on promastigote and amastigote axenic were determined. To determine the survival index peritoneal macrophage were infected with promastigotes of Leishmania amazonensis and treated with copaiba oil. The cytotoxic effect of copaiba oil was assessed on macrophage strain J774G8 by assay of sulforhodamine B. RESULTS: Copaiba oils showed variable levels of activity against promastigote forms with IC(50) values in the range between 5 and 22microg/mL. The most active oil was that from Copaifera reticulata (collected in Pará State, Brazil) with IC(50) values of 5, 15, and 20microg/mL for promastigote, axenic amastigote and intracellular amastigote forms, respectively. Amphotericin B showed IC(50) of 0.058 and 0.231microg/mL against promastigote and amastigote forms, respectively. Cytotoxicity assay showed that this copaiba oil obtained from Copaifera reticulata showed low cytotoxicity against J774G8 macrophages. CONCLUSION: Copaiba oils showed significant activity against the parasite Leishmania amazonensis.

In vitro antifungal activity of extracts and neolignans from Piper regnellii against dermatophytes.

The present study was designated to evaluate the in vitro antidermatophyte activity of extracts from leaves of Piper regnellii as well as of the bioactivity-directed isolation of neolignans. The antifungal assay was performed by microdilution techniques. The hydroalcoholic extract of Piper regnellii leaves presented a strong activity against the dermatophyte fungi Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis and Microsporum gypseum with MICs of 15.62, 15.62, 15.62 and 62.5 microg/ml, respectively. On light microscopy and scanning electron microscopy of nail fragments not exposed to hydroalcoholic extract of Piper regnelli leaves, well-formed and extensive mycelial growth was seen. On nail fragments exposed to hydroalcoholic extract at concentrations more than 1.2mg/ml and then inoculated with spore suspension, growth was not seen. The hydroalcoholic extract was fractionated on silica gel in to nine fractions. The active chloroform fraction was lyophilized and chromatographed by column chromatography on silica gel. Structures were established by comparison with literature data and identified as eupomatenoid-3 and eupomatenoid-5. The pure compounds showed strong activity on Trichophyton rubrum with MIC of 50 and 6.2 microg/ml, respectively. Comparing the activity of the active chloroform fraction obtained from hydroalcoholic crude extract with that of isolated compound eupomatenoid-5, it is clear that this showed the same results against Trichophyton rubrum. The results showed that the plant could be explored for possible antifungal agents and provides preliminary scientific validation for the traditional medicinal use of this plant.

Virulence and antibiotic susceptibility of Aeromonas spp. isolated from drinking water.

Aeromonas isolates from tap water, mineral water, and artesian well water were investigated for their ability to produce different potential virulence factors or markers such as hemolysins, cytotoxins, phospholipase, DNase, hydrophobicity and their ability to adhere to epithelial cells and to abiotic surfaces. The susceptibility to antibiotics of Aeromonas isolates was also examined. Majority of the isolates displayed hemolytic activity against sheep erythrocytes, while only 7 of the 23 Aeromonas strains displayed DNase activity and 4 of the 23 Aeromonas strains tested were regarded as positive for phospholipase production. Most of the isolates showed cytotoxic activities in culture filtrate dilutions at titer of 1/8 or lower. No general relation between the strain isolated and the ability to interact with epithelial cells could be established. Using the bacterial adherence to hydrocarbons method, most of the strains were classified as highly hydrophilic. All five Aeromonas jandaei strains isolates, 9 of the 12 Aeromonas sp strains and four of the five Aeromonas hydrophila were multidrug resistant. The most active antimicrobial was ciprofloxacin (susceptible in 100% of the isolates), and the least active antibiotic was ampicillin (resistance in 92% of the isolates). The majority of the isolates tested were not killed by chlorine at 1.2 mg/l. Whether the high tolerance to chlorine of Aeromonas isolates can be linked to greater virulence is not know.

Atribuição dos deslocamentos químicos dos átomos de ¹H e 13C do acetato de acantoaustralida / 1H and 13C NMR assignments of acanthoaustralide-1-O-Acetate

Do extrato hidroetanólico das partes aéreas de Acanthospermum australe (Asteraceae) foram identificados uma lactona diterpênica, o acetato de acantoaustralida (1) e dois flavonoides: quercetina (2) e crisosplenol D (3). As estruturas foram identificadas através de técnicas espectroscópicas de RMN de ¹H e 13C, gHSQC, gHMBC, TOCSY, gNOESY, EM e pela comparação com dados da literatura.

From the hydroethanolic extract of the aerial parts of Acanthospermum australe (Asteraceae) a diterpene lactone, acanthoaustralide-1-O-Acetate (1) and two flavonoids: quercetin (2) and chrysosplenol D (3) were identified. The structures were determined though the use of spectroscopic techniques such as NMR (¹H, 13C{¹H}, gHSQC, gHMBC, TOCSY, gNOESY), MS and compared with the literature data.

Estudo morfoanatômico preliminar do caule e da folha de Acanthospermum australe (Loefl.) Kuntze (Asteraceae-Heliantheae) / A preliminary morphological and anatomical study of the stem and leaf of Acanthospermum australe (Loefl.) Kuntze (Asteraceae-Heliantheae)

Descrições morfoanatômicas preliminares do caule e da folha de Acanthospermum australe (Loefl.) Kuntze foram realizadas a partir de material vegetal fresco e fixado. Secções transversais e paradérmicas, coradas com safranina/azul de astra e azul de toluidina foram analisadas ao microscópio. As superfícies do caule e da folha foram observadas através de microscopia eletrônica de varredura. A. australe apresenta, tanto no caule quanto na folha, tricomas tectores pluricelulares, unisseriados e de extremidades afiladas, e glandulares discóides, com pedúnculo curto unicelular, no qual se prendem cinco séries de células. A folha é anfiestomática e os estômatos são do tipo anomocítico, levemente proeminentes em relação ao nível das demais células epidérmicas. O caule apresenta crescimento secundário inicial, destacando-se os ductos secretores no parênquima cortical e medular.

Preliminary morphological and anatomical descriptions of stem and leaf of Acanthospermum australe (Loefl.) Kuntze were made in fresh and fixed material. Cross and superficial sections, stained with safranin/astra blue and Toluidine blue were analyzed at the light microscopic and the images were acquired by means the program Image Pro-Plus, version 4.0 (Media Cybertecnics). The surface of the stem and leaf were observed through scanning electronic microscopy. The species presents in the stem and leaf surface multicellular nonglandular trichome, uniseriate with a pointed edge and multicellular glandular trichome like a disk with a unicellular peduncle, in which is attached a series of five cells. The leaf presents anomocytic stomata in both surfaces a little prominent in relation to the level of the epidermal cells. The stem presents an initial secondary growth with evident secretory ducts in the cortex and pith.

Atividade antileishmania do extrato hidroalcoólico e de frações obtidas de folhas de Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck / Antileishmanial activity of hydroalcoholic extract and fractions obtained from leaves of Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.)

A atividade biológica do extrato bruto e as várias frações obtidas de folhas de Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck foi avaliada em Leishmania amazonensis. Este estudo incluiu o processo de extração e o fracionamento biomonitorado pelo método de cromatografia de adsorção. Um aumento progressivo no efeito antileishmania foi observado durante o processo de purificação. O extrato hidroalcoólico solúvel em água (EBA) apresentou concentração inibitória 50 por cento (IC50) igual a 167 µg/mL enquanto o extrato hidroalcoólico solúvel em acetato de etila (EBAcOEt) mostrou um IC50 de 30 mg/mL sobre o crescimento de formas promastigotas após 48 h de cultivo. A fração hexano (FHex) apresentou uma atividade antileishmania maior que o EBAcOEt com IC50 de 21,5 µg/mL. Análises de citotoxicidade indicaram que as concentrações tóxicas do EBA, EBAcOEt e das frações foram maiores para macrófagos J774G8 do que para os protozoários.

Biological activity of the crude extract and several fractions obtained from Piper regnellii var. pallescens was assessed on Leishmania amazonensis. This study included the extraction process and bioassay-guided fractionation by the adsorption chromatography method. A progressive increase in the antileishmanial effect was observed in the course of the purification process. The hydroalcoholic extract water soluble (EBA) had a 50 percent inhibitory concentration (IC50) at 167 µg/mL whereas the hydroalcoholic extract acetate soluble (EBAcOEt) showed an IC50 of 30 µg/mL against the growth of promastigote forms after 48 h of culturing. The hexan fraction (FHex) showed an antileishmanial activity greater than EBAcOEt with IC50 at 21.5 µg/mL. Analysis of cytotoxicity indicated that the toxic concentrations of the EBA, EBAcOEt, and fractions were higher for J774G8 macrophages than for the protozoans.

Antimicrobial and phytochemical studies on Pedilanthus tithymaloides.

The antibacterial and antifungal properties of ethanolic extract of the leaves of Pedilanthus tithymaloides and some of its constituents were investigated by the dilution method.

Influence of extracts of Stryphnodendron polyphyllum Mart. and Stryphnodendron obovatum Benth. on the cicatrisation of cutaneous wounds in rats.

Stem bark of the two species Stryphnodendron polyphyllum Mart. and Stryphnodendron obovatum Benth., Leguminosae, was investigated for wound healing, antibacterial and antioxidant activity. These plants contain 12 and 19% tannins in their stem bark, respectively, and are widely used in traditional medicine in Brazil. The total content of phenolics of the crude extract (CE) of Stryphnodendron obovatum was 76.95 +/- 2.98% (CV = 3.87%) and of the ethyl-acetate fraction (EAF) was 89.13 +/- 0.34% (CV = 0.38%); whereas in Stryphnodendron polyphyllum the CE phenolics content was 51.62+/-1.53% (CV = 2.96%) and the EAF phenolics content was 59.00 +/- 1.91% (CV = 3.24%). The tannin content of CE from Stryphnodendron obovatum [36.58 +/- 0.35% (CV = 0.98%)] was about 11% higher than in CE from Stryphnodendron polyphyllum [25.43 +/- 0.96% (CV = 3.77%)]. The difference between the species was even greater in the EAF: in Stryphnodendron obovatum the EAF phenolics content was 55.01 +/- 0.36% (CV = 0.65%), whereas in Stryphnodendron polyphyllum the content was 36.16 +/- 0.42% (CV = 1.16%). The healing effect of ointments containing 2.5% crude lyophilised extract (PCE) and 2.5% ethyl-acetate lyophilised fraction (PEA) of the stem bark of Stryphnodendron polyphyllum and Stryphnodendron obovatum was studied in cutaneous wounds of Wistar rats after 4, 7 and 10 days of treatment. Epithelial cell proliferation in the area of re-epithelialisation of the wounds was evaluated by counting the metaphases blocked by vincristine sulfate. With PCE an increase in epidermal growth was observed after 4 and 7 days of treatment with Stryphnodendron polyphyllum, and after 7 and 10 days of treatment with Stryphnodendron obovatum. Wounds treated with PEA of Stryphnodendron obovatum showed increased epidermal growth only 4 days after the treatment; for Stryphnodendron polyphyllum, epidermal growth was observed after 4 and 7 days of treatment. Both the CE and the EAF fractions of Stryphnodendron polyphyllum and Stryphnodendron obovatum showed antibacterial activity against Staphylococcus aureus with MIC values of 125 and 250 microg/ml, respectively. Gram-negative bacteria tested were not inhibited by extracts and fractions at concentrations > 1000 microg/ml. The antioxidant activity through reduction of the DPPH radical in TLC, confirmed the anti-radical properties of these extracts in both species. CE and EAF of both species showed a radical scavenging activity (RSA) and protected DPPH from discolouration, already at 0.032 microg/ml. The extract from Stryphnodendron polyphyllum were more effective than those Stryphnodendron obovatum, although the former had a lower tannin content.

Microbiological quality of drinking water of urban and rural communities, Brazil.

OBJECTIVE: To evaluate the microbiological quality of treated and untreated water samples came from urban and rural communities and to examine the relationship between coliforms occurrence and average water temperature, and a comparison of the rainfall levels. METHODS: A sample of 3,073 untreated and treated (chlorinated) water from taps (1,594), reservoir used to store treated water (1,033), spring water (96) and private well (350) collected for routine testing between 1996 and 1999 was analyzed by the multiple dilution tube methods used to detect the most probable number of total and fecal coliforms. These samples were obtained in the region of Maring , state of Paran , Brazil. RESULTS: The highest numbers water samples contaminated by TC (83%) and FC (48%) were found in the untreated water. TC and FC in samples taken from reservoirs used to store treated water was higher than that from taps midway along distribution lines. Among the treated water samples examined, coliform bacteria were found in 171 of the 1,033 sampling reservoirs. CONCLUSIONS: Insufficient treatment or regrowth is suggested by the observation that more than 17% of these treated potable water contained coliform. TC and FC positive samples appear to be similar and seasonally influenced in treated water. Two different periods must be considered for the occurrence of both TC and FC positive samples: (i) a warm-weather period (September-March) with high percentage of contaminated samples; and (ii) cold-weather period (April-August) were they are lower. Both TC and TF positive samples declined with the decreased of water temperature.

Microbiological quality of drinking water of urban and rural communities, Brazil

OBJETIVO: Avaliar a qualidade microbiológica de amostras de água tratada e näo-tratada proveniente de comunidades urbanas e rurais e examinar a relaçäo entre ocorrência de coliformes e a média de temperatura da água, e uma comparaçäo dos níveis de precipitaçäo de chuva. MÉTODOS: No período de 1996 a 1999, foram analisadas 3.073 amostras de água tratada (clorada) e näo-tratada pelo método dos tubos múltiplos para determinar o número mais provável de coliformes totais e fecais. Destas, 1.594 provenientes de água de torneiras, 1.033 de reservatório para estocar água tratada, 96 de água de minas e 350 de poços particulares. Tais amostras foram obtidas na regiäo de Maringá, Paraná, Brasil. RESULTADOS: O maior número de amostras contaminadas por TC (coliformes totais) (83 por cento) e FC (coliformes fecais) (48 por cento) foi observado em água näo tratada. O índice de TC e FC foi maior nas amostras de reservatórios do que nas torneiras ao longo de sistema de distribuiçäo. Entre as amostras de água tratada, foram encontradas bactérias do grupo coliforme em 171 dos 1.033 reservatórios amostrados. CONCLUSOES: A observaçäo de que mais de 17 por cento da água potável tratada contêm coliformes sugere tratamento insuficiente ou recrescimento. Em água tratada, amostras positivas para TC e FC parecem ser similares e sazonalmente influenciadas. Dois diferentes períodos podem ser considerados para a ocorrência de amostras positivas para TC e FC: (i) período quente e úmido (Setembro-Março) com alta percentagem de amostras contaminadas; e (ii) período frio e úmido (Abril-Agosto) onde a positividade é baixa. Amostras positivas para TC e FC diminuem com o decréscimo da temperatura da água

Treatment of sewage sludge generated in municipal wastewater treatment plants.

This study was designed to evaluate the performance of a cylindrical anaerobic digester in treating secondary sewage sludge. A series of three independent batch experiments was performed for a total operation time of 60 d. The system of anaerobic digestion showed stability conditions, with no noticeable scum or foaming problems. The chemical oxygen demand reduction reached 29,21, and 45% in sludge and 95,85, and 82% in supernatant for the three experiments, respectively. Total coliform bacteria levels in the digester ranged from 10(4) to 10(5) in influent sludge and from 10(4) to 10(3) in effluent sludge, with an average reduction of 90%. Fecal coliforms of the order of 10(4) were enumerated in influent sludge and those of the order of 10(0) were enumerated in effluent sludge, with an average reduction of 99.9%. The studied system had satisfactory results, showing that both organic matter and indicator bacteria levels substantially decrease when the sludge is submitted to anaerobic digestion.