Biological activities of marine invertebrates extracts from the northeast brazilian coast / Atividades biológicas de extratos de invertebrados marinhos da costa nordeste do Brasil

Abstract This paper reports the in vitro antiproliferative effects, antiprotozoal, anti-herpes and antimicrobial activities of 32 organic extracts of 14 marine sponges and 14 corals collected in northeast Brazilian coast. The ethanolic extracts of the sponges Amphimedon compressa and Tedania ignis, and the acetone extract of Dysidea sp. showed relevant results concerning the antiproliferative effects against A549, HCT-8, and PC-3 cell lines by sulforhodamine B assay, but also low specificity. Concerning the antiprotozoal screening, the ethanolic extract of Amphimedon compressa and the acetone and ethanolic extracts of Dysidea sp. were the most active against Leishmania amazonensis and Trypanosoma cruzi expressing β-galactosidase in THP-1 cells. In the preliminary anti-HSV-1 (KOS strain) screening, the ethanolic extracts of the sponges Amphimedon compressa, Haliclona sp. and Chondrosia collectrix inhibited viral replication by more than 50%. The most promising anti-herpes results were observed for the ethanolic extract of Haliclona sp. showing high selective indices against HSV-1, KOS and 29R strains (SI> 50 and >79, respectively), and HSV-2, 333 strain (IS>108). The results of the antibacterial screening indicated that only the ethanolic extract of Amphimedon compressa exhibited a weak activity against Enterococcus faecalis, Pseudomonas aeruginosa and Escherichia coli by the disk diffusion method. In view of these results, the extracts of Amphimedon compressa, Tedania ignis and Dysidea sp. were selected for further studies aiming the isolation and identification of the bioactive compounds with antiproliferative and/or antiprotozoal activities. The relevant anti-herpes activity of the ethanolic extract of Haliclona sp. also deserves special attention, and will be further investigated.

Resumo Este artigo reporta as atividades in vitro antiproliferativa, atiprotozoárica, anti-herpética e antimicrobiana de 32 extratos orgânicos provenientes de 14 esponjas marinhas e 14 corais coletados no litoral nordestino brasileiro. Os extratos etanólicos das esponjas Amphimedon compressa e Tedania ignis, e o extrato acetônico de Dysidea sp. demonstraram resultados promissores em relação aos efeitos antiproliferativos frente as linhagens celulares A549, HCT-8, PC-3 pelo método da sulforrodamina B, mas sem especificidade. Em relação à atividade antiprotozárica, os extratos etanólico de Amphimedon compressa e acetônico e etanólico de Dysidea sp. apresentaram atividade contra Leishmania amazonensis e Trypanosoma cruzi através do método de expressão de β-galactosidase em células THP-1. Na investigação preliminar de atividade antiviral frente ao vírus Herpes simplex tipo 1 (cepa KOS), os extratos etanólicos das esponjas Amphimedon compressa, Haliclona sp. e Chondrosia collectrix inibiram mais de 50% da replicação viral. O extrato etanólico da esponja Haliclona sp. demonstrou resultados promissores para atividade anti-herpética com altos índices de seletividade para as cepas KOS (IS >50) e 29R (IS>79) frente ao VHS-1 e cepa 333 (IS>108) frente ao VHS-2. O extrato etanólico da esponja Amphimedon compressa exibiu uma pequena atividade contra Enterococcus faecalis, Pseudomonas aeruginosa and Escherichia coli pelo método de difusão em disco. De acordo com os resultados apresentados, os extratos das esponjas Amphimedon compressa, Tedania ignis e Dysidea sp. serão selecionados para futuros estudos de isolamento e identificação dos compostos bioativos para as atividades antiproliferativa e antiprozoárica. O extrato etanólico de Haliclona sp. será investigado por possuir atividade relevante anti-herpética.

Biological activities of marine invertebrates extracts from the northeast brazilian coast.

This paper reports the in vitro antiproliferative effects, antiprotozoal, anti-herpes and antimicrobial activities of 32 organic extracts of 14 marine sponges and 14 corals collected in northeast Brazilian coast. The ethanolic extracts of the sponges Amphimedon compressa and Tedania ignis, and the acetone extract of Dysidea sp. showed relevant results concerning the antiproliferative effects against A549, HCT-8, and PC-3 cell lines by sulforhodamine B assay, but also low specificity. Concerning the antiprotozoal screening, the ethanolic extract of Amphimedon compressa and the acetone and ethanolic extracts of Dysidea sp. were the most active against Leishmania amazonensis and Trypanosoma cruzi expressing ß-galactosidase in THP-1 cells. In the preliminary anti-HSV-1 (KOS strain) screening, the ethanolic extracts of the sponges Amphimedon compressa, Haliclona sp. and Chondrosia collectrix inhibited viral replication by more than 50%. The most promising anti-herpes results were observed for the ethanolic extract of Haliclona sp. showing high selective indices against HSV-1, KOS and 29R strains (SI> 50 and >79, respectively), and HSV-2, 333 strain (IS>108). The results of the antibacterial screening indicated that only the ethanolic extract of Amphimedon compressa exhibited a weak activity against Enterococcus faecalis, Pseudomonas aeruginosa and Escherichia coli by the disk diffusion method. In view of these results, the extracts of Amphimedon compressa, Tedania ignis and Dysidea sp. were selected for further studies aiming the isolation and identification of the bioactive compounds with antiproliferative and/or antiprotozoal activities. The relevant anti-herpes activity of the ethanolic extract of Haliclona sp. also deserves special attention, and will be further investigated.

Biological activities of marine invertebrates extracts from the northeast brazilian coast

Abstract This paper reports the in vitro antiproliferative effects, antiprotozoal, anti-herpes and antimicrobial activities of 32 organic extracts of 14 marine sponges and 14 corals collected in northeast Brazilian coast. The ethanolic extracts of the sponges Amphimedon compressa and Tedania ignis, and the acetone extract of Dysidea sp. showed relevant results concerning the antiproliferative effects against A549, HCT-8, and PC-3 cell lines by sulforhodamine B assay, but also low specificity. Concerning the antiprotozoal screening, the ethanolic extract of Amphimedon compressa and the acetone and ethanolic extracts of Dysidea sp. were the most active against Leishmania amazonensis and Trypanosoma cruzi expressing -galactosidase in THP-1 cells. In the preliminary anti-HSV-1 (KOS strain) screening, the ethanolic extracts of the sponges Amphimedon compressa, Haliclona sp. and Chondrosia collectrix inhibited viral replication by more than 50%. The most promising anti-herpes results were observed for the ethanolic extract of Haliclona sp. showing high selective indices against HSV-1, KOS and 29R strains (SI> 50 and >79, respectively), and HSV-2, 333 strain (IS>108). The results of the antibacterial screening indicated that only the ethanolic extract of Amphimedon compressa exhibited a weak activity against Enterococcus faecalis, Pseudomonas aeruginosa and Escherichia coli by the disk diffusion method. In view of these results, the extracts of Amphimedon compressa, Tedania ignis and Dysidea sp. were selected for further studies aiming the isolation and identification of the bioactive compounds with antiproliferative and/or antiprotozoal activities. The relevant anti-herpes activity of the ethanolic extract of Haliclona sp. also deserves special attention, and will be further investigated.

Resumo Este artigo reporta as atividades in vitro antiproliferativa, atiprotozoárica, anti-herpética e antimicrobiana de 32 extratos orgânicos provenientes de 14 esponjas marinhas e 14 corais coletados no litoral nordestino brasileiro. Os extratos etanólicos das esponjas Amphimedon compressa e Tedania ignis, e o extrato acetônico de Dysidea sp. demonstraram resultados promissores em relação aos efeitos antiproliferativos frente as linhagens celulares A549, HCT-8, PC-3 pelo método da sulforrodamina B, mas sem especificidade. Em relação à atividade antiprotozárica, os extratos etanólico de Amphimedon compressa e acetônico e etanólico de Dysidea sp. apresentaram atividade contra Leishmania amazonensis e Trypanosoma cruzi através do método de expressão de -galactosidase em células THP-1. Na investigação preliminar de atividade antiviral frente ao vírus Herpes simplex tipo 1 (cepa KOS), os extratos etanólicos das esponjas Amphimedon compressa, Haliclona sp. e Chondrosia collectrix inibiram mais de 50% da replicação viral. O extrato etanólico da esponja Haliclona sp. demonstrou resultados promissores para atividade anti-herpética com altos índices de seletividade para as cepas KOS (IS >50) e 29R (IS>79) frente ao VHS-1 e cepa 333 (IS>108) frente ao VHS-2. O extrato etanólico da esponja Amphimedon compressa exibiu uma pequena atividade contra Enterococcus faecalis, Pseudomonas aeruginosa and Escherichia coli pelo método de difusão em disco. De acordo com os resultados apresentados, os extratos das esponjas Amphimedon compressa, Tedania ignis e Dysidea sp. serão selecionados para futuros estudos de isolamento e identificação dos compostos bioativos para as atividades antiproliferativa e antiprozoárica. O extrato etanólico de Haliclona sp. será investigado por possuir atividade relevante anti-herpética.

Strychnos pseudoquina A. St. Hil.: a Brazilian medicinal plant with promising in vitro antiherpes activity.

AIMS: To investigate the anti-HSV and anti-inflammatory effects of a standardized ethyl acetate extract (SEAE) prepared with the stem bark of Strychnos pseudoquina, along with two isolated compounds: quercetin 3-O-methyl ether (3MQ) and strychnobiflavone (SBF). METHODS AND RESULTS: The mechanisms of action were evaluated by different methodological strategies. SEAE and SBF affected the early stages of viral infection and reduced HSV-1 protein expression. Both flavonoids elicited a concentration-dependent inhibition of monocyte chemoattractant protein-1 (MCP-1), whereas 3MQ reduced the chemokine release more significantly than SBF. Conversely, both compounds stimulated the production of the cytokines TNF-α and IL-1-ß in LPS-stimulated cells, especially at the intermediate and the highest tested concentrations. CONCLUSIONS: SEAE and SBF interfered with various steps of HSV replication cycle, mainly adsorption, postadsorption and penetration, as well as with ß and γ viral proteins expression; moreover, a direct inactivation of viral particles was observed. Besides, both flavonoids inhibited MCP-1 selectively, a feature that may be beneficial for the development of new anti-HSV agents. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicated that the samples present anti-HSV and anti-inflammatory activities, at different levels, which is an interesting feature since cold and genital sores are accompanied by an inflammation process.

Antiherpes evaluation of soybean isoflavonoids.

The antiviral effects of soybean isoflavonoids have been investigated recently, especially those of genistein. It has been reported that this isoflavone is able to inhibit herpes simplex virus (HSV) replication, which is associated with skin and epithelial mucosa infections. The treatment of these infections with antiherpes drugs has resulted in the emergence of resistant viral strains. Based on this evidence, the aim of this study was to investigate the anti-HSV effects of soybean isoflavonoids: daidzein, genistein, glycitein, and coumestrol. Genistein and coumestrol inhibited HSV-1 (KOS and 29R strains, which are acyclovir sensitive and acyclovir resistant, respectively) and HSV-2 (333 strain) replication, whereas no antiviral effects were detected for daidzein and glycitein. The mechanisms of action were evaluated by different methodological strategies. Coumestrol affected the early stages of viral infection, and both compounds were able to reduce HSV-1 protein expression, as well as HSV-2 cell-to-cell spread.

Simultaneous quantification of flavonoids from Achyrocline satureioides by a polar-reversed phase LC method--application to skin permeation/retention studies.

A selective and sensitive polar-reversed phase LC method was validated for simultaneous quantification of the main Achyrocline satureioides flavonoids (quercetin, luteolin, and 3-O-methylquercetin) in skin samples after permeation/retention studies from topical nanoemulsions. The method was linear in a range of 0.25 to 10.0 microg/mL exhibiting a coefficient of determination higher than 0.999 for all flavonoids. No interference of the nanoemulsion excipients or skin components was observed in the retention times of all flavonoids. The R.S.D. values for intra- and inter-day precision experiments were lower than 6.73%. Flavonoids recovery from nanoemulsions and skin matrices was between 90.05 and 109.88%. In a permeation/retention study with porcine ear high amount of 3-O-methylquercetin was found in the skin sample (0.92 +/- 0.22 microg/g) after two hours. The proposed method was suitable to quantify the main flavonoids of A. satureioides in skin permeation/retention studies from topical nanoemulsions.

Characterization and cytotoxic activity of sulfated derivatives of polysaccharides from Agaricus brasiliensis.

Agaricus brasiliensis cell-wall polysaccharides isolated from fruiting body (FR) and mycelium (MI) and their respective sulfated derivatives (FR-S and MI-S) were chemically characterized using elemental analysis, TLC, FT-IR, NMR, HPLC, and thermal analysis. Cytotoxic activity was evaluated against A549 tumor cells by MTT and sulforhodamine assays. The average molecular weight (Mw) of FR and MI was estimated to be 609 and 310 kDa, respectively. FR-S (127 kDa) and MI-S (86 kDa) had lower Mw, probably due to hydrolysis occurring during the sulfation reaction. FR-S and MI-S presented ~14% sulfur content in elemental analysis. Sulfation of samples was characterized by the appearance of two new absorption bands at 1253 and 810 cm(-1) in the infrared spectra, related to S=O and C-S-O sulfate groups, respectively. Through (1)H and (13)C NMR analysis FR-S was characterized as a (1→6)-(1→3)-ß-D-glucan fully sulfated at C-4 and C-6 terminal and partially sulfated at C-6 of (1→3)-ß-D-glucan moiety. MI-S was shown to be a (1→3)-ß-D-gluco-(1→2)-ß-D-mannan, partially sulfated at C-2, C-3, C-4, and C-6, and fully sulfated at C-6 of the terminal residues. The combination of high degree of sulfation and low molecular weight was correlated with the increased cytotoxic activity (48 h of treatment) of both FR-S (EC50=605.6 µg/mL) and MI-S (EC50=342.1 µg/mL) compared to the non-sulfated polysaccharides FR and MI (EC50>1500 µg/mL).

In vivo anti-herpes simplex virus activity of a sulfated derivative of Agaricus brasiliensis mycelial polysaccharide.

Agaricus brasiliensis (syn. A. subrufescens), a basidiomycete fungus native to the Atlantic forest in Brazil, contains cell walls rich in glucomannan polysaccharides. The ß-(1 → 2)-gluco-ß-(1 → 3)-mannan was isolated from A. brasiliensis mycelium, chemically modified by sulfation, and named MI-S. MI-S has multiple mechanisms of action, including inhibition of herpes simplex virus (HSV) attachment, entry, and cell-to-cell spread (F. T. G. S. Cardozo, C. M. Camelini, A. Mascarello, M. J. Rossi, R. J. Nunes, C. R. Barardi, M. M. de Mendonça, and C. M. O. Simões, Antiviral Res. 92:108-114, 2011). The antiherpetic efficacy of MI-S was assessed in murine ocular, cutaneous, and genital infection models of HSV. Groups of 10 mice were infected with HSV-1 (strain KOS) or HSV-2 (strain 333). MI-S was given either topically or by oral gavage under various pre- and posttreatment regimens, and the severity of disease and viral titers in ocular and vaginal samples were determined. No toxicity was observed in the uninfected groups treated with MI-S. The topical and oral treatments with MI-S were not effective in reducing ocular disease. Topical application of MI-S on skin lesions was also not effective, but cutaneously infected mice treated orally with MI-S had significantly reduced disease scores (P < 0.05) after day 9, suggesting that healing was accelerated. Vaginal administration of MI-S 20 min before viral challenge reduced the mean disease scores on days 5 to 9 (P < 0.05), viral titers on day 1 (P < 0.05), and mortality (P < 0.0001) in comparison to the control groups (untreated and vehicle treated). These results show that MI-S may be useful as an oral agent to reduce the severity of HSV cutaneous and mucosal lesions and, more importantly, as a microbicide to block sexual transmission of HSV-2 genital infections.

Production of polysaccharide from Agaricus subrufescens Peck on solid-state fermentation.

The interest upon products obtained from fungi has increased during the recent years. Among the most noticeable, nutraceuticals, enzymes, and natural drugs occupy a privileged position. Fungal biomass for the obtainment of those products can be produced either by solid-state fermentation (SSF) or submersed fermentation. SSF has been employed for the production of spawn on pretreated wheat grains with the objective of increasing the fungal polysaccharide (glucomannans) contents. Among the important factors for the production of spawn, time of cooking, time of resting after grain cooking, consequently grain moisture, substrate pH, temperature of incubation, and initial inoculum amount are among the most significant. For wheat grains, cooking time of 21 min followed by a 24-min resting time has been shown as optimal for the production of glucomannans by the fungus Agaricus subrufescens (=Agaricus brasiliensis). Amendments of CaSO(4) (up to 3 %) and CaCO(3) (up to 1 %) had an important influence on the substrate pH. In general, better results for glucomannan production were obtained when no supplement was added or when up to 0.25 % CaCO(3) (pH 6.6) has been added to the mix. Our results demonstrate that the inoculum amount necessary for the best polysaccharide levels is around 10.3 %, while the best temperature is around 27.2 °C. Besides using the spawn for its main purpose, it could potentially and alternatively be used as nutraceutical due to the high levels of glucomannan observed (6.89 %), a compound technically proven to be a potent immunostimulatory and antitumoral agent.

Microbiological and physicochemical analysis of the coastal waters of southern Brazil.

The aim of this study was to assess the impact of sewage discharge on coastal waters by evaluating the influence of physicochemical parameters on the presence of enteric microorganisms in seawater samples collected from 11 beaches in Florianopolis, Santa Catarina, Brazil, over a one-year period (August 2009 to July 2010). Samples were assessed for the presence of human adenoviruses (HAdV), polyomavirus (JCPyV), hepatitis A virus (HAV), and noroviruses (HuNoV GI and GII). Escherichia coli and physicochemical parameters (salinity, temperature, pH and dissolved oxygen) were also evaluated. From the 132 samples analyzed, 55% were positive for HAdV, 51.5% for HAV, 7.5% for HuNoV GI, 4.5% for HuNoV GII, and 3% for JCPyV. E. coli levels ranged from 8 to 1325 CFU/100mL at all sites. The overall results highlight the problem of sewage discharge into coastal waters and confirm that there is no correlation between viral presence and bacterial contamination.

An HPLC-UV method for the measurement of permeability of marker drugs in the Caco-2 cell assay

The Caco-2 cell line has been used as a model to predict the in vitro permeability of the human intestinal barrier. The predictive potential of the assay relies on an appropriate in-house validation of the method. The objective of the present study was to develop a single HPLC-UV method for the identification and quantitation of marker drugs and to determine the suitability of the Caco-2 cell permeability assay. A simple chromatographic method was developed for the simultaneous determination of both passively (propranolol, carbamazepine, acyclovir, and hydrochlorothiazide) and actively transported drugs (vinblastine and verapamil). Separation was achieved on a C18 column with step-gradient elution (acetonitrile and aqueous solution of ammonium acetate, pH 3.0) at a flow rate of 1.0 mL/min and UV detection at 275 nm during the total run time of 35 min. The method was validated and found to be specific, linear, precise, and accurate. This chromatographic system can be readily used on a routine basis and its utilization can be extended to other permeability models. The results obtained in the Caco-2 bi-directional transport experiments confirmed the validity of the assay, given that high and low permeability profiles were identified, and P-glycoprotein functionality was established.

An HPLC-UV method for the measurement of permeability of marker drugs in the Caco-2 cell assay.

The Caco-2 cell line has been used as a model to predict the in vitro permeability of the human intestinal barrier. The predictive potential of the assay relies on an appropriate in-house validation of the method. The objective of the present study was to develop a single HPLC-UV method for the identification and quantitation of marker drugs and to determine the suitability of the Caco-2 cell permeability assay. A simple chromatographic method was developed for the simultaneous determination of both passively (propranolol, carbamazepine, acyclovir, and hydrochlorothiazide) and actively transported drugs (vinblastine and verapamil). Separation was achieved on a C18 column with step-gradient elution (acetonitrile and aqueous solution of ammonium acetate, pH 3.0) at a flow rate of 1.0 mL/min and UV detection at 275 nm during the total run time of 35 min. The method was validated and found to be specific, linear, precise, and accurate. This chromatographic system can be readily used on a routine basis and its utilization can be extended to other permeability models. The results obtained in the Caco-2 bi-directional transport experiments confirmed the validity of the assay, given that high and low permeability profiles were identified, and P-glycoprotein functionality was established.

Viability of human periodontal ligament fibroblasts in milk, Hank's balanced salt solution and coconut water as storage media.

AIM: To evaluate the effectiveness of various storage media at 5 °C for maintaining the viability of human periodontal ligament fibroblasts (PDLF). METHODOLOGY: Plates with PDLF were soaked in recently prepared Hank's balanced salt solution (HBSS), skimmed milk, whole milk, Save-A-Tooth(®) system's HBSS (Save), natural coconut water, industrialized coconut water or tap water (negative control) at 5 °C for 3, 6, 24, 48, 72, 96 and 120 h. Minimum essential medium (MEM) at 37 °C served as the positive control. PDL cell viability was determined by MTT assay. Data were statistically analysed by Kruskal-Wallis test complemented by the Scheffé test (α=5%). RESULTS: The greatest number of viable cells was observed for MEM. Skimmed and whole milk, followed by natural coconut water and HBSS, were the most effective media in maintaining cell viability (P<0.05). From 24 to 120 h, Save, industrialized coconut water and tap water were the worst storage media. CONCLUSIONS: Skimmed and whole milk had the greatest capacity to maintain PDLF viability when compared with natural coconut water, HBSS, Save, industrialized coconut water and tap water.

Assessment of adenovirus, hepatitis A virus and rotavirus presence in environmental samples in Florianopolis, South Brazil.

AIMS: To assess the presence of human adenovirus (HAdV), hepatitis A (HAV) virus and rotavirus A (RV-A) in environmental samples from the Southern region of Brazil and to provide viral contamination data for further epidemiological studies and governmental actions. METHODS AND RESULTS: Water samples from various sources (seawater, lagoon brackish water, urban wastewater, drinking water sources-with and without chlorination and water derived from a polluted creek) and oysters of two growing areas were analysed by enzymatic amplification (nested PCR and RT-PCR), quantification of HAdV genome (qPCR) and viral viability assay by integrated cell culture-PCR (ICC-PCR). From June 2007 to May 2008 in a total of 84 water samples, 54 (64·2%) were positive for HAdV, 16 (19%) for RV-A and 7 (8·3%) for HAV. Viability assays showed nonpositive samples for HAV; though, infectious viruses were confirmed for RV-A (12·5%) and HAdV (88·8%). Oyster samples by PCR were positive for HAdV (87·5%) and RV-A (8·3%), but none for HAV. Quantitative PCR in oysters showed means loads in genomic copies (gc) of 9·1 × 10(4) gc g(-1) (oyster farm south) and 1·5 × 10(5) gc g(-1) (oyster farm north) and in waters ranging from 2·16 × 10(6) (lagoon water) to 1·33 × 10(7) gc l(-1) (untreated drinking water). CONCLUSIONS: This study has shown a widespread distribution of the analysed viruses in this particular region with high loads of HAdV in the environment which suggests the relevance of evaluating these viruses as positive indicators of viral contamination of water. SIGNIFICANCE AND IMPACT OF THE STUDY: The environmental approach in this study provides data concerning the prevalence, viability and quantification of enteric viruses in environmental waters and oysters in the South region of Brazil and has indicated that their presence might pose a risk to population in contact with the environmental samples searched.

In vitro antiherpes effects of a C-glycosylflavonoid-enriched fraction of Cecropia glaziovii Sneth.

AIMS: To investigate the in vitro antiherpes effects of the crude aqueous extract obtained from Cecropia glaziovii leaves and their related fractions, the n-butanol fraction (n-BuOH) and the C-glycosylflavonoid-enriched fraction (MeOH(AMB)), and to determine the viral multiplication step(s) upon which this C-glycosylflavonoid-enriched fraction acts. METHODS AND RESULTS: The antiviral activity was evaluated against human herpes virus types 1 and 2 (HHV-1, HHV-2) by plaque reduction assay. The mode of action of the most active fraction was investigated by a set of assays, and the results demonstrated that MeOH(AMB) fraction exerts anti-herpes action by the reduction of viral infectivity (only against HHV-2); by the inhibition of virus entry into cells; by the inhibition of cell-to-cell virus spread as well as by the impaired levels of envelope proteins of HHV-1. The high-performance liquid chromatography (HPLC)-photo-diode array (PDA) analysis showed that the C-glycosylflavonoids are the major constituents of this fraction. CONCLUSIONS: These data showed that the MeOH(AMB) fraction has an antiviral activity against HHV types 1 and 2. The C-glycosylflavonoids are the major constituents of this fraction, which suggests that they could be one of the compounds responsible for the detected anti-herpes activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The MeOH(AMB) fraction can be regarded as a phytopharmaceutical candidate for the treatment of herpetic infections.

Detection of enteric viruses in sewage sludge and treated wastewater effluent.

Sewage sludge and treated wastewater when contaminated with enteric virus and discharged into the environment, could pose a human health risk. The aim of study was to verify the presence and viability of enteric viruses in sewage sludge and treated wastewater at a local sewage plant in Florianopolis city, Brazil. Sewage sludge was concentrated by organic flocculation and polyethylene glycol precipitation and wastewater by electronegative membrane filtration and ultrafiltration by Centriprep Concentrator. Adenovirus (AdV), hepatitis A virus (HAV), and Rotavirus (RV) were examined for all samples for 12 months and Poliovirus (PV) was also tested for in sewage sludge samples. AdV was the most prevalent in both kind of samples, followed by RV, PV (in sludge) and HAV. Viral viability by cell culture (ICC-PCR) was: AdV: 100%, HAV: 16.7%, PV: 91.7%, RV: 25% in sludge and AdV: 66.6%, HAV: 66.6% and RV: 0% in wastewater. IFA for AdV in sludge ranged from 70 to 300 FFU/ml. QPCR for AdV ranged from 4.6 x 10(4) to 1.2 x 10(6) and from 50 to 1.3 x 10(4) gc/ml in sludge and wastewater, respectively. HAV quantification in sludge ranged from 3.1 x 10(2) to 5.4 x 10(2) gc/ml. In conclusion, it was possible to correlate presence and viability of enteric viruses in the environmental samples analyzed.

Evaluation of HA negatively charged membranes in the recovery of human adenoviruses and hepatitis A virus in different water matrices.

Human adenoviruses (HAdV) and hepatitis A virus (HAV) are shed in the faeces and consequently may be present in environmental waters, resulting in an increase in pathogen concentration that can affect water quality and human health. The aim of this study was to evaluate an adsorption-elution method which utilizes negatively charged membrane HA to determine the efficient recovery of HAdV and HAV from different water matrices and to combine this procedure with a qualitative molecular method (nested RT-PCR and nested PCR). The best efficiency recovery was achieved in distilled water and treated wastewater effluent (100%) for both viruses and in recreational lagoon water for HAV (100%). The efficiency recovery was 10% for HAdV and HAV in seawater and 10% for HAdV in lagoon water. The viral detection limit by nested PCR for HAV in water samples ranged between 20-0.2 FFU/mL and 250 and 25 TCID50/mL for HAdV. In conclusion, these results suggest that the HA negatively charged membranes vary their efficiency for recovery of viral concentration depending upon the types of both enteric viruses and water matrices.

Antiviral activity-guided fractionation from Araucaria angustifolia leaves extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Araucaria angustifolia (Bert.) O. Kuntze (Araucariaceae) is a Brazilian medicinal plant traditionally used for the treatment of various illnesses including dried skin, wounds, shingles, and sexually transmitted diseases. AIM OF THE STUDY: The rationale of the study was to provide evidence of its antiherpes activity in order to confirm its popular use that could be related to herpes disease. MATERIALS AND METHODS: The crude hydroethanolic extract (HE) obtained from Araucaria angustifolia leaves was submitted to a sequential liquid-liquid extraction with solvents of increased polarity. The HE and fractions obtained were evaluated for cytotoxicity and antiherpes activity (Herpes Simplex Virus type 1) by MTT assay. The most active fractions were selected to perform an in vitro antiviral activity-guided chromatographic fractionation. RESULTS: The ethyl acetate (EA) and n-butanol (NB) fractions have shown the best results for antiherpetic activity and their further fractionation yielded 22 subfractions. From these subfractions, 14 were active, and the most potent antiherpetic activity was obtained for NB1-4 subfraction with selectivity index (SI) of 57.51. Chemical analysis of NB1-4 subfractions revealed the presence of proanthocyanidins and the known biflavonoids (bilobetin, II-7-O-methyl-robustaflavone and cupressuflavone). The same biflavonoids have been detected in EA subfractions. CONCLUSION: The present study has shown that the hydroethanolic extract from Araucaria angustifolia leaves as well as many different fractions and subfractions exhibited antiherpes activity, supporting the use of this plant species in folk medicine.

Comparative study of two extraction methods for enteric virus recovery from sewage sludge by molecular methods.

The aim of this study was to compare two nucleic acid extraction methods for the recovery of enteric viruses from activated sludge. Test samples were inoculated with human adenovirus (AdV), hepatitis A virus (HAV), poliovirus (PV) and rotavirus (RV) and were then processed by an adsorption-elution-precipitation method. Two extraction methods were used: an organic solvent-based method and a silica method. The organic-based method was able to recoup 20% of the AdV, 90% of the RV and 100% of both the PV and HAV from seeded samples. The silica method was able to recoup 1.8% of the AdV and 90% of the RV. These results indicate that the organic-based method is more suitable for detecting viruses in sewage sludge.

Comparative study of two extraction methods for enteric virus recovery from sewage sludge by molecular methods

The aim of this study was to compare two nucleic acid extraction methods for the recovery of enteric viruses from activated sludge. Test samples were inoculated with human adenovirus (AdV), hepatitis A virus (HAV), poliovirus (PV) and rotavirus (RV) and were then processed by an adsorption-elution-precipitation method. Two extraction methods were used: an organic solvent-based method and a silica method. The organic-based method was able to recoup 20 percent of the AdV, 90 percent of the RV and 100 percent of both the PV and HAV from seeded samples. The silica method was able to recoup 1.8 percent of the AdV and 90 percent of the RV. These results indicate that the organic-based method is more suitable for detecting viruses in sewage sludge.