Microbiological characterization of stormwater in a high-income neighborhood in Rio de Janeiro, Brazil.

Stormwater harvesting and reuse in the urban environment is emerging as an alternative water source, despite human pathogens in the stormwater may represent a hazard to public health. This study presents the results of 1-year monitoring to evaluate the quality of stormwater obtained in a high-income neighborhood in Rio de Janeiro for a set of microbiological parameters as total coliforms, Escherichia coli (E. coli), human adenovirus (HAdV), human JC polyomavirus (JCPyV), Group A rotavirus (RVA), and norovirus GI and GII. Forty-eight stormwater samples obtained from two multiplex units presented total coliforms and E. coli in 91.7% (n = 44) and 58.3% (n = 28) of samples, while HAdV and JCPyV were detected in 20.8% (n = 10) and 12.5% (n = 6), respectively. Viral quantification ranged from 103 to 104 genomic copies/liter (GC/L) for HAdV and from 101 to 104 GC/L for JCPyV. Neither RVA nor norovirus GI and GII was detected. Fifteen out of sixteen (93.8%) samples containing viruses were compliant as per fecal indicator bacteria (FIB) according to Brazilian standards for rainwater reuse and US EPA Guidelines for Water Reuse, suggesting that viruses monitoring should complement the study of bacterial indicators.

Anti-inflammatory effect of leaves of Vernonia zeylanica in lipopolysaccharide-stimulated RAW 264.7 macrophages and carrageenan-induced rat paw-edema model.

ETHNOPHARMACOLOGICAL RELEVANCE: Vernonia zeylanica (L.) Less (Family: Compositae) is a medicinal plant used as external applications for boils, bone fractures, eczema and internally for asthma in traditional medicine in Sri Lanka. Anti-nociceptive, anti-bacterial and anti-proliferative activities have been reported previously. AIM OF THE STUDY: To investigate the anti-inflammatory activity of methanol/dichloromethane extract (MDE) of leaves of V. zeylanica by assessing in vivo inhibition of rat paw-edema, in vitro inhibition of the production of nitric oxide (NO) and superoxide and inhibitory effect on inducible nitric oxide synthase (iNOS) gene expression. MATERIALS AND METHODS: In vivo anti-inflammatory activity of MDE was tested at the dose of 1500 mg/kg using rat paw-edema model. Indomethacin and Gum acacia was used as the positive and vehicle control respectively. In vitro NO inhibitory activity of 7.8-250 µg/ml MDE was tested using lipopolysaccharide (LPS)-stimulated (1 µg/ml) mouse macrophages (RAW264.7 cells) and rat peritoneal cells (RPC) obtained following carrageenan-induction (5 mg/Kg). Griess method was used to quantify the nitrite levels in culture supernatants. In vitro inhibition of superoxide production of Phorbal 12-myristate 13-acetate (PMA)-stimulated RAW cells was determined by quantitative Nitroblue Tetrazolium (NBT) assay. N-monomethyl-L-arginine acetate (NMMA) (1 mM) and Diphenyleneiodonium chloride (DPI) (10 µM) were used as the positive controls for inhibitory activity of NO and superoxide production respectively. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis was carried out to test the inhibitory effect on mRNA expression of iNOS gene. RESULTS: Treatment with MDE of V. zeylanica at 1500 mg/kg showed significant inhibition of paw-edema from 1st-5th hour (P < 0.01) compared with the control. The reference drug, indomethacin showed a biphasic pattern and its highest inhibition was (98.3 ± 7.1%) at 4th h (P < 0.01). MDE of V. zeylanica showed similar inhibition of paw-edema with highest inhibition recorded as 94.5 ± 5.28%, at 5th h (P < 0.01). The inhibitory concentration (IC50) of MDE for in vitro NO inhibitory activity was 105 µg/ml for RAW cells and 80 µg/ml for RPCs. Both NO inhibitory activities showed significant dose-dependency (r = 0.998 and r = 0.915 respectively; p < 0.05). MDE concentration of 250 µg/ml showed 55% inhibition of ROS production in RAW cells. NMMA showed 78% and 70.1% inhibition of NO production with RAW cells and RPCs whereas DPI showed 61% superoxide inhibitory activity with RAW cells. NO inhibitory activity of MDE on RAW cells was confirmed by the significant reduction (99.1%) in iNOS gene expression. CONCLUSION: These results demonstrated potent anti-inflammatory activity of MDE of V. zeylanica reflected by its significant in vivo inhibition of rat paw-edema, in vitro inhibition of NO and superoxide production, and the reduction of iNOS gene expression. Thus, further purification and isolation of bioactive compounds from V. zeylanica are emphasized.

Screening for bioactive secondary metabolites in Sri Lankan medicinal plants by microfractionation and targeted isolation of antimicrobial flavonoids from Derris scandens.

ETHNOPHARMACOLOGICAL RELEVANCE: Sri Lanka is known to have very diverse flora. Many of these species are used for plant-based remedies, which form the integral part of two Sri Lankan systems of traditional medicine, Ayurveda and Deshiya Chikitsa. Despite their widespread use, only a limited number of studies have probed into the scientific evidence for bioactivity of these medicinal plants. Such studies rarely progress to the identification of bioactive natural products. AIM OF THE STUDY: The primary aim was to develop a bioactivity screening method and apply it to 50 Sri Lankan medicinal plants where antimicrobial properties could be relevant for its traditional use. The subsequent aim was the progression into defining and characterising potent isolates within targeted compound classes from such plants, i.e. Derris scandens and its antimicrobial flavonoids. MATERIAL AND METHODS: The plant collection comprised 24 species of Fabaceae, 15 Rubiaceae, 7 Solanaceae and 4 Cucurbitaceae plants. These 50 species were collected based on their ethnopharmacological importance and use in Sri Lankan traditional medicine. Crude extracts from each species were initially subjected to radial disc diffusion and microdilution assays. Subsequently, aqueous extracts of all plants were microfractionated in deep well plates using reversed-phase HPLC. Fractions were tested for antibacterial and cytotoxic activities and masses of target bioactive compounds were identified using mass spectrometry. Bioactive compounds with the masses identified through microfractions were isolated from Derris scandens using reversed-phase HPLC. The isolated pure compounds were characterised using LC-MS and NMR. RESULTS: Crude aqueous extracts from 19 species showed activity against Gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) in the radial disc diffusion assay. Crude aqueous extracts from 34 plant species and organic extracts from 46 plant species were active against S. aureus (≤4 mg mL-1) in the microdilution assay. Microfractionation demonstrated antibacterial activity for 19 plants and cytotoxicity for 6 plants. Furthermore, target bioactive compounds and their molecular ions were identified during microfractionation. Dalpanitin and vicenin-3, two of the flavonoids isolated from Derris scandens gave MICs of 23 µg mL-1 against S. aureus. Dalpanitin also exhibited relevant MICs on Gram-negative bacteria (94 µg mL-1 against Escherichia coli and Pseudomonas aeruginosa). CONCLUSION: The microfractionation protocol developed in this study enabled time-efficient screening of many plants species, using a small quantity of sample material. In addition, microfractionation served as a guiding tool for identifying individual antimicrobial compounds. Through this process, flavonoids were isolated from Derris scandens, out of which dalpanitin and vicenin-3 showed activity in the low micromolar range. The high hit rate for in vitro antibacterial properties from this ethnopharmacologically guided sample collection gives credence to Sri Lankan traditional herbal medicine as a source for drug discovery.

Antifeedant, contact toxicity and oviposition deterrent effects of phyllostine acetate and phyllostine isolated from the endophytic fungus Diaporthe miriciae against Plutella xylostella larvae.

BACKGROUND: Larvae of the diamond back moth (DBM), Plutella xylostella, are destructive cabbage pests causing economic losses worldwide. Continuous application of synthetic pesticides to control this pest has resulted in environmental pollution and resistant pest strains. Thus, there is a crucial need to seek natural alternatives with minimal detrimental effects. This study was designed to investigate the antifeedant activities of endophytic fungi of Cyperus iria and to determine the antifeedant, contact toxicity and oviposition deterrent activities of phyllostine acetate and phyllostine of the endophytic Diaporthe miriciae fungus. RESULTS: Two cyclohexeneoxidediones, phyllostine acetate (1) and phyllostine (2), isolated from an ethyl acetate extract of D. miriciae exhibited strong antifeedant, contact toxicity, and oviposition deterrent activities against P. xylostella. Phyllostine acetate (1) and phyllostine (2) showed feeding deterrent indexes of 100% at 50 µg cm-2 in the no-choice leaf disc assay and 50% feeding deterrence (DC50 ) values of 9 and 4.7 µg cm-2 respectively. The median lethal concentration (LC50 ) values of phyllostine acetate (1) and phyllostine (2) were 4.38 and 6.54 µg/larva in the contact toxicity assay. The oviposition deterrent indexes of the two compounds were 100% for phyllostine acetate (1) and 28.6% for phyllostine (2) at 50 µg cm-2 . CONCLUSION: Phyllostine acetate and phyllostine show promise as compounds for the control of P. xylostella. This study encourages further investigation of endophytic fungi of the family Cyperacea, for the development of natural pest control agents in agriculture. © 2019 Society of Chemical Industry.

Stable isotope feeding studies reveal a steroid 5(6→7)abeo ring contraction biogenesis for the antibiotic solanioic acid produced by cultures of the fungus Rhizoctonia solani.

Culture feeding experiments with [1-13C]-acetate, [2-13C]- acetate, and [1,2-13C]-acetate have shown that the steroid ring B contraction involved in the biogenesis of the unprecedented carbon skeleton of the antibiotic solanioic acid (1) by the fungus Rhizoctonia solani involves cleavage of the C-5/C-6 bond. The study revealed that 9-epi-solanioic acid (4), which spontaneously converts to solanioic acid (1), is also produced by the cultures and it may be the actual natural product.

Identification of Novel HIV-1 Latency-Reversing Agents from a Library of Marine Natural Products.

Natural products originating from marine and plant materials are a rich source of chemical diversity and unique antimicrobials. Using an established in vitro model of HIV-1 latency, we screened 257 pure compounds from a marine natural product library and identified 4 (psammaplin A, aplysiatoxin, debromoaplysiatoxin, and previously-described alotaketal C) that induced expression of latent HIV-1 provirus in both cell line and primary cell models. Notably, aplysiatoxin induced similar levels of HIV-1 expression as prostratin but at up to 900-fold lower concentrations and without substantial effects on cell viability. Psammaplin A enhanced HIV-1 expression synergistically when treated in combination with the protein kinase C (PKC) activator prostratin, but not the histone deacetylase inhibitor (HDACi) panobinostat, suggesting that psammaplin A functions as a latency-reversing agent (LRA) of the HDACi class. Conversely, aplysiatoxin and debromoaplysiatoxin synergized with panobinostat but not prostratin, suggesting that they function as PKC activators. Our study identifies new compounds from previously untested marine natural products and adds to the repertoire of LRAs that can inform therapeutic "shock-and-kill"-based strategies to eliminate latent HIV-infected reservoirs.

Serpulanines A to C, N-Oxidized Tyrosine Derivatives Isolated from the Sri Lankan Fungus Serpula sp.: Structure Elucidation, Synthesis, and Histone Deacetylase Inhibition.

Serpulanines A (1), B (2), and C (3) have been isolated from extracts of the rare Sri Lankan macrofungus Serpula sp. The structures of 1, 2, and 3 were elucidated by a combination of spectroscopic and single-crystal X-ray diffraction analyses. Serpulanines A (1) and B (2) both contain the rare (E)-2-hydroxyimino hydroxamic acid functional group array. A proposed biogenesis for serpulanine B (2) suggests that its (E)-2-hydroxyimino hydroxamic acid moiety arises from a diketopiperazine precursor. Synthetic serpulanine A (1) inhibited class I/II histone deacetylases in murine metastatic lung carcinoma cells with an IC50 of 7 µM.

Protective Effects of Six Selected Dietary Compounds against Leptin-Induced Proliferation of Oestrogen Receptor Positive (MCF-7) Breast Cancer Cells.

BACKGROUND: Obesity is considered as one of the risk factors for breast cancer. Leptin has been found to be involved in breast cancer progression. Therefore, novel approaches to antagonize biological effects of leptin are much needed. The objective of this study was to evaluate the protective effects of six dietary compounds (quercetin, curcumin, gallic acid, epigallocatechin gallate (EGCG), ascorbic acid and catechin) and assess the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in leptin-stimulated MCF-7 breast cancer cells in vitro. METHODS: MCF-7 cells were exposed to leptin, leptin and compound and compound alone for 48 h. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT and fluorometric assays after 48 h incubation. Phosphorylation of ERK1/2 was quantified by ELISA. RESULTS: Only quercetin, curcumin and EGCG showed significant protective effects against leptin-induced proliferation of MCF-7 cells. Increase in ERK1/2 phosphorylation in response to leptin was reduced by the addition of quercetin, curcumin and EGCG. CONCLUSIONS: Considering the high prevalence of obesity, this observation provides a rationale for use of curcumin, quercetin and EGCG as antagonists of leptin in the treatment of obese breast cancer patients.

Chemical Transformations of the Fungal Meroterpenoid Dhilirolide A Reveal Skeletal Degradation and Rearrangement Reactions with Biosynthetic Implications.

Treatment of the fungal meroterpenoid dhilirolide A (1) with either sodium azide or perchloric acid results in conversion of the dhilirane carbon skeleton of 1 to the 14,15-dinordhilirane carbon skeleton of the products 5-7, with and without concomitant transfer of an acetyl residue to form a C-9 acetate ester. The discovery of these transformations, which are vinylogous retro-Claisen-type condensations, suggests an efficient biogenetic route to 14,15-dinordhiliranes such as dhilirolide K (3).

Anti-hepatocarcinogenic and Anti-oxidant Effects of Mangrove Plant Scyphiphora hydrophyllacea.

CONTEXT: Scyphiphora hydrophyllacea is a shrub mangrove plant of the family Rubiaceae and not yet been studied for anti-hepatocarcinogenic effects. OBJECTIVES: We investigated possible in vitro anti-hepatocarcinogenic and antioxidant properties of S. hydrophyllacea. MATERIALS AND METHODS: Dried leaves of S. hydrophyllacea were sequentially extracted into hexane, chloroform, ethyl acetate, and methanol and tested for cytotoxicity on HepG2 cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and sulforhodamine B assays, and for antioxidant activities by the free radical 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) assays. Total phenolic and flavonoid contents were estimated in all four extracts. The hexane and chloroform extracts were tested for pro-apoptotic properties in HepG2 cells, and bioactive components were identified by gas chromatography-mass spectrometry (GC-MS) analysis. RESULTS: The hexane and chloroform extracts showed dose-dependent and time-dependent cytotoxic effects. Morphological changes observed under fluorescence microscope related to apoptosis, and significant (P < 0.001) increases in caspase 3 and 9 levels were observed in hexane and chloroform extract-treated cells. Slight DNA fragmentation was observed only in response to the chloroform extract. mRNA expressions of p53 and Bax were significantly upregulated by low doses of hexane and chloroform extracts. Highest antioxidant activity was observed in the methanol extract. GC-MS profiles identified 24 and four major compounds in the hexane and chloroform extracts, respectively. These included some known anticancer compounds such as lupeol. CONCLUSION: Cytotoxicity, antioxidant effects, and apoptosis-related changes exerted by hexane and chloroform extracts of S. hydrophyllacea concluded that these two extracts are good source for isolation of possible anticarcinogenic compounds. SUMMARY: The hexane and chloroform extracts of Scyphiphora hydrophyllacea showed dose-dependent and time-dependent cytotoxic effects.Morphological changes related to apoptosis and significant (P < 0.001) increases in caspase 3 and 9 levels were observed in hexane and chloroform extract-treated cells.mRNA expressions of p53 and Bax were significantly upregulated by low doses of hexane and chloroform extracts.Highest antioxidant activity was observed in the methanol extract.GC-MS profiles identified 24 and four major compounds in the hexane and chloroform extracts, respectively. Abbreviation used: DPPH: 1,1-diphenyl-2-picryl-hydrazyl, ABTS: 2,2'-azinobis-3-ethylbenzthiazoline-6-sulfonic acid, GC-MS: gas chromatography-mass spectrometry, DNA: deoxyribonucleic acid, HCC: Hepatocellular carcinoma, GAE: gallic acid equivalents, SRB: sulforhodamine B, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, AO/EB: acridine orange/ethidium bromide, GAPDH: Glyceraldehyde 3-phosphate dehydrogenase, IC50: half maximal inhibitory concentration; QE: quercetin equivalents, HE: hexane extract, CE: chloroform extract, EAE: ethyl acetate extract, ME: methanolic extract, TPC: total polyphenol content, TFC: total flavonoid content, ANOVA: Analysis of variance.

Oleanolic acid from antifilarial triterpene saponins of Dipterocarpus zeylanicus induces oxidative stress and apoptosis in filarial parasite Setaria digitata in vitro.

Absence of a drug that kills adult filarial parasites remains the major challenge in eliminating human lymphatic filariasis (LF); the second leading cause of long-term and permanent disability. Thus, the discovery of novel antifilarial natural products with potent adulticidal activity is an urgent need. In the present study, methanol extracts of leaves, bark and winged seeds of Dipterocarpus zeylanicus (Dipterocarpaceae) were investigated for macro and microfilaricidal activity. Two antifilarial triterpene saponins were isolated from winged seed extracts by bioactivity guided chromatographic separation and identified using Nuclear Magnetic Resonance and mass spectroscopic analysis as oleanolic acid 3-O-ß-D- glucopyranoside (1) (IC50 = 20.54 µM for adult worms, 19.71 µM for microfilariae ) and oleanolic acid 3-O-α-L-arabinopyranoside (2) (IC50 = 29.02 µM for adult worms, 25.99 µM for microfilariae). Acid hydrolysis of both compounds yielded oleanolic acid (3) which was non or least toxic to human peripheral blood mono nuclear cells (Selectivity index = >10) while retaining similar macrofilaricidal (IC50 = 38.4 µM) and microfilaricidal (IC50 = 35.6 µM) activities. In adult female worms treated with 50 and 100 µM doses of oleanolic acid, condensation of nuclear DNA, apoptotic body formation and tissue damage was observed by using Hoechst 33342 staining, TUNEL assay and Hematoxylin and Eosin staining respectively. A dose dependent increase in caspase 3/CED3 activity and decrease in total protein content were also observed in these parasites. A dose dependant DNA fragmentation was observed in adult parasites and microfilariae. Decreased levels of reduced glutathione (GSH) and elevated levels of glutathione S transferase (GST), superoxide dismutase (SOD) and reactive oxygen species (ROS) were also observed in parasites treated with oleanolic acid indicating an oxidative stress mediated apoptotic event. Compound 3/oleanolic acid was thus identified as a potent and safe antifilarial compound in vitro.

Isolation of a new resorcinolic lipid from Mangifera zeylanica Hook.f. bark and its cytotoxic and apoptotic potential.

Mangifera zeylanica is a plant endemic to Sri Lanka and its bark has been used in traditional medicine to treat some cancers. This study was aimed to isolate potentially cytotoxic compound/s from the hexane extract of the bark of M. zeylanica by bio-activity guided fractionation. The structure of the isolated compound (1) was elucidated using 1H, 13C NMR and mass spectrometric techniques. Compound 1 was identified as a new resorcinolic lipid (5-((8Z, 11Z, 14Z)-hexatriaconta-8, 11, 14-trienyl) benzene-1,3-diol). Apoptotic potential of the isolated compound was determined only in MCF-7 (estrogen receptor positive) breast cancer cells to which it was more cytotoxic than to normal mammary epithelial cells. Oxidative stress markers [reactive oxygen species (ROS), glutathione levels (GSH) and glutathione-S-transferase (GSH)] were also determined in MCF-7 cells treated with compound 1. Treatment with compound 1 led to an increase in caspase 7 activity, morphological features of apoptosis and DNA fragmentation in MCF-7 cells. Furthermore, it also led to an increase in ROS and GST levels while depleting GSH levels. Results of this study suggest that isolated new resorcinolic lipid can induce apoptosis in MCF-7 cells, possibly via oxidative stress mechanism.

Cytotoxic effects of ergone, a compound isolated from Fulviformes fastuosus.

BACKGROUND: Mushrooms inspired the cuisines of many cultures and conventional medicaments for cancer. However, a substantial number of mushroom species are yet unexplored, possessing an unknown chemical, biological and pharmacological profiles. Fulviformes fastuosus is a terrestrial mushroom, which is commonly found in Sri Lankan woodlands. The current study was aimed at isolation and characterization of a potent cytotoxic compound from F. fastuosus and investigating the apoptotic effect induced by the active principle against cancer and normal cell lines. METHODS: Bioactivity guided isolation of active principles from the methanol extract of F. fastuosus was performed by a rapid extraction and isolation method using different chromatographic techniques. Potential cytotoxic compound was identified using one and two dimensional nuclear magnetic resonance spectroscopy and mass spectrometry. Isolated compound was screened for in vitro cytotoxicity against Hepatocellular carcinoma (HepG-2), Muscle rhabdomyosarcoma (RD) and Rat Wistar liver normal (CC-1) cell lines using 3 4, 5-(dimethylthiazol-2-yl) 2-5-diphenyl tetrazolium bromide (MTT) cell viability assay. Apoptotic features of cells were observed via microscopic examination and ethidium bromide/acridine orange fluorescent staining. RESULTS: The interpretation of spectral data resulted in the identification of the chemical structure as ergosta-4,6,8 (14),22-tetraen-3-one (ergone). Ergone exhibited promising cytotoxic properties against RD cells with less cytotoxicity effect on CC-1 cells. In addition, ergone also possesses a strong cytotoxic effect against HepG-2 cells showing low toxic level for CC-1 cells. Apoptotic features of treated cells were detected via morphological characterization and ethidium bromide/acridine orange staining. CONCLUSION: The present study elaborates the isolation of a potent cytotoxic compound; ergone, from F. fastuosus via a rapid and efficient isolation method. Importantly, ergone has exhibited greater cytotoxic activity against RD cells with high selectivity index compared to cytotoxicity against HepG-2 cells. Ergone can be used in the development of therapeutic strategies for curbing rhabdomyosarcoma.

Rhizome extracts of Curcuma zedoaria Rosc induce caspase dependant apoptosis via generation of reactive oxygen species in filarial parasite Setaria digitata in vitro.

Human lymphatic filariasis (LF) is mainly caused by filarial parasite Wuchereria bancrofti and is the second leading cause of long term and permanent disability in tropical countries. To date, incapability to eliminate long lived adult parasites by current drugs remains the major challenge in the elimination of LF. Hence, in the current study, the efficacy of rhizome extracts of Curcuma zedoaria (a plant traditionally used in Sri Lanka in the management of LF) was evaluated as an effective filaricide in vitro. Sequential solvent extracts of C. zedoaria rhizomes were screened for in vitro antifilarial activity at 0.01-1 mg/mL concentrations by motility inhibition assay and 3-(4, 5 dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium bromide (MTT) reduction assay using cattle parasite Setaria digitata as a model organism. Exposure of parasites to hexane and chloroform extracts of C. zedoaria caused a dose dependant reduction in motility and viability of microfilariae (IC50 = 72.42 µg/mL for hexane extract, 191.14 µg/mL for chloroform extract) and adult parasites (IC50 = 77.07 µg/mL for hexane extract, 259.87 µg/mL for chloroform extract). Both extracts were less toxic to human peripheral blood mononuclear cells when compared to filariae. A dose dependant increase in caspase 3/CED 3 and a decrease in total protein content, cyclooxygenase (COX) and protein tyrosine phosphatase (PTP) activities were observed in adult parasites treated with hexane or chloroform extract. A significant degree of chromatin condensation and apoptotic body formation were also observed in these worms by Hoechst 33342 and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) staining respectively. Dose dependant chromosomal DNA laddering was observed in treated adult worms but not in microfilariae in response to both extracts. Oxidative stress parameters such as reduction in reduced glutathione (GSH) levels and increase in glutathione s transferase (GST), superoxide dismutase (SOD) and catalase activities, increased reactive oxygen levels (ROS) and lipid peroxidation were also observed indicating that an apoptotic event is induced by reactive oxygen species.

New halogenated constituents from Mangifera zeylanica Hook.f. and their potential anti-cancer effects in breast and ovarian cancer cells.

ETHNOPHARMACOLOGICAL RELEVENCE: Mangifera zeylanica Hook.f. (Anacardiaceae) is a plant endemic to Sri Lanka. Its bark has been used in traditional and Ayurvedic medicine for the treatment of various diseases including some cancers. AIM OF THE STUDY: This study was planned to isolate and identify potentially cytotoxic compounds from the bark of M. zeylanica, which may have contributed to its ethno pharmacological use in the treatment of cancer. MATERIALS AND METHODS: The chloroform extract of M. zeylanica bark which is cytotoxic to breast and ovarian cancer cells was fractionated using column chromatography and preparative reversed phase high performance liquid chromatography to isolate four compounds. Structures of the isolated compounds were elucidated by means of (1)H- and (13)C NMR spectroscopy, and mass spectrometric techniques. Cytotoxic potential of the isolated compounds was tested in MDA-MB-231 (triple negative breast cancer), MCF-7 (estrogen receptor positive breast cancer), SKOV-3 (ovarian epithelial cancer) and MCF-10A (normal mammary epithelial) cells by SRB assay. Human cancer drug target real-time PCR array was carried out to analyze regulation of possible cancer drug target genes in compound 2 treated triple negative breast cancer cells. DPPH radical scavenging and caspase 3 and 7 induction in response to isolated compounds were also studied. RESULTS: Two new halogenated compounds, bromomangiferic acid (1), and chloromangiferamide (2) along with two known compounds quercetin (3), and catechin (4), were isolated from the bark of Mangifera zeylanica for the first time. Interestingly, chloromangiferamide showed cytotoxicity only to triple negative breast cancer cells [IC50:73.19±0.87µM (24h), 56.29±0.86µM (48h)] with no cytotoxicity to other two cancer cell lines or to normal mammary epithelial cells. Quercetin and catechin were cytotoxic to all three cancer cell lines while bromomangiferic acid had no effect. Chloromangiferamide significantly regulated expression of genes associated with apoptosis, drug metabolism, cell cycle, receptor tyrosine kinase signaling, protein kinases, histone deacetylases, growth factors and receptors, topoisomerases, PI-3 kinases and phosphatases in triple negative breast cancer cells. CONCLUSION: Selective cytotoxic activity in triple negative breast cancer cells and regulation of some cancer drug target genes by chloromangiferamide indicate that it can be used to develop a potential chemotherapeutic agent for triple negative breast cancer cells.

Antimicrobial activities of endophytic fungi of the Sri Lankan aquatic plant Nymphaea nouchali and chaetoglobosin A and C, produced by the endophytic fungus Chaetomium globosum.

Twenty distinct endophytic fungi were isolated from the surface-sterilized plant parts of Nymphaea nouchali and were identified using morphological and molecular techniques. At 300 µg/disc concentration, eight of the 20 fungal extracts exhibited antimicrobial activities against Staphylococcus aureus (ATCC 25923) and Bacillus cereus (ATCC 11778) while two within the eight showed activity against Pseudomonas aeruginosa (ATCC 9027) and Escherichia coli (ATCC 35218). Furthermore, investigation of the crude extract of Chaetomium globosum resulted in the isolation of two known cytochalasans, chaetoglobosin A and C, and their structures were elucidated and confirmed by mass and nuclear magnetic resonance (NMR) (1H, 13C, COSY, HSQC, HMBC and tROESY) spectral data. Chaetoglobosin A showed antibacterial activities against Bacillus subtilis (MIC 16 µg mL-1), Staphylococcus aureus (MIC 32 µg mL-1) and methicillin-resistant Staphylococcus aureus (MRSA, MIC 32 µg mL-1). This is the first study to report the isolation, identification and antimicrobial properties of endophytic fungi of N. nouchali in Sri Lanka.

Antimicrobial activities of endophytic fungi obtained from the arid zone invasive plant Opuntia dillenii and the isolation of equisetin, from endophytic Fusarium sp.

BACKGROUND: Opuntia dillenii is an invasive plant well established in the harsh South-Eastern arid zone of Sri Lanka. Evidence suggests it is likely that the endophytic fungal populations of O. dillenii assist the host in overcoming biotic and abiotic stress by producing biologically active metabolites. With this in mind there is potential to discover novel natural products with useful biological activities from this hitherto poorly investigated source. Consequently, an investigation of the antimicrobial activities of the endophytes of O. dillenii, that occupies a unique ecological niche, may well provide useful leads in the discovery of new pharmaceuticals. METHODS: Endophytic fungi were isolated from the surface sterilized cladodes and flowers of O. dillenii using several nutrient media and the antimicrobial activities were evaluated against three Gram-positive and two Gram-negative bacteria and Candida albicans. The two most bioactive fungi were identified by colony morphology and DNA sequencing. The secondary metabolite of the endophyte Fusarium sp. exhibiting the best activity was isolated via bioassay guided chromatography. The chemical structure was elucidated from the ESIMS and NMR spectroscopic data obtained for the active metabolite. The minimum inhibitory concentrations (MICs) of the active compound were determined. RESULTS: Eight endophytic fungi were isolated from O. dillenii and all except one showed antibacterial activities against at least one of the test bacteria. All extracts were inactive against C. albicans. The most bioactive fungus was identified as Fusarium sp. and the second most active as Aspergillus niger. The structure of the major antibacterial compound of the Fusarium sp. was shown to be the tetramic acid derivative, equisetin. The MIC's for equisetin were 8 µg mL(-1) against Bacillus subtilis, 16 µg mL(-1) against Staphylococcus aureus and Methicillin Resistant Staphylococcus aureus (MRSA). CONCLUSIONS: O. dillenii, harbors several endophytic fungi capable of producing antimicrobial substances with selective antibacterial properties. By producing biologically active secondary metabolites, such as equisetin isolated from the endophytic Fusarium sp., the endophytic fungal population may be assisting the host to successfully withstand stressful environmental conditions. Further investigations on the secondary metabolites produced by these endophytes may provide additional drug leads.

Solanioic Acid, an Antibacterial Degraded Steroid Produced in Culture by the Fungus Rhizoctonia solani Isolated from Tubers of the Medicinal Plant Cyperus rotundus.

Solanioic acid (1), a degraded and rearranged steroid that exhibits in vitro antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), has been isolated from laboratory cultures of the fungus Rhizoctonia solani obtained from tubers of the plant Cyperus rotundus collected in Sri Lanka. The structure of solanioic acid (1) was elucidated by detailed analysis of NMR data, a single crystal X-ray diffraction analysis of a reduction product 2, and Mosher ester analysis on a derivative of the natural product. Solanioic acid (1) has an unprecedented carbon skeleton.

Helvolic acid, an antibacterial nortriterpenoid from a fungal endophyte, Xylaria sp. of orchid Anoectochilus setaceus endemic to Sri Lanka.

An endophytic fungus was isolated from surface sterilized leaf segments of Anoectochilus setaceus, an orchid endemic to Sri Lanka, and was identified as Xylaria sp. by morphological characters and DNA sequencing. Bioassay-guided chromatographic fractionation of the organic extract of a laboratory culture of this fungus led to the isolation of the known antibacterial helvolic acid. Helvolic acid was active against the Gram-positive bacteria, Bacillus subtilis [minimal inhibitory concentrations (MIC), 2 µg mL-1] and methicillin-resistant Staphylococcus aureus (MIC, 4 µg mL-1).

Dhilirolides E-N, meroterpenoids produced in culture by the fungus Penicillium purpurogenum collected in Sri Lanka: structure elucidation, stable isotope feeding studies, and insecticidal activity.

Extracts of laboratory cultures of the fungus Penicilium purpurogenum obtained from rotting fruit of the tree Averrhoa bilimbi growing in Sri Lanka have yielded 10 new meroterpenoids, dhilirolides E-N (5-14). The structures of the new dhilirolides have been elucidated by analysis of spectroscopic data and a single-crystal X-ray diffraction analysis of dhilirolide L (12). Dhilirolides A-N (1-14) represent the four unprecedented and rearranged dhilirane, isodhilirane, 14,15-dinordhilirane, and 23,24-dinorisodhilirane meroterpenoid carbon skeletons. Stable isotope feeding studies have confirmed the meroterpenoid biogenetic origin of the dhilirolides and provided support for a proposed genesis of the new carbon skeletons. Dhilirolide L (12) showed significant feeding inhibition and sublethal developmental disruption in the cabbage looper Trichoplusia ni, an important agricultural pest, at low concentrations.