Saleem Shahzad

Antiproliferative Activity and Characterization of Metabolites of Aspergillus nidulans: An Endophytic Fungus from Nyctanthes arbor-tristis Linn. against Three Human Cancer Cell Lines

BACKGROUND Endophytic fungi are receiving attention as sources of structurally novel bioactive secondary metabolites towards drug discovery from natural products. This study reports the isolation and characterization of secondary metabolites from an endophytic fungus Aspergillus nidulans, associated with Nyctanthes arbor-tristis Linn., a plant which has a traditional use to cure many ailments including cancer. OBJECTIVE The objective of this study was to evaluate the antiproliferative activity of the metabolites of A. nidulans from N. arbor-tristis on three human cancer cell lines, lung (NCI-H460), breast (MCF-7) and uterine cervix (HeLa), and carry out their characterization. METHODS The extracts of the endophytic fungus cultured on potato dextrose agar were subjected to various chromatographic techniques. Structures of pure compounds were determined using spectroscopic techniques. The non-polar constituents were analyzed by GC-MS. Antiproliferative activity was determined by sulforhodamine B (SRB) assay. RESULTS The extracts and fractions showed moderate to good growth inhibition of the aforementioned human cancer cell lines. The broth extract was most potent (IC50 = 10 ± 3.1 μg/mL and LC50= 95 ± 3.9) against HeLa whereas petroleum ether insoluble fraction of mycelium was most active against NCI-H460 and MCF-7 (IC50 = 10 ± 2.1 µg/mL and 18 ± 3.1 µg/mL respectively). GC-MS led to identify 12 compounds in mycelium and 19 compounds in broth. Four pure compounds were isolated and characterized one compound 5, 10-dihydrophenazine-1-carboxylic acid (1) from broth and three 1-hydroxy-3-methylxanthone (2), ergosterol (3) and sterigmatocystin (4) from mycelium. 1 has not been reported earlier as a plant/fungal metabolite while 2-4 are new from this source. Sterigmatocystin exhibited growth inhibitory effect (IC50 = 50 ± 2.5 µM/mL) against only MCF-7 cell line whereas other compounds had IC50 > 100. CONCLUSIONS In this paper, the cytotoxicity of mycelium and broth constituents of endophytic fungus Aspergillus nidulans from Nyctanthes arbor-tristis is reported for the first time. The study shows that fungus Aspergillus nidulans from Nyctanthes arbor-tristis is capable of producing biologically active natural compounds and provides a scientific rationale for further chemical investigations of endophyte-producing natural products.

Valuable additions to the diversity of mushrooms in Gilgit-Baltistan, Pakistan

T prevalence of invasive fungal infections (IFIs) has grown in last decades. With the increasing number of immunocompromised patients, Aspergillus fungi have become known as one of the major causes of human infection. The pathogenesis of invasive aspergillosis is intrinsically linked to the competency of the host immune system and its ability to recognize and destroy the fungus. Innate immunity plays an essential role in host defense against invasive aspergillosis. Developing new antifungal agents is restricted cause of eukaryotic nature of fungi. Therefore interest in application of natural immunostimulators to increase immune responses against pathogens has expanded. Honey is a natural substance produced by honey bees. Studies indicated that honey has different health-beneficial properties including gastro protective, hepatoprotective, reproductive, hypoglycemic, antioxidant, antihypertensive, antibacterial, anti-fungal, anti-inflammatory and wound healing effects. This work was designed to study the immunological effects of three Iranian honeys on some parts of innate immunity as well as survival rate in mice infected with Aspergillus fumigatus. For this purpose, mice were divided into 10 groups (honey alone, honey and infection, negative and positive controls) each containing 10 individuals. Mice were treated with honey (1.5 g/kg BW/orally) for 10 days. At day 6, Aspergillus fumigatus conidia (5×105/ml) were injected intravenously to the infection groups. The animals were sacrificed at day 11 and their peritoneal macrophages and spleen cells were cultured. Macrophage killing and nitric oxide production along with some proinflammatory cytokines (IL6, IL1β) were measured as well. TLR2 and TLR4 genes expression in macrophages was determined by semi-quantitative RT-PCR. Ten mice from infectious groups were kept for 30 days and survival rate was monitored. Based on the obtained results, all honeys could significantly increase killing of Aspergillus fumigatus conidia by macrophages and mean survival rate compared with control group (p<0.05). Honeys alone had immunomodulatory effect on proinflammatory cytokines, NO production and understudied genes. However by inducing infection, groups under honey treatment had higher cytokine and NO production and TLR2 gene expression compared to infection alone group (p<0.05). LPS stimuli had no significant effect on macrophages neither killing nor nitric oxide production (p>0.05). It is concluded that honey could boost the immune system and increase survival time in invasive aspergillosis; in order to illuminate the mechanism of action of honey on natural and adoptive immune responses during invasive aspergillosis, this study should be continued into the future.T use of embryonated egg as an alternative in the study of the pathogenesis of fungi is evolving, although murine models are “gold standard” however, these models are used to screen determinants of virulence among fungi species. This study was aimed at the study of the virulence potential of clinical (WM626) and environmental (EN028) isolate of Cryptococcus neoformans both belong to molecular types VNII in chick embryo infected via Chorio-allantioc membrane (CAM). At a concentration of 108 cfu per ml, the two strains had varying virulence potential on the embryo. Environmental strain EN028 achieved 100% mortality on day 5 of experiment whereas strain WM 626 did not cause 100% mortality. Histopathology of CAM from both strains revealed massive disruption of CAM cells in EN028 when compared to WM626. Also with decrease in the concentration of infectious does, infection without mortality was observed. Our study suggests that embryonated eggs is a useful alternative tool to pre-screen Cryptococcus neoformans strains to select strains for subsequent testing in murine models.

Effect of seed pelleting with biocontrol agents on growth and colonisation of roots of mungbean by root-infecting fungi.

BACKGROUND Mungbean (Vigna radiata (L.) Wilczek) is a leguminous pulse crop that is a major source of proteins, vitamins and minerals. Root-infecting fungi produce severe plant diseases like root rot, charcoal rot, damping-off and stem rot. The soil-borne pathogens can be controlled by chemicals, but these chemicals have several negative effects. Use of microbial antagonist such as fungi and bacteria is a safe, effective and eco-friendly method for the control of many soil-borne pathogens. Biological control agents promote plant growth and develop disease resistance. Application of bacteria and fungi as seed dressing suppressed the root-infecting fungi on leguminous crops. RESULTS Seeds of mungbean were pelleted with different biocontrol agents to determine their effect on plant growth and colonisation of roots by root-infecting fungi, viz. Fusarium solani, Macrophomina phaseolina, Pythium aphanidermatum, Rhizoctonia solani and Sclerotium rolfsii. Treatment of mungbean seeds with fungal antagonists showed more shoot and root length as compared to bacterial antagonists, whereas seed treated with bacterial antagonists showed maximum shoot and root weight. Trichoderma harzianum and Bacillus subtilis were the best among all the biocontrol agents since they provided the highest plant growth and greater reduction in root colonisation by all root-infecting fungi. Bacillus cereus, Trichoderma virens, Pseudomonas fluorescens and Micrococcus varians were also effective against root-infecting fungi but to a lesser extent. T. harzianum, T. virens, B. subtilis and P. fluorescens were found to be best among all biocontrol agents. CONCLUSION The root-infecting fungi can be controlled by pelleting seeds with biocontrol agents as it is safe and effective method. Additionally, plant growth was promoted more by this method.