Nighat Fatima

Encapsulation of Ellipticine in poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) based nanoparticles and its in vitro application.

Biodegradable, biocompatible, renewable and non-toxic polyhydroxyalkanoates (PHAs) based nanoparticles are the novel nanotherapeutic tool which are used for the encapsulation of antineoplastic drugs for cancer therapy. In this study, poly-3-hydroxybutyrate-co-5 mol% 3-hydroxyvalerate (PHBV-S), poly-3-hydroxybutyrate-co-11 mol% 3-hydroxyvalerate (PHBV-11) and poly-3-hydroxybutyrate-co-15 mol% 3-hydroxyvalerate (PHBV-15) were used as a nanocarrier for encapsulation of Ellipticine (EPT). EPT is a model anticancer drug. Physicochemical characteristics such as particle size, its morphology and zeta potential of blank and EPT loaded PHBV-S, PHBV-11 and PHBV-15 nanoparticles were studied. In vitro cytotoxicity tests confirmed that the blank PHBV-S, PHBV-11 and PHBV-15 nanoparticles were demonstrating significant biocompatibility without affecting the survival of cancer cell line A549. The loading efficiency of EPT in PHBV nanoparticles was observed in the range of 39.32 to 45.65%. The % inhibition of cancer cell line A549 ranged from 64.28 to 67.77% in comparison to EPT alone in which % inhibition found to be ≤45.11%. The IC50 value for each of three different formulations of EPT loaded PHBV nanoparticles ranged from 1.00 to 1.31 μg/mL. The order of % inhibition of cancer cell line A549 for drug loaded nanoparticles was EPT-PHBV-15>EPT-PHBV-S>EPT-PHBV-11. This system had demonstrated a great potential to increase the cytotoxic effect of EPT by increasing its bioavailability.

Isolation and characterization of a biosurfactant-producing Fusarium sp. BS-8 from oil contaminated soil

This study reports characterization of a biosurfactant‐producing fungal isolate from oil contaminated soil of Missa Keswal oil field, Pakistan. It was identified as Fusarium sp. BS‐8 on the basis of macroscopic and microscopic morphology, and 18S rDNA gene sequence homology. The biosurfactant‐producing capability of the fungal isolates was screened using oil displacement activity, emulsification index assay, and surface tension (SFT) measurement. The optimization of operational parameters and culture conditions resulted in maximum biosurfactant production using 9% (v/v) inoculum at 30°C, pH 7.0, using sucrose and yeast extract, as carbon and nitrogen sources, respectively. A C:N ratio of 0.9:0.1 (w/w) was found to be optimum for growth and biosurfactant production. At optimal conditions, it attained lowest SFT (i.e., 32 mN m−1) with a critical micelle concentration of ≥ 1.2 mg mL−1. During 5 L shake flask fermentation experiments, the biosurfactant productivity was 1.21 g L−1 pure biosurfactant having significant emulsifying index (E24, 70%) and oil‐displacing activity (16 mm). Thin layer chromatography and Fourier transform infrared spectrometric analyses indicated a lipopeptide type of the biosurfactant. The Fusarium sp. BS‐8 has substantial potential of biosurfactant production, yet it needs to be fully characterized with possibility of relatively new class of biosurfactants.

Therapeutic potential of Taraxacum officinale against HCV NS5B polymerase: In-vitro and In silico study

Discovery of alternative and complementary regimens for HCV infection treatment is a need of time from clinical as well as economical point of views. Low cost of bioactive natural compounds production, high biochemical diversity and inexistent/milder side effects contribute to new therapies. Aim of this study is to clarify anti-HCV role of Taraxacum officinale, a natural habitat plant rich of flavonoids. In this study, methanol extract of T. officinale leaves was initially analyzed for its cytotoxic activity in human hepatoma (Huh-7) and CHO cell lines. Hepatoma cells were transfected with pCR3.1/Flagtag/HCV NS5B gene cloned vector (genotype 1a) along with T. officinale extract. Considering NS5B polymerase as potential therapeutic drug target, twelve phytochemicals of T. officinale were selected as ligands for molecular interaction with NS5B protein using Molecular Operating Environment (MOE) software. Sofosbuvir (Sovaldi: brand name) currently approved as new anti-HCV drug, was used as standard in current study for comparative analysis in computational docking screening. HCV NS5B polymerase as name indicates plays key role in viral genome replication. On the basis of which NS5B gene is targeted for determining antiviral role of T. officinale extract and 65% inhibition of NS5B expression was documented at nontoxic dose concentration (200μg/ml) using Real-time PCR. In addition, 57% inhibition of HCV replication was recorded when incubating Huh-7 cells with high titer serum of HCV infected patients along with leaves extract. Phytochemicals for instance d-glucopyranoside (-31.212 Kcal/mol), Quercetin (-29.222 Kcal/mol), Luteolin (-26.941 Kcal/mol) and some others displayed least binding energies as compared to standard drug Sofosbuvir (-21.0746 Kcal/mol). Results of our study strongly revealed that T. officinale leaves extract potentially blocked the viral replication and NS5B gene expression without posing any toxic effect on normal fibroblast cells of body.

In silico analysis and molecular docking studies of potential angiotensin-converting enzyme inhibitor using quercetin glycosides.

The purpose of this study was to analyze the inhibitory action of quercetin glycosides by computational docking studies. For this, natural metabolite quercetin glycosides isolated from buckwheat and onions were used as ligand for molecular interaction. The crystallographic structure of molecular target angiotensin-converting enzyme (ACE) (peptidyl-dipeptidase A) was obtained from PDB database (PDB ID: 1O86). Enalapril, a well-known brand of ACE inhibitor was taken as the standard for comparative analysis. Computational docking analysis was performed using PyRx, AutoDock Vina option based on scoring functions. The quercetin showed optimum binding affinity with a molecular target (angiotensin-converting-enzyme) with the binding energy of −8.5 kcal/mol as compared to the standard (−7.0 kcal/mol). These results indicated that quercetin glycosides could be one of the potential ligands to treat hypertension, myocardial infarction, and congestive heart failure.

Chaetomium endophytes: a repository of pharmacologically active metabolites

Fungal endophytes are group of fungi that grow within the plant tissues without causing immediate signs of disease and are abundant and diverse producers of bioactive secondary metabolites. The Chaetomium genus of kingdom fungi is considered to be a rich source of unique bioactive metabolites. These metabolites belong to chemically diverse classes, i.e., chaetoglobosins, xanthones, anthraquinones, chromones, depsidones, terpenoids and steroids. Cheatomium through production of diverse metabolites can be considered as a potential source of antitumor, cytotoxic, antimalarial, antibiotic and enzyme inhibitory lead molecules for drug discovery. This review covers isolation of Cheatomium endophytes, extraction and isolation of metabolites and their biological activities.

Molecular cloning and sequence analysis of a PVGOX gene encoding glucose oxidase in Penicillium viticola F1 strain and it\'s expression quantitation

The PVGOX gene (accession number: KT452630) was isolated from genomic DNA of the marine fungi Penicillium viticola F1 by Genome Walking and their expression analysis was done by Fluorescent RT-PCR. An open reading frame of 1806bp encoding a 601 amino acid protein (isoelectric point: 5.01) with a calculated molecular weight of 65,535.4 was characterized. The deduced protein showed 75%, 71%, 69% and 64% identity to those deduced from the glucose oxidase (GOX) genes from different fungal strains including; Talaromyces variabilis, Beauveria bassiana, Aspergillus terreus, and Aspergillus niger, respectively. The promoter of the gene (intronless) had two TATA boxes around the base pair number -88 and -94 and as well as a CAAT box at -100. However, the terminator of the PVGOX gene does not contain any polyadenylation site (AATAAA). The protein deduced from the PVGOX gene had a signal peptide containing 17 amino acids, three cysteine residues and six potential N-linked glycosylation sites, among them, -N-K-T-Y- at 41 amino acid, -N-R-S-L- at 113 amino acid, -N-G-T-I- at 192 amino acid, -N-T-T-A at 215 amino acid, -N-F-T-E at 373 amino acid and -N-V-T-A- at 408 amino acid were the most possible N-glycosylation sites. Furthermore, the relative transcription level of the PVGOX gene was also stimulated in the presence of 4% (w/v) of calcium carbonate and 0.5 % (v/v) of CSL in the production medium compared with that of the PVGOX gene when the fungal strain F1 was grown in the absence of calcium carbonate and CSL in the production medium, suggesting that under the optimal conditions, the expression of the PVGOX gene responsible for gluconic acid biosynthesis was enhanced, leading to increased gluconic acid production. Therefore, the highly glycosylated oxidase enzyme produced by P. viticola F1 strain might be a good producer in the fermentation process for the industrial level production of gluconic acid.

Endophytic fungi associated with Taxus fuana (West Himalayan Yew) of Pakistan: potential bio-resources for cancer chemopreventive agents.

Abstract Context: Endophytic fungi, being a prolific source of bioactive secondary metabolites, are of great interest for natural product discovery. Objective: Isolation and partial characterization of endophytic fungi inhabiting the leaves and woody parts of Taxus fuana Nan Li & R.R. Mill. (Taxaceae) and evaluation of biological activity. Materials and methods: Endophytic fungal isolates were identified by molecular analysis of internal transcribed spacer (ITS) regions of 18S rDNA. Extracts of the endophytic fungi cultured on potato dextrose agar and modified medium were evaluated using cancer chemoprevention bioassays [inhibition of TNF-α-induced NFκB, aromatase and inducible nitric oxide synthase (iNOS); induction of quinone reductase 1 (QR1)] and growth inhibition with MCF-7 cells. Results: Nine of 15 fungal isolates were identified as belonging to Epicoccum, Mucor, Penicillium, Chaetomium, Paraconiothriym, Plectania or Trichoderma. Five of the 15 extracts inhibited NFκB activity (IC50 values ranging between 0.18 and 17 μg/mL) and five inhibited iNOS (IC50 values ranging between 0.32 and 12.9 μg/mL). In the aromatase assay, only two isolates mediated inhibition (IC50 values 12.2 and 10.5 μg/mL). With QR1 induction, three extracts exhibited significant activity (concentrations to double activity values ranging between 0.20 and 5.5 μg/mL), and five extracts inhibited the growth of MCF-7 cells (IC50 values ranging from 0.56 to 17.5 μg/mL). Six active cultures were derived from woody parts of the plant material. Conclusion: The endophytic fungi studied are capable of producing pharmacologically active natural compounds. In particular, isolates derived from the wood of Taxus fuana should be prioritized for the isolation and characterization of bioactive constituents.

Prioritizing and modelling of putative drug target proteins of Candida albicans by systems biology approach

Candida albicans (Candida albicans) is one of the major sources of nosocomial infections in humans which may prove fatal in 30% of cases. The hospital acquired infection is very difficult to treat affectively due to the presence of drug resistant pathogenic strains, therefore there is a need to find alternative drug targets to cure this infection. In silico and computational level frame work was used to prioritize and establish antifungal drug targets of Candida albicans. The identification of putative drug targets was based on acquiring 5090 completely annotated genes of Candida albicans from available databases which were categorized into essential and non-essential genes. The result indicated that 9% of proteins were essential and could become potential candidates for intervention which might result in pathogen eradication. We studied cluster of orthologs and the subtractive genomic analysis of these essential proteins against human genome was made as a reference to minimize the side effects. It was seen that 14% of Candida albicans proteins were evolutionary related to the human proteins while 86% are non-human homologs. In the next step of compatible drug target selections, the non-human homologs were sequentially compared to the human microbiome data to minimize the potential effects against gut flora which accumulated to 38% of the essential genome. The sub-cellular localization of these candidate proteins in fungal cellular systems indicated that 80% of them are cytoplasmic, 10% are mitochondrial and the remaining 10% are associated with the cell wall. The role of these non-human and non-gut flora putative target proteins in Candida albicans biological pathways was studied. Due to their integrated and critical role in Candida albicans replication cycle, four proteins were selected for molecular modeling. For drug designing and development, four high quality and reliable protein models with more than 70% sequence identity were constructed. These proteins are used for the docking studies of the known and new ligands (unpublished data). Our study will be an effective framework for drug target identifications of pathogenic microbial strains and development of new therapies against the infections they cause.

Biological Evaluation of Endophytic Fungus Chaetomium sp. NF15 of Justicia adhatoda L.: A Potential Candidate for Drug Discovery.

Background The endophytes of medicinal plants, such as Justicia adhatoda L., represent a promising and largely underexplored domain that is considered as a repository of biologically active compounds. Objectives The aim of present study was isolation, identification, and biological evaluation of endophytic fungi associated with the J. adhatoda L. plant for the production of antimicrobial, antioxidant, and cytotoxic compounds Materials and Methods Endophytic fungi associated with the J. adhatoda L. plant were isolated from healthy plant parts and taxonomically characterized through morphological, microscopic, and 18S rDNA sequencing methods. The screening for bioactive metabolite production was achieved using ethyl acetate extracts, followed by the optimization of different parameters for maximum production of bioactive metabolites. Crude and partially purified extracts were used to determine the antimicrobial, antioxidant, and cytotoxic potential Results Out of six endophytic fungal isolates, Chaetomium sp. NF15 showed the most promising biological activity and was selected for detailed study. The crude ethyl acetate extract of NF15 isolate after cultivation under optimized culture conditions showed promising antimicrobial activity, with significant inhibition of the clinical isolates of Staphylococcus aureus (87%, n=42), Pseudomonas aeruginosa (> 85%, n = 41), and Candida albicans (62%, n = 24). Conclusions The present study confirms the notion of selecting endophytic fungi of medicinal plant Justicia for the bioassay-guided isolation of its bioactive compounds, and demonstrates that endophytic fungus Chaetomium sp. NF15 could be a potential source of bioactive metabolites

Antioxidants, antimicrobial and cytotoxic potential of Swertia chirata

The current study was designed to evaluate the properties of various fractions of methanolic extract prepared from aerial parts of Swertia chirata. Five fractions, RA-78, RA-91, RA-112, RA-14 and RA-15 showed significantly high DPPH scavenging capacity. In finding out antimicrobial activities, different fractions have shown mild activities against Bordetella bronchiseptica and Micrococcus luteus, while all fractions showed significant results against Aspergillus fumigatus followed by Mucor species and five fractions were active against Aspergillus niger. In measurement of brine shrimp cytotoxic potential of different fractions, three fractions RA-12, RA-34 and RA-78 had shown the best cytotoxic results with zero LD50. In determining antileishmanial properties of different fractions, the best results were exhibited by fraction RA-78 and RA-112 with zero percent survival of Leishmania parasite. Various fractions of methanolic extract prepared from aerial parts of Swertia chirata promisingly possess antioxidant, antimicrobial, brine shrimp cytotoxic, and antileishmanial activities.