In vitro phytochemical and anticancer activity of Misopates orontium L. and Dicliptera bupleuroides Nees.

In the present study phytochemical analysis and anticancer activity of Misopates orontium L. and Dicliptera bupleuroides Nees was carried out. Methanolic extracts of M. orontium and D. bupleuroides were selected for phytochemical analysis. The present analysis showed the presence of phytochemical such as carbohydrates, proteins, tannins, glycosides, alkaloids, saponins, phenols and flavonoids in M. orontium and D. bupleuroides. Anticancer assays including MTT, Alamar Blue (AB), Neutral Red (NR) and lactate dehydrogenase (LDH) were employed on whole herb methanolic extract and all other fractions of both plants to calculate the % age of cell viability and cell cytotoxicity. The percentage of cell viability was highly significant in all anticancer assays for all fractions. Therefore, ethyl acetate and aqueous fractions showed the excellent profile in evaluation of cytotoxicity in each assay. All above findings indicated that the whole herb of both selected plants have strong anticancer activity.

Mutation in pvcABCD operon of Pseudomonas aeruginosa modulates MexEF-OprN efflux system and hence resistance to chloramphenicol and ciprofloxacin.

Pseudomonas aeruginosa harbors pvcABCD operon that is responsible for the synthesis of paerucumarin. Here we report the involvement of pvcABCD operon in chloramphenicol and ciprofloxacin resistance. P. aeruginosa mutant defective in pvcB (PW4832) was more sensitive to chloramphenicol and ciprofloxacin in comparison with its parent strain (MPAO1). A mutation in pvcA gene in MPAO1 (PW4830) did not alter the sensitivity to either antibiotic. As chloramphenicol and ciprofloxacin are substrates of MexEF-OprN efflux pump, so we decided to investigate the modulation of MexEF-OprN and its transcriptional regulator MexT in PW4832, PW4830 and MPAO1 strains. We isolated and sequenced mexT gene from MPAO1, PW4830 and PW4832. The nucleotide sequence of mexT gene in all three strains was identical. Expression levels of mexEF-oprN, mexT and mexS genes were checked via quantitative real-time RT-PCR. All these genes showed significant repression in mRNA levels in PW4832 as compared to MPAO1. These results indicate that chloramphenicol and ciprofloxacin sensitivity in PW4832 is due to transcriptional repression of mexT and mexEF-oprN genes. Exogenous addition of paerucumarin resumed the expression of mexT and mexEF-oprN genes as well as resistance against chloramphenicol and ciprofloxacin in PW4832 strain. This is a novel finding linking pvcB gene of P. aeruginosa with chloramphenicol and ciprofloxacin resistance and MexEF-OprN pump modulation which needs to be further explored.

In vitro anticancer and antioxidant potential of Cestrum species.

The methanolic extract of leaves and stem of Cestrum nocturnum and Cestrum diurnum were investigated for their antioxidant and anticancer attribute through standard methods. 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was carried out to estimate the antioxidant activity of the extracts. Whereas, anticancer potential of extracts were tested against colon cancer cell line, HCT 116 and acute myeloid leukemia (AML) cell lines, THP-1. Results showed that extracts of both plants exhibited a very strong antioxidant activity in a dose dependent manner. In addition, both extracts efficiently increased the cell death in two different cancer cell lines. Moreover, DNA fragmentation analysis further strengthens the anticancer potential of extracts of both types of plants. Current study, therefore, provide a preliminary data highlighting the antioxidant and anticancer activities of methanolic extract of leaves and stem of Cestrum nocturnum and Cestrum diurnum.

Functional probiotic attributes and gene encoding plantaracin among variant Lactobacillus Plantarum strains.

Fourteen Lactobacillus plantarum strains isolated from various food sources and two different climatic regions, Ireland and Pakistan were analyzed for their probiotic functions. RecA gene based multiplex PCR amplifications and pulsed-field gel electrophoresis (PFGE) were performed to genetically characterize these strains at subspecies level. All the strains were tested for bacteriocin activity against major food borne pathogens (L. innocua and L. monocytogenes). Bacteriocin was further purified using HPLC and identified with MALDI TOF MS analysis. Identification of gene encoding plantaracin 423 along with its enzyme sensitivity assays to protinease K and pepsin were also performed. Probiotic properties of bacteriocin producing strains were also analyzed by acid tolerance, bile salt tolerance, survival in simulated gastric juice and antibiotic resistance tests. Ten distinctly different strains of PFGE patterns were identified following ApaI digestion. Antimicrobial screening showed five L. plantarum strains as the potential producers of bacteriocin, expressing GIZ (Growth inhibition zone) up to 12, 12, 14, 11 and 13 mm, respectively against L. innocua. Molecular characterization of these strains further exhibited that plantaracin gene was present in the genome of L. plantarum strains AS-4, AS-6, AS-7, AS-13 and AS-14. All strains presented significant in vitro functional probiotic potential. Current study therefore, not only highlights bacteriocin regulated probiotic potential of L. plantarum strains isolated from different sources and climatic regions but also designates high heterogeneity in functional properties of the L. plantarum strains.

Purification and Characterization of a recombinant ß-Xylosidase from Bacillus licheniformis ATCC 14580 into E. coli Bl21.

Present research work is aimed to purify and characterize a recombinant ß-xylosidase enzyme which was previously cloned from Bacillus licheniformis ATCC 14580 in to Escherichia coli BL21. Purification of recombinant enzyme was carried out by using ammonium sulphate precipitation method followed by single step immobilized metal ion affinity chromatography. Specific activity of purified recombinant ß-xylosidase enzyme was 20.78 Umg-1 with 2.58 purification fold and 33.75% recovery. SDS-PAGE was used to determine the molecular weight of recombinant purified ß-xylosidase and it was recorded as 52 kDa. Purified enzyme showed stability upto 90°C within a pH range of 3-8 with and optimal temperature and pH, 55ºC and 7.0, respectively. The enzyme activity was not considerably affected in the presence of EDTA. An increase in the enzyme activity was found in the manifestation of Mg+2. Enzyme activity was also increased by 6%, 18% and 22% in the presence of 1% Tween 80, ß-mercaptoethanol and DTT, respectively. Higher concentrations (10 - 40%) of organic solvents did not show any effect upon activity of enzyme. All these characteristics of the recombinant enzyme endorsed it as a potential candidate for biofuel industry.

Phytochemical screening and antioxidant potential of Parthenocissus quinquefolia (L.) planch extracts of bark and stem.

The phytochemical screening and antioxidant potential of bark and stem of Parthenocissus quinquefolia (L.) planch was assessed in order to verify its ethnopharmacological significance. All major secondary metabolites e.g. alkaloids, flavonoids, saponins, terpenoids, tannins, reducing sugars, cardiac glycosides and anthraquinones were present. Antioxidant activity was analysed by using five techniques which included DPPH Free Radical Scavenging Activity, FRAP (Ferric Reducing Antioxidant Power), TAA (Total Antioxidant Activity, TPC (Total Phenolic Content and MC (Metal Chelating) Activity. Ethanolic extract of bark showed the highest scavenging effects of 90.01±0.01%, with IC50 value of 24.32mg/ml. Aqueous stem extract showed best activity with IC50 value of 13.6±0.34mg/ml. The significance antioxidant potential indicates the effectiveness of bark and stem of P. quinquefolia in treatment of many diseases.

Adsorption of Eosin B from Wastewater onto the Prepared Porous Anion Exchange Membrane.

This research describes the fabrication of the porous trimethylamine (TMA)-grafted anion exchange membrane (AEM) over a phase inversion process. The synthesis of the generated AEM was verified using Fourier transform infrared (FTIR) spectroscopy. The fabricated porous AEM showed 240% water uptake (WR), 1.45 mg/g ion exchange capacity (IEC), and a 9.0% linear expansion ratio (LER) at 25 °C. It exhibited a porous structure and higher thermal stability. It was utilized to remove eosin B (EB) from wastewater via the process of adsorption. The adsorption capacity of EB increased with time and the starting concentration of EB while decreasing with temperature and the AEM dosage. Adsorption isotherm investigation results showed that EB adsorption onto the porous AEM followed the Langmuir isotherm because the value of correlation coefficient (R2 = 0.992) was close to unity. Because the correlation coefficient was close to one, it was determined through adsorption kinetic experiments that the adsorption of EB on the produced porous AEM was suitable for a pseudo-second-order model. Thermodynamic study about process of EB adsorption on the porous AEM revealed that there was an exothermic (ΔH° = -16.60 kJ/mol) and spontaneous process.

Next-generation sequencing to elucidate adaptive stress response and plantaricin genes among Lactobacillus plantarum strains.

Aim: The objective of this study was to identify the genes involved in plantaricin synthesis and adaptive stress response in four Lactobacillus plantarum strains (AS-6, AS-8, AS-9 and AS-10) and one Lactobacillus paraplantarum strain (AS-7) for their usage in medicine and industry. Materials & methods: Whole genomes of these strains were sequenced by a high-throughput sequencing technique known as next-generation sequencing via Ilumina MiSeq platform and the genes were identified by using various bioinformatics tools and software. Results: Plantaricin genes (plnD, plnE, plnF, plnG, plnI) and genes regulating response to temperature, pH, bile salt, osmotic and oxidative stress were identified in all strains. Conclusion: Lactobacilli could be an option to combat antimicrobial resistance and might replace harmful antibiotics in future.

Citrullus colocynthis regulates de novo lipid biosynthesis in human breast cancer cells.

BACKGROUND: Reprogrammed energy metabolism is considered a hallmark of cancer and is proposed as an important target for therapy. Uncontrolled and infinite cell proliferation needs efficient energy sources. To meet the demands of cancer cells lipid metabolism is activated. Citrullus colocynthis is a traditional medicinal plant known for its anticancer and hypolipidemic effects. AIMS: Aim of the current study was to assess the effect of C. colocynthis leaves on regulation of lipid metabolism in MCF-7, a human breast cancer cell line. METHODS: Methanolic extract of leaves and its fractions in increasing polarity-based solvents (n-hexane, chloroform, ethyl acetate and n-butanol) were prepared and analyzed for the presence of secondary metabolites in each fraction. Bioassays and apoptosis genes expression analysis was conducted to evaluate the anticancer and cytotoxic effect of breast cancer cells treated with extract and its fractions, separately. Lipid quantification and gene expression regulation of genes involve in lipid metabolism was performed to evaluate regulation of lipid metabolism. RESULTS: Results showed a significant anticancer activity of methanolic extract of C. colocynthis and two of its fractions prepared with chloroform and ethyl acetate. Quantification of lipids depicted significant increase in cholesterol and increase in triglycerides of treated cells compared to control untreated cells. Expression regulation of genes further confirmed the lipid regulation through significant down regulation of genes involve in lipid metabolism (FASN, HMGCLL1, ACSL5 and ELOVL2). CONCLUSION: The present study concludes that C. colocynthis holds strong anticancer potential through regulation of lipid metabolism and with further studies can be proposed for novel therapeutic approaches.

Significantly enhanced biomass production of a novel bio-therapeutic strain Lactobacillus plantarum (AS-14) by developing low cost media cultivation strategy.

BACKGROUND: Probiotic bacteria are becoming an important tool for improving human health, controlling diseases and enhancing immune responses. The availability of a cost effective cultivation conditions has profound effect on the efficiency and role of probiotic bacteria. Therefore the current study was conducted with an objective to develop a low cost growth medium for enhancing the biomass production of a bio-therapeutic bacterial strain Lactobacillus plantarum AS-14. In this work the isolation of Lactobacillus plantarum AS-14 bacterial strain was carried out from brinjal using cheese whey as a main carbon source. Moreover, the effect of four other nutritional factors besides cheese whey was investigated on the enhanced cell mass production by using response surface methodology (RSM). RESULTS: The best culture medium contained 60 g/l cheese whey, 15 g/l glucose and 15 g/l corn steep liquor in addition to other minor ingredients and it resulted in maximum dry cell mass (15.41 g/l). The second-order polynomial regression model determined that the maximum cell mass production (16.02 g/l) would be obtained at temperature 40°C and pH 6.2. Comparative studies showed that cultivation using cheese whey and corn steep liquor with other components of the selected medium generated higher biomass with lower cost than that of De Man, Rogosa and Sharpe (MRS) medium under similar cultivation conditions (pH 6.2 and temperature 40°C). CONCLUSION: It is evident that the cell biomass of L. Plantarum AS-14 was enhanced by low cost cultivation conditions. Moreover, corn steep liquor and ammonium bisulphate were perceived as low-cost nitrogen sources in combination with other components to substitute yeast extract. Of all these factors, cheese whey, corn steep liquor, yeast extract and two operating conditions (temperature and pH) were found to be the most significant parameters. Thus the cost effective medium developed in this research might be used for large-scale commercial application where economics is quite likely important.

Efficacy of Locally Isolated Lactic Acid Bacteria Against Antibiotic-Resistant Uropathogens.

BACKGROUND: Antibiotic resistance represents a serious global health threat to public health, so infections such as pneumonia and urinary tract infection (UTI) are becoming harder to treat. Therefore, it is necessary to develop an action plan to restrain the problem of antibiotic resistance. One approach in UTI control could be the use of lactobacilli because these indigenous inhabitants in human intestine have been found to play an important role in protecting the host from various infections. OBJECTIVES: We sought to check the efficacy of locally isolated Lactobacillus species to eradicate antibiotic-resistant pathogenic bacteria causing UTI. MATERIALS AND METHODS: Lactic acid bacteria isolated from spoiled fruits and vegetables and grown in MRS medium were screened against multi-drug-resistant Candida albicans, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, and Enterococcus fecalis. RESULTS: Fifty-four lactic acid bacteria were isolated from spoiled fruits and vegetables, of which 11 Gram-positive and catalase-negative Lactobacillus isolates were identified by carbohydrate assimilation profiles as Lactobacillus acidophilus, L. paracasei, L. delbrueckii, L. casei, L. helveticus, L. brevis, L. salivarius, L. fermentum, L. rhamnosus, L. animalis, and L. plantarum. The latter organism had the highest abundance of all the samples, so its isolates were also verified through 16S rRNA gene sequencing. The isolated Lactobacilli were screened against multi-drug-resistant uropathogens, viz. C. albicans, P. aeruginosa, K. pneumoniae, E. fecalis, and E. coli. The growth inhibition zone (GIZ) was over 10 mm against all the uropathogenic test organisms, where L. fermentum and L. plantarum strains demonstrated remarkable inhibitory activities against E. coli and E. faecalis, with a GIZ up to 28 mm. The susceptibility test to 16 antibiotics showed multidrug resistance (3 to 5 antibiotics) among all the tested uropathogens. CONCLUSIONS: The obtained results revealed that all the Lactobacillus isolates displayed antimicrobial activity against 6 out of 7 antibiotic-resistant uropathogens, indicating that these bacteria could represent a good ecological plan for the control and prevention of UTI.