Identification of Efflux Pump Mutations in Pseudomonas aeruginosa from Clinical Samples.

Efflux pumps are a specialized tool of antibiotic resistance used by Pseudomonas aeruginosa to expel antibiotics. The current study was therefore conducted to examine the expression of MexAB-OprM and MexCD-OprJ efflux pump genes. In this study, 200 samples were collected from Khyber Teaching Hospital (KTH) and Hayatabad Medical Complex (HMC) in Peshawar, Pakistan. All the isolates were biochemically identified by an Analytical Profile Index kit and at the molecular level by Polymerase Chain Reaction (PCR) utilizing specific primers for the OprL gene. A total of 26 antibiotics were tested in the current study using the guidelines of the Clinical and Laboratory Standard Institute (CLSI) and high-level resistance was shown to amoxicillin-clavulanic acid (89%) and low-level to chloramphenicol (1%) by the isolates. The antibiotic-resistant efflux pump genes MexA, MexB, OprM, MexR, MexC, MexD, OprJ, and NfxB were detected in 178 amoxicillin-clavulanic acid-resistant isolates. Mutations were detected in MexA, MexB, and OprM genes but no mutation was found in the MexR gene as analyzed by I-Mutant software. Statistical analysis determined the association of antibiotics susceptibility patterns by ANOVA: Single Factor p = 0.05. The in silico mutation impact on the protein structure stability was determined via the Dynamut server, which revealed the mutations might increase the structural stability of the mutants. The docking analysis reported that MexA wild protein showed a binding energy value of -6.1 kcal/mol with meropenem and the mexA mutant (E178K) value is -6.5 kcal/mol. The mexB wild and mutant binding energy value was -5.7 kcal/mol and -8.0 kcal/mol, respectively. Efflux pumps provide resistance against a wide range of antibiotics. Determining the molecular mechanisms of resistance in P. aeruginosa regularly will contribute to the efforts against the spread of antibiotic resistance globally.

Molecular Characterization and Epidemiology of Antibiotic Resistance Genes of ß-Lactamase Producing Bacterial Pathogens Causing Septicemia from Tertiary Care Hospitals.

Septicemia is a systematic inflammatory response and can be a consequence of abdominal, urinary tract and lung infections. Keeping in view the importance of Gram-negative bacteria as one of the leading causes of septicemia, the current study was designed with the aim to determine the antibiotic susceptibility pattern, the molecular basis for antibiotic resistance and the mutations in selected genes of bacterial isolates. In this study, clinical samples (n = 3389) were collected from potentially infected male (n = 1898) and female (n = 1491) patients. A total of 443 (13.07%) patients were found to be positive for bacterial growth, of whom 181 (40.8%) were Gram-positive and 262 (59.1%) were Gram-negative. The infected patients included 238 males, who made up 12.5% of the total number tested, and 205 females, who made up 13.7%. The identification of bacterial isolates revealed that 184 patients (41.5%) were infected with Escherichia coli and 78 (17.6%) with Pseudomonas aeruginosa. The clinical isolates were identified using Gram staining biochemical tests and were confirmed using polymerase chain reaction (PCR), with specific primers for E. coli (USP) and P. aeruginosa (oprL). Most of the isolates were resistant to aztreonam (ATM), cefotaxime (CTX), ampicillin (AMP) and trimethoprim/sulfamethoxazole (SXT), and were sensitive to tigecycline (TGC), meropenem (MEM) and imipenem (IPM), as revealed by high minimum inhibitory concentration (MIC) values. Among the antibiotic-resistant bacteria, 126 (28.4%) samples were positive for ESBL, 105 (23.7%) for AmpC ß-lactamases and 45 (10.1%) for MBL. The sequencing and mutational analysis of antibiotic resistance genes revealed mutations in TEM, SHV and AAC genes. We conclude that antibiotic resistance is increasing; this requires the attention of health authorities and clinicians for proper management of the disease burden.

Microbial degradation of low density polyethylene by Exiguobacterium sp. strain LM-IK2 isolated from plastic dumped soil.

Low-Density Polyethylene (LDPE) is one of the significant environmental pollutants as it is resistant to natural degradation. In this study, we reported the LDPE-degrading bacterial strain i.e., Exiguobacterium sp. strain LM-IK2 isolated from plastic dumped soil which shows potential degradation capability. The percent weight loss of LDPE was calculated as - 5.70 ± 0.7 after 90 days of incubation in a carbon-free MSM medium. The Field Emission Scanning Electron Microscopy (FE-SEM) analysis shows that LDPE films show slight surface disruption after treatment with bacteria. The Fourier Transform Infrared Spectroscopy (FTIR) revealed the chemical changes in LDPE films e.g., formation and reduction of typical carbonyl peaks after incubation with bacteria. The X-Ray Diffraction (XRD) analysis displayed an increase in percent crystallinity, with a slight change in total carbon content. Genetic analysis showed the presence of Laccase (167 bp) and Alkane Hydroxylase (330 bp) genes that are responsible for LDPE degradation. Thus, Exiguobacterium sp. strain LM-IK2 has the potential to degrade LDPE and could be further explored to improve its efficiency in the bioremediation of LDPE.

Isolation and molecular characterization of extended spectrum beta lactamase producing Escherichia coli from chicken meat in Pakistan.

The goal of this study was to find E. coli, a prevalent pathogen that causes food-borne illnesses, in chicken samples (n = 500) collected from three districts in KhyberPukhtunkhwa: Mardan, Swabi, and Swat. The E. coli isolates were identified by Gram staining, API strips and Universal Stress Protein. A total of 412 samples tested positive for E. coli and were sensitive to MEM, TZP, and FOS as evidenced by disc diffusion method. The isolates were resistant to TE, NOR, and NA with statistically significant results (P≤0.05). The isolates showed the presence of different antibiotic resistance genes; blaOXA-1, blaCTX-M15, blaTEM-1, QnrS, TetA, AAC, AAD, Sul1 and Sul2. The results revealed mutations in blaOXA-1 gene (H81Q), blaTEM-1 (C108Y, T214A, K284E and P301S), QnrS (H95R) and Sul2 (E66A). The findings of this study may be helpful in better management of E. coli infections by physicians.

Molecular epidemiology of antibiotic-resistant genes and potent inhibitors against TEM, CTX-M-14, CTX-M-15, and SHV-1 proteins of Escherichia coli in district Peshawar, Pakistan.

The Extended Spectrum Beta-Lactamases (ESBLs) producing bacteria is an issue of concern for clinicians resulting in minimize the treatment options. To overcome resistance mechanisms, novel inhibitors with good Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) properties must inhibit the ESBLs resistant genes. The current study aimed to identify the antibiotic resistance genes of ESBLs producing E. coli and a single inhibitor was designed to inhibit all the resistant proteins. The results showed that 42.9% ESBL producers had CTX-M (69.9%), TEM (63.4%), SHV (34.5%) and CTX-M-14 (17.5%) genes. The ESBLs producing isolates were resistant to cephalosporins, quinolones, and sulfonamide with Minimum Inhibitory Concentration (MICs) ranging from 64 to >256 µg/ml. To design multi inhibitory ligands, RECAP synthesis was used for the de-novo discovery of 1000 inhibitors database. Protein crystal structures were retrieved from Protein Data Base (PDB). Lipinski's rules of five were applied to the novel inhibitors database to improve the ADMET properties. The novel inhibitors database was selected for docking simulations. Placement of the ligand was used by the London dG algorithm implemented in Molecular Operating Environment (MOE), while GBVI/WSA dG algorithm was used for final refinement. Based on docking score, visual inspection of ligands interaction with key residues, binding affinity, and binding energy of ligands with proteins, ten compounds were selected for ESBLs proteins with best ADMET properties, binding energy, and binding affinity the reported ones. These hits compounds have unique scaffolds and are predicted to be a starting point for developing potent inhibitors against antibiotic-resistant proteins.

Potential cytotoxic and mutagenic effect of Pinus wallichiana, Daphne oleiodes and Bidens chinensis.

The Pinus wallichiana, Daphne oleiodes and Bidens chinensis have a long history of being used traditionally for treatment of various types of disorders. Most of the uses have been without any scientific evidence and toxicological assessment. We evaluated the mutagenic and cytotoxic capabilities of various parts of P. wallichiana, D. oleoides and B. chinensis. Ames Salmonella mutagenicity assay determined the mutagenicity activity against TA 98 and TA 100 bacterial strains of Salmonella typhimurium without metabolic activator S9 system. The number of mutant colonies in negative control was considered as limit to determine the mutagenicity effects of every extract. Brine shrimps lethality bioassay was used to determine the cytotoxic capabilities of the selected plants. The P. wallichiana, D. oleiodes and B. chinensis did not showed any mutagenic activity both for frameshift mutation (TA98) and base-pair substitution (TA100) without S9 mixture. The crude methanolic extract of P. wallichiana stem showed moderate cytotoxicity (53.33%) at 1000 µg/ml with LD50 value 599.634. The D. oleoides fruit showed a toxicity of 60% at 1000 µg/ml with LD50 value 367.730. The B. chinensis (whole plant) showed lethality of 63.3% at 1000 µg/ml, with LD50 204.833. The absence of any mutagenic activity of crude extract of the tested plants in both bacteria strains, TA 98 and TA 100 without the S9 mix confirms the safety of these plants to the consumers.

Phenotypic and molecular characterization of virulence factors of extra-intestinal pathogenic Escherichia coli isolated from patients of Peshawar, Pakistan.

Extra-Intestinal Escherichia coli (ExPEC) are important cause of Urinary Tract Infections (UTIs) and systemic infections. The purpose of this study was to investigate numerous ExPEC bacterial isolates for phenotypic virulence characteristics including hemolytic activity and resistance pattern and to observe their association with genetic traits via Polymerase Chain Reaction (PCR). A total of 367 ExPEC isolates were collected from patients admitted in Khyber Teaching Hospital (KTH) Peshawar, Pakistan. Standard techniques were used for identification of isolates, determination of hemolytic potential and antimicrobial susceptibility testing. PCR was used for screening of virulence genes using specific primers. A total of 367 ExPEC isolates were characterized, among which 62.7, 24.3, 7.1 and 6% were isolated from urine, pus, sputum and wound specimens, respectively. Majority of the isolates (82.8%) were hemolysin positive. Multi drug resistance pattern was shown by 41% of the isolates and harbored at least one virulence gene (71.7%), of which sat was the most prevalent (64.3%). The highest resistance was found to cefotaxime (99.2%), ampicillin (97.5%) and aztreonem (89.6%). 15 different virulence genes combinations were observed in the current study. A total of 16 virotypes (15 of positive virulence genes and one of no virulence gene) were observed in the current study. The current investigation showed a high prevalence of sat and hlyA genes among ExPEC isolate, suggesting a role of these genes in the pathogenesis of ExPEC.

Designing of potent inhibitors for metallo-beta-lactamases producing Escherichia coli in molecular specification hereto.

The rapid spread of Metallo-ß-Lactamases producing Gram-negative bacteria in Pakistan is alarming and novel inhibitors with multi inhibition potential are required. In the current study, an effort was made to identify the resistance genes of MBLs producing E. coli and single inhibitor was designed having the potential to block all resistant proteins. Results showed that out of 573 clinical isolates, 14.1% MBLs producers have NDM-1 (27.2%) and VIM (13.6%) gene. The isolates were resistant to MEM, AMP, AMC, FEP, CTX, LEV and ATM, while effective antibiotics were TGC, CO, FOS and AK with MICs ranging from 4 to >32µg/ml. RECAP synthesis was used for de-novo discovery of 1000 inhibitors and protein crystal structures were retrieved from PDB. Active sites were identified in each protein and to improve ADMET properties, Lipinski's rules of five was applied. Placement of the ligand was done by London dG algorithm implemented in MOE. For final refinement, GBVI/WSA dG algorithm was used. Based on docking score, visual inspection of ligands interaction with key residues, binding affinity and binding energy of ligands with proteins, 10 compounds were selected for MBLs proteins which presented best ADMET properties, binding energy and affinity than the reported ones.

Pathogens constancy, harbinger of nosocomial infection cum identification of resistant genes and drug designing.

Hospitals are the most prominent places for the growth and spread of bacteria which are resistant to the available antibiotics. This antibiotic resistance is due to the over and misuse of antibiotic dosages of a high-density of patient population which are in frequent interaction with inanimate items of the hospitals and the consequent risk of cross infection. Out of 1010 samples of the current study, 510 (50.49%) were culture positive of which 329 (64.5%) were Gram-positive while 181 (35.49%) Gram negative. The Gram positive bacterial isolates in the current study were; S. aureus and S. epidermidis while Gram negative were; Citrobacter, P. aeruginosa, Provedencia, E. coli, Proteus, Klebsiella, Shigella and Serratia. In the current study, 50 ESBL and 10 MBL producing isolates were obtained from inanimate objects. The ESBL positive isolates were highly resistant to MEM. Out of 50 ESBL, 2 isolates were resistant to DO, SXT and FEP while 1 was resistant to TZP and CN. The BlaTEM gene was detected in 22 and BlaCTX was detected in 19 ESBL producing isolates. Six of the MBL producing isolates were NDM positive while all of the isolates were AmpC negative. Most of the isolates had more than two resistant genes. Several classes of inhibitors have been reported for ß-lactamase TEM, Metallo-ß-lactamase and ß-lactamase proteins. Complex-base pharmacophore models were generated on the bases of co-crystalline ligands attached in the 3-D structures of the proteins. The validated pharmacophore models were used for the screening of ZINC drug like database. As a screening results 571 structurally diverse hits of Ethyl Boronic Acid, 866 on L-Captopril and 1020 of Nacubactam were mapped and filtered via Lipinski's rule of five. In conclusion, 30 hits (10 to each protein) having diverse structure and binding modes with all the three proteins active sites were selected as leading novel inhibitors. These selected novel inhibitors have different scaffolds and a strong possibility to act as an additional starting opinion in the development of new and potential inhibitors.

Pharmacological activities of Justicia adhatoda.

The current study focused on the pharmacological activities of Justicia adhatoda; including antibacterial, antifungal, phytotoxic, cytotoxic, haemagglutination, insecticidal, in vitro antiglycation, DPPH antioxidant and anti-termite. The crude methanolic extract (Crd. Met. Ext) showed 46.4 % antibacterial activity against M. morganii while the n-hexane fraction showed good (71.4%) and moderate (55.1%) activity against M. morganii and A. baumannii respectively. The EtOAc and aqueous fractions, in most of the cases, showed low to no activity against the selected bacterial pathogens, against A. niger, T. harzianum, A. parasiticus and V. dahliae. The Crd. Met. Ext and fractions showed low activity, against P. notatum and P. digitatum, Crd. Met. Ext. and all fractions were inactive. The percent growth regulation, in case of phytotoxic activity, by Crd. Met. Ext was 25 and 16.6, n-hexane fraction 16.6, 16.6 and 0, CHCl3 25, 8.33 and 0 % and EtOAc fractions 8.33, 8.33 and 0% at 1000 and 100 and 10µg/ml respectively. The aqueous fraction was inactive at all the test concentrations. The results of brine shrimp cytotoxic activity for Crd. Met. Ext was 13.33% and n-hexane fraction 20% at 1000, µg/ml respectively. All of the other fractions showed low to no activity at different test concentrations. All of the test samples were inactive against RBC's of the blood groups at all concentration indicating that the selected plant lack phytolectins and haemagglutination activity. The Crd. Met. Ext and various fraction showed low activity against the test insects i.e. C. pulicaria, C. chinensis and T. castaneum. The absorbance value of plant extract for anti-glycation activity at various concentration were: 0.08, 0.067, 0.053 and 0.04 in comparison with Aminoguanidine 0.04, 0.035, 0.03 and 0.02 respectively at 10, 50, 90 and 130µl. The DPPH radical scavenging activities were proportional to the concentration of the fractions, as the concentration of these increased, the percent scavenging activity also increased. The CHCl3 and EtOAc fractions killed all the termites in 24 hours while Crd. Met. Ext, n-hexane and aqueous fractions took 2-3 days.

Prevalence of Hepatitis C virus and its risk factors in blood donors in district Peshawar.

The current study was designed in order to elucidate the most sensitive method for daily practice as well as to evaluate the risk factors for HCV infection associated with blood transfusion in District Peshawar. A total of 1400 healthy volunteer blood donors were tested for Anti-HCV. A questionnaire was used to evaluate the risk factors. Initial testing of all blood samples was done by Immuno Chromatographic Technique (ICT) and confirmed by micro particle enzyme immunoassay (MEIA) and Enzyme Linked Immunosorbent Assay (ELISA). The comparison among ICT, ELISA and MEIA techniques was also evaluated for the purpose of sensitivity. Among 1400 blood donors, 26 (1.85%) cases were found positive for Anti-HCV. These 26 cases were positive on MEIA, 16 individuals were positive on ELISA while 14 were positive on ICT. These 26 cases had different histories of dental treatment (50%), traveled abroad (23.07%), surgery (11.53%), blood transfusion (7.69%) and unknown reason (7.69%). Among all these different histories of dental treatment and blood transfusion were the main risk factors for HCV infection. The results revealed that MEIA is a quick and reliable technique for routine screening of blood donors particularly for controlling the spread of HCV.

Green synthesis, characterisation and biological evaluation of AgNPs using Agave americana, Mentha spicata and Mangifera indica aqueous leaves extract.

The current study was performed to synthesize stable, eco-friendly and bio-compatible silver nano-particles (AgNPs) of Agave americana, Mentha spicata and Mangifera indica leaves and to screen them for biological activities. The ultraviolet-visible spectroscopic analysis revealed that λ-max for AgNPs range from 350-500 nm. All AgNPs possessed polycrystalline structure as notified as intense graphical peaks in complete spectrum of 20 values ranging from 10-80° in X-ray diffraction measurements and supported by scanning electron microscopy data. The size of the nano-particles was confirmed by transmission electron microscopy (30-150 nm). Mass loss at variable temperatures was evaluated by simultaneous thermogravimetric and differential thermal analysis revealed reduction in mass and activity of compounds was notified by temperature increase from 200 to 800 °C, thus concluding it as thermally sensitive compounds. A. americana AgNPs showed significant (96%) activity against Methicillin resistant Staphylococcus aureus, Escherichia coli (95%) and Fusarium oxysporum (89%). Good antioxidant activity was shown by M. spicata AgNPs at 300 µl (79%). M. indica AgNPs showed significant phytotoxic activity (88%) at highest concentration. No haemagglutination reaction was observed for the test samples. The above results revealed that AgNPs synthesized from selected plant species possesses significant antimicrobial and phytotoxic effect.

Isolation and Structure Elucidation, Molecular Docking Studies of Screlotiumol from Soil Borne Fungi Screlotium rolfsii and their Reversal of Multidrug Resistance in Mouse Lymphoma Cells.

A new compound namely (13-(3,3-dihydroxypropyl)-1,6-dihydroxy-3,4-dihydro-1H-isochromen-8(5H)-one (1) was isolated from an ethyl acetate extract of the borne fungi Screlotium rolfsii. Its chemical structure was elucidated by spectroscopic analysis. Screlotiumol 1 were evaluated for their effects on the reversion of multidrug resistant (MDR) mediated by P-glycoprotein (P-gp) of the soil borne fungi. The multidrug resistant P-glycoprotein is a target for chemotherapeutic drugs in cancer cells. In the present study rhodamine-123 exclusion screening test on human mdr1 gene transfected mouse gene transfected L5178 and L5178Y mouse T-cell lymphoma which showed excellent MDR reversing effect in a dose dependent manner against mouse T-lymphoma cell line. Moreover, molecular docking studies of compound-1 also showed better results as compared with the standard. Therefore the preliminary results obtained from this study suggest that screlotiumol 1 could be used as a potential agent for the treatment of cancer.

Antibacterial, antifungal, phytotoxic, antioxidant and hemagglutination activities of organic fractions of Arisaema tortuosum.

In the current study, the antimicrobial, phytotoxic, haemagglutination and antioxidant potential of crude methanolic extract (Crd. MeOH Ext.) and four organic fractions of Arisaema tortuosum was investigated. All fractions have been screened for antimicrobial properties against eight bacterial pathogens and six fungal pathogens using agar well diffusion and tube dilution method, respectively. Furthermore, the organic fractions were also screened for its phytotoxicity against Lemna minor. Haemagglutination was performed against all human blood groups while free radical scavenging activity was performed to investigate the antioxidant potential of A. tortuosum. Results obtained for antibacterial activity exhibited various degree of zone of inhibition and significant activity was observed for Pseudomonas aeruginosa (27.16±0.60) followed by Bacillus cereus (18.55±0.69) for Crd. MeOH Ext. and chloroform (CHCl3) fraction, respectively while some strains showed resistant at same concentration. Similarly, non-significant antifungal activity was observed for the plant extracts. However, the highest activity among the strains was observed for Alternaria alternata (22±1.24%) and Aspergillus niger (20±1.00%) for ethyl acetate (EtOAc) fraction and Crd. MeOH Ext., respectively. The plant extracts showed good phytotoxic activity with 77.06% inhibition for n-hexane fraction at 1000µg/mL. The result of Nitric Oxide (NO) reducing assay revealed that the plant has less antioxidant activity with 46.06% inhibition for CHCl(3) fraction at 900µg/mL. For haemagglutination assay, the result displayed no agglutination in all the testing concentration. Based on the current results, it can be concluded that A. tortuosum has significant antimicrobial and moderate phytotoxic potential and therefore can leads to antibiotics and herbicide production.

Antibacterial activity of crude methanolic extract and various fractions of Vitex agnus castus and Myrsine africana against clinical isolates of Methicillin Resistant Staphylococcus aureus.

Staphylococcus aureus is a nosocomial pathogen that resides in the soft tissues causing many diseases. The current study was conducted to determine the prevalence of Methicillin Resistant S. aureus (MRSA) in ear discharge and pus of patients and antibacterial activity of crude methanolic extract (Cr. MeOH Ext.) and various fractions of M. Africana and V. agnus castus against clinical isolates of MRSA. A total of 40 samples were collected from ear, nose and throat (ENT) outpatient department and wards of Khyber Teaching Hospital (KTH), Peshawar. Out of 40 samples, 36 (90%) samples showed growth on Mannitol Salt Agar (MSA) media out of which 9(25%) were MRSA and the remaining 27(75%) were methicillin susceptible S. aureus (MSSA). A good antibacterial activity was observed for the Cr. MeOH Ext. (76.1%) and ethyl acetate (EtOAc) fraction of V. agnus castus against S11 (71.4%). The n-hexane fraction also showed good antibacterial effect (70%) against S26. The chloroform (CHCl3), butanol (BuOH) and aqueous fractions of M. africana showed good antibacterial activity against S11 (71.4%), S32 (70%) and S26 (75%), respectively. The above results revealed that the selected plants can be further utilized for isolation of the active ingredients as the crude extracts were found good for inhibition of MRSA.

Response rates of standard interferon therapy in chronic HCV patients of Khyber Pakhtunkhwa (KPK).

BACKGROUND: Interferon based therapy is used to eradicate the Hepatitis C Virus from the bodies of the infected individuals. HCV is highly prevalent in Khyber Pakhtunkhwa (KPK) that is why it is important to determine the response of standard interferon based therapy in Chronic HCV patients of the region. STUDY DESIGN: A total of 174 patients were selected for interferon based therapy. The patients were selected from four different regions of KPK. After confirmation of active HCV infection by Real Time PCR, standard interferon with ribavirn was given to patients for 6 months. After completion of therapy, end of treatment virologic response (ETR) was calculated. RESULTS: Out of total 174 patients, 130 (74.71%) showed ETR and 44 (25.28%) did not show ETR. In district Bunir, out of 52 patients, 36 (69.23%) showed ETR and 16 (30.79%) did not show ETR. In district Mardan, out of the total 74 patients, 66 (89.18%) were negative for HCV RNA and 8 (10.81%) were resistant to therapy. In Peshawar, out of 22, 16 (60%) were negative and 6 (40%) were positive for HCV RNA at the end of 6 months therapy. In the Federally Administered Tribal Area (FATA), out of 18 only 10 (55.5%) were negative and 8 (44.45%) were positive for active HCV infection. CONCLUSION: It is concluded that the response of antiviral therapy against HCV infection in chronic HCV patients of KPK province is 74.71%. The high response rate may be due to the prevalence of IFN-responsive HCV genotypes (2 and 3) in KPK.

The antifungal, cytotoxic, antitermite and insecticidal activities of Zizyphus jujube.

Plants are very useful, self-generating machines, producing a variety of useful bioactive products. Keeping in view this idea, the crude methanolic extract and various fractions of Zizyphus jujuba were screened for antifungal, cytotoxic, antitermite and insecticidal activities. Low activity was shown by the crude methanolic extract (12%), n-hexane (9%), chloroform (20%) and ethyl acetate (14%) fraction against Penicillium notatum. Low activity was shown by the n-hexane fraction against Aspergillus niger (10%) and Trichoderma harzianum (13%) and inactive against Aspergillus flavus, Fusarium oxysporum and Rhizopus stolonifer. The CHCl(3) fraction exhibited low activity of 10% against F. oxysporum while showing no activity against the rest of the test fungi. All the test samples were inactive against Rhizopus stolonifer. The crude methanolic extract was highly cytotoxic (73.33%) at the concentration of 1000 (µg/ml) while the rest of the test samples were low in toxicity at the same concentration. The crude methanolic extract of Zizyphus jujuba showed significant antitermite activity against Heterotermes indicola, among the test samples. Against Tribolium castaneum, Rhizopertha dominica and Callosbruchus analis the insecticidal activity was determined. All the test samples except n-hexane showed low activity (20%) against T. castaneum. The n-hexane fraction showed low activity (20%) against R. dominica while the rest of the fractions were inactive against it. Low activity of 40% and 20% was shown by the chloroform and n-hexane fraction respectively against C. analis. The results of the present study revealed that the plant could be as potent source of cytotoxic drugs.

Anti-spasmodic action of crude methanolic extract and a new compound isolated from the aerial parts of Myrsine africana.

BACKGROUND: Myrsine africana is an herbaceous plant that is traditionally used as appetizer and carminative. Locally, it is used for the treatment of pulmonary tuberculosis, rheumatism and diarrhea by healers. The aims of the current study were to screen the crude methanol extract obtained from the aerial parts (leaves and stem) of M. africana, for antispasmodic actions on isolated tissues and further to subject the ethyl acetate (EtOAc) fraction of plant to column chromatography for isolation of pure compounds. METHODS: The antispasmodic action of the crude methanol extract was measured on the spontaneous rabbit's jejunum preparations at concentration 0.01, 0.03, 0.1, 0.3, 1.0, 5.0 and 10.0 mg/ml. The crude extract was also applied, in similar concentrations, on KCl (80 mM) induced contractions to explain its possible mode of action. RESULTS: A new compound Myrsigenin was isolated from the EtOAc fraction of M. africana. The structure of the compound was identified with the help of 13C-NMR, 1H-NMR, HMBC, HMQC, NOESY and COSY. The plant crude methanol extract showed a significant antispasmodic action on rabbit jejunum and abolished the tissue contraction completely at concentration of 5.0 mg/ml. CONCLUSION: The study concludes that the methanol crude extract of aerial parts of M. africana has antispasmodic action possibly through the calcium channel blocking mechanisms. A new compound Myrsigenin was isolated from the EtOAc fraction of the plant.

Frequency distribution of HCV genotypes among chronic hepatitis C patients of Khyber Pakhtunkhwa.

BACKGROUND: Hepatitis C Virus (HCV) genotypes frequency is important for the predication of response to therapy and duration of treatment. Despite variable response rates experienced in the case of Interferon (IFN) -based therapies, there was scarcity of data on HCV genotypes frequency in Khyber Pakhtunkhwa (KPK). STUDY DESIGN: A total of 200 blood samples were collected from chronic HCV patients prior to the initiation of anti-viral therapy. The study population included patients from 6 districts of KPK. Active HCV infection was confirmed in case of all the patients by real time PCR. HCV genotypes were determined in each case by Type-specific PCR. RESULTS: The analysis revealed that out of 200 PCR positive samples; 78 (39%) were 2a, 62 (31%) were 3a, 16 (8%) were 3b, 34 (17%) were untypable while 1a, 2b and 1b were 3 (1.5%), 2 (1%) and 5 (2.5%), respectively. CONCLUSION: Genotype determination is not carried out prior to therapy in KPK. Although, the abundantly prevalent types (2a and 3a) of HCV in KPK are susceptible to combination therapy, yet resistance experienced in some of the chronic HCV patients may partly be attributed to the prevalence of less prevalent resistant genotypes (1a, 1b) of HCV among the population.

Anti-inflammatory and enzyme inhibitory activities of a crude extract and a pterocarpan isolated from the aerial parts of Vitex agnus-castus.

A new compound, 6a,11a-dihydro-6H-[1] benzofuro [3,2-c][1,3]dioxolo[4,5-g]chromen-9-ol was isolated from the ethyl acetate fraction of Vitex agnus-castus. The structure of this compound was identified with the help of spectroscopic techniques ((13)C NMR, (1)H NMR, HMBC, HMQC, NOESY and COSY). The compound showed low urease- (32.0%) and chymotrypsin- (31.4%) inhibitory activity, and moderate (41.3%) anti-inflammatory activity. The crude extract and various fractions obtained from the aerial parts of the plant were also screened for possible in vitro hemagglutination, antibacterial and phytotoxic activities. No hemagglutination activity against human erythrocytes was observed in crude extracts and fractions of V. agnus-castus. The fractions and crude methanolic extract showed moderate and low antibacterial activity. Exceptions were the CHCl(3) fraction, which showed significant antibacterial activity against Klebsiella pneumonia (81% with MIC(50)=2.19 mg/mL), the n-hexane fraction, which exhibited no activity against Salmonella typhi, and the CHCl(3) and aqueous fractions, which showed no activity against Bacillus pumalis. Moderate phytotoxic activity (62.5%) was observed by n-hexane fraction of V. agnus-castus against Lemna minor L at 1000 µg/mL.