Molecular characterization of mycotoxin-producing Aspergillus parasiticus and sensitivity pattern to different disinfectants

Aims@#The study was aimed to isolate and characterize the mycotoxin-producing filamentous Aspergillus parasiticus from the feed samples. The sensitivity pattern of the isolates was assessed against different disinfectants.@*Methodology and results@#Fifty different feed samples were screened for A. parasiticus isolation. Isolates were subjected to macroscopic and microscopic characterization. Polymerase chain reaction was performed to confirm the isolates at the genomic level. Mycotoxin producing potential of the isolates was assessed by thin-layer chromatography (TLC). To quantify the toxins, high performance liquid (HPLC) was employed. The antifungal potential of disinfectants was determined by the well diffusion method followed by minimum inhibitory concentration (MIC) calculation. Out of twenty isolates of A. parasiticus, 11(55%) isolates were observed positive for toxin production. Three toxigenic isolates (AspP2, AspP4 and AspP8) were selected to evaluate their susceptibility against disinfectants by well diffusion method. AspP2 produced maximum (5.90 ng/mL) toxin, followed by AspP4 (3.11 ng/mL) and AspP8 (18.47 ng/mL). Terralin showed maximum fungicidal activity with 29.66 ± 8.08 mm zone of inhibition at 0.42 μg/mL MIC. Hypochlorite and Instru Star showed 99% disinfection with 30, 60 and 90 min contact time (6 mean log reduction) for all A. parasiticus isolates. Alpha Guard inhibited growth after 15 min contact time for all the isolates.@*Conclusion, significance and impact of study@#This study provides data indicating the contamination of feed samples with mycotoxin-producing A. parasiticus isolates and their sensitivity against commercially available disinfectants. Use of these disinfectants in appropriate concentrations and time could help prevent the contamination of food, feed and healthcare settings with the fungal species.

Phytochemical analysis of Berberis lyceum methanolic extract and its antiviral activity through the restoration of MAPK signaling pathway modulated by HCV NS5A

Objective: To evaluate the antiviral activity and phytochemicals of selected plant extracts and their effect on the mitogen-activated protein kinase (MAPK) signaling pathway modulated by hepatitis C virus (HCV) nonstructural protein 5A (NS5A). Methods: A total of ten plant extracts were initially screened for their toxicities against HepG2 cells. The non-toxic plants were tested for their inhibitory effect on the expression of HCV NS5A at both mRNA and protein levels using real-time PCR and Western blotting assays, respectively. The differential expression of the genes associated with MAPK pathway in the presence of NS5A gene and plant extract was measured through real-time PCR. Subsequently, the identification of secondary metabolites was carried out by phytochemical and HPLC analysis. Results: The phytochemical profiling of Berberis lyceum revealed the presence of alkaloids, phenols, saponins, tannins, flavonoids, carbohydrates, terpenoids, steroids, and glycosides. Similarly, quercetin, myricetin, gallic acid, caffeic acid, and ferulic acid were identified through HPLC analysis. The methanolic extract of Berberis lyceum strongly inhibited HCV RNA replication with an IC50 of 11.44 μg/mL. RT-PCR and Western blotting assays showed that the extract reduced the expression of HCV NS5A in a dosedependent manner. Berberis lyceum extract also attenuated NS5Ainduced dysregulation of the MAPK signaling pathway. Conclusions: Our findings suggest that Berberis lyceum extract strongly inhibits HCV propagation by reducing HCV NS5Ainduced perturbation of MAPK signaling.

Phytochemical analysis of Berberis lyceum methanolic extract and its antiviral activity through the restoration of MAPK signaling pathway modulated by HCV NS5A

Objective: To evaluate the antiviral activity and phytochemicals of selected plant extracts and their effect on the mitogen-activated protein kinase (MAPK) signaling pathway modulated by hepatitis C virus (HCV) nonstructural protein 5A (NS5A). Methods: A total of ten plant extracts were initially screened for their toxicities against HepG2 cells. The non-toxic plants were tested for their inhibitory effect on the expression of HCV NS5A at both mRNA and protein levels using real-time PCR and Western blotting assays, respectively. The differential expression of the genes associated with MAPK pathway in the presence of NS5A gene and plant extract was measured through real-time PCR. Subsequently, the identification of secondary metabolites was carried out by phytochemical and HPLC analysis. Results: The phytochemical profiling of Berberis lyceum revealed the presence of alkaloids, phenols, saponins, tannins, flavonoids, carbohydrates, terpenoids, steroids, and glycosides. Similarly, quercetin, myricetin, gallic acid, caffeic acid, and ferulic acid were identified through HPLC analysis. The methanolic extract of Berberis lyceum strongly inhibited HCV RNA replication with an IC50 of 11.44 μg/mL. RT-PCR and Western blotting assays showed that the extract reduced the expression of HCV NS5A in a dosedependent manner. Berberis lyceum extract also attenuated NS5Ainduced dysregulation of the MAPK signaling pathway. Conclusions: Our findings suggest that Berberis lyceum extract strongly inhibits HCV propagation by reducing HCV NS5Ainduced perturbation of MAPK signaling.

Pharmaceutical evaluation of mefenamic acid tables available in local market of Rawalpindi Islamabad, Pakistan

Mefenamic acid is a commonly prescribed non-steroidal anti-inflammatory drug in mild to moderate pains. Different multinational and national brands of mefenamic acid are available and are being frequently prescribed in Rawalpindi and Islamabad, Pakistan. Pharmacies and dispensaries associated with clinics usually dispense local brands which are cost effective as compared to multinational brands. Present study was conducted to assess pharmaceutical equivalence of one multinational [XI] and four national brands namely, X2, X3, X4 and X5, so as to prove their use as an alternative to one another. All the brands were evaluated for their quality control parameters of hardness, friability, weight variation, disintegration, dissolution and assay of active ingredient. All the brands fulfilled quality control criteria's of USP while 2 of the national brands did not meet B.P standards of assay of active ingredients by brands XI, X4 and X5 were pharmaceutical equivalents and can be substituted one another. Results of the study reveal that good manufacturing practice guidelines are dominantly being followed in multinational as well as national pharmaceutical companies?

Determination of S- and R-warfarin enantiomers by using modified HPLC method

Warfarin is a commonly prescribed anticoagulant existing in two enantiomeric forms S- and R-warfarin. Many techniques have been used to analyze warfarin in plasma but less frequently for enantiomeric analysis. One of the HPLC method employed was further simplified and made economical. Method was validated according to ICH guidelines and was found to be sensitive, reliable and less time consuming. For both enantiomers, LLOQ was 12.5ng/mL. The CV% and accuracy for method were in the range of 0.8-14.6% and 92-107% respectively. The recoveries for both enantiomers were in the range of 86-103.8%. Blood samples were collected from 170 stable patients taking warfarin and S- and R-warfarin levels were determined by this method. Majority of subjects were found to have S/R-warfarin ratio of about 1:2 as reported in previous studies due to rapid clearance of S-enantiomer than R-enantiomer. However individual subjects data was suggestive of presence of slow metabolizers of S-warfarin leading to altered S/R ratio. Previous studies have also pointed out CYP2C9 polymorphism being responsible for such inter-individual differences in S-warfarin metabolism. So plasma warfarin S/R ratio may serve as a useful phenotypic test for CYP2C9 polymorphism

Assessment of sex differences in Pharmacokinetics of carvedilol in human

Carvedilol is an anti-hypertensive agent capable of blocking both alph [a] and beta [p] receptors used to preclude cardiac arrhythmias and angina

The study was designed to evaluate the Pharmacokinetics of carvedilol in human male and female volunteers

Healthy male and female [twenty each] volunteers were finalized for the study after preliminarily clinical examination. Blood samples were collected at specific time intervals after giving an oral dose of 12.5mg carvedilol, separated the plasma and placed at -80°C until analysis. Estimation of carvedilol in human plasma was accomplished by High performance liquid chromatographic [HPLC] method using fluorescent detector

Plasma concentration-time curve was used for calculation of pharmacokinetic parameters using two-compartment open model

Mean [SD] values of AUC and C[max] 0.076+/-0.021microg.h/ml and 0.024+/-0.005microg/mL, respectively] in male differ significantly [P<0.05] from the female 0.197+/-0.042jug.h/ml and 0.048+/-0.02microg/mL, respectively]

Overall, bioavailability of carvedilol was somewhat higher in females than in males, but these differences could be expounded by the lower body weight of female

Conversely, no significant differences were found for tmax, clearance and half-life in male and female

Moreover the ethnicity had significant impact on the Pharmacokinetics of carvedilol in human

Bioequivalence study of montelukast tablets in healthy Pakistani volunteers

Montelukast is a leukotrien receptor antagonist used for asthma treatment. Objective of this study was to evaluate the bioequivalence of two montelukast 10mg tablets, Innovator drug [Singulair] as reference and other locally manufactured drug [Montiget] in 12 healthy volunteers. It was randomized, single dose, two-period crossover study with 1 week washout period. Blood samples [4-5 ml] were collected before and after drug administration and plasma was separated for analysis. Concentrations of montelukast at different time intervals were determined by validated UV-HPLC method at 345nm wavelength. Bioequivalence was assessed by using non compartmental approach and also calculated the 90% confidence interval of the least-squared pharmacokinetic parameters [C[max], AUC[0-t] and AUC[0-infinity]]. On average, C[max], AUC[0-t], AUC[0-inf], was 2.35 micro g/ml, 1.28 micro g.h./ml, 1.67 micro g.h./ml, for innovator drug and 2.53 micro g/ml, 1.53 micro g.h./ml, 1.96 micro g.h./ml, for test drug, respectively. Confidence interval [90%] for C[max], AUC[0-t] and AUC[0-inf] was 89-97%, 85-91% and 81-98% respectively. No statistical difference was found between the C[max] and AUC values of test and reference drugs. The confidence intervals for C[max], AUC[0-t] and AUC[0-infinity] are fully laid within the acceptable range of FDA [80-125%], thus two formulations are considered to be bioequivalent

Comparative bioavailability and pharmacokinetics of two oral formulations of flurbiprofen: a single-dose, randomized, open-label, two-period, crossover study in Pakistani subjects

Comparative bioavailability studies are conducted to establish the bioequivalence of generic formulation with that of branded reference formulation, providing confidence to clinicians to use these products interchangeably. This study was carried out to compare a locally manufactured formulation of flurbiprofen with that of a branded product. Twenty two healthy male adults received a single dose of flurbiprofen [100mg] either generic or branded product according to randomization scheme on each of 2 periods. Blood samples were collected and plasma flurbiprofen concentration was determined by a validated HPLC method. Pharmacokinetic parameters like AUC[0-t], AUC[0- Infinity], C[max], T[max], t[1/2] Vd and clearance were determined. The 90% CI for the ratio of geometric means of test to reference product's pharmacokinetic variables was calculated. Pharmacokinetic parameters for two formulations were comparable. Ratio of means of AUC[0-24], AUC[0- Infinity] and C[max] for test to reference products and 90% CI for these ratios were within the acceptable range. The p-values calculated by TOST were much less than the specified value [p-0.05]. ANOVA gave p-values which were more than the specified value [p-0.05] for sequence, subject, period and formulation. Test formulation of flurbiprofen [tablet Flurso] was found to meet the criteria for bioequivalence to branded product [tablet Ansaid] based on pharmacokinetic parameters

Quantitative determination of deferiprone in human plasma by reverse phase high performance liquid chromatography and its application to pharmacokinetic study

Deferiprone [1, 2 dimethyl-3-hydroxypyrid-4-one] is considered to be the standard iron chelator. Pharmacokinetic studies of generic formulations are required in local condition before placed on the market. High performance liquid chromatographic [HPLC] method was used for quantification of deferiprone in human plasma using UV/VIS detector. Chromatographic separation was carried out on C[18] column, with a mobile phase of methanol-buffer [18:82, v/v], pH 3.5, and caffeine was used as an internal standard. The calibration curve was linear over the range 0.25-10 micro g/mL in human plasma [R[2] = 0.9994]. After oral administration of deferiprone [500 mg] to human, the plasma concentration-time curve of deferiprone was conformed to two-compartment open model. The deferiprone plasma concentration showed a rapid absorption and average area under the plasma concentration-time curve [AUC] of deferiprone was 17.0 +/- 1.23 h. micro g/mL. Average absorption and elimination half-life values of deferiprone of 24 volunteers were 0.62 +/- 0.12 and 2.65 +/- 0.43 hours. This study confirms the rapid absorption of deferiprone in humans. AUC was similar to that previously reported but C[max] was slightly lower than that stated in the literature