Archive for the ‘Faculty of Pharmaceutical Sciences’ Category

DEVELOPMENT AND VALIDATION OF A 96-MICROWELL BASED ASSAY OF AZITHROMYCIN BY CHARGE TRANSFER COMPLEXATION WITH 2,3- DICHLORO-5,6-DICYANO-1,4-BENZOQUINONE

January 25, 2017

Author: Ejiofor Innocentmary Ifedibaluchukwu
Department: Pharmaceutical and Medicinal Chemistry
Affiliation: Nnamdi Azikiwe University Awka

A 96-microwell-based visible spectrophotometric assay was developed and validated for the determination of azithromycin in tablets. The formation of a coloured charge-transfer complex between azithromycin as n-electron donor, and 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone, as π-electron acceptor, was investigated, and employed as a basis for the development of the proposed assay. The proposed assay was carried out in a 96-microwell plate. The absorbance of the coloured charge transfer complex was measured at 490 nm wavelength using a microwellplate absorbance reader. The optimum conditions of the reaction and the analytical procedures of the assay were established. Under the optimum conditions, linear relationship with good correlation coefficient was found between the absorbance and the concentration of azithromycin in the range of 0.076 – 0.180 mg/ml, with the equation y=1.048x and R2 = 0.993. The ΔG at different temperatures were found to be -8429.8, -9342.6, -13011.9, -12678, and -15124.8 Kcal/mol. The limits of detection and quantitation were found to be 0.023 and 0.069 mg/ml respectively. The procedure gave good precision. The assay was applied successfully to the analysis of azithromycin in tablets with satisfactory result. The assay described has high throughput property, consumes little volume of organic solvent, offers reduction in analysis cost and the exposure of the analyst to the toxic effects of organic solvent.

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WOUND-HEALING ACTIVITY OF THE AQUEOUS LEAF EXTRACT AND FRACTIONS OF FICUS EXASPERATA (MORACEAE) AND SAFETY EVALUATION IN ALBINO RATS

January 12, 2017

Author: Umeh Victoria Nonyelum
Department: Pharmacy and Toxicology
Affiliation: Nnamdi Azikiwe University Awka

The leaf of Ficus exasperata Vahl Enum (Moraceae) is used in various parts of Nigeria and some parts of Africa for the treatment of various pathological states including cutaneous wounds. The present study was undertaken to justify the use of the aqueous extract of the leaf of Ficus exasperata for the treatment of cutaneous wounds and to evaluate the effect of its prolonged oral administration. Shade-dried leaves of Ficus exasperata were grounded, extracted using cold maceration method and freeze-dried to a constant weight. The acute and sub-chronic toxicity studies of the crude extract were carried out in rats. The water extract was fractionated with n-hexene, methanol, ethyl acetate and chloroform. The anti-microbial, qualitative phytochemistry as well as the wound-healing effect of the water extract and fractions was studied. For the acute toxicity, no death was recorded up to 5000mg/kg.There were a significant increase(p ˂0 .05) in ALT and AST on days 61 and 91 (4000 mg/kg group). There was significant increase (p ˂0.05) in serum ALP on day 31 (4000 mg/kg) and on days 61 and 91(1000, 2000 and 4000 mg/kg). Hematological analysis revealed no significant changes (p˃0.05) in PCV, RBC and HB value in all the groups. Significant decrease (p˂0.05) in the WBC occurred in groups 1000, 2000 and 2000 mg/kg on day 91. Histopathological data revealed extensive damage in the kidney architecture with pronounced loss of glomeruli in 4000 mg/kg group. There was hemorrhage within the central vein of the liver for 4000 mg/kg group. A significant wound contraction (p˂0.05) was observed in 5, 15, 20, and 25%w/w and the positive control groups on the 4th, 8th, 12th and 16th days of the treatment. Of all the fractions tested, significant contraction (p<0.05) was only noticed in chloroform fraction though lower than that of the aqueous extract. The phytochemical analysis of the extract revealed the presence of alkaloid, saponins, flavonoids, glycosides, tanins, phlobatannins, terpenoids, fats and oil. The extract exhibited a concentration-dependent inhibition against the tested micro-organisms with minimum inhibitory concentration ranging from 10 to 250 mg/ml of the extract. In conclusion, the present study justified the use of the leaf extract of F. exasperata in the treatment of cutaneous wounds. However, prolonged oral ingestion of the extract can adversely affect the liver and the kidney.

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ANTIOXIDANT ACTIVITY OF THE LEAF EXTRACT AND FRACTIONS OF SPONDIAS MOMBINLINN ANARCADIACAE

January 11, 2017

Author: Duru Chioma Adaeze
Department: Pharmacology and Toxicology
Affiliation: Nnamdi Azikiwe University Awka

Spondias mombin is used ethnomedicinally in Nigeria and other parts of the world in the treatment of various diseases including epilepsy, arthritis, anxiety, wounds and schizophrenia. In this study, we investigated acute and subchronic toxicity of ethanol extract as well as in vitro and in vivo antioxidant activities of ethanol extract and fractions of leaves of Spondias mombin harvested fresh and shed dried. The ability of the extract and fractions to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (0.6 mMol) was used to assess their in vitro antioxidant activities while in vivo antioxidant activity was evaluated using carbon tetrachloride (CCL4) induced oxidative stress. Acute toxicity studies of the extract showed that the median lethal dose (LD50) was above 5000mg/kg body weight and no death was recorded throughout the 90 days subchronic toxicity test. The test extract and fractions exhibited potential scavenging effects on DPPH radical with methanol fraction exhibiting the highest scavenging activity (IC50 2μg/ml), followed by chloroform (IC50) and ethanol (IC5044μg/ml). Methanol showed a higher DPPH scavenging activity than the standard, ascorbic acid (IC50 4μg/ml). CCL4 decreased serum levels of antioxidant enzymes (catalase, superoxide dismutase) and increased the formation of malondialdehyde (MDA) in untreated animals while significant increases in these enzymes and decreases in MDA were observed in the treatment groups, with the methanol fraction exhibiting the highest in vivo antioxidant effect even more than the positive control. The extract (750 mg/kg, 1500mg/kg and 3000mg/kg) showed no significant effect on haematological and biochemical parameters. Significance between control and extract treated groups were determined using students t-test and two way ANOVA. Differences between means were considered significant at p<0.05.Histopathological studies showed no obvious pathological changes in the liver and kidney architecture of treated rats when compared to those of the control group. These findings substantiate the high safety profile and the free radical scavenging/antioxidant activity of Spondias mombin leaves and it could be a valuable herbal alternative to synthetic drugs for myriad of diseases associated with oxidative stress.

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CHARACTERIZATION OF ORGANIC CATION AND MULTIDRUG AND TOXIN EXTRUSION TRANSPORTERS IN RESPIRATORY EPITHELIAL CELLS

January 11, 2017

Author: Ugwu Malachy Chigozie
Department: Pharmaceutical Microbiology and Biotechnology
Affiliation: Nnamdi Azikiwe University Awka

To fully exploit membrane transporters for targeted drug delivery in the lung, the use of a readily
available and well-characterized tissue culture model and cheap/ easily detectable substrates are indispensable. Human multidrug and toxin extrusion proteins (hMATEs) are organic cation /H+ antiporters, which have been identified to be involved in the secretory transport of various organic cations across the cell membranes. Fluorescent dyes are important class of sub-cellular probes which allow the examination of cellular processes in real-time with minimal impact upon the processes. They are, therefore widely used in bio-analysis. One of primary objectives of the present study is therefore to screen for protein expression, localization and transport characteristics of hMATEs in Calu-3 cell line using a fluorescent cationic dye: 4,6 di-amidino-2-phenyhlindole (DAPI). The study equally evaluated the suitability and relevance of four fluorescent dyes as non-radioactive probes for characterizing organic cation transporters using fluorometry. Also the suitability of human nasal epithelial (HNE_248 ) cell as a physiologically adequate and relevant cell culture model for investigating the interactions of inhaled drugs with the organic cations transporters (OCT) in the respiratory epithelium was evaluated. Substrate uptake, inhibition, and transport were performed to establish active transport mechanisms. MATEs and OCTs genes expression were also determined with quantitative polymerase chain reaction (qPCR). PCR studies revealed the expression of hMATE1 gene in Calu-3 cells. The uptake of DAPI followed Michealis-Menteen kinetics. It was concentration (Vmax=6.663  2.610M/mg protein/ 5mins, Km =2252  1632M), temperature (uptake at 37 C4C) and pH (highest uptake at an alkaline pH of 8)-dependent. Cimetidine, famotidine, and propidium iodide (PI ) significantly inhibited DAPI uptake with IC50 of 204.1M, 287.2M and 324.6M respectively while probenecid had no observable inhibition on the dye’s uptake. Transport of the dye across the cells was polarized {apicalbasolateral (APBL) transport was 3fold  BLAP), saturable (Km = 44.76  39.41 μM, Vmax = 0.0647  0.01451 nmol/cm2/s.}. Intracellular uptake of the cell-permeant fluorescent dyes was demonstrated to be enhanced in Chinese hamster ovary (CHO) cells stably expressing hOCT2 when compared with control CHO-wild cells. This increase of dyes influxes was found to be concentration –dependent and a saturable carriermediated process (except for rhodamine 123) .Their kinetic parameters were: Km = 414.6  141M, Vmax=0.0219  0.005 M/mg protein/ 7 mins (4-Di-1-ASP), Km = 72.63  12.02M, Vmax=0.131 0.007M/mg protein/ 7 mins (amiloride) and Km = 82.47  29.15M, Vmax=0.952  0.17 M/mg protein/ 7 mins (rhodamine 6G) respectively. Their uptake was pH -dependent (higher uptake at alkaline pH range of 8.0 – 8.5 and the uptake of dyes was significantly (P < 0.05) decreased in acidic conditions in all the transfected cells.The uptake was reduced by various known inhibitors of hOCT2 activity. Quinine, verapamil, corticosterone and quinidine markedly reduced the uptakes of the dyes across board. RT-PCR analysis showed high mRNA levels for two polyspecific organic cation/carnitine transporters, OCT3 and OCTN2, in the human nasal epithelia of adenocystic fibrotic (HNE-248) cells. The uptake of the dye was saturable/ concentration- dependent and with a Km of 2640 ± 224 M and a Vmax of 0 .0753 ± 0.008 M/ mg protein/15 min. Significantly Statistical difference was observed between total uptake at 37°C and 4°C (p < 0.05). The intracellular accumulation of the compound was inhibited by organic cation transporters (OCTs) and carnitine/organic cation transporter (OCTNs) inhibitors {quinine,quinidine &verapamil with IC50s of 226,1306 and 1131M respectively.}. The uptake mechanism was found to be independent of sodium and glucose.

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CIPROFLOXACIN HYDROCHLORIDE– SODIUM CHOLATE COMPLEX FOR ENHANCED ANTIBACTERIAL ACTIVITY

January 11, 2017

Author: Ugochukwu Jane Ijeoma
Department: Pharmaceutical Microbiology and Biotechnology
Affiliation: Nnamdi Azikiwe University Awka

This research work considers ciprofloxacin hydrochloride sodium-cholate complex for enhanced and prolonged anti-bacterial activity. Ciprofloxacin hydrochloride was reacted with sodium cholate to develop a hydrophobic ion-pair complex. The complex was characterized by octanol water partitioning, melting point determination, UV-visible and Fourier Transform Infra Red (FTIR) spectrometry. The activity of the complex formed was determined against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Streptococcus pneumoniae by the agarwell diffusion method. The safety profile of the complex was determined in white male albino mice. Pharmacokinetic evaluation of the drugs was done by agar-well diffusion method of bioassay, using 6 groups of white albino rats (n=4) administered with 7.1 mg/kg and 14.2 mg/kg uncomplexed drug; 7.1 mg/kg and 14.2 mg/kg of complex and 7.1 mg/kg or 14.2 mg/kg PEGYlated complex in a single dose by intraperitoneal route. Raw data were subjected to statistical analyses by t-test and ANOVA, using the SPSS software. Also, data from the pharmacokinetic studies were analyzed using the WINonlin software. Significance was accepted at 5 % level. The complex was whitish to off-white in color and crystalline, with a melting point of 238 0C. Maximum absorption was obtained at 409 nm. The FTIR analysis of the complex as contrasted to the uncomplexed drug revealed significant bond changes. Best complexation was achieved at molar ratio of the drug to complexing agent of 1:1 and pH of 9. The percentage weight of ciprofloxacin base in the complex was determined as 44.9 %. Weight by weight, the complex showed comparable antibacterial activity to the uncomplexed drug. The LD50 of the complex was above 5000 mg/kg. The biochemical, hematological and histology results showed complexation did not increase adverse effects of ciprofloxacin, and any such slight effects were reversible upon discontinuation of administration. The PEGYlated complex showed higher AUC and Cmax peak than the uncomplexed drug. Thus, we could conclude that the PEGYlated complex would be therapeutically more beneficial than the uncomplexed drug.

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