1.
Chemical Equivalence Of Different Brands Of Oxytertacycline Hydrochloride And Its Minimum
by Sadaf Hina | Prof. Dr. Muhammad Ashraf | Dr. Aftab | Dr. Muhammad Adil Rasheed.
Material type: ; Format:
print
; Nature of contents: ; Literary form: Publisher: 2012Dissertation note: This project was designed to study the chemical equivalence of various brands of Oxytetracycline hydrochloride (long acting, short acting & PVP) approved by the ministry of health and available in the local market for veterinary use. Oxytetracycline was measured by HPLC method developed and standardized in the laboratory. Limit of detection (LOD) and limit of quantification (LOQ) of the Oxytetracycline by HPLC assay method were determined. From stock solution of working standard (Oxytetracycline hydrochloride) different concentrations 0.05, 0.1, 0.5, 1.0, 10, 25, 50 and 100µg per ml were prepared for the determination of LOD. The LOD calculated was 0.100(µg/ml) and LOQ was 0.5 (µg/ml). Correlation coefficient was 0.99994050. Concentration of the active ingredient (Oxytetracycline hydrochloride) in all preparations was same as mentioned on the label except Oxytetracycline (74%), Terrasym PVP-100 (81%), and Onyx-LA (72%).
MIC of Oxytetracycline hydrochloride against following bacterial isolates determined by micro-broth dilution test was Bacillus subtilis (50µg), Staphylococcus aureus (100µg), Eschericiha coli (50µg), Salmonella enterica (1000µg) and Pasturella multocida (50µg).It showed that all these bacterial cultures have developed resistance against Oxytetracycline hydrochloride.
Availability: Items available for loan: UVAS Library [Call number: 1468,T] (1).
2.
Isolation, Characterization Of Chondroitin Sulphate And Its Efficacy In Osteoarthritis
by Humaira Majeed Khan | Prof. Dr. Muhammad Ashraf | Prof. Dr. Mansur-ud-Din Ahmad.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2012Dissertation note: Chondroitin sulphate (CS) and Glucosamine sulphate (GS) are two main components of articular cartilage. It is believed that these molecules slow down wear and tear of cartilage. Moreover, if administered exogenously as drugs, these may initiate synthesizing capacity of cartilage. Among these, GS promotes the formation and repair of cartilage, whereas CS promotes elasticity and prevent cartilage breakdown by inhibiting degradative enzymes. Concurrent use of both structural units of cartilage as drugs in osteoarthritis (OA) may lessen the progression of disease.
The present study was conducted to elucidate the chicken keel cartilage as an alternate and potential source for this endogenous component that may be used exogenously to repair or prevent damage to joints. Chicken keel cartilages were collected from healthy broilers. CS was extracted using MgCl2 solution (3M), dialyzed and digested with papain. The extracted material was purified by ethanol precipitation, centrifugation and then freeze dried. Proximate analysis of semi-purified polysaccharides revealed the presence of carbohydrates (65.49±0.10), crude protein (12.82±0.26), ash (11.12±.56), moisture (9.88±0.32) and fat (0.69±0.14). Fiber contents were found to be nil in the processed samples. Dimethylmethylene blue binding (DMMB) assay was performed for determination of percent contents of CS in extracted semi-purified samples and mean concentration was found to be 70.77±2.35. Semi-purified polysaccharides were further characterized by FTIR (Fourier Transform Infrared Spectrometer) technique and characteristic Peaks of CS molecules were recorded at 854, 854 and 853 cm-1 and then compared with spectrum of standard CS. Protein content being a major impurity in extracted samples was determined by Bradford method quantitatively (4.64±0.29). Two protein impurities having 77.8 and 50.5 kDa molecular weights were revealed by SDS-PAGE.
Efficacy of semi-purified CS from chicken keel cartilage, standard CS from shark source and GS, alone and in combination in experimental OA rat model was evaluated. To develop OA similar to spontaneous OA, 10mg papain/0.5mL (Sigma, Cat # P 3125) in buffered solution of 0.05 M sodium acetate pH 4.5 was injected intra-articularly in each right knee joint of fifty five albino rats (pre-anesthetized with anesthetic ether). Ten rats (n= 10) were injected with 0.5mL of normal saline (0.9%) in right knee joint that served as control group. Then from fifty five papain injected rats, twenty five were divided into five groups (n=5) for development and assessment of OA model (OA groups). Progression of disease was monitored by clinical scores, histopathological scores and concentration of CTX-II as biomarker in sera samples of experimental rats by ELISA using a commercial kit (serum preclinical CartiLaps ® ELISA kit) for control and OA groups (n=5) on day 0 (control group) and days 1st, 7th, 14th, 21st and 28th post papain injection (OA groups). Highest mean clinical score (10.38±1.1) was observed on 1st day and least on 28th day post papain injection i.e. 5.00±.34. Highest mean histopathological score and CTX-II concentration was recorded on 28th day i.e. 12.82±1.64 and 36.82±3.81. Values of clinical scores, histopathological scores and CTX-II concentration reached to maximum on 21st day and then sustained thereon. Second phase of experiment is comprised of evaluating and comparing the efficacy of extracted CS samples (chicken keel cartilages), standard CS (shark source) alone and in combination with GS. For this purpose, remaining five rats out of ten injected with normal saline intra-articularly served as control groups along with treated and non treated groups of experimental rats. Remaining thirty OA induced rats were divided into six groups (five rats /group). Group 1 (n=5) called non treated group received only placebo till 60th day and served as negative control group.
Treated Group 2 received GS alone, Group 3 CS (standard) and Group 4 were given extracted CS. Group 5 was treated with combination of GS plus CS (standard) and Group 6 with GS plus CS (sample). Doses of glycosaminoglycans (GAGs) were administered as 1.2g/kg/day CS and 1.5g/kg/day GS alone and in combinations. Drugs were offered early in the morning in bolus form with feed (10g) after overnight fasting while non-treated group received only placebo (without any drug).
Anti-arthritis activities of CS standard and extracted alone and in combination with GS were assessed clinically, analyzed statistically by using one way ANOVA. Level of significance (P<0.05) was recorded by using Duncan's Multiple Range (DMR) Post hoc Test. Mean scores of clinical, histopathology and CTX-II concentrations observed at 60th day in control rats (without OA) were 0.00, 0.00 and 2.55, respectively. OA induced untreated group showed mean score for clinical signs, histopathological scores and CTX-II concentrations 4.15, 12.24 and 36.70 and GS treated group 3.19, 3.96 and 6.12 at 60th day of treatment, respectively. For CS (standard), mean scores of clinical signs, histopathological lesions and CTX-II concentrations were recorded as 2.64, 2.44 and 4.48 and for CS (extracted) were 2.26, 2.28 and 4.40 in sera correspondingly at 60th day of treatment. The lowest mean values of clinical signs, histopathology and CTX-II concentrations in sera of treated group with standard CS plus GS were found to be 0.94, 0.94 and 2.62 followed by extracted CS plus GS treated groups 01.05, 1.27 and 2.74, respectively. Clinical, histopathological scores and CTX-II concentrations in group of rats treated with combinations were found to reverse the diseased condition after 60th days of treatment as the values were close to that of normal rats and far away from OA rats. It is concluded that extracted CS from poultry has comparable efficacy with CS standard from shark source alone and in combination with GS. Poultry by-product (keel cartilage) is found to be an alternate and cheap source for CS (chondroprotective agent) as compare to expensive, less available and religiously prohibited source for Islamic countries particularly.
Availability: Items available for loan: UVAS Library [Call number: 1544,T] (1).
3.
Proteomic And Genomic Analysis Of Methicillin-Resistant Staphylococcus Aureus And Efficacy Of Indigenous Medicinal Plants Essential Oils
by Sarwat Ali Raja | Prof. Dr. Muhammad Ashraf | Dr. Tayyaba Ijaz | Dr. Aqeel Javeed | Prof. Dr. Aftab Ahmed Anjum.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: A Cohort study (prospective and observational) was performed to study the prevalence of Methicillin resistant Staphylococcus aureus from the healthy individuals of community, hospitalized patients and associated health-care workers and indigenous plants essential oils were screened as new, improved & potent antibacterial/s against resistant strains of MRSA.
The method involved isolation and identification of MRSA from surgical wounds of hospitalized patients & associated health care workers in a tertiary care hospital in Lahore and healthy volunteers from the community. Plant essentials oils & extracts were evaluated for their antibacterial activity against selected MRSA isolates. Oils were recovered by steam distillation using an all-glass distillation assembly. Then in vitro sensitivity and MICs of plant essential oils were determined using vancomycin and linezolid as commercial standards. The essential oils were screened further for the active constituents by column chromatography using various solvents and identification of compounds were performed by GC/MS analysis and the fractions which showed prompt results were evaluated for antimicrobial activity against the MRSA isolates in quest to find new therapeutic options. Finally effective essential oils and their active fractions were studied for their toxicity using in vitro Genotoxic assays such as Ames and Comet assays. To further ensure their beneficial effects antimutagenic effect of the essential oils were also studied.
Prevalence of S. aureus among patients was 52.9%, in HCWs 86.5% and in community 74% with an overall percentage of 72.6%. Among S. aureus those declared as MRSA were 91.8% from patients, 50.6% from HCWs and 59.5% from community with an overall percentage of 62.2% MRSA. Among the isolated MRSA overall 90.6% were Coagulase positive and 75.2% were biofilm positive.
SUMMARY
211
The pattern of MRSA resistance against current antibiotics have shown an overall increase in the resistance with maximum shown for lincomycin followed by tetracycline, ampicillin, fusidic acid, amoxicillin and piperacillin with tazobactam. The most effective options among current regime were tigecyclin, amikacin and meropenem showing an overall least resistance. Resistance against linezolid was observed with an overall percentage of 25.6 % and vancomycin 33.3% by disc diffusion method.
The MRSA isolates resistant to one or more groups of antibiotics were declared as MDRs. Among patients and health-care workers all were declared as MDRs where as in community 93.1% isolates were MDRs.
Upon Protein profiling using whole cell proteins 44 bands of the polypeptides were produced with molecular size 10-200kDa from the three sampling groups and were categorized into 5 clusters showing an overall significance correlation with each other explaining an interesting fact that all these strains were interlinked establishing the fact of flow of hospital acquired MRSA in the community and vice versa. This analysis also gave an insight in explaining the fact of horizontal transmission of infection within the hospital.
Keeping in view the raise in resistance among current available antibiotics indigenous medicinal plants essential oils were screened for active constituents exhibiting anti-bacterial effects against MRSA isolates.
Maximum yield was obtained from Carum copticum followed by Cuminum cyminum and minimum yield was obtained in case of Zingiber officinale. Upon qualitative analysis of all five essential oils Carum copticum essential oil showed zones of inhibition greater than the standards vancomycin and linezolid followed Cuminum cyminum and Zingiber officinale in all three
SUMMARY
212
sampling groups. Anethum sowa and Myristica fragrans essential oils showed no activity against MRSA.
Minimum inhibitory concentration of the three essential oils determined by micro broth dilution method indicated that Carum copticum showed least value in all three types of MRSA isolates followed by Zingiber officinale and Cuminum cyminum.
Effective essential oils were further fractioned using silica gel gravity columns. All the fractions obtained were screened for the anti-bacterial activity against all three types of MRSA isolates. Only fraction F1 of Carum copticum showed activity greater than pure essential oil and the two commercial standards of vancomycin and linezolid.
For the identification of active constituents GC/MS analysis was performed on all three essential oils and their respective fractions. In case of fraction F1 the most dominant constituents were Carvacrol, p-Cymene, Ʈ-Terpinene and Apiol. In other two plants none of the fractions were effective. Therefore it was concluded to use pure essential oils in case of Zingiber officinale and Cuminum cyminum rather than their individual fractions and incase of Carum copticum Fraction F1 has shown superior activity.
Finally these essential oils were tested for possible mutagenic effect using bacterial reversion mutation assay and Comet assay. No mutagenic effects were observed at MIC and above doses. These effective essential oils were also evaluated for possible antimutagenic effect. Both Carum copticum and Zingiber officinale essential oils showed strong antimutagenic effects and weak antimutagenic effect by Cuminum cyminum. Upon analysis of nuclear damage none of the plants essential oils and fraction F1 of Carum copticum showed genotoxic effects and indicated to be safe. Thus from the study it was concluded that Carum copticum essential oil and its fraction F1 were the most effective to be further investigated as an alternative treatment for MRSA infections. Availability: Items available for loan: UVAS Library [Call number: 2410-T] (1).
