Investigation of sweeping as a sample enrichment method in micellar electrokinetic chromatography in the analysis of pharmaceutical preparations and biological fluids

The present thesis deals with the study of sweeping as one of the most important sample preconcentration techniques in micellar electrokinetic chromatography (MEKC). The work includes the study of the fundamentals of sweeping as well as the application in the pharmaceutical field. The thesis is divided into four main parts. In the first part of the thesis, the processes involved in sweeping under homogeneous and under inhomogeneous electric field conditions are theoretically discussed. These processes include stacking or destacking of micelles when entering the sample zone, sweeping of analytes by the stacked or destacked micelles, and destacking or stacking of the swept analyte zone. A new robust and reliable method for the assessment of the sweeping efficiency is developed based on recording the peak height dependent on the injected sample volume. The values obtained via this method agree well with theoretically predicted ones. The results obtained show that the sweeping efficiency for neutral analytes is independent of the electric conductivity of the sample matrix. It is also shown that under specific conditions unexpectedly high enrichment factors are obtained which are attributed to the focusing of neutral analytes by micellar transient isotachophoresis. In the second part of the thesis, we extend our developed method for the assessment of sweeping efficiency to the general case, in which the distribution coefficient and the electric conductivity is varied in the sample and BGE compartments. It is shown that in the general case – in contrast to the classical description of sweeping – the obtainable enrichment factor is not only dependent on the retention factor of the analyte in the sample zone but also dependent on the retention factor in the BGE. An additional focusing/defocusing step is confirmed and the term “Retention factor gradient effect (RFGE)” is introduced. The validity of the derived equation is confirmed experimentally and theoretically under variation of the organic solvent content (in the sample and/or the BGE), the type of organic solvent (in the sample and/or the BGE), the electric conductivity (in the sample), the pH (in the sample), and the concentration of surfactant (in the BGE). In the third part of the thesis, the processes involved in sweeping in cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) are theoretically discussed with a special focus on dynamic pH junction and adsorption of the analyte onto the capillary wall. Ethylparaben (pharmaceutical preservative) as an example of acidic analytes and desloratadine (antihistaminic drug) as an example of basic analytes are investigated using different types of ß-cyclodextrins. The presence of RFGE as an additional focusing/defocusing effect in sweeping-CD-MEKC is confirmed under the conditions of different content of cyclodextrin and different pH between the sample and the BGE. Desloratadine shows an unexpectedly low enrichment factor compared to the less hydrophobic ethylparaben. This unexpected behavior is ascribed to the strong adsorption of the protonated species of this drug onto the inner capillary wall in the sample zone that significantly counteracts the sweeping process. This effect is confirmed by the improvement in the enrichment factor achieved by the addition of a dynamic coating agent to the sample solution. In the fourth part of the thesis, a CD-MEKC method is developed for the simultaneous determination of the antihistaminic drugs loratadine and desloratadine (the major metabolite and an impurity of loratadine). The tendency of these drugs, as hydrophobic basic analytes, to be adsorbed onto the inner capillary wall and the difficulty to separate them due to the extremely high retention factors make the present study challenging. The use of a low pH sample solution overcome problems associated with the low solubility of the studied analytes in aqueous solution while having advantages with regard to online focusing. In addition, the use of a basic BGE and the presence of cyclodextrin reduce the adsorption of these analytes in the separation compartment. The separation is achieved in less than 7 min using a BGE consisting of 10 mmol L-1 sodium borate buffer, pH 9.30 containing 40 mmol L-1 SDS and 20 mmol L-1 hydroxypropyl-β-CD while the sample solution is composed of 10 mmol L-1 phosphoric acid, pH 2.15. All validation parameters are thoroughly investigated. The developed method is successfully applied to the analysis of the studied drugs in tablets and in spiked human urine. Moreover, desloratadine is detected at the stated pharmacopeial limit (0.1%) as an impurity in loratadine bulk powder. The obtained results are compared with those of the official liquid chromatographic method and are found in a good agreement

Evaluation of the genetic effects of the in vitro antimicrobial activities of Rhazya stricta leaf extract using molecular techniques and scanning electron microscope

Rhazya stricta plants have always played a major role in the treatment of human and animal diseases and it has main role in the folk medicine. The aim of this study was to explore the potential antimicrobial activities of the aqueous leaves extract of R. stricta on Gram-negative and Gram-positive food-borne bacteria and evaluate the antimicrobial effect at the molecular level. The results indicate that the aqueous leaves extract of R. stricta exhibited the antimicrobial activity against tested microorganisms. A clear, but significantly smaller, inhibition zones were formed after the treatment of two Gram-negative bacteria (Escherichia coli and Aeromonas hydrophila) and one Gram-positive bacteria (Staphylococcus aureus) with the aqueous leaves extract of R. stricta (50 mg) comparing with those formed after the treatment with streptomycin (15 mg). Moreover, the results obtained after the treatments of bacterial strains with elevated concentrations of aqueous extracts of the wild plant of R. stricta leaves reveled that the extract has potent lethal activities as the growth turbidity decreased as the concentration or time of exposure increased. In addition, the observation by the scanning electron microscope showed that cells of the bacterial strains were damaged after the treatment with plant extracts. The noticed antimicrobial effect was explored at the molecular level, using restriction fragment length polymorphism (RFLP) analysis of the plasmid DNA and random amplification of polymorphic DNA (RAPD) analysis of the genomic DNA extracted from the control (untreated) and R. stricta leaf extract-treated bacterial strains. The results demonstrate polymorphic band pattern for most treated microbes compared with the wild type (untreated) strain. Concerning gene expression under the same conditions, total protein contents of the three treated bacteria showed significantly gradual increase in all of the treatment doses compared to control. In addition, the SDS-PAGE of the bacterial cellular proteins resulted in the induction of some protein bands under the treatment conditions. All these results strongly point out the mutagenicity, lethal and antimicrobial effect of the leaves extract of R. stricta. The results indicate the possibility of using the leaves extract of R. stricta as a source of antibacterial compounds for treatment of infections caused by multi-drug resistant (MDR) bacterial pathogens.Keywords: Medicinal plants, Rhazya stricta, antimicrobial, mutagenicity, RAPD, RFLP, SEM, E. coli, S. aureus, A. hydrophilaAfrican Journal of Biotechnology Vol. 12(21), pp. 3171-318

Genetic diversity and DNA fingerprint study in tomato (Solanum lycopersicum L) cultivars grown in Egypt using simple sequence repeats (SSR) markers

A collection of ten cultivars of tomato grown in Egypt were screened with 20 simple sequence repeat (SSR) primers in order to determine genetic identities, genetic diversity and genetic relationships among these  cultivars. On an average, 38 alleles were amplified using SSR primers with scorable fragment sizes ranging  from approximately 75 to 275 bp. 23 alleles were polymorphic thus revealing 60.5% of polymorphism. The  genetic similarity estimated according to SSR data was scaled between 17.6 and 93.2%, suggesting the  potential of SSR markers in discriminating among plants of close or distant genetic backgrounds. Unweighted pair group method with arithmetic mean (UPGMA) clustering grouped the cultivars into two groups where the  two Egyptian cultivars Edkawy and Giza 80 were clustered in different group. In addition, clustering was found  consistent with the known information regarding growth habit. The genetic distance information obtained in  this study might be useful to breeder for planning crosses among these cultivars.Key words: Tomato cultivars, diversity, Simple sequence repeats (SSR), Egypt

Clinical, biochemical and inflammatory predictors of mortality in patients with spontaneous bacterial peritonitis

Background: Spontaneous bacterial peritonitis (SBP) is a serious complication of liver cirrhosis. It contributes to high morbidity and mortality in this population. In-hospital mortality of SBP ranges between 20% and 40%, suggesting that further refinements are essential in managing SBP. Early recognition of high-risk patients would enable us to reduce the short-term mortality.Objective: The current study aimed to evaluate the value of clinical, biochemical and inflammatory markers in the prediction of 1-month and 3-month cumulative mortality in patients with SBP.Patients and methods: Two hundred patients with a confirmed diagnosis of SBP were enrolled. They were admitted and received the proper treatment at the National Liver Institute Hospital-Menoufia University, Egypt. Patients were prospectively followed up for mortality over a period of three months. Predictors of mortality were assessed and analyzed.Results: Mortality rates were 20% and 41% at 1 month and 3 month respectively. Our findings showed that low blood pressure, abdominal pain, fever, higher Child-Pugh score, MELD score, serum bilirubin, INR, serum creatinine, C-reactive protein to albumin (CRP/Albumin) ratio, neutrophil–lymphocyte ratio (NLR), massive splenomegaly and large ascites have been demonstrated as risk factors associated with short-term mortality.Conclusion: SBP carries a high risk of mortality among cirrhotic patients. Clinical parameters (low blood pressure, abdominal pain, fever, massive splenomegaly and large ascites), prognostic scores (Child-Pugh and MELD) and inflammatory markers (CRP, CRP/albumin ratio, and NLR) seem to be accurate and reliable tools that could independently predict short-term mortality in patients with SBP

A reproducible protocol for regeneration and transformation in canola (Brassica napus L.)

The objective of the present study is to develop an efficient protocol for shoot and plant regeneration using five commercial canola cultivars grown under the Egyptian agricultural conditions. The regeneration efficiency from hypocotyl explants was examined. The data indicated that embryonic calli were formed within two weeks in the presence of 1 mgl-1 2,4-D. Adventitious shoots emerged from the embryonic callus in the presence of 4.5 mgl-1 BA. The cultivars showed a varied response to shoot regeneration. Regeneration frequency was high in the cultivar Sarow-4 (68%) followed by Masrri L-16 (64%) compared with the other cultivars tested. Hypocotyl explants from the cultivars Sarow-4 and Semu-249 were inoculated and co-cultivated with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pBI-121 containing the neomycin phosphotransferase-II gene (NPT-II). The resulted putative transgenic plantlets were able to grow under knanamycin containing medium. The stable integration of the NPT-II gene into the plant genomes was tested by PCR using NPT-II -specific primers. The GUS gene expression can be detected only in the transgenic plants. The reported protocol in the present study is repeatable and can be used to regenerate transgenic canola plants expressing the genes present in A. tumifaciens binary vectors.Keywords: Agrobacterium, canola, GUS assay, regeneration, fransformation, NPT II gen

Conserved peptides within the E2 region of Hepatitis C virus induce humoral and cellular responses in goats

The reason(s) why human antibodies raised against hepatitis C virus (HCV) E2 epitopes do not offer protection against multiple viral infections may be related to either genetic variations among viral strains particularly within the hypervariable region-1 (HVR-1), low titers of anti E2 antibodies or interference of non neutralizing antibodies with the function of neutralizing antibodies. This study was designed to assess the immunogenic properties of genetically conserved peptides derived from the C-terminal region of HVR-1 as potential therapeutic and/or prophylactic vaccines against HCV infection. Goats immunized with E2-conserved synthetic peptides termed p36 (a.a 430–446), p37(a.a 517–531) and p38 (a.a 412–419) generated high titers of anti-p36, anti-p37 and anti-P38 antibody responses of which only anti- p37 and anti- p38 were neutralizing to HCV particles in sera from patients infected predominantly with genotype 4a. On the other hand anti-p36 exhibited weak viral neutralization capacity on the same samples. Animals super-immunized with single epitopes generated 2 to 4.5 fold higher titers than similar antibodies produced in chronic HCV patients. Also the studied peptides elicited approximately 3 fold increase in cell proliferation of specific antibody-secreting peripheral blood mononuclear cells (PBMC) from immunized goats. These results indicate that, besides E1 derived peptide p35 (a.a 315–323) described previously by this laboratory, E2 conserved peptides p37 and p38 represent essential components of a candidate peptide vaccine against HCV infection

Systemic Antibiotic Therapy Reduces Circulating Inflammatory Dendritic Cells and Treg-Th17 Plasticity in Periodontitis

Periodontitis (PD) is a common dysbiotic inflammatory disease that leads to local bone deterioration and tooth loss. PD patients experience low-grade bacteremias with oral microbes implicated in the risk of heart disease, cancer, and kidney failure. Although Th17 effectors are vital to fighting infection, functional imbalance of Th17 effectors and regulatory T cells (Tregs) promote inflammatory diseases. In this study, we investigated, in a small pilot randomized clinical trial, whether expansion of inflammatory blood myeloid dendritic cells (DCs) and conversion of Tregs to Th17 cells could be modulated with antibiotics (AB) as part of initial therapy in PD patients. PD patients were randomly assigned to either 7 d of peroral metronidazole/amoxicillin AB treatment or no AB, along with standard care debridement and chlorhexidine mouthwash. 16s ribosomal RNA analysis of keystone pathoge