No description available.

Background: Staphylococcus aureus readily colonises human epithelia despite the presence of innate defences including mild acidity and cationic antimicrobial peptides (CAMPs). Reduced sensitivity of S. aureus to these defences appears partly due to the increased biosynthesis of lysyl-phosphatidylglycerol (L-PG) in its plasma membrane. The precise mechanisms by which L-PG facilitates tolerance to acidity and CAMPs remain under-investigated, due to the lipid’s lability in mild aqueous conditions. This study examines the role of L-PG in responses to epithelial defences and describes the synthesis and characterisation of a stable L-PG analogue designed for use in biophysical experiments to investigate membrane defences in S. aureus. Methods: The genetic regulation of L-PG biosynthesis was studied by assessing changes in graXRS, vraFG and mprF expression in response to mild acidity. The effect of acidity on S. aureus membrane lipid composition was quantified by 31P NMR. Monolayers and bilayers formed from S. aureus lipid extracts and synthetic lipid models were employed in investigations into the effects of low pH and CAMPs on membrane structure and physicochemical properties. The experimental techniques included neutron diffraction, small-angle neutron scattering, neutron reflectivity, pressure–area isotherms, zeta potential measurements and 2H-NMR. Results: Increased L-PG synthesis at pH 5.5, to ~50% total phospholipid, correlated with mprF and graRS expression. The L-PG concentrations at pH 5.5 produced a condensing effect on the bacterial membrane (with both natural and synthetic lipids), making it cationically charged and less permeable to solvent. These membrane changes reduced electrostatic attraction of CAMPs and retarded their ability to partition into the membrane. Conclusions: L-PG plays an important role in S. aureus tolerance to mild acidity by altering plasma membrane charge and lipid packing properties. These membrane alterations also facilitate tolerance to other epithelial defences such as CAMPs, making L-PG biosynthesis a putative target for antimicrobial therapeutics.

Essential oils derived from dried and fresh leaves of Aloysia citrodora were obtained by hydrodistillation, and were investigated for a range of pharmacological properties: receptor binding, in vitro acetylcholinesterase (AChE) inhibitory, antioxidant activities, and neuroprotection properties relevant to neurodegenerative diseases. Fresh leaf A. citrodora essential oil inhibited [3H] nicotine binding to well washed rat forebrain membranes, with mean apparent IC50 of 0.0018 mg/ml. No significant binding activity was observed for A. citrodora essential oil derived from fresh or dried leaves, for GABAAR and NMDARs. A. citrodora essential oil, both dried and fresh, exhibited radical scavenging activity (up to 100%, IC50 < 0.0001 mg/ml) and iron (II) chelating properties (approx. IC50 = 0.05 mg/ml), and showed neuroprotective characteristics against the toxic effects of H2O2 (100%, 0.001 mg/ml) and β-amyloid (approx. 50%, 0.01 mg/ml) in CAD neuronal cell culture. Both EOs from dried and fresh leaves also displayed effective AChE inhibitory activity, with the dried leaves oil displaying more clear AChE inhibitory activity than fresh oil, which could be related to the higher respective levels of caryophyllene oxide. Recombinant human anticholinesterase enzyme was used for structure based in silico screening of A. citrodora essential oil constituents for AChE Inhibitors, and the top scoring hits with highest pharmacophore fit values showed common interactions with residues at the active site of that of donepezil. The top seven hits in order of fit score, were β-curcumene, curcumene bisabolene, trans-calamenene, caryophyllene oxide, β-sesquiphellandrene and geranyl acetate. This indicates that plants may yield novel effective and safe AChE inhibitors, other than alkaloids. To begin to identify the chemicals underpinning the pharmacological properties of A. citrodora, GC/MS analysis of the chemical composition of the essential oil from leaves of A. citrodora identified eighty three major chemicals, including the presence of terpenoids, monoterpenes and sesquiterpenes, and 6-methyl-5-hepten-2-one, the main constituents being limonene, caryophyllene oxide, curcumene, spathulenol, 1,8-cineole constituting 47% of the total oil. Finally, a simple, inexpensive solid phase extraction method was developed for fractionation of essential oils. Collectively, this thesis provides a better understanding of the pharmacology of the Aloysia essential oil and its constituents relating to its potential use in the treatment neurodegenerative disease.

No description available.

The literature, Vietnamese health statistics reveal problems with the antibiotic use with misunderstanding leading to the irrational and inappropriate use of these drugs resulting in bacterial resistance together with its consequences. In Vietnam the public healthcare service is provided at community level based on a system of communes. Here it is accepted that health centres are located in each rural area but that, public health workers are disadvantaged especially with regard to their educated/training, but are still mainly responsible for provision of healthcare including administration of antibiotics. The main aim of this study was to develop a conceptual framework for an education and training model for public health workers to reduce antibiotic misuse. It was piloted among the population in Myloc district, Nam Dinh province Vietnam but could be transferable to other rural areas in Vietnam. Thus, as a starting point baseline measures were taken using method triangulation in order to evaluate the current situation of antibiotic use in this study location. This survey revealed a very high rate of antibiotic administration (79.8%) of which more than half (54%) were incorrectly prescribed for non-infectious conditions. It also revealed misunderstandings andlimited knowledge and perceptions regarding the use of antibiotics, and that staff had received littlepost basic training and education.These findings provided baseline data for the development of the training programme. Through reviewing theories of learning, principles of adult learning and teaching, the basic philosophies of experiential learning from the western world were taken into account then adapted to the Vietnamese context, especially to the situation of the commune health workers. The model was developed, based on Kolb’s (1984) experiential learning cycle, with modifications to fit with Vietnamese condition. The model named the ‘Modified Kolb’s Model for Vietnam’ (MKMVN) then was used to design and implement the training programme, taken place in each commune health centre.

Increased advanced glycation endproducts (AGEs) formation and oxidative stress are believed to underlie the pathogenesis of diabetic vascular complications including the impairment of the wound healing. In most developing countries, diabetic treatment is expensive and plants provide a cheap potential natural source of anti-diabetic remedies. Several studies have examined the beneficial effects of using Momordica charantia (MC) because of its hypoglycaemic properties in diabetic subjects. Here, for the first time, the anti-glycation and antioxidant properties of aqueous extracts of Momordica charantia pulp (MCP), flesh (MCF) and charantin were assessed in vitro. Since wound-healing is one of the most costly complications and affecting 15% of diabetic patients, the potential angiogenic activities of MCP, MCF and charantin in the presence or absence of AGEs were investigated. Lysozyme was glycated using either glucose or methylglyoxal in the presence or absence of 5 to 15 mg/ml of Momordica charantia extracts in 0.1 M sodium phosphate buffer (pH 7.4) at 37°C for three days. The formation of glucose or methylglyoxal-derived AGE crosslinks was assessed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by Coomassie blue staining. A non-competitive ELISA method was used to investigate the effect of Momordica charantia extracts on carboxymethyllysine (CML) concentrations. Antioxidant activities of all extracts of Momordica charantia were evaluated using the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radical-scavenging activity, metal chelating activity and reducing power. The phenolic, flavonols and flavonoid contents of all extracts were also measured. In vitro angiogenic assays including cell proliferation, migration and endothelial tube formation in Matrigel™ were used to assess the potential angiogenic effects of the natural extracts. By Western blotting, the angiogenic signalling pathways induced by AGEs and potentially modulated by MCP, MCF and charantin were also investigated. Furthermore, the neutralization of the receptor for AGEs (RAGE) was performed using a monoclonal anti-RAGE antibody to highlight the role of RAGE in the modulation of AGE-induced signalling pathways followed addition of MCP, MCF and charantin. All extracts inhibited the formation of MG-derived AGEs in a dose-dependent manner and the MCF extract showed the most potent inhibitory effect on both AGE and CML formation. Antioxidant capacity of MCF was significantly higher than MCP based upon the DPPH and hydroxyl radical-scavenging activity (p < 0.005); however, MCP shows higher metal-chelating activity in comparison to other extracts. The content of phenolic compounds was expressed in gallic acid equivalents (GAE), whereas flavonols and flavonoid contents were expressed in rutin equivalents (RE). In addition, all Momordica charantia extracts increased bovine aortic endothelial cell (BAEC) proliferation, migration and tube formation with induction of p-ERK1/2 expression through RAGE. Moreover, these natural extracts decreased the anti-angiogenic effects of high concentration of BSA-AGEs. Momordica charantia does not only have established hypoglycaemic effects but this study shows that crude extracts are capable of preventing MG-derived cross-linked AGEs and Glyoxalic acid-derived CML at least in vitro. This anti-glycation activity might be due to their antioxidant properties from their phenolic content. Furthermore, because of its pro-angiogenic effects and its ability to reduce the AGEs-induced anti-angiogenic effect, Momordica charantia presents a promising natural product for the development of a new strategy to accelerate wound-healing especially in diabetic foot. Thus, use of Momordica charantia deserves more attention in particular its ability to reduce AGE formation and oxidative stress in diabetic subjects and as a pro-angiogenic therapy.

Millions of Yemenis and East Africans habitually chew daily the fresh leaves and twigs of Catha edulis commonly known as khat; it produces a stimulating amphetamine-like effect. Most people believe that khat leaves relieve fatigue and reduce body temperature; therefore it is a common practice to chew khat during sickness and during treatment. The effect of khat chewing on the bioavailability of certain antibiotics has been studied; the outcome of these studies showed that khat chewing significantly reduced the bioavailability of the antibiotics, however, khat and its effect on anti-malarial drugs has not been studied. The aim of this study was to evaluate the effect of khat on antimalarial chemotherapy and to explore possible pharmacokinetic interactions between chloroquine (CQ) and khat duri!1g co-administration in healthy adult Yemeni volunteers. Furthermore, we wanted to determine the effect of khat on CQ concentration, parasitaemia and parasite clearance in Plasmodium falciparum infected patients. In addition, anti-malarial activity was tested in vitro against CQ sensitive-resistant strains. In a two-phase cross-over study, 15 healthy adult male volunteers were given a single dose of 600 mg of CQ with and without khat. Plasma concentrations ofCQ were determined during a 24 h period following drug administration on both occasions. CQ plasma concentrations were determined by a validated HPLC-UV method. Pharmacokinetic parameters ofCQ were calculated using compartmental analysis. In the two periods of treatment, the mean (SD) peak plasma concentrations (Cmax) were 415 (103) nglml (CQ with khat) and 508 (106) ng/ml (CQ alone). The total areas under the curve (ACUO-24) were 2108 (682) ng/h/ml (CQ with khat) and 2797 (845) ng/h Iml (CQ alone). The time taken to reach Cmax (Tmax) was 3.8 (0.41) hand 3.6 (0.51) h. and elimination half-life (tv,) was 7.7 (2.10) hand 7.5 (2.77) h respectively. Statistically significant differences were observed for both (Cmax) (p < .001) and (ACUO-24) (p< .003) of CQ when comparing values with or without khat. These results demonstrat~ a pharmacokinetic interaction between CQ and khat and suggest that the observed interaction may be clinically significant, although the CQ levels were above the therapeutic level for P.falciparum.

Cationic liposomes have been extensively explored for their efficacy in delivering nucleic acids, by offering the ability to protect plasmid DNA against degradation, promote gene expression and, in the case of DNA vaccines, induce both humoural and cellular immune responses. DNA vaccines may also offer advantages in terms of safety, but they are less effective and need an adjuvant to enhance their immunogenicity. Therefore, cationic liposomes can be utilised as delivery systems and/or adjuvants for DNA vaccines to stimulate stronger immune responses. To explore the role of liposomal systems within plasmid DNA delivery, parameters such as the effect of lipid composition, method of liposome preparation and presence of electrolytes in the formulation were investigated in characterisation studies, in vitro transfection studies and in vivo biodistribution and immunisation studies. Liposomes composed of 1,2-dioleoyl-sn-glycero 3-phosphoethanolamine (DOPE) in combination with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-stearoyl-3- trimethylammonium-propane (DSTAP) were prepared by the lipid hydration method and hydrated in aqueous media with or without presence of electrolytes. Whilst the in vitro transfection efficiency of all liposomes resulted to be higher than Lipofectin, DSTAP-based liposomes showed significantly higher transfection efficiency than DOTAP-based formulations. Furthermore, upon intramuscular injection of liposomal DNA vaccines, DSTAP-based liposomes showed a significantly stronger depot effect at the injection site. This could explain the result of heterologous immunisation studies, which revealed DSTAP-based liposomal vaccines induce stronger immune responses compared to DOTAP-based formulations. Previous studies have shown that having more liposomally associated antigen at the injection site would lead to more drainage of them into the local lymph nodes. Consequently, this would lead to more antigens being presented to antigen presenting cells, which are circulating in lymph nodes, and this would initiate a stronger immune response. Finally, in a comparative study, liposomes composed of dimethyldioctadecylammonium bromide (DDA) in combination with DOPE or immunostimulatory molecule of trehalose 6,6-dibehenate (TDB) were prepared and investigated in vitro and in vivo. Results showed that although DDA:TDB is not able to transfect the cells efficiently in vitro, this formulation induces stronger immunity compared to DDA:DOPE due to the immunostimulatory effects of TDB. This study demonstrated, while the presence of electrolytes did not improve immune responses, small unilamellar vesicle (SUV) liposomes induced stronger humoural immune responses compared to dehydration rehydration vesicle (DRV) liposomes. Moreover, lipid composition was shown to play a key role in in vitro and in vivo behaviour of the formulations, as saturated cationic lipids provided stronger immune responses compared to unsaturated lipids. Finally, heterologous prime/boost immunisation promoted significantly stronger immune responses compared to homologous vaccination of DNA vaccines, however, a single immunisation of subunit vaccine provoked comparable levels of immune response to the heterologous regimen, suggesting more immune efficiency for subunit vaccines compared to DNA vaccines.

No description available.

In many countries, Phyllanthus niruri L is one of the most popular alternatives of natural herbal remedy to overcome many symptoms due to its wide range of therapeutic uses. Even though a considerable number of research projects have been conducted in order to reveal the pharmacological activities of Phyllanthus niruri L, and that a number of reports have been produced mentioning its pharmacological effect, the rich constituents of this plant are yet to be comprehensively studied, particularly with regards to the nature of the biological activities that the compounds have. Accordingly, the main focus of this study is the exploration of Phyllanthus niruri L as a new candidate of natural compound. This was done through a series of bioassay - guided plant extraction and isolation protocols. Phyllanthus niruri L crude extracts were tested against plasmodium, cancer cell lines, and platelet aggregation . Guided by the bioassay results, the isolation procedures were performed using advance chromatography techniques, in order to purify the most - active substances that represent the final candidate natural product. This study also employed flow cytometry and proteomics stud ies, as an attempt to identify the fundamental principles of the mechanism of action of the isolated compounds. The results demonstrated that Phyllanthus niruri L extracts showed a potency as antiplasmodial, anti-cancer, as well as anti-platelet agent. In inhibiting plasmodium falciparum growth, the potency of the extracts from the most to the least potent activity was methanol > water > ethanol > chloroform > hexane (IC 50values were 1.6 μg/ml, 9.6 μg/ml, 25 μg/ml, and 141 μg/ml, respectively). With regards to its anti-cancer effect, Phyllanthus niruri L extracts showed a significant cytotoxic effect on human Caucasian lung large cell carcinoma (COR-L23), human acute T lymphoblastic leukaemia (MOLT-4), and human caucasian chronic myelogenous leukaemia (K562). Among all extracts, methanol extract demonstrated the strongest cytotoxicity effect with a low IC 50 values for all cell lines tested (IC 50 values for COR-L23, MOLT - 4, and K562 was 48.92 ± 0.52 μg/ml,42.21 ± 4.98 μg/ml , and 139.28 ± 19.02 μg/ml, respectively). Methanol, water, ethanol, and hexane extracts did not show any toxicity towards normal fibroblast cell line (3T3). However, chloroform extract demonstrated a toxic effect to the normal cells line (IC 50 value 164.3 ± 8.4 μg/ml). x The antiplatelet activity of Phyllanthus niruri L was further explored in this study due to a remarkable inhibitory effect of methanolic extract observed on ADP - induced platelet aggregation. With the aid of bioassay - guided isolation protocol, the study has isolated four compounds from the methanolic extract, which demonstrated a potency in preventing in-vitro platelet aggregation induced by ADP (compound 1, 2 , 3, and 6). The IC 50 of each compound was 179.9 ± 2.67 μg/ml (compound 1), 31.91 ± 1.86 μg/ml (compound 2),77.68±6.44 μg/ml (compound 3), and 43.35 ± 6.44 μg/ml (compound 6). Compound 2 was identified as corilagin or [3,5 -dihydroxy-2-(3,4,5-trihydroxybenzoyl)oxy -6-[(3,4,5- trihydroxybenzoyl)oxymethyl]oxan-4-yl]3,4,5-trihydroxybenzoate. The finding of this study demonstrated that corilagin altered the G-protein signalling pathway in a selective manner by impeding Gq-protein cascade. Corilagin might act through G13-mediated signalling; however it showed no significant effect on Gi-mediated signalling pathway. Consequently, corilagin inhibited platelet shape changes, granule secretion, and platelet aggregate formation, which was suggested to take place by the inhibition of the elevation of intracellular Ca 2+ level due to the inactivation of PLCβ. Although corilagin showed no observable inhibitory effect on the initial activation of the major platelet glycoprotein, GPIIb/IIIa, the findings suggested that the interaction between the activated integrin and the related ligands is affected, which results in the inhibition of further amplification of platelet aggregation. Overall,this study confirmed the antiplatelet activity of corilagin and explained its coherent mode of actions, which support the future development of corilagin, isolated from Phyllanthus niruri L as a natural-sourced antiplatelet compound.