Honey is an old remedy recently rediscovered as a possible alternative to modern antibiotics in wound management but its mode of action is not fully understood. The antibacterial activity of honey can be divided into hydrogen peroxide and non-hydrogen peroxide-derived activity. This later type of activity is characteristic of honeys from Australasia (e.g. manuka honey) and preferred for wound management, although historically local honeys have been used. The main aim of this study was to investigate the mechanisms of antibacterial action of manuka honey. The stability of antibacterial action of manuka honey under different conditions was determined, it was observed that manuka honey lost its antibacterial activity when pH was increased and that it remained the same with heating. Storage seemed to increase the potency of manuka honey. The effects of honey on Staph. aureus and Pseud. aeruginosa, were investigated using MIC/MBC detenninations, time-kill studies, commitment to death, resistance training, electron microscopy, effects on respiration rates, leakage of intracellular material, and for Staph. aureus the proteome of treated and non-treated cells were compared. It was observed that the effect of manuka honey on Gram-positive and Gram-negative cells is different. Gram-positive bacteria had a lower MIC than Gram-negative bacteria, but the time-kill experiments and the commitment-to-deaths howed that Gram negative were inhibited more rapidly. Clinical strains of both bacteria showed different time-kill profiles to type strains. The methodology used for MIC determination was found to affect to the results obtained. No honey-resistant ram-positive bacteria were recovered, but Gramnegative bacteria were found to be able to become phenotypically resistant to manuka honey. Electron microscopy showed that honey inflicted physical damages in both types of cells, and in Gram-positive bacteria led to an increase in the proportion of population of cells with a complete septum. Gram-positive cells incubated in honey increased their endogenousre spiration rate whilst this was decreased in Gram-negative, major leakage was observed in Gram-negative bacteria whilst only minor leakage was observed in Gram-positive bacteria, which is consistent with the amount of damage observed with electron microscopy. The proteome analysis of Staph aureus, revealed a general down regulation of protein synthesis. Thirty Portuguese honeys were assayed for their antibacteriaal ativity and honeys derived from Lavandulas toechas(lavender) were found to possess non-peroxide activity. A selection of manuka honeys was screened for antimicrobial producing bacteria. In total 106 bacteria were recovered (85% were identified as Bacillus sp. ) and of those, 76 were capable of inhibiting the growth of at least one strain of bacteria tested, meaning that some of the antibacterial activity in manuka honey could be due to the presence of antimicrobial agents of bacterial origin. The antibacterial activity of manuka honey has previously been claimed to be due to hydrogen peroxide production and not to a non-peroxide source of activity. A study of free radical production and antioxidant potential demonstrated that manuka honey did not produce any hydroxyl radicals via the Fenton reaction. Thus hydrogen peroxide could not be present. It was also observed that even free radical-producing honeys were able to quench radical production in vitro. In conclusion this study has demonstrated that the non-peroxide activity of manuka honey is not exclusive to Australasia honeys, that it is not derived from hydrogen peroxide generation and may have a microbial origin. Furthermore the action of manuka honey on Gram-negative bacteria seems to be more physical than in Gram positive where it appears to interfere with the cell physiology, perhaps by stopping the cell cycle before cytokinesis.

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The present study describes the preliminary evaluation of Philippine medicinal plants <italic>Artemisia vulgaris, Chrysanthemum coronarium, Moringa oleifera, Sesbania grandiflora</italic> and <italic>Vitex negundo</italic> for their antagonistic activity at selected biogenic amine receptors on smooth muscle of the airways, gastrointestinal tract and vascular system. The antagonistic activity of these plants were studied against dose-response curves for contractions of the guinea pig ileum, trachea and aorta to 5-hydroxytryptamine (5-HT2 receptors), methacholine (M3 muscarinic receptors), histamine (Hi receptors), phenylephrine (d -adrenoceptors) and P-phenylethylamine (trace amine-associated receptors, TAARi). The methanolic extracts of <italic>S. grandiflora</italic> (flowers and leaves) revealed the presence of histamine Hi receptor and muscarinic M3 receptor antagonist in the ileum. The <italic>A. vulgaris</italic> chloroform (AV-CHC13) and methanol (AV-MeOH) extracts, and the acid-base extract of <italic> V. negundo</italic> (VN-E) showed histamine Hi antagonism in the ileum and trachea. Further analysis of AV-CHCI3 isolated two major components yomogin and l,2,3,4-diepoxy-ll(13)-eudesmen-12,8-olide. Yomogin a sesquiterpene lactone exhibited a novel histamine Hi receptor antagonism in the ileum. Repeated exposure of aortic rings to phenylephrine and (3-PEA CRCs produced significant increases in maximum vascular tension due to enhanced intracellular Ca2+ mobilization. Both the AV-CHCI3 and VN-E inhibited this enhanced response. Further analysis of AV-CHCI3 revealed that it is probably inhibiting the increase of vascular tone mediated via intracellular Ca2+ release regulated by ryanodine. This study further validates the traditional use of <italic>S. grandiflora, A. vulgaris</italic> and <italic>V. negundo</italic> in the treatment of hyperactive gut, asthma and hypertension.

Inflammation is a general term that is related to diseases such as rheumatoid arthritis and psoriasis. Harpagophytum procumbens (H. procumbens), commonly known as Devil's Claw, is a popular natural anti-inflammatory product. Relatively high proportions of active glycosides in its secondary tubers are of particular interest. Therefore, in this thesis four of the major glycosides of H. procumbens (harpagoside, harpagide, verbascoside and S-O-p-coumaroyl harpagide) were analysed for their topical and trans-cutaneous delivery, anti-inflammatory and virucidal effects. The delivery depended on their physiochemical characteristics and the vehicles used. They were all successfully delivered across the skin layers into subcutaneous compartments and achieved steady state flux of (20.0, 129.7, 30.6, 26.7 x 10" umol cm" hr") in water. H. procumbens and three glycosides showed decreased amounts of expressed inflammatory components using western blotting, immunocytochemistry and ELISA assays. Interestingly, harpagide was discovered to be pro-inflammatory. Amounts of H. procumbens delivered trans-cutaneously (receptor phase) were re-applied on ex-vivo porcine skin. Despite the low amounts of permeated glycosides, they successfully inhibited the expression and pathway of the inflammatory enzyme COX-2. The amounts of the active glycosides were determined in different formulations and very low amounts were found. Hence, standardizing the proportional amounts of active components is important in order to optimise the effects of H. procumbens. The delivery of H. procumbens into ex-vivo joint capsule led to undetectable amounts of glycosides. This was expected due to the multi layers the polar compounds had to cross before reaching the synovial fluid. Finally, cytotoxic and plaque assays were carried out for H. procumbens and its glycosides. The data obtained showed significant virucidal effects towards Herpes simplex virus (HSV-1). H. procumbens used trans-cutaneously proved to be effective in-vitro and a new mechanism proposed whereby active glycosides can work either synergistically or antagonistically within the extract towards inflammation and infection (eg. cold sores).

Garlic extract is very complex, yielding a number of organic sulphur constituents that are thought to be responsible for its anticandidal properties. Many of these are now being investigated in an attempt to determine the mechanisms by which they act. The effects of fresh and freeze dried extracts of <italic> Allium sativum</italic> (garlic) on the physiology and morphology of <italic> Candida albicans</italic> were compared. Inhibition of growth and loss of structural integrity was observed for both fresh garlic extract (FGE) has a greater efficacy than garlic powder extract (GPE) as indicated both by its effects on morphology and inhibition of growth. Gas chromatography-mass spectrometry of extracts was employed to separate and quantify putative inhibitory sulphur-containing components fresh and freeze-dried extracts yielded the same components but fresh garlic yielded ten times more sulphur constituents. Cell death mechanisms were investigated by flow cytometry. Low concentrations of allyl alcohol (AA) triggered a necrotic response, whereas an apoptotic type of cell death was observed at higher concentrations (>6mM). Conversely, low concentrations of diallyl disulphide (DADS) induced apoptosis, whereas higher concentrations (>6mM) resulted in a necrotic response. Further investigations with using 2-photon microscopy determined that a short 30 min exposure to 0.5mM DADS and then removal, induced 70% cell death (50% necrotic, 20% apoptotic) within 2h this figure increased to 75% after 4h. Intracellular levels of reactive oxygen species (ROS), were increased with >10mM menadione, 2mg ml"1 GPE, ImM AA or DADS as measured using dihydrofluorescein and detected by flow cytometry. Two-photon laser scanning microscopy was employed to monitor the intracellular responses of individual <italic>C. albicans</italic> cells after treatment. Changes typical of oxidative stress NADH oxidation, glutathione depletion and increased reactive oxygen species (ROS), were observed. Additionally, DADS induced a marked enhancement of mitochondrial membrane potential and low respiration rates as could be verified in cell suspensions. The plasma membrane was monitored by use of the Bis-oxonol dye, DiBaC4(3). Calculation of the electrochemical potential was achieved by application of the Nernst equation. Complete depolarisation was observed with low concentrations of AA, suggesting that for this constituent, the plasma membrane may be a primary target. Effects of garlic extract and diallyl disulphide on plasma membrane were less obvious. Putative targets for DADS are glutathione-S-transferase as determined by in vitro kinetics using cell-free extracts additional targets are likely to be a component prior to Site II in the respiratory electron transport chain as well as ATPsynthase as determined by decreased oxygen consumption and proton production respectively. Known targets for allyl alcohol are alcohol dehydrogenases Adhl and 2 (in the cytosol) and Adh3 (mitochondrial), although the significant decrease in NAD(P)H after addition of AA is indicative of another mechanism of action.

Oxidative damage is implicated in the pathogenesis of a number of diseases. Scientific research shows positive links between accumulated free radical damage and age-related diseases such as atherosclerosis and osteoarthritis. There is great interest in the possibility that the antioxidant potential of plant-derived compounds such as flavonoids may reduce the risk of developing these conditions. The aim of this study was to evaluate the antioxidant activity of selected non-food plants, traditionally used by herbalists in their treatment of osteoarthritis, using crude plant extracts and herbal tinctures, the most commonly used form of plant extract. As herbalists traditionally argue that herbs used in combinations or formulae will increase in efficacy when used together, an exploratory study was further carried out to investigate whether the antioxidant activity of two herbs tested in combination was greater than the sum of both herbs tested singly. Eight plants were selected for phytochemical analysis and investigation for antioxidant activity, based on discussions with clinic supervisors from four herbal medicine training clinics and a review of patient's case notes. The prescriptions from a pilot study investigating outcomes for the herbal treatment of osteoarthritis were used as selection criteria. Chromatographic analysis of each plant by TLC, HPLC and GCMS confirmed the presence of a number of flavonoids reported in the literature and of other compounds which were not possible to identify. Previous studies have established that certain flavonoids in vitro can exert pro-oxidant or antioxidant effects according to the concentration and presence of transition metal ions such as copper and iron. In view of the pro-oxidant effects observed for some extracts during biochemical analysis, metal analysis by ICP was carried out on the selected plant material to test for the presence of selected metal ions known to catalyse free radical reactions. ICP analysis showed the presence of most of the selected metals in all the plant samples. Several pathways, by which flavonoids and other plant phenolics may exert their effects on chemical oxidation have been identified, one of which is their free radical scavenging capacity to halt the propagation stage of lipid peroxidation. Since lipid peroxidation is implicated in the pathogenesis of osteoarthritis, assays to measure this in vitro were investigated and the following two assays selected: - lipid peroxide assay using the ferric thiocyanate method for the detection of peroxides and an assay using the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) an established method for investigating the potential free radical scavenging activity of plant extracts. The lipid peroxide assay and method of analysis was re-evaluated and a standardised procedure established. All eight crude plant extracts showed marked antioxidant activity in both assays. Results for the crude plant extract in the lipid peroxide assay varied according to concentration, with 0.1% w/v giving the best results. The crude plant extracts in almost all cases seemed to be more active as antioxidants than tinctures (fluid extracts). When combinations of crude plant extracts were tested in pairs for antioxidant activity, results demonstrated synergy from five of the pairs and antagonism from three, approximately one third of the possible 28 two-herb combinations tested. The synergistic interactions observed could form the foundation for the future development of an antioxidant formula to offset the effects of free radical damage.

Oxidatively damaged DNA is thought to be important in both the initiation and development of bladder carcinoma. Phytochemical compounds are thought to promote optimal health, partly via their antioxidant effects in protecting cellular components against damaging free radicals. The objective of this study is to assess the effect of a standardised bilberry extract, mirtoselect, on the level of endogenous and induced oxidatively damaged DNA in bladder cancer cells, as assessed by the Comet assay. The Comet assay, also known as single-cell gel electrophoresis, represents a simple method for measuring DNA strand break damage in eukaryotic cells. The sensitivity and specificity of the assay is greatly enhanced by the addition of bacterial repair endonucleases that recognise specific types of damage in the DNA and converts these lesions into additional DNA breaks. - Studies of mirtoselect against three bladder cancer cell lines (RT112, RT4 and HT1376) have shown significant antiproliferative activities against RT112 cells and against RT4 cells (p<0.05), but not against HT1376 cells. The treatment of all bladder cancer cell lines with mirtoselect (50 µg/ml) for a duration of seven days did not lower the level of endogenous oxidatively damaged DNA as detected by the modified endonuclease-alkaline Comet assay. However, a significant level of protection was observed when exogenous hydrogen peroxide was used to induce oxidatively damaged DNA in all the bladder cancer cell lines studied. Further studies revealed that mirtoselect may possibly mediate its antioxidant property through metal chelation, rather than free radical scavenging. - Our studies demonstrated that mirtoselect was potent enough to reduce levels of exogenously-induced oxidatively damaged DNA in the bladder cancer cell lines studied. Additionally, the ability of mirtoselect to reduce bladder cancer cells proliferation further highlights anthocyanins as promising future chemopreventive agents against bladder cancer.

Functional selectivity, which highlights the ability of ligands to differentially activate the signalling pathways linked to G protein-couple receptors (GPCRs) has provided an avenue for developing ligands with greater safety profiles. Pilocarpine (Pilo), a non-selective muscarinic acetylcholine receptor (mAChR) agonist has been shown to differentially activate G protein subtypes linked to the M3 mAChR. In this study the pharmacology of Pilo was further investigated using a number of readouts. When compared to methacholine (MCh), a reference agonist, Pilo appeared to preferentially stimulate inositol phosphates production than global receptor phosphorylation. The ligand also appeared to preferentially promote phosphorylation of Ser412 at the third intracellular loop of the receptor than Ser577 at the C-terminal tail. This differential phosphorylation may be linked to the fact that these residues are phosphorylated by distinct protein kinases. However, such preferential phosphorylation was not evident at the mutant M3 RASSL receptor that was engineered to respond to Clozapine-N-oxide (CNO). This mutant receptor was phosphorylated in response to CNO stimulation in a similar manner as the wild-type M3 mAChR responding to ACh. Allosteric modulation has been considered an attractive approach to selectively target GPCR subtypes for multiple disease indications. BQCA and LY2033298 have been shown to act allosterically at the M1 and M4 mAChR, respectively. In this study, we provided evidence that BQCA is probe dependent and the compound is more potent as an affinity modulator of ACh than Pilo. However BQCA did not significantly potentiate the phosphorylation state of the M1 mAChR following stimulation with a sub-maximal concentration of ACh. Similar results were obtained for LY2033298 at the M4 mAChR which suggest that allosteric modulators do not promote a receptor conformation that increases the accessibility of phosphorylation sites to protein kinases.

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