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Over the last 4 years, our research interests were focused on the development of new methodologies for the synthesis of 5-substituted-3-carbomethoxy-3-sulfolenes, never reported before, and their application in total synthesis of natural products.

C-glycosyl flavonoids differ from their O-glycosylated cousins by possessing a stronger carbon-carbon covalent bond between the flavonoid backbone and sugar moiety, but relative rarity in the human diet has led to their neglect in the literature. Accordingly, the fate of dietary C-glycosylated flavonoids in humans is unknown, although such a seemingly minor structural divergence is predicted to strongly modulate their pharmacokinetics and bioactivity. Whilst a variety of mono- and di-C-glycosylated flavones and dihydrochalcones are found in common food crops such as tomatoes and wheat, reviewed in chapter 1, the dietary burden is unusually high in consumers of the popular South African tisane, rooibos tea, due to high concentrations of the unique C-glycosyl dihydrochalcone, aspalathin. Aspalathin is shown to be absorbed as an intact C-glycosyl dihydrochalcone, excreted in low quantities in the urine of human volunteers as a methylated derivative, 3-0- methylaspalathin (3-0-MA), with and without glucuronidation following consumption of green rooibos tea (O.74±0.26 % total dose, n=6). Kinetics ofeOMT-catalysed aspalathin methylation are derived (3-0-MA, App KM 39±5 IlM, App V max 6.67±0.40 Kat mol COMTi), and both human intestinal and hepatic cytosolic fractions also shown to catalyse aspalathin methylation.

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Peptides derived from food proteins after enzymatic treatment and/or processing, are known to be bioactive in both biological and food systems; for this reason fish muscle peptides were investigated for their antihypertensive and antioxidant activity. Atlantic mackerel muscle proteins were hydrolysed with pepsin and pancreatine and the resultant hydrolysate was sequentially fractionated on 2 kDa membrane ultrafilters and further by gel filtration, ion exchange and high performance liquid chromatography and the resultant peptide fraction contained the amino acids histidine, proline, tyrosine, methionine, leucine, tryptophan and lysine. For ACE inhibitory activity, the peptide fraction (MFPH-V-JPA) had an inhibitory concentration (IC50) of 0.15 mg/ml and showed competitive inhibition for ACE with an inhibition constant (Ki) of 0.32 mg/ml. In terms of antioxidant activity, the HPLC isolated peptide fraction (LC1-Z) contained the amino acids serine, histidine, tyrosine, phenylalanine, tryptophan and lysine. It inhibited the formation of both peroxides and malonaldehyde in a linoleic acid model emulsion in a dose dependent manner with lipid oxidation inhibitory concentration (IC50) of 1.80 mg/ml. At concentration of 8 mg/ml, the inhibition of linoleic acid oxidation was more than that of 0.01 % butylated hydroxytoluene (BHT) and trolox (p < 0.001). The mechanism of antioxidant activity of the peptide (LC1-Z) was by carbon centered radical scavenging (5.34 %), hydroxyly radical scavenging (IC50 value of 1.60 mg/ml), metal chelating (5.72 %) and reducing ability. In caco-2 cells, 1 mg/ml of the peptide (LC1-Z) was not toxic to the cells seeded at 2 x 104 cells/well. Proxidant tBHP (2.5 mM) reduced cell viability significantly (79.3 %) but this increased to 94.7 % in the presence of the peptide or trolox. The peptide (1 mg/ml) also reduced TBARS formation (33.18 mug/ml) in cells compared to cells treated with tBHP alone (38.18 mug/ml). The activity of caspases-3 and -7, was higher in caco-2 cells treated with tBHP only (157.5 +/- 7.99 %) compared with those treated with the peptide (25.7 + 3.92 %). Morphological modification of the caco-2 cells treated with tBHP was evident as the cells appeared detached from the flask surface compared to those treated with the peptide (LC1-Z) which were healthy and attached to the flask surface. In Ea.hy 926 cells, reactive oxygen species were reduced by 26 % and 39 % in the lucigenin-chemiluminscence and flourescence methods respectively in cells treated with the peptide. In a caco-2 cell monolayer, transepithelial transport of the peptide was observed in both directions with a basolateral to apical apparent permeability of 0.95 +/- 0.12 cm-1 and apical to basolateral flux of 0.74 + 0.20 cm-1. HPLC chromatograms of the buffer solution taken from the apical and basolateral side showed the presence of the peptide in both sides i.e. 11.5% for apical to basolateral flux and 12.2 % for basolateral to apical. These results demonstrate that peptides with antihypertensive and antioxidant activity can be derived from Atlantic mackerel muscle proteins, with potential for neutraceutical applications.

As well as peptic ulcers, Helicobacter pylori is associated with the development of gastritis, gastric adenocarcinoma and lymphoma, and has been classified as a class I carcinogen in humans (International Agency for Research on Cancer Working Group, 1994). Although the bacteria can be eradicated in up to 90% of patients, side effects, poor compliance and the resistance of the bacteria to antibiotics are common causes of frequent treatment failure. Celery seed extracts (CSE) from a unique source in India has been used as herbal medicine since antiquity and found to have anti-inflammatory and gastroprotective properties (Butters et al., 2004; Whitehouse et al., 2001). This study followed on observations that crude extracts exhibited anti-helicobacter activity (Rainsford & Liu, 2006).CSE was selectively fractionated followed by HPLC. Fractions were collected and bio-assayed against different strains of H. pylori using conventional culture methods. The most potent component that was obtained from HPLC and purified was designated celery seed with anti-Helicobacter activity (CAH). This component has strong bactericidal effects against H. pylori; the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 3.15 mug ml[-1] and 6.25 - 12.5 mug ml[-1], respectively. This compares favourably with the MIC and MBC of tetracycline, which are in the region of 3.15 mug ml[-1]. The isolated compound has highly specific inhibitory effect on H. pylori, since no inhibitory activity was detected against Campylobacter jejuni or Escherichia coli at these levels. The molecular ion of CAH was measured as 384.23 by mass spectrometry, giving the empirical formula as C[24]H[32]O[4]. The MS and NMR data strongly suggest this compound is a phthalide dimer. From radioactive bioassay, CAH inhibits RNA synthesis by 50% of that seen in a negative control in 3 days, while DNA and protein synthesis were unchanged. These suggested that the new compound may be suitable for further investigation as an agent for treating H. pylori infections.

Norditerpenoid alkaloids have important biological activities. Many of them are potent ligands and therefore promising leads for novel selective antagonists and/or agonists of subtypes of nicotinic acetylcholine receptors (nAChR) and voltage-gated sodium channels. Aconitum, Consolida, and Delphinium are important sources of norditerpenoid alkaloids. This thesis is focussed on the chemistry of norditerpenoid alkaloids from these three genera, starting with a review of those recently isolated from Aconitum, Consolida, and Delphinium with brief aspects of taxonomy, biological activities, and modes of action. Selected aspects of the uses in traditional medicine of Delphinium and Aconitum are presented, including data on both toxicity and detoxification. In this thesis, chemical constituents of the seeds of Delphinium cultivar Pacific Giant and the seeds ofAconitum lycoctonum were investigated. To extract the crude alkaloidal material, Soxblet extraction was used for Delphinium cv Pacific Giant seeds and room temperature extraction for A. lycoctonum seeds. The crude extracts were purified by repeated column chromatography (over silica and alumina gels), yielding five known norditerpenoid alkaloids from Delphinium cv Pacific Giant (delavaines A and B, delpheline, methyllycaconitine (MLA), and pacinine) and two from A. lycoctonum (lycaconitine and N-succinylanthranoyl lycoctonine) which were analysed by detailed spectroscopic methods. X-Ray crystallographic analysis of aconitine, mesaconitine, lycoctonine, and delpheline was also studied. Three compounds were obtained from semisynthesis starting with MLA: lycoctonine by basic hydrolysis, inuline by acidic hydrolysis and by esterification of lycoctonine, and elatine by methylenedioxy acetal fonnation. These known alkaloids were structurally elucidated by a variety of spectroscopic techniques. MLA is a selective competitive antagonist at 0.7 sub-type nAChR. From collaborative studies, the results of biological activity for methyl esters delavaines A and B (a 3:2 mixture), delpheline'iand pacinine (the B-ring C6-ketone of delpheline) are reported in this thesis. Their biological activities were detennined in competitive o.-bungarotoxin binding assays for 0.7 nAChR in rat brain membranes. Delavaines A and B were potent ligands (ICso = 50 nM, cfMLA ICso =-1-2 nM), whereas delpheline and pacinine displayed only modest activity at 0.7 nAChR (ICso = -1 JlM). After studying N-ethylpiperidine as a model alkaloid, the pKa (a key physico-chemical parameter) ofMLA was measured by IH NMR as 7.15. A brief comparison with pKa data reported in the literature IS made across closely related norditerpenoid alkaloids.