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The aim of this Ph.D. project was to investigate the effects of Colostrinin (CLN) on cells in culture, and its ability to prevent or alleviate cytotoxicity induced by reactive oxygen species (ROS) and beta-amyloid (Aβ). Two cell culture systems were used to analyse the effects of CLN; rat primary hippocampal cultures and the B50 rat neuronal cell line. Bovine CLN was found to have no adverse effects on cell morphology or survival and to have a small, non-significant effect, on both menadione-induced, oxidative stress-mediated toxicity and Aβ₁-₄₂-induced toxicity of neurons in primary hippocampal cultures. This protective effect was found to potentially related to the antioxidant effects of CLN. CLN was demonstrated to prevent a menadione-induced increase in ROS in the B50 cell line. Furthermore CLN was able to reverse a significant Aβ₁-₄₂ mediated increase in the protein levels of the antioxidant enzyme Cu/Zu superoxide dismutase (SOD) in primary hippocampal cultures, although CLN alone caused an increase in SOD1 protein in the B50 cell line. Bovine CLN was shown, in both B50 cells and primary hippocampal cells, not to significantly decrease the protein levels of cyclin dependent kinase 5 (Cdk5) which has previously been demonstrated to be involved in the mechanism of tumour necrosis factor (TNF) α-mediated protection against Aβ-induced toxicity in primary hippocampal cultures. Furthermore CLN did not consistently alter the expression of activated caspase 3 in either B50 or primarytippocampal cells. This study adds to the knowledge and understanding of the mechanism of action of CLN.

BACKGROUND: Alzheimer's disease (AD) is a complex, multifactorial neurological disorder characterized by an insidious onset with progressive symptoms of memory impairment, language deficit, behavioural problems including agitation, mood disturbances and poor judgment. AD involves multiple pathogenetic factors such as A~ production and aggregation, oxidative stress, tau protein aggregation, metal ions (e.g. Cu2+, Zn2+, Fe2+) and reduced levels of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes. All these factors play important roles in neurodegeneration associated with AD. These provide diverse multiple targets for examining AD-modifying drugs. In view of this, the study was focussed on finding natural plant extracts used traditionally for centuries to treat memory and cognition related disorders specifically AD. Attention was also focussed on certain phytochemicals to find their therapeutic values in AD. AIMS: To find the neuroprotective, anti-cholinesterase and antioxidant activities of an aqueous extract of Withania somnifera, Bacopa monniera, Salvia patens, Salvia elegans, Capsicum annum, Uncaria tomentosa, Melissa officinalis and Centella asiatica plants. Four phytochemicals namely allicin, ajoene, capsaicin and asiatic acid were also examined for these activities. The targets chosen in this study were AChE and BuChE, neurotoxic A~ fibrils and oxidative stress pathway using in vitro models. METHDOLOGY: The aqueous extracts were obtained by boiling dried powdered plant materials in deionized water for 25 min. Enzyme assay were performed to determine anti-ChE activity by Ellman's method, while kinetics (Km and Vmax) were analyzed using Lineweaver-Burk plot method. Antioxidant capacities of these plant extracts and phytochemicals were determined using FRAP and TEAC assays. The neuroprotective activity of plant extracts and phytochemicals were determined under in vitro condition in differentiated pheochromocytoma (PC12) cell culture against H202 and A~ induced toxicities. X111 The anti-aggregation property was determined by transmission electron microscopy (TEM) and the Thioflavin T (ThT) fluorescence assay. RESUL TS: An aqueous extract of W somnifera showed dual inhibitory activities against both AChE and BuChE. The ICso values for AChE and BuChE were O.l5±0.007 and 0.80±0.005mg/mL, respectively. The potency of inhibition was greater for AChE compared with BuChE. The mode of inhibition was non- competitive mixed inhibition. Other plant extracts displayed either weak or negligible enzyme inhibition activity. In contrast, allicin demonstrated potent dual enzyme inhibitory activity. The ICso values for AChE and BuChE inhibition were 0.01±0.009 and 0.05±0.018mg/mL, respectively, while ajoene demonstrated weak enzyme inhibitory activity compared with allicin. The ICso values for AChE and BuChE inhibition of ajoene were 0.55±0.012 and 0.07±0.015mg/mL, respectively. Most of the plant extracts possessed high antioxidant capacities; FRAP values: 4242±112 to 1364644±130J,!mol Fe2+E/g dried weight; TEAC values: 1.89±0.12 to 26.20±0.05 mmol Trolox E/g dried weight. In contrast, allicin, ajoene and capsaicin showed weak antioxidant capacities; FRAP values: 1615±90, 934±35 and 1423±75 umol Fe2+E/g dried weight, respectively, whereas asiatic acid showed no apparent antioxidant capacity. These results led to the second part of the study in which these aqueous extracts and phytochemicals were examined for their neuroprotective properties under in vitro conditions in differentiated PC12 cells against H202 and A~ induced toxicity. The neuroprotective effect varied from plant to plant. An aqueous extract of W somnifera, B. monniera, S. elegans, U tomentosa, C. asiatica and pure compounds asiatic acid and capsaicin showed up to 70-90% protection of PC 12 cells against H202 and A~ induced toxicity whereas, allicin and M ofjicinalis extract showed no protective effects. Anti-aggregation measured by TEM and ThT fluorescence assay showed that an aqueous extract of W somnifera and allicin strongly inhibited fibril formation compared with control samples. These results suggest that the aqueous extract of W somnifera root and allicin have an ability to inhibit formation of mature fibrils which leads to plaque formation. XIV CONCLUSION: These finding demonstrate that that an aqueous extract prepared from these medicinal plants and phytochemicals have significant neuroprotective activities against different targets including AChE, BuChE, ROS and A~ implicated in AD. Hence, these results provide evidence of the usefulness of these medicinal plants and phytochemicals which may be used in the future to develop new therapeutic strategies for the prevention of and treatment of AD.

BACKGROUND: Dementia is a neurodegenerative disease of the brain associated with cognitive and memory impairments. Despite recognition of several types of dementia, the Alzheimer type is the most studied and understood. The cholinergic theory of Alzheimer's disease led to the development of licensed drugs based on the inhibition of the enzyme acetylcholinesterase. Extracts of Salvia (sage) species have been reported to have cholinergic activities relevant to the treatment of Alzheimer's disease. AIMS: Lack of information on a chemical fingerprint of the extracts responsible for inhibition of the enzymes butyrylcholinesterase and acetylcholinesterase prompted this in vitro investigation of sage species for anti-cholinesterase activity. Cholinergic receptor binding activity, inhibition of ß-secretase, and a pro-inflammatory cytokine suppressive activity of extracts of sage species were also studied as relevant treatment targets. METHODS: The extracts were obtained by methods of supercritical fluid extraction using 1,1,1,2-tetrafluoroethane (Phytosol A) and steam distillation. Dose-dependant inhibition of human cholinesterases by the extracts and constituents was determined using the method of Ellman, while inhibition of ß-secretase via a fluorometric method. The nicotinic acetylcholine receptors binding activity was measured as an amount of [3H]-nicotine displaced from human acetylcholine receptors, whereas the muscarinic activity was assessed using the displacement of [3H]-scopolamine. Determination of interleukin 8 inhibitory activity by the extracts was performed via a quantitative sandwich enzyme immunoassay using a commercially available kit. RESULTS: Inhibition of butyrylcholinesterase by the Phytosol extracts of S. apiana, S. fruticosa and S. officinalis var. purpurea was non-competitive. In contrast, inhibition of acetylcholinesterase by S. officinalis var. purpurea oil was competitive. S. corrugata extract was the most potent inhibitor of acetylcholinesterase with an IC50 value of 0.009±0.004 mg ml", while S. officinalis var. purpurea oil was the most active inhibitor of butyrylcholinesterase with an IC50 value of 0.015±0.004 mg ml''. Time dependent increase in inhibition of butyrylcholinesterase by steam distilled oils of S. fruticosa and S. officinalis var. "purpurea" was also evident. IC50 values decreased from 0.15±0.007 and 0.14±0.007 mg ml-1 with 5 minutes to 0.035±0.016 and 0.06±0.018 mg ml-1 with 90 minutes incubation time respectively. Phytosol A extracts were more potent than steam distilled oils with respect to anti-cholinesterase activity. Minor synergy in inhibition of bovine acetylcholinesterase was apparent in 1,8-cineole/a-pinene and 1,8- cineole/caryophyllene oxide combinations, whereas a combination of camphor and 1,8- cineole was antagonistic. Oil of S. apiana displaced [3H]-nicotine from human nicotinic acetylcholine receptors and [3H]-scopolamine from muscarinic acetylcholine receptors in a dose dependent manner with IC50 values of 0.02 mg ml" and IC50 0.1 mg ml" respectively. This oil also showed a modest suppression of interleukin 8 secretions from goblet cells. None of the tested oils and constituents had anti-ß secretase activity. CONCLUSION: These findings demonstrate that the cholinergic activity of the extracts results from a complex interaction between their constituents. Thus, inhibition of acetylcholinesterase is mainly due to the activity of the main constituents with some degree of synergy, whereas anti-butyrylcholinesterase activity is down to major synergistic interactions and identification of a chemical fingerprint responsible for the overall activity is therefore challenging. A synergistic combination of extracts or their standardised fractions with multiple activities is may be a candidate for clinical trials in Alzheimer's disease.

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