Dissertations / Theses on the topic 'Antioxidants – Physiological effect'

Ultraviolet (UV) radiation damages both eukaryotic and prokaryotic cells by causing the formation of free radicals which damage cell membranes and DNA. Antioxidant vitamins have been shown to protect cells from UV-induced damage by scavenging free radicals. The protection of skin and its normal flora is necessary for the health of individuals in resisting diseases caused by microorganisms and delaying the long-term damage caused by UV radiation.This research investigated the effects of the antioxidants vitamin A and ascorbic acid, as well as UV-irradiation on both prokaryotic (Staphylococcus epidermidis) cells and eukaryotic (human fibroblast skin) cells. This information is important in determining the effects of vitamin treatment on skin and its normal flora.Results indicate that ascorbic acid is rapidly (within six hours) degraded after being dissolved in water or medium. Treatment of cells with ascorbic acid must take into account this rapid degradation. S.epidermidis cells were protected from UV-induced damage by treatment with ascorbic acid but were more sensitive to UV-irradiation when treated with vitamin A. Human fibroblast cells treated with ascorbic acid did not exhibit morphological changes when compared to untreated cells.
Department of Biology

The amino acid, tryptophan, obtained through dietary means, is metabolised by the enzymes tryptophan 2,3-dioxygenase (TDO), indoleamine 2,3-dioxygenase (IDO) and tryptophan hydroxylase. All the enzymes have an effect on circulating tryptophan levels, especially TDO, since it is the major site of tryptophan catabolism in the liver and results in the production of kynurenine metabolites, viz. kynurenine, kynurenic acid, 3-hydroxyanthranilic acid and quinolinic acid. Extrahepatically, IDO is responsible for the synthesis of the kynurenine metabolites. Tryptophan 2,3-dioxygenase and IDO activity is increased by hormones or substrates such as tryptophan, and inflammation, in the case of IDO. Tryptophan availability for serotonin (5-HT) synthesis by the enzyme tryptophan hydroxylase is primarily dependent on TDO activity. A study was attempted in order to ascertain whether any of the endogenous metabolites of the kynurenine and serotonergic pathways would be able to inhibit TDO activity. Results showed that although the kynurenines had no effect, the indoleamines, except for the indoleacetic acids, were able to reduce TDO activity. 6-Methoxy-2-benzoxazolinone (6-MBOA), a structural analogue to melatonin, was the most potent inhibitor with a reduction in activity of 55 % compared with the control. The pineal gland in the rat brain has been shown to have the highest IDO activity. With induction, the kynurenine metabolite concentrations of kynurenic acid and quinolinic acid are increased. The effects of both compounds were determined on the serotonergic pathway. Although kynurenic acid produced no significant effect, quinolinic acid significantly reduced N-acetylserotonin and melatonin synthesis at concentrations of lOJLM and 100 JLM respectively. Many authors have implicated oxygen derived species as causative agents in the important neurodegenerative disorders such as Parkinson's and Huntington's disease. Increased radical generation and lipid peroxidation have been suggested to be responsible for the toxic destruction of neurons, especially in the brain because of its high lipid content and oxygen demand. The brain is therefore vulnerable to oxidative attack. During inflammatory diseases, IDO is induced with a resultant increase in kynurenines. This study was also an attempt at determining the effect of kynurenines on lipid peroxidation. All metabolites of the kynurenine pathway were able to induce lipid peroxidation significantly. The antioxidative potential of various tryptophan analogues, viz. serotonin, melatonin and 6-methoxy-2-benzoxazolinone, was determined using quinolinic acid-induced lipid peroxidation. Serotonin, melatonin and 6-MBOA were able to significantly reduce quinolinic acid-induced lipid peroxidation.

[Truncated abstract. Please see the pdf format for the complete text.] Interest in the role of peroxynitrite in the pathogenesis of atherosclerosis has increased due to many in vitro studies which have demonstrated its potent oxidising and nitrating capability and immunohistochemical staining studies which demonstrate nitration of tyrosine in vivo. It is frequently suggested that the production of nitric oxide and superoxide at sites of inflammation implicates peroxynitrite as the major damaging reactive nitrogen species in vivo. Evidence for a role for peroxynitrite is often demonstrated by measurement of 3-nitrotyrosine yet even this cannot distinguish peroxynitrite from other nitrating species. Clearly, however, if peroxynitrite is important in atherogenesis, then identification of mechanisms for its detoxification could provide a means of preventing such effects. Therefore, this Thesis has sought to determine whether phenolic compounds of dietary origin can be preferentially nitrated by reactive nitrogen species thereby protecting endogenous structures, such as low density lipoproteins, from atherogenic modifications. This Thesis focuses upon phenolic acids as they have received relatively less attention than other classes of phenolic compounds, such as flavonoids, yet they are quite abundant in socially important beverages such as red wine. In order to complete the required analyses, the development of methods to detect phenolic acids and their nitration products together with 3-nitrotyrosine, dityrosine and 5-nitro-γ-tocopherol was necessary. The initial in vitro experiments described herein sought to determine the products of reaction of peroxynitrite with phenolic acids of the 4-hydroxy and 3,4-dihydroxy type and then to examine whether these products could account for a protective effect upon tyrosine, lipids and endogenous anti-oxidants, if any was observed, when isolated LDL was treated with SIN-1, which releases peroxynitrite through the simultaneous generation of nitric oxide and superoxide. A concurrent minor focus was to examine the relationship between structure and activity of these phenolic acids under various regimes of oxidative insult. These experiments indicate that, at least in this in vitro model, oxidation is a dominant mechanism over nitration. Peroxynitrite was shown to nitrate coumaric acid in moderate yields but exclusive oxidation of caffeic acid appeared to occur. Although a potential role for γ-tocopherol as an anti-nitration agent was inferred, all types of chemical treatment of LDL in the presence of phenolic acids yielded oxidation as the primary end point. In fact, nitration of tyrosine was not detected and nitration of coumaric acid was at the limit of detection. Since nitration of tyrosine is generally regarded as important in many disease states, a more physiological nitrating mechanism involving artificially stimulated neutrophils was used. This system demonstrated that although physiologically relevant reactive nitrogen species can result in nitration of phenolic compounds, in a complex system including biological structures (LDL) and phenolic compounds, oxidation but not nitration of all species appears to occur. As a consequence of the results above, an examination of carotid plaque was undertaken to determine to what extent nitration occurred relative to oxidation in atherosclerotic tissue. These studies applied methods developed herein to detect 3-nitrotyrosine and dityrosine in complex biological matrices as markers of nitration and oxidation respectively. The data obtained demonstrated that nitration was a minor modification of protein (0.01%) compared to oxidation (0.3%) even in a highly diseased tissue such as carotid artery plaque. A secondary study examining plasma revealed that dityrosine, which has been implicated in irreversible albumin aggregation in chronic renal failure and more recently in heart disease, is elevated in chronic renal failure subjects compared to well matched controls. A separate examination of plasma from healthy subjects revealed that in both the fasting and post prandial state 3-nitrotyrosine could not be detected and, in fact, interfering species could be problematic in the GC-MS analysis of 3-nitrotyrosine. The lack of nitration of any substrate observed in vitro using reactive nitrogen species generated in the aqueous phase, the relative lack of nitration of tyrosine in plaque proteins and the lipophilicity of nitric oxide, the precursor of all reactive nitrogen species, suggested that nitration could be more closely associated with lipid structures. The known ability of γ-tocopherol to form 5-nitro-γ-tocopherol was used to probe this concept. The 5-nitro-γ-tocopherol content of lipid extracts obtained from carotid artery plaques was very high (30%). This indicated that nitration is predominantly a lipid phase phenomenon and that nitrating species are present in much greater abundance than oxidising species in vivo.

Reduction of milk production following acute bovine mastitis causes important economic losses. In this study, two experiments were conducted to asses the ability of different antioxidants to prevent neutrophil (PMN)-induced mammary damage in acute bovine mastitis. First, a co-culture model composed of bovine mammary epithelial cell line (MAC-T cells) and bovine PMN activated by phorbol myristate acetate was used. Activated PMN release reactive oxygen species that are cytotoxic for bovine epithelial cells. Addition of dimethylthiourea or bathocuproinic acid did not induce any protective effect. On the other hand, addition of catechin, deferoxamine or glutathione ethyl ester (GEE) significantly reduced PMN-induced cytotoxicity in a dose-dependent manner as demonstrated by lower levels of released lactate dehydrogenase (LDH). The second experiment was undertaken with the last three antioxidants to evaluate their protective effects in vivo. A model of LPS-induced mastitis on dairy cows was used. The extent of cell damages was evaluated by measuring quarter milk levels of LDH and 4-methylumbelliferyl N-acetyl beta-D-glucosaminidase ( NAGase) at varying intervals before and after intramammary infusions of LPS, with or without antioxidants. Milk levels of haptoglobin and bovine serum albumin were also analysed. Catechin and GEE did not induce any protective effect whereas infusions of deferoxamine, a chelator of iron, decreased milk levels of LDH, NAGase and haptoglobin hence suggesting a protective effect against PMN-induced damage. Deferoxamine did not interfere with PMN migration into the mammary gland. Additionally, deferoxamine inhibited bacterial growth in vitro but did not affect PMN's ability to phagocytize live Escherichia coli. Overall, our results suggest that local infusion of deferoxamine may be an effective tool to protect mammary tissue against PMN-induced oxidative stress during bovine mastitis.

Whey protein isolates (WPI) have been indicated to have potent cholesterol lowering and antioxidative properties. Such effects, however, are not consistently observed, which could be the result of major differences in the processing, isolation and composition of WPI. Moreover, the mechanisms of action or the bioactive component(s) in WPI are poorly understood although the relatively high cysteine content in WPI has been suggested to play an important role. Although high dietary cysteine has been shown to lower plasma homocysteine concentrations, the impact of WPI in this regard has not been investigated. The overall objective of this thesis was to examine the antioxidative and plasma cholesterol and homocysteine lowering properties of two WPI that were produced via different industrial processing and isolation techniques with the milk protein, casein, used as the control protein. We also examined for the mechanism(s) of action of WPI in terms of possible antioxidative, and plasma cholesterol and homocysteine lowering effects. In this regard, the intake of bovine serum albumin (BSA), a major cysteine-rich whey protein was also studied since this protein has been implicated as a key bioactive component for the antioxidant effects of WPI. Four studies were performed. The first involved the characterization of a variety of commercially prepared WPI by high performance capillary electrophoresis for identification of two WPI products that showed major differences in protein composition for subsequent feeding trials. Most of the WPI had similar characteristic electrophoretic profiles, however, significant differences in protein and macronutrient (Ca, Mg, P) composition were noted in two commercial WPI that were chosen as the test proteins in subsequent feeding trials. In the first two feeding studies, hamsters were fed different commercial WPI or milk protein (BSA or casein) containing diets that were either matched or unmatched in terms of macro

Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Biomedical Technology in the Faculty of Health and Wellness Sciences at the Cape Peninsula University Of Technology 2014
Oxidative stress (OS) plays a central role in the progression of diabetes mellitus (DM). Prevention of DM and its complications is a challenging health problem as it impacts on various organ functions, including reproduction. Diabetes mellitus with hyperglycaemic condition generates high production of reactive oxygen species. An imbalance between antioxidant mechanism and reactive oxygen species generates oxidative stress. OS damages the sperm membrane by oxidation of polyunsaturated fats which in turn reduces the sperm motility and ability to fuse with the oocyte and OS directly damage sperm DNA, compromising the paternal genomic contribution to the embryo development. Recent experimental evidence shows that modulation of oxidative stress and natural antioxidants may determine the outcome of male reproductive function. Previous investigations indicate that the supplementation and treatment with phytomedicine might play role in the prevention and management of DM and its subsequent complications on male reproductive function. This study explored the pharmacological potential of kolaviron (KV) on testicular and epididymal tissue in diabetic and non- diabetic Wistar rats. All experiments were conducted for a period of six weeks. Male Wistar rats (240–290 g) were randomly divided into 5 groups (n=12) where all the rats received a standard diet. Non diabetic rats control group and other four groups injected with different treatments. Non diabetic rat (N) received vehicle: Dimethylsulfoxide. Diabetes rats (D) were induced by a single intraperitoneal injection of freshly prepared streptozotocin (STZ) solution, 50mg/kg body weight. The N and D were treated with kolaviron (100 mg/kg body weight) orally, five times a week .The last group, diabetic rats were given subcutaneously injection of the standard anti-diabetic drug, insulin (0.2 u/kg) every second day. After the feeding period, testicular and epididymal tissues were collected and were analysed. All parameters were determined using appropriate methods in homogenized tissues. Data were expressed as mean ± SD. Plasma glucose as well as malondialdehyde (MDA) was significantly higher, while body, testicular and epididymal weights were lower in the D group compared to the N group and N+KV. Both kolaviron and insulin were able to ameliorate these effects. Testicular and epididymal antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in induced diabetic rats were significantly (p<0.05) low compared to diabetic control group. However, KV treated group shown significantly higher SOD, CAT and GPx activities compared D group. In conclusion, our findings demonstrated that KV could improve antioxidant enzymes and modulate STZ induced diabetic related oxidative stress in the male reproductive system. Kolaviron can potentially be used as an anti-diabetic treatment, however further studies are needed. Key words: Oxidative stress, Diabetes mellitus, antioxidants, kolaviron, epididymal tissue, testicular tissue, superoxide dismutase, catalase, glutathione peroxidase, lipid peroxidation, streptozotocin

Thesis (MTech (Consumer Science: Food and Nutrition))--Cape Peninsula University of Technology, 2016.
Background: The different types and forms of rooibos and the ways in which it is prepared and flavoured for consumption influences its total polyphenol content and total antioxidant capacity (TAC) and hence depends on its consumer practices. Design: Phase 1 of the study entailed the selection and preparation of different rooibos types and forms; rooibos brewed for different times; and with different household and commercially added flavourings to determine the total polyphenol content, TAC, flavonol and flavanol content; and subsequent identification of the optimal cup of rooibos based on the first two biochemical parameters. For Phase 2 a questionnaire was used to obtain information on the profile of the adult rooibos herbal tea consumer, as well as of those consuming the optimal cup of rooibos. Results: The following prepared rooibos samples delivered the higher biochemical parameter content: green / unfermented (type representative); green / unfermented leaves and powdered extract (form representatives); that brewed for 10 minutes or longer; and those with added honey. The optimal cup of rooibos was identified as the one brewed for 10 minutes or longer. The older respondents and those with a lower level of education consumed a higher daily amount of rooibos (p < 0.05) and those who brewed rooibos in a teapot consumed the optimal cup (p < 0.05). However, very few respondents consumed the advised number of cups per day (< 1%) and the identified optimal cup (15.9%). Conclusions: Rooibos consumers in this study did not consume it in sufficient amounts and did not brew it for long enough to fully gain from its attributed health benefits.

Thesis (MSc)--University of Stellenbosch, 2007.
ENGLISH ABSTRACT: Human studies on the response of muscle to contusion injury are limited, probably due to the large variability in injury severity and the non-specificity of clinical symptoms reported. To circumvent this problem, several experimental animal models have been designed to study muscle damage and regeneration after contusion injuries. However, the majority of techniques currently used to induce contusion injury are very invasive and therefore not optimal. Furthermore, published studies regarding clinical treatment of such injuries are limited. The main aims of this study were therefore: a) to establish and characterise an in vivo model of non-invasive contusion injury, and b) to assess the effect of pre-injury chronic administration of the antioxidant supplement Oxiprovin™ - a natural grape seed extract (GSE) - on skeletal muscle recovery after experimentallyinduced injury. Two groups of male Wistar rats were subjected to 14 days of oral administration of isovolaemic placebo (sterile isotonic saline) or GSE (20 mg/kg/day) prior to induced contusion. Contusion injury was induced with the mass-drop technique, and recovery parameters assessed for up to 14 days post-injury. Placebotreated rats on average exhibited a 56 % higher creatine kinase (CK) activity when compared to the GSE-treated rats when area under the curve (AUC) was calculated for 14 days post-injury (p < 0.001). In the placebo group, plasma oxygen radical absorbance capacity (ORAC) was unchanged over time, but muscle ORAC was significantly increased by day 7 post-injury (p < 0.001). In the GSE group, a significant decrease in both plasma (p < 0.01) and muscle ORAC (p < 0.001) was evident 4 hr after injury, followed by a significant increase by day 3 (p < 0.05 and p < 0.001 respectively). CD34+ satellite cell (SC) numbers (quiescent and activated) peaked earlier in GSE-treated rats when compared to placebo-treated rats (4 hours vs. day 7 post-injury). Total satellite cell number (CD56+) also peaked earlier in GSE-treated rats than in placebo-treated rats (4 hours vs. 3 days post-injury), while M-cadherin+ SC numbers (quiescent, activated or proliferating) in both treatment groups were significantly increased 4 hours post-injury (p < 0.001), but more so in the placebo group. In GSE-treated rats when compared to placebo-treated rats, newly generated muscle fibres (displaying central nuclei and MHCf +) both appeared (day 3 vs. day 7 post-injury) and peaked in number (day 3 vs. day 7 post-injury; increase from baseline p < 0.001 for both) earlier. The results of this study demonstrate that we have successfully established an in vivo model for non-invasive contusion injury in rats. Furthermore, we have shown that Oxiprovin™: a) increased the ability to scavenge reactive species generated after injury and b) resulted in the activation of satellite cells and formation of newly generated muscle fibres at an earlier time point, thus accelerating the recovery of skeletal muscle after a standardised contusion injury.
AFRIKAANSE OPSOMMING: Eksperimente aangaande die reaksie van spier op kneusings in mense is beperk, waarskynlik as gevolg van die groot verskeidenheid simptome wat mag voorkom en die verskille in die ernstigheid van beserings. Ten einde hierdie problem te oorbrug, is verskeie eksperimentele diermodelle opgestel om kneusings en die herstel van spier daarna te ondersoek. Die tegnieke wat grootendeels vandag gebruik word om kneusings te veroorsaak, maak inbraak op die spier deur die spier te ontbloot voor besering, en is dus nie ideaal nie. Daar is ook nie baie bewyse aangaande die mees geskikte manier om so ‘n besering klinies te behandel nie. Die doel van hierdie studie was dus om: a) ‘n in vivo model van kneusings op te stel en te omskryf, en b) die effek van chroniese toediening van die antioksidant Oxiprovin™ - ‘n natuurlike druifsaad ekstrak (DSE) – op die herstel van skeletspier na ‘n kneusing te ondersoek. Twee groepe manlike Wistar rotte is onderwerp aan mondelikse toediening van isovolemiese plasebo (steriele isotoniese soutoplossing) of DSE (20 mg/kg/dag) vir ‘n tydperk van 14 dae voor kneusing. Kneusing is geïnduseer met die “massdrop” tegniek, en parameters van herstel is ondersoek tot en met 14 dae na besering. Plasebo-behandelde rotte het gemiddeld 56 % hoër kreatien kinase (KK) aktiwiteit in vergelyking met DSE-behandelde rotte (p < 0.001), toe die oppervlak onder die kurwe (OOK) tot en met 14 dae na besering bereken is. Geen verskil oor tyd is in die plasebo groep opgemerk toe plasma suurstof radikaal absorpsie kapasiteit (SRAK) bepaal is nie, maar ‘n betekenisvolle toename in spier SRAK teen dag 7 (p < 0.001) is waargeneem. ‘n Betekenisvolle afname in beide plasma (p < 0.01) en spier (p < 0.001) SRAK van die DSE is teen 4 hr waargeneem, gevolg deur ‘n betekenisvolle toename teen dag 3 na besering (p < 0.05 en p < 0.001 onderskeidelik). Die aantal CD34+ satelliet selle (SS – rustend en geaktiveerd) het beduidend vroeër in die DSE groep gestyg in vergelyking met die plasebo groep (4 uur vs. 7 dae na besering). Die totale aantal SS (CD56+) het ook vroeër in die DSE-behandelde rotte as die plasebobehandelde rotte gestyg (4 uur vs. 3 dae na besering), terwyl die aantal Mcadherin+ SS (rustend, geaktiveerd of prolifererend) betenisvol gestyg het in beide groepe teen 4 uur (p < 0.001) na besering, maar hoër in die plasebo groep was. Die aantal nuutgevormde spiervesels (met sentraal geleë nukleï en MHCf +) het beide vroeër verskyn en gepiek in die DSE-behandelde rotte in vergelyking met die plasebo-behandelde rotte (dag 3 vs. dag 7 na besering). Die resultate van hierdie studie dui aan dat ons instaat was om ‘n in vivo model van nie-indringende kneusing in rotte op te stel. Verder, het ons ook bewys dat Oxiprovin™ toediening die vermoë verleen het om: a) reaktiewe spesies wat na beserings gevorm word, meer doeltreffend te verwyder en b) satelliet selle vroeër te aktiveer en die vorming van nuwe skeletspiervesels te vervroeg, om sodoende die herstel van skeletspier na ‘n gestandardiseerde kneusing vinniger te bewerkstellig.

Weight loss is a common occurrence in patients with human immunodeficiency virus (HIV) and contributes to further debilitation in the acquired immune deficiency syndrome (AIDS). Wasting syndrome (WS) is defined as 10% or more unintentional weight loss from usual body weight. The etiology of WS includes alterations in metabolism, which contribute to loss of lean body mass. Cytokine driven oxidative stress may play a critical role in the metabolic pathways that lead to HIV wasting. Studies have shown that that patients infected with HIV may have a depleted antioxidant (AO) defense system, the integrity of which is needed to efficiently scavenge reactive oxygen species (ROS). It has been theorised that low AO intake may contribute to a depressed AO defense system, which drives oxidative stress (OS). In this study we examined 16 subjects who had documented WS but no active infectious process, stratified into 10 to 15% weight loss (n = 7) and over 15% weight loss (n = 9) groups, and reported on oxidative stress measures and AO intake. (Abstract shortened by UMI.)

Thesis (MSc)--University of Stellenbosch, 2007.
ENGLISH ABSTRACT: The human body has developed an integrated antioxidant defence system to protect against free radical damage. Acute exercise may result in the increased generation of free radicals, including reactive oxygen species, and this may overwhelm antioxidant defence systems resulting in oxidative stress. However, it has been shown that individuals who undergo regular exercise training may have improved antioxidant capacity when compared to sedentary controls. Results from research regarding the association between antioxidant capacity and exercise training are however not conclusive and further investigation is required. Therefore, the aim of this study was to investigate the association between the total plasma antioxidant status and selected plasma indicators of antioxidant status and the MnSOD Ala-9Val (-28C®T) polymorphism in trained male athletes (rugby players) and sedentary male students while controlling for dietary intake of the major antioxidants using a validated dietary assessment method. In order to address the potential confounding effect of dietary antioxidant intake on antioxidant status in the main study, a FFQ that measures vitamin C, vitamin E, carotenoid and flavonoid intake was developed. The reproducibility was assessed by the repeat administration of the FFQ (n = 38), while the va lidity was assessed using a 28-day closeended dietary record and repeated plasma vitamin C values (n = 18). Several statistical tests were conducted to compare the values obtained from the FFQ with values obtained from the various reference methods. While results from Bland-Altman plots suggested that the reproducibility and validity of FFQ was not completely satisfactory, similar mean values, moderate to strong correlation coefficients, and a high percentage of individuals classified correctly according to quartiles of intake indicated satisfactory reproducibility and validity of the FFQ in assessing antioxidant intake. Furthermore, moderate to strong validity coefficients obtained from the method of triads also indicated satisfactory validity for the FFQ. The main study involved a cross-sectional study that compared plasma vitamin C and carotenoid levels as well as total plasma antioxidant status in trained rugby players (n = 76) and sedentary male subjects (n = 39) with different MnSOD genotypes, while controlling for dietary antioxidant intake. Rugby players had significantly higher plasma vitamin C and carotenoid levels compared to sedentary students, which indicated more satisfactory plasma antioxidant status. This was also reflected in the tendency for total plasma antioxidant status (ORAC assay) to be higher in rugby players than sedentary students. MnSOD genotype did not influence plasma vitamin C and carotenoid levels or plasma total antioxidant status, with or without control for dietary antioxidant intake. Dietary vitamin C, vitamin E, carotenoid an flavonoid intake (from foods + supplements) was similar for rugby players and sedentary students and was adequate for both groups. Thus the association between antioxidant status and MnSOD genotype in rugby players and sedentary students seemed not to be influenced by dietary antioxidant intake. In conclusion therefore, rugby players undergoing regular exercise training had a more satisfactory antioxidant status compared to sedentary students. Based on this conclusion, the widespread use of antioxidant supplements by athletes is questioned.
AFRIKAANSE OPSOMMING: Die menslike liggaam beskik oor ‘n geintegreerde antioksidantmeganisme om dit teen vryradikaalskade te beskerm. Akute oefening kan bydra tot ‘n verhoogde produksie van vry radikale, insluitend reaktiewe suurstofspesies, wat kan veroorsaak dat die antioksidantbeskermingsmeganisme oorlaai word, wat dan kan aanleiding gee tot die ontstaan van oksidatiewe stress. Dit is aangetoon dat persone wat gereeld oefening doen verbeterde antioksidantkapasiteit toon in vergelyking met persone wat geen oefening doen nie. Die resultate van navorsingstudies wat die verband tussen antioksidantkapasiteit en oefening ondersoek is egter teenstrydig en verdere navorsing op hierdie gebied is essensieël om uitsluitsel te kry oor kontensieuse vraagstukke. Die doel van hierdie studie was dus om ondersoek in te stel na die verband tussen plasma antioksidant status, die MnSOD Ala-9Val (-28C T) polimorfisme en geselekteerde plasma antioksidantmerkers in geoefende manlike atlete (rugby spelers) en ‘n onaktiewe manlike kontrolegroep terwyl gekontroleer word vir die dieetinname van die vernaamste antioksidante. Om vir die potensiële invloed van dieetantioksidantinname op die antioksidantstatus van proefpersone in die hoofstudie te kontroleer, is ‘n voedsel frekwensievraelys wat vitamien C-, vitamien E-, karotenoïed- en flavinoïedinname meet, ontwikkel. Die herhaalbaarheid (betroubaarheid) van die vraelys is getoets deur herhaalde voltooiing daarvan deur ‘n toetsgroep (n=38), terwyl die geldighied getoets is deur gebruik te maak van ‘n 28-dag geslote dieetrekord en herhaalde plasma vitamien C bepalings as verwysingswaardes (n=18). Verskeie statistiese toetse is uitgevoer om die frekwensievraelys waardes met die verskillende verwysingswaardes te vergelyk. Alhoewel die Bland -Altman grafieke nie dui op bevredigende herhaalbaarheid en geldigheid van die voedselfrekwensie vraelys nie, dui gelyke gemiddelde waardes, matig tot sterk en betekenisvolle korrelasiekoeffisiënte en ‘n hoë persentasie individue korrek geklassifiseer volgens kwartiele van inname, wel op bevredigende herhaalbaarheid en geldigheid. Matige tot sterk geldigheidskoeffisiënte is ook verkry met die toepassing van “The method of Triads”, wat verdere steun bied vir bevredigende geldigheid. In die hoofstudie is plasma vitamien C, karotenoïedvlakke en totale plasma antioksidantstatus in manlike rugby spelers (n=76) vergelyk met dié van onaktiewe manlike kontroles (n=39). Vergelykings tussen MnSOD genotipes binne die aktiwiteitsgroepe is ook getref. Al genoemde analises is gekontroleer vir dieet antioksidantinname. Resultate dui daarop dat die plasma vitamien C en karotenoïedvlakke van rugby spelers betekenisvol hoër was as dié van die kontrolegroep, wat dui op ‘n meer bevredigende antioksidantstatus. Hierdie resultaat is ook weerspieël in die feit dat totale plasma antioksidantstatus (ORAC) in die rugby spelers oog geneig was om hoër te wees as dié van die kontrole groep. Dit het ook geblyk dat MnSOD genotipe nie ‘n effek gehad het op plasma vitamien C-, karotenoïed- of totale antioksidantstatus nie, met of sonder kontrole vir dieet antioksidantinname. Die dieet vitamien C-, vitamien E-, karotenoïed- en flavinoïedinname (vanaf voedsel en supplemente) was dieselfde vir rugby spelers en kontrole en was toereikend vir beide groepe. Dit blyk dus dat dat die verband tussen antioksidantstatus en MnSOD genotipe in die twee groepe nie beinvloed is deur antioksidantinname nie. Ten slotte kan die gevolgtrekking gemaak word dat manlike rugby spelers ‘n meer bevredigende antioksidant status het as onaktiwe manlike kontroles. Op grond van hierdie gevolgtrekking word die algemene gebruik van antioksidant supplemente deur atlete bevraagteken.

Mediterranean species have to be in constant acclimation to seasonal environmental changes, including variations in water availability, air temperature and solar radiation. Due to global warming, these environmental changes are predicted to increase in frequency and intensity. Shrubs have a crucial role in plant communities such as avoiding soil erosion and, therefore, the study of shrub responses to abiotic stresses is essential to predict future changes in populations. Here, we evaluated the performance of the Mediterranean shrub, Cistus albidus, under several abiotic stresses in natural and controlled conditions. It was found that C. albidus plants have to face several combinations of abiotic stressors, with drought and cold being the main environmental cues that threaten their performance during winter. As a first line of defence, C. albidus diurnally changed the angle of their most apical leaves together with a decussate leaf disposition to avoid photoinhibition. Structural mechanisms were not always effective for the whole plant, and carotenoids had a primordial role during the day, dissipating excess energy as a second line of defence. They also had an important function under sustained winter stress, but α-tocopherol was the crucial antioxidant responding to stress, as a third line of defence. Jasmonates seemed to be involved in many abiotic stress responses, including cold stress and drought. High jasmonates contents, especially those of 12-oxo-phytodienoic acid, jasmonic acid and jasmonoyl-isoleucine, were found in natural conditions during a particularly cold day and after a winter drought event in C. albidus seedlings. Furthermore, it is shown that α-tocopherol and jasmonates have an important role in drought tolerance and survival of seedlings in natural field conditions. Even though seedlings have evolved numerous mechanisms to tolerate stress, there was a 30% decrease in survival after summer drought. It is concluded, given the variability in stress responses and the number of mechanisms to withstand abiotic stress, that C. albidus is a highly tolerant and resilient plant to abiotic stress. However, there are still tipping points that could become more frequent in the wake of global change that could cause shifts in C. albidus populations.

Thesis (MSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Contusion injuries cause significant muscle damage, activating a series of cellular events. Satellite cells (SC), the key role players in muscle regeneration, are activated to proliferate and develop into mature myoblasts, which could fuse to form new myotubes or to repair damaged fibres. Evidence suggests that anti-oxidants, such as those found in grape seed extract (GSE), enhance repair, but their effect on SCs is still unclear. This study aimed to harvest and culture primary rat myoblasts to investigate the effect of chronic in vivo GSE supplementation on SCs following a standardised crush injury. Using a modified pre-plate technique, myoblasts were harvested from rat muscle and then compared with the immortal C2C12 cell line for proliferation and differentiation competence. Several media options were compared: i) DMEM with or without L-glutamine, ii) Ham‘s F10 or iii) DMEM with L-glutamine and Ham‘s F10 combined. Primary myoblasts proliferated and differentiated at a much slower rate than C2C12 cells. The combined media was selected for further use. To investigate the effects of GSE on the recovery, rats were supplemented daily with GSE or placebo 14 days prior to a standardised mass-drop crush injury to the gastrocnemius. SCs were isolated and cultured from uninjured (NI, baseline) and from injured rats 4 hours (4h), 3 days (3d) or 14 days (14d) post-injury. Expression of myogenic proteins Pax7, M-cadherin, MyoD, CD56, desmin and CD34 was determined by flow cytometry. Myoblasts were sorted according to their CD56 and CD34 expression and three sub-sets were collected and re-cultured, namely CD56+/CD34-, CD56-/CD34+ and CD56+/CD34+. After 24 hours, sorted cells were stained for desmin expression. Pax7, M-cadherin and MyoD were present in 100% of isolated cells from all groups confirming their myogenic SC identity. For all groups, desmin was expressed only in ~80% of SCs. Lower adhesion competency in GSE supplemented groups resulted in lower yield obtained for culturing. Expression of CD56 increased significantly 3d post-injury in the placebo group. In contrast, with GSE, CD56 already increased 4h post-injury and decreased again 3d post-injury. Although CD34 expression did not differ dramatically, expression pattern resembled that of CD56. Immunocytochemistry revealed a range in morphology and desmin expression of sorted myoblasts. More myoblasts with high desmin expression were observed in the two CD56+ sub-sets (irrespective of CD34 expression), indicating that CD56 is still expressed in more mature myoblasts. Flow cytometry revealed a population of myoblasts expressing particularly high levels of desmin, primarily in the non-injured baseline GSE group. We hypothesise that this result is an indication of preparedness of myoblasts to respond earlier to injury, enabling quicker repair. This cell population with high desmin content is restored in skeletal muscle after repair (14d), only when supplemented with GSE. In conclusion, GSE attenuated adhesion competence of primary myoblasts in culture, but resulted in earlier maturation of SCs, possibly due to baseline preparedness of myoblasts in uninjured muscle for a quick response. Both reduced adhesion competence and early progression of myoblasts could enhance wound healing in skeletal muscle.
AFRIKAANSE OPSOMMING: Kneuswonde veroorsaak aansienlike skade aan skeletspier, wat ‘n reeks sellulêre prosesse in werking stel. Satellietselle, die hoofrolspelers tydens spierregenerasie, vermenigvuldig en ontwikkel tot volwasse mioblaste, wat saamsmelt om nuwe spiervesels te vorm. Antioksidante, soos die wat in druiwepit-ekstrak voorkom, bespoedig herstel, maar hul uitwerking op satellietselle is steeds onduidelik. Die doel van hierdie studie was om mioblaste uit rotspiere te isoleer en te kweek om die effek van langdurige in vivo aanvulling van druiwepit-ekstrak op satellietselle na ‘n kneusbesering te bepaal. 'n Aangepaste protokol is gebruik om primêre mioblaste te isoleer, wat daarna met C2C12 selle, ten opsigte van hul vermenigvuldigings- en differensiasievermoë vergelyk is. Verskeie groeimedia is gebruik: i) DMEM met of sonder L-glutamien, ii) Ham F10 en iii) ‘n kombinasie van DMEM, L-glutamien en Ham F10. Primêre mioblaste het stadiger vermenigvuldig en gedifferensieer as C2C12 selle. Die gekombineerde medium is vir verdere gebruik gekies. Om die uitwerking van druiwepit-ekstrak op spierherstel te ondersoek, is rotte vir 14 dae onderwerp aan daaglikse aanvullings van druiwepit-ekstrak of placebo voor ‘n gestandardiseerde kneusbesering aan die gastrocnemius. Satellietselle is geïsoleer vanuit onbeseerde spier (basiskontrole) en vanuit beseerde spier 4 ure (4h), 3 dae (3d) en 14 dae (14d) na die besering. Die uitdrukking van spierverwante proteïene Pax7, M-cadherin, MyoD, CD56, desmin en CD34 is vasgestel met 'n vloeisitometer. Mioblaste is daarna gesorteer op grond van hul CD56- en CD34-uitdrukking. Drie sub-groepe is versamel en verder gekweek, nl. CD56+/CD34-, CD56-/CD34+ en CD56+/CD34+. Na 24 uur is gesorteerde selle gekleur om desmin-uitdrukking te bepaal. Pax7, M-cadherin en MyoD is deur 100% satellietselle in alle groepe uitgedruk, wat hul spierverwante identiteit bevestig, alhoewel slegs 80% selle in alle groepe desmin uitgedruk. Druiwepit-ekstrak het die vermoë van selle om aan plate te heg onderdruk, wat gelei het tot ‘n laer opbrengs van mioblaste. Drie dae na die besering in die placebo groep het die CD56-uitdrukking beduidend toegeneem. In teenstelling hiermee het CD56-uitdrukking in die druiwepit-ekstrak groep 4 ure na die besering beduidend toegeneem en weer afgeneem na 3 dae. Hoewel daar nie sulke dramatiese verskille was tussen groepe ten opsigte van CD34-uitdrukking nie, was daar ‘n soortgelyke tendens as vir CD56-uitdrukking. Immunositochemie het ‘n verskeidenheid van morfologieë en variërende desminvlakke in gesorteerde mioblaste blootgestel. In die twee CD56+ groepe is meer mioblaste wat hoë desmin vlakke uitdruk gevind, wat aandui dat CD56 uitgedruk word deur meer volwasse mioblaste, ongeag van CD34-uitdrukking. Tydens vloeisitometrie is ‘n populasie selle wat hoë desminvlakke uitdruk, hoofsaaklik in die onbeseerde en 14d druiwepit-ekstrak groepe gevind. Dit is ‘n aanduiding dat sommige mioblaste voorbereid is om na 'n besering vinniger te reageer. Na die herstelproses word hierdie groep selle hernu in die teenwoordigheid van druiwepit-ekstrak-aanvulling. Die resultate het gevolglik daartoe gelei dat druiwepit-ekstrak die hegtingsvemoë van mioblaste verlaag, maar dat die aanvulling in vivo tot vroeër ontwikkeling van mioblaste lei, waarskynlik deur satellietselle voor te berei vir 'n vinnige respons na ‘n besering. Beide die onderdrukking van aanhegting aan kultuurplate en die vroeë ontwikkeling van mioblaste, kan die herstel van die skeletspier verbeter.
NRF and the Harry Crossley bursary for funding

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
Lead (Pb) is one of the most toxic anthropogenic pollutants of widespread occurrence in both terrestrial and aquatic ecosystems. The phytotoxicity of lead is a complex phenomenon that involves changes in development as well as in physiological, biochemical and molecular mechanisms according to different plant survival strategies. It is well known that heavy metals accelerate the production of reactive oxygen species (ROS) which can lead to changes in the concentration of antioxidant enzymes. The aim of this study was to investigate the effect of Pb on growth, physiology, absorption and translocation of Pb, activity and gene expression of antioxidant enzymes in "Sempre Verde" (SV) and "SetentÃo" (SET), Vigna unguiculata cultivars. The seeds were germinated in vermiculite and, after seven days, the seedlings were transferred to Hoagland solution. After seven days in a hydroponic system the plants were supplemented with EDTA-Pb 0.5 mM. Physiological parameters were measured using an infrared gas analyzer. The Pb content in roots and shoots was measured by ICP OES. The activities of catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.1) and superoxide dismutase (SOD, EC 1.15.1.1) was determined. The expression of the CAT gene (VuCAT 1 and 2), the APX (VuAPX 1 to 6) and SOD (VuSOD Cu / Zn 1 to 3, VuSODMn1, VuSODCu / chap1, VuSODFe 1 to 3) was evaluated by RT- qPCR using specific primers. Total RNA was extracted from the roots and leaves of control plants and treated. The results showed that the leaf areas were reduced by 60 (SV) and 88% (SET). The inhibition of net photosynthesis was 40 (SV) and 72% (SET). The cultivars showed differences in the accumulation and distribution of Pb, as well as in enzyme activity. SV had more Pb in roots than in leaves unlike SET that accumulates more Pb in leaves than in roots. The activity of the antioxidant enzymes CAT, APX and SOD was enhanced in SET, regardless of the tissue. In SV roots, on the other hand, the CAT activity was not modified whereas in SV leaves the SOD activity underwent an inhibition. All the remaining antioxidant activities in SV were stimulated. The CAT 1 and 2 genes were differently expressed in leaves and roots of SV and SET. The relative expression of the APX multigene family in leaves and roots in these cultivars was partially distinct. Likewise genes SOD in leaves showed a partially distinct profile in the two cultivars. Rather, the relative expression of SOD genes in the roots and SV SET is completely different. Taken together, the two Vigna unguiculata cultivars showed a different behavior towards tolerance, accumulation and detoxification of Pb. These results suggest that Pb induces oxidative stress in SV and SET cultivars and that the high activity of antioxidant enzymes could serve as an important component of defense against the damage caused by Pb, and SV more tolerant to stress than SET
O chumbo (Pb) à um dos poluentes antropogÃnicos mais tÃxicos de ocorrÃncia generalizada em ambos os ecossistemas terrestres e aquÃticos. A fitotoxicidade do chumbo à um fenÃmeno complexo que envolve mudanÃas no desenvolvimento, bem como nos mecanismos fisiolÃgicos, bioquÃmicos e moleculares, de acordo com as diferentes estratÃgias de sobrevivÃncia das plantas. à bem conhecido que os metais pesados aceleram a produÃÃo de espÃcies reativas de oxigÃnio (EROs) e que podem provocar mudanÃas na concentraÃÃo das enzimas antioxidantes. O objetivo desse trabalho foi investigar o efeito do Pb no crescimento, fisiologia, absorÃÃo e translocaÃÃo de Pb, a atividade e expressÃo gÃnica das enzimas antioxidantes em "Sempre Verde" (SV) e "SetentÃo" (SET), cultivares de Vigna unguiculata. As sementes foram germinadas em vermiculita e, apÃs sete dias, as plÃntulas foram transferidas para soluÃÃo de Hoagland. Depois de sete dias em um sistema hidropÃnico as plantas foram suplementadas ou nÃo com EDTA-Pb 0,5 mM. Os parÃmetros fisiolÃgicos foram medidos atravÃs de um analisador de gÃs infravermelho. O conteÃdo de Pb nas raÃzes e na parte aÃrea foi medido por ICP OES. As atividades das enzimas catalase (CAT, EC 1.11.1.6), ascorbato peroxidase (APX, EC 1.11.1.1) e superÃxido dismutase (SOD, EC 1.15.1.1) foram determinadas. A expressÃo dos genes da CAT (VuCAT 1 e 2), da APX (VuAPX 1 a 6) e da SOD (VuSOD Cu/Zn 1 a 3, VuSODMn1, VuSODCu/Chap1, VuSODFe 1 a 3), foi avaliada por RTqPCR utilizando iniciadores especÃficos. O RNA total foi extraÃdo a partir das raÃzes e das folhas nas plantas controle e tratadas. Os resultados revelaram que as Ãreas foliares diminuÃram 60 (SV) e 88% (SET). A inibiÃÃo da fotossÃntese lÃquida foi 40 (SV) e 72% (SET). Os cultivares apresentaram diferenÃas na acumulaÃÃo e distribuiÃÃo de Pb, bem como nas atividades enzimÃticas. SV apresentou mais Pb nas raÃzes do que nas folhas ao contrÃrio de SET que acumula mais Pb nas folhas do que nas raÃzes. A atividade das enzimas antioxidantes CAT, APX e SOD foram estimuladas no cultivar SET independentemente do tecido. No entanto, a atividade da CAT em raÃzes do cultivar SV nÃo sofreu modificaÃÃo e a atividade da SOD em folhas, do mesmo cultivar, foi inibida, sendo as demais atividades em SV estimuladas. O aumento das atividades das enzimas em raÃzes e folhas de SV e SET estÃo de acordo com suas expressÃes gÃnicas. Os genes CAT 1 e 2 foram diferentemente expressos nas folhas e nas raÃzes de SV e SET. A expressÃo relativa da famÃlia multigÃnica da APX em raÃzes e folhas nesses cultivares foi parcialmente distinta. Da mesma forma os genes da SOD em folhas apresentaram um perfil parcialmente distinto nos dois cultivares. Ao contrÃrio, a expressÃo relativa dos genes da SOD nas raÃzes de SV e de SET foi completamente diferente. Analisados em conjunto, os dois cultivares de Vigna unguiculata apresentaram um comportamento diferente em relaÃÃo a tolerÃncia, acumulaÃÃo, e desintoxicaÃÃo do Pb. Estes resultados sugerem que o Pb induz estresse oxidativo nos cultivares SV e SET e que a elevada atividade das enzimas antioxidantes poderia servir como um importante componente de defesa contra os danos provocados pelo Pb, sendo SV mais tolerante a esse estresse do que SET.

Thesis (PhD) -- Macquarie University, Division of Environmental and Life Sciences, Dept. of Chemistry and Biomolecular Sciences, 2007.
Bibliography: leaves 295-322.
Generation of peroxide groups in proteins exposed to a wide variety of reactive oxygen species (ROS) requires an initial formation of protein carbon-centred or peroxyl free radicals, which can be reduced to hydroperoxides. Both protein radicals and protein hydroperoxides are capable of oxidizing important biomolecules and thus initiate biological damage. In this study, we investigated the inhibition of protein hydroperoxide formation by ascorbate and GSH in gamma-irradiated HL-60 cells.--We used HL-60 cells as a model for general protection of living organisms by ascorbate (Asc) and glutathione (GSH) from the deleterious effects of protein hydroperoxides generated by radicals produced by gamma radiation. Measurement by HPLC indicated that incubation of HL-60 cells with Asc in the presence of ascorbate oxidase resulted in the accumulation of intracellular Asc. The intracellular Asc levels were lowered by irradiation, demonstrating intracellular consumption of Asc by the radiation-generated radicals. Exposure of HL-60 cells to increasing gamma irradiation doses resulted in increasing accumulation of protein peroxides in the cells. This was measured by the FOX assay. A significant decrease in intracellular protein hydroperoxides was noted when the cells were treated with ascorbic acid before irradiation. A dose-dependent protective effect of Asc was observed. Asc loading also provided strong protection from radiation-generated protein hydroperoxides independently of the composition of the external medium, showing that only the radicals formed within the cells were effective in oxidizing the cell proteins. Similarly, protein peroxidation was inhibited in cells with enhanced levels of GSH and increased when the intracellular GSH concentration was reduced. These findings indicate that ascorbate and GSH are important antioxidants in protecting cells from oxidative stress associated with the generation of protein hydroperoxide.
Mode of access: World Wide Web.
xxix, 322 leaves ill

"A thesis submitted in partial fulfillment of the requirements for the award of the degree of Doctor of Philosophy".
Thesis (PhD) -- Macquarie University, Division of Environmental and Life Sciences, Dept. of Chemistry and Biomolecular Sciences, 2007.
Bibliography: p. 243-277.
Introduction -- A convenient synthesis of 30HKG -- Facile synthesis of the UV filter compounds 30HKyn and AHBG -- Synthesis, identification and quantification of novel human lens metabolites -- Modification of bovine lens protein with UV filters and related metabolites -- Effect of UV light on UV filter-treated lens proteins -- Conclusions and future directions.
The kynurenine-based UV filters are unstable under physiological conditions and undergo side chain deamination, resulting in α,β-unsaturated carbonyl compounds. These compounds can react with free or protein bound nucleophiles in the lens via Michael addition. The key sites of the UV filters kynurenine (Kyn) and 3-hydroxykynurenine (3OHKyn) modification in human lenses include cysteine (Cys), and to a lesser extent, lysine (Lys) and histidine (His) residues. Recent in vivo studies have revealed that 3-hydroxykynurenine-O-β-D-glucoside (3OHKG) binds to Cys residues of lens crystallins in older normal human lenses. As a result of this binding, human lens proteins become progressively modified by UV filters in an age-dependent manner, contributing to changes that occur with the development of age-related nuclear (ARN) cataract. Upon exposure to UV light, free UV filters are poor photosensitisers, however the role of protein-bound species is less clear. It has been recently demonstrated that Kyn, when bound to lens proteins, becomes more susceptible to photo-oxidation by UV light. Therefore, the investigation of 3OHKG binding to lens proteins, and the effect of UV light on proteins modified with 3OHKG and 3OHKyn, were major aims of this study. As a result of the role of these compounds as UV filters and their possible involvement in ARN cataract formation, it is crucial to understand the nature, concentration and modes of action of the UV filters and their metabolites present in the human lenses. Therefore, an additional aim was to investigate human lenses for the presence of novel kynurenine-based human lens metabolites and examine their reactivity.--As 3OHKG is not commercially available, to conduct protein binding studies, an initial aim of this study was to synthesise 3OHKG (Chapter 2). Through the expansion and optimisation of a literature procedure, 3OHKG was successfully synthesised using commercially available and inexpensive reagents, and applying green chemistry principles, where toxic and corrosive reagents were replaced with benign reagents and solvent-free and microwave chemistry was used. A detailed investigation of different reaction conditions was also conducted, resulting in either the improvement of reaction yields or reaction time compared to the literature method. Applying the same synthetic strategy, and using key precursors from the synthesis of 3OHKG, the UV filters 3OHKyn and 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid-O-β-D-glucoside (AHBG), were also successfully synthesised (Chapter 3).
Chapter 4 describes the investigation of both normal and cataractous human lenses in an attempt to identify novel human lens metabolites derived from deaminated Kyn and 3OHKyn (Chapter 4, Part A). Initially, 4-(2-aminophenyl)-4-oxobutanoic acid (AHA), glutathionyl-kynurenine (GSH-Kyn), kynurenine yellow (Kyn yellow), 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid (AHB), glutathionyl-3-hydroxykynurenine (GSH-3OHKyn) and 3-hydroxykynurenine yellow (3OHKyn yellow) were synthesised and human lenses were examined for their presence. AHA and AHB were synthesised from similar precursors to those used in the synthesis of 3OHKG, while the GSH adducts and yellow compounds were synthesised from Kyn and 3OHKyn via base induced deamination. Following isolation and structural elucidation, AHA, AHB and GSH-Kyn were confirmed as novel human lens metabolites. They were quantified in low pmol/mg lens (dry mass) levels in normal and cataractous lenses of all ages, while GSH-3OHKyn, Kyn yellow and 3OHKyn yellow were not detected. In contrast to AHA, the lens metabolites AHB, GSH-Kyn and GSH-3OHKyn were found to be unstable at physiological pH. The spectral properties of these compounds suggest that they may act as UV filters. --Chapter 4 (Part B) also describes the identification and characterisation of a novel human lens UV filter, cysteinyl-3-hydroxykynurenine -O-β-D-glucoside (Cys-3OHKG). An authentic standard was synthesised via Michael addition of cysteine to deaminated 3OHKG. Cys-3OHKG was detected in low pmol/mg lens (dry mass) levels in normal lenses only after the 5th decade of life and was absent in cataractous lenses. Cys-3OHKG showed rapid decomposition at physiological pH.
Chapter 5 describes the identification and quantification of amino acids involved in covalent binding of 3OHKG to lens proteins. Model studies with bovine lens proteins and 3OHKG at pH 7.2 and 9.5 were undertaken. The amino acid adducts were identified via total synthesis and spectral analysis, and subsequently quantified upon acid hydrolysis of the modified lens proteins. Under both pH conditions, 3OHKG was found to react with lens proteins predominantly via Cys residues with low levels of binding also detected at Lys residues. Comparative studies with Kyn (pH 9.5) and 3OHKyn (pH 7.2 and 9.5) resulted in modified lens proteins at Cys residues, with only minor modification at Lys residues at pH 9.5. The extent of modification was found to be significantly higher at pH 9.5 in all cases. His adducts were not identified. 3OHKG-, Kyn- and 3OHKyn-modified lens proteins were found to be coloured and fluorescent, resembling those of aged and ARN cataractous lenses. In contrast, AHB and AHA, which can not form α,β-unsaturated carbonyl compounds, resulted in non-covalent modification of lens proteins. AHB may contribute to lens colouration and fluorescence as further reactions of this material yielded species that have similar characteristics to those identified from 3OHKyn modification. These species are postulated to arise via auto-oxidation of the o-aminophenol moiety present in both 3OHKyn and AHB.--In Chapter 6, the potential roles of 3OHKG and 3OHKyn, and the related species AHA and AHB, in generating reactive oxygen species and protein damage following illumination with UV light was examined. The UV filter compounds were examined in both their free and protein-bound forms. Kyn-modified proteins were used as a positive control. Exposure of these compounds to UV light (λ 305-385 nm) has been shown to generate H2O2 and protein-bound peroxides in a time-dependent manner, with shorter wavelengths generating more peroxides. The yields of peroxides were observed to be highly dependent on the nature of the UV filter compound and whether these species were free or protein bound, with much higher levels being detected with the bound species. Thus, protein-bound 3OHKyn yielded higher levels of peroxide than 3OHKG, with these levels, in turn, higher than for the free UV filter compounds. AHB-treated lens proteins resulted in formation of low but statistically significant levels of peroxides, while AHA-treated lens proteins resulted in insignificant peroxide formation. The consequences of these photochemical reactions have been examined by quantifying protein-bound tyrosine oxidation products (3,4-dihydroxyphenylalanine [DOPA], di-tyrosine [di-Tyr]) and protein cross-linking. 3OHKG-modified proteins gave elevated levels of di-Tyr, but not DOPA, whereas 3OHKyn-modified protein gave the inverse. DOPA formation was observed to be independent of illumination and most likely arose via o-aminophenol auto-oxidation. AHB- and AHA-treated lens proteins resulted in statistically insignificant di-Tyr formation, while a light independent increase in DOPA was observed for both samples. Both reducible (disulfide) and non-reducible cross-links were detected in modified proteins following illumination. These linkages were present at lower levels in modified, but non-illuminated proteins, and absent from unmodified protein samples.
This work has provided an optimised synthetic procedure for 3OHKG and other lens metabolites (Chapters 2 and 3). Four novel lens metabolites have been identified and quantified in normal and cataractous human lenses (Chapter 4). Subsequent experiments, described in Chapter 5, identified the major covalent binding sites of 3OHKG to lens proteins, while AHA and AHB showed non-covalent binding. Further work described in Chapter 6 showed that protein-bound 3OHKG, Kyn and 3OHKyn were better photosensitisers of oxidative damage than in their unbound state. Together, this research has provided strong evidence that post-translational modifications of lens proteins by kynurenine-based metabolites and their interaction with UV light appear, at least in part, responsible for the age-dependent colouration of human lenses and an elevated level of oxidative stress in older lenses. These processes may contribute to the progression of ARN cataract.
Mode of access: World Wide Web.
xxxix, 308 p. ill. (some col.)

Experiments were conducted to examine the role of dietary selenium (Se) and changes in antioxidant capacity during DMBA-induced mammary carcinogenesis. Weanling rats were fed graded amounts of Se in an AIN-76 diet, modified to approximate the amount and type of dietary fat consumed in Canada. Animals fed 3-4 ppm Se had a reduced tumor incidence but there was evidence of chronic Se toxicity. Low Se did not elevate tumor incidence. Thus, supplemental dietary Se did not offer any protection in reducing the incidence of cancer when rats were fed a diet representative of North American intakes of fat. Blood GSHPx activity was elevated in rats that remained free of tumors (NT group) compared to animals that would eventually develop tumors (WT group). SOD activity was reduced in WT and NT rats, but appeared to be related to carcinogen administration. These differences were observed before tumor development and over a wide range of Se intakes. WT rats excreted more $ sp{75}$Se compared to both control and NT rats. These changes, however, were not reflected in elevated lipid peroxidation. Thus, one of the protective mechanisms during carcinogenesis may be the capacity of the animal to elevate GSHPx activity.

Com o objetivo de avaliar o desempenho de diferentes doses e produtos fitossanitários utilizados no tratamento de sementes de soja, foi efetuada a caracterização do efeito fisiológico na germinação e no desenvolvimento das plântulas por intermédio dos testes de germinação e de vigor (índice de velocidade de germinação, envelhecimento acelerado, comprimento e massa de matéria seca de plântula, teste de frio e análise computadorizada de imagem) em laboratório, além da determinação do teor de clorofila (por intermédio do valor SPAD), fotossíntese líquida, condutância estomática, transpiração, temperatura foliar, conteúdo de peróxido de hidrogênio, peroxidação de lipídios, atividade da catalase (enzima antioxidante), e teor de proteína e de nitrogênio total. Com base nos resultados obtidos observou-se que todos os tratamentos apresentaram desempenho satisfatório no que diz respeito à germinação. Não se observaram diferenças em termos de índice de velocidade de germinação, envelhecimento acelerado, comprimento e massa de matéria seca de raiz, índice de vigor e de crescimento (análise de imagem) e peroxidação de lipídeos. O tratamento com Fipronil + Piraclostrobina + Tiofanato Metílico apresentou tendência de melhor desempenho em termos de comprimento de parte aérea, enquanto o tratamento Controle apresentou tendência de melhor desempenho, mas não diferiu dos tratamentos: Fipronil + Piraclostrobina + Tiofanato Metílico, Fluxapiroxade e Piraclostrobina em termos de massa de matéria seca de parte aérea. Os tratamentos Fipronil e Fluxapiroxade apresentaram melhor desempenho que o Controle, em termos de relação massa de matéria seca de raiz e de parte aérea. O tratamento com Fipronil apresentou melhor tendência de desempenho pelo teste de frio e juntamente com Fipronil + Piraclostrobina + Tiofanato Metílico e Fluxapiroxade apresentou desempenho melhor que o Controle no de índice de uniformidade. A Piraclostrobina apresentou desempenho melhor que o Controle em termos de teor de clorofila, fotossíntese líquida e temperatura foliar, enquanto o tratamento com Fipronil foi melhor que o Controle e os demais tratamentos em termos de condutância estomática e transpiração. Os tratamentos com Fipronil e Piraclostrobina apresentaram desempenho melhor que o Controle, em termos de conteúdo de peróxido de hidrogênio e os tratamentos com Fipronil + Piraclostrobina + Tiofanato Metílico e Fluxapiroxade e Piraclostrobina apresentaram desempenho melhor que o Controle, em termos da atividade da catalase. O Fluxapiroxade (50 g por 100 kg de sementes) apresentou melhor desempenho, mas não diferiu do Controle e dos tratamentos com Fipronil, Fluxapiroxade (25 e 75 g por 100 kg de sementes) e Piraclostrobina em termos do teor de proteína e nitrogênio total. Com os resultados apresentados, conclui-se que não houve prejuízo ao vigor das plântulas quando realizado o tratamento de sementes. Além disso, pode-se concluir que esta constitui prática agronômica eficiente não só no que diz respeito ao controle de doenças e pragas, mas também apresenta benefícios no que diz respeito à fisiologia das plântulas de soja, sobretudo para tratamento realizado com estrobilurinas. Esses benefícios podem favorecer um melhor estabelecimento das plântulas no campo.
This study aimed to evaluate the physiological effects of soybean seed treatment with different pesticides and doses on germination and seedling development. Germination and vigor tests, such as speed of germination index, accelerated aging, length and dry weight of seedling, cold test and computerized image analysis were performed in the laboratory, besides the determination of chlorophyll content (SPAD value), net photosynthesis, stomatal conductance, leaves transpiration and temperature, content of hydrogen peroxide, lipid peroxidation, catalase (an antioxidant enzyme) activity, protein and total nitrogen. The results obtained revealed that all treatments showed satisfactory performance in germination. No differences in terms of speed index, accelerated aging, root length and dry weight, vigor and growth index (image analysis) and lipid peroxidation were observed. Treatment with Fipronil + Pyraclostrobin + Methyl Tiophanate tended to perform better in terms of shoot, while control treatment tended to perform better, but did not differ from treatments with Fipronil + Pyraclostrobin + Methyl Tiophanate, Fluxapyroxad and Pyraclostrobin in terms of shoot dry matter. Fipronil and Fluxapyroxad treatments performed better than the control in terms of relationship between root and shoot dry weight. Treatment with Fipronil showed better performance trend for the cold test and along with Fipronil + Pyraclostrobin + Methyl Tiophanate and Fluxapyroxad performed better than control on the uniformity index. Pyraclostrobin showed better performance than the control in terms of chlorophyll content, net photosynthesis and leaf temperature, whereas treatment with Fipronil was better than the control and other treatments in terms of stomatal conductance and transpiration. Treatments with Fipronil and Pyraclostrobin performed better than the control in terms of content of hydrogen peroxide and treatments with Fipronil + Pyraclostrobin + Methyl Tiophanate, Fluxapyroxad and Pyraclostrobin performed better than the control in terms of catalase activity. Fluxapyroxad (50 g per 100 kg of seeds) performed better, but did not differ from control, Fipronil, Fluxapyroxad (25 and 75 g per 100 kg of seeds) and Pyraclostrobin in terms of protein content and total nitrogen. With the results presented, we conclude that there was no damage to seedling vigor when seed treatment was applied. Furthermore, it can be concluded that this is an efficient agronomic practice with regard to the control of diseases and pests and it also has benefits to the physiology of soybean seedlings, mainly when treatment is done with Strobilurins. These benefits can favorable to a better seedling establishment in the field.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Universidade Estadual Paulista (UNESP)
A temperatura ambiente elevada representa o principal fator limitante do desenvolvimento da produção avícola em regiões de clima quente. O objetivo do trabalho foi verificar se a dieta suplementada com vitaminas C e E, seria capaz de neutralizar, ou reduzir, os efeitos do estresse por calor, aplicado dos 28 aos 42 dias de idade, sobre a resposta fisiológica, o desempenho, rendimento de carcaça e qualidade da carne de frangos. Foram utilizados 384 frangos de corte machos distribuídos num delineamento inteiramente casualizado, com arranjo fatorial 2 x 3 (com e sem suplementação da dieta com vitaminas e temperaturas ambientais associadas ao pair-feeding) e 16 repetições. As aves foram mantidas em termoneutralidade até os 28 dias. A partir desta idade, foram alojadas em grupos de quatro por gaiola, em três salas climatizadas: duas termoneutras (22,6 e 22,5ºC) e uma de estresse por calor (31,7ºC). Metade das aves recebeu dieta suplementada com vitaminas C (536 mg/kg) e E (127 mg/kg). Na sala de estresse por calor as aves tiveram livre acesso à ração; nas salas termoneutras metade das aves recebeu ração à vontade e a outra metade recebeu quantidade limitada, no sistema pair-feeding. Foram avaliados a temperatura retal e da superfície da pele e características de desempenho, rendimento de carcaça e qualidade instrumental da carne. As análises de qualidade de carne foram realizadas no músculo pectoralis major (peito) 24 horas após o abate. A suplementação da dieta com vitaminas C e E não foi capaz de neutralizar, nem de reduzir, os efeitos negativos do estresse por calor sobre as características de desempenho, rendimento de carcaça e qualidade de carne das aves. A semelhança nos resultados de desempenho entre aves em estresse por calor e em pair-feeding sugere que a queda no desempenho sob estresse por calor deveu-se, principalmente, à redução no consumo de alimentos
Elevated ambient temperature represents the main limiting factor for the development of chicken production in hot climate regions. The objective of this study was to evaluate if the diet supplemented with vitamins C and E would be able to reduce, or neutralize the negative effects of heat stress applied between 28 and 42 days of age, on the physiological response, performance, slaughter yield and meat quality of chickens. A total of 384 male broiler chickens were assigned to a completely randomized design, with a 2 x 3 factorial arrangement (diet with or without vitamins supplementation and ambient temperatures associated with pair-feeding) and 16 replications. The chickens were kept in thermoneutral conditions up to 28 days of age. From this age and on, were housed in groups of four per cage, in three environmentally controlled chambers: two were thermoneutral (22,6 e 22,5ºC) and one for heat stress (31,7ºC). Half the chickens were offered a diet supplemented with vitamins C (536 mg/kg) and E (127 mg/kg). In the heat stress chamber, the chickens had free access to the feed; in the thermoneutral chambers half the chickens had free access to the feed and the other half received a limited amount, in a pair-feeding system. Rectal and body surface temperatures and performance, slaughter yield and meat quality traits were evaluated. Meat quality analyses were performed in the pectoralis major muscle 24 hours after slaughter. Diet supplementation with vitamins C and E were not able to neutralize, neither to reduce, the negative effects of heat stress on the performance, slaughter yield and meat quality traits. The similarity of performance results between the chickens submitted to heat stress and pair-feeding, suggested that the reduction in performance under heat stress were due mainly to the drop in feed consumption

Orientador: Ana Silvia Alves Meira Tavares Moura
Banca: José Roberto Sartori
Banca: Dirlei Antonio Berto
Banca: Luiz Lehmann Coutinho
Banca: José F. M. Mendes
Resumo: A temperatura ambiente elevada representa o principal fator limitante do desenvolvimento da produção avícola em regiões de clima quente. O objetivo do trabalho foi verificar se a dieta suplementada com vitaminas C e E, seria capaz de neutralizar, ou reduzir, os efeitos do estresse por calor, aplicado dos 28 aos 42 dias de idade, sobre a resposta fisiológica, o desempenho, rendimento de carcaça e qualidade da carne de frangos. Foram utilizados 384 frangos de corte machos distribuídos num delineamento inteiramente casualizado, com arranjo fatorial 2 x 3 (com e sem suplementação da dieta com vitaminas e temperaturas ambientais associadas ao pair-feeding) e 16 repetições. As aves foram mantidas em termoneutralidade até os 28 dias. A partir desta idade, foram alojadas em grupos de quatro por gaiola, em três salas climatizadas: duas termoneutras (22,6 e 22,5ºC) e uma de estresse por calor (31,7ºC). Metade das aves recebeu dieta suplementada com vitaminas C (536 mg/kg) e E (127 mg/kg). Na sala de estresse por calor as aves tiveram livre acesso à ração; nas salas termoneutras metade das aves recebeu ração à vontade e a outra metade recebeu quantidade limitada, no sistema pair-feeding. Foram avaliados a temperatura retal e da superfície da pele e características de desempenho, rendimento de carcaça e qualidade instrumental da carne. As análises de qualidade de carne foram realizadas no músculo pectoralis major (peito) 24 horas após o abate. A suplementação da dieta com vitaminas C e E não foi capaz de neutralizar, nem de reduzir, os efeitos negativos do estresse por calor sobre as características de desempenho, rendimento de carcaça e qualidade de carne das aves. A semelhança nos resultados de desempenho entre aves em estresse por calor e em pair-feeding sugere que a queda no desempenho sob estresse por calor deveu-se, principalmente, à redução no consumo de alimentos
Abstract : Elevated ambient temperature represents the main limiting factor for the development of chicken production in hot climate regions. The objective of this study was to evaluate if the diet supplemented with vitamins C and E would be able to reduce, or neutralize the negative effects of heat stress applied between 28 and 42 days of age, on the physiological response, performance, slaughter yield and meat quality of chickens. A total of 384 male broiler chickens were assigned to a completely randomized design, with a 2 x 3 factorial arrangement (diet with or without vitamins supplementation and ambient temperatures associated with pair-feeding) and 16 replications. The chickens were kept in thermoneutral conditions up to 28 days of age. From this age and on, were housed in groups of four per cage, in three environmentally controlled chambers: two were thermoneutral (22,6 e 22,5ºC) and one for heat stress (31,7ºC). Half the chickens were offered a diet supplemented with vitamins C (536 mg/kg) and E (127 mg/kg). In the heat stress chamber, the chickens had free access to the feed; in the thermoneutral chambers half the chickens had free access to the feed and the other half received a limited amount, in a pair-feeding system. Rectal and body surface temperatures and performance, slaughter yield and meat quality traits were evaluated. Meat quality analyses were performed in the pectoralis major muscle 24 hours after slaughter. Diet supplementation with vitamins C and E were not able to neutralize, neither to reduce, the negative effects of heat stress on the performance, slaughter yield and meat quality traits. The similarity of performance results between the chickens submitted to heat stress and pair-feeding, suggested that the reduction in performance under heat stress were due mainly to the drop in feed consumption
Doutor

Thesis (MTech (Biomedical Technology))--Cape Peninsula University of Technology, 2015.
Diabetes mellitus (DM) has been reported to be one of the greatest global public health threats. Statistics of the fertility status of modern society has linked increased DM to a decrease in fertility rates. Hyperglycaemia is characteristic of DM that results in a disturbance of proteins, lipids and carbohydrate metabolism leading to an increase production of reactive oxygen species (ROS). In the case where ROS overwhelms antioxidant mechanisms, the body goes into state of oxidative stress (OS). OS plays a vital role in the progression of DM which leads to dysfunction and damage of various organs including that of the reproductive system. Os has shown to cause damage to the sperm membraneby oxidation of polyunsaturated fatty acids (PUFA’s) as the sperm membrane are rich in PUFA’s. This damage contributes to reduced sperm motility, concentration, morphological abnormalities and the sperms ability to fuse with the ZP of the oocyte. DM has been observed to cause testicular degeneration by interrupting sertoli cell production and maintenance thus resulting in a disturbance of the normal functioning of the reproductive system. Experimental studies have targeted more natural sources for treating DM and its complications of the reproductive system. Plants and natural dietary substances have shown to have high antioxidant contents that combat DM induced oxidative stress. This study explored the effect the Hypoxis hemerocallidea (H. hemerocallidea) supplementation on testicular and epididymal tissue, sperm motility and reproductive hormones in male wistar rats. The experiment were conducted for 6 weeks and the rats (230-260 grams) were randomly divided into 5 groups (n=12 per group). Diabetes was induced in 3 of the 5 groups. The first group was the normal control group (A), second the diabetic control group (B), third was the diabetic group treated with 800mg/kg H. hemerocallidea (group C), fourth the diabetic group treated with 200mg/kg H. hemerocallidea (group D) and fifth the non-diabetic group supplemented with 800mg/kg H. hemerocallidea (group E). Blood glucose showed a significant increase in the diabetic group when compared to the normal control and treated groups. H. hemerocallidea showed improvement in sperm motility and sperm morphology more at 800mg/kg when compared to diabetic group and diabetic group treated with 200mg/kg. Body, testicular and epipidymal weights of diabetic control were significantly lower when compared to the other groups. Testicular and epididymal Malondialdehyde levels were decreased in normal control, diabetic groups treated with different doses of H. hemerocallidea and the non-diabetic group supplemented with H. hemerocallideaon comparing with the diabetic control group. Antioxidants such as Superoxide dismutase, Catalase and total Glutathione activity was observed to be dosage dependent in certin groups but most showed a significant increase when compared to the diabetic control group. The total antioxidant capacity was measured using Oxygen radical absorbance capacity (ORAC) and Ferric ion reducing antioxidant power (FRAP); increase was observed when normal control group and treated groups were compared to the diabetic group. Testosterone and estradiol levels were also increased when the normal control group and treated groups were compared to the diabetic control group. Based on our findings it can be concluded that H. hemerocallidea supplementation can potentially be used to counteract deleterious effects of DM on the male reproductive system.

Lee Chung-hung.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2003.
Includes bibliographical references (leaves 113-133).
Abstracts in English and Chinese.
Chapter Chapter 1 --- General Introduction
Chapter 1.1 --- History of soybean --- p.1
Chapter 1.2 --- Health benefits of soybean --- p.2
Chapter 1.3 --- Introduction to flavonoids --- p.3
Chapter 1.4 --- Bioavailability of flavonoids --- p.5
Chapter 1.5 --- Chemistry of isoflavones --- p.6
Chapter 1.6 --- Estrogenic property of isoflavones --- p.8
Chapter 1.7 --- Nutritional significance of isoflavones and their glycosides --- p.8
Chapter 1.7.1 --- Anticarcinogenic activity --- p.9
Chapter 1.7.2 --- Antioxidative activity --- p.10
Chapter 1.7.3 --- Cardioprotective activity --- p.13
Chapter 1.7.4 --- Osteoprotective activity --- p.14
Chapter 1.7.5 --- Neuroprotective activity --- p.15
Chapter 1.7.6 --- Antiangiogenic activity --- p.16
Chapter Chapter 2 --- Composition of Soybean Isoflavones
Chapter 2.1 --- Introduction --- p.17
Chapter 2.2 --- Objective --- p.19
Chapter 2.3 --- Materials and Methods --- p.20
Chapter 2.3.1 --- Extraction and isolation --- p.20
Chapter 2.3.1.1 --- Preparation of soybean butanol extract --- p.20
Chapter 2.3.1.2 --- Preparation of isoflavones and their glycosides from soybean butanol extract --- p.20
Chapter 2.3.2 --- HPLC analysis --- p.21
Chapter 2.3.2.1 --- Sample preparation for the HPLC analysis --- p.21
Chapter 2.3.2.2 --- HPLC analysis --- p.22
Chapter 2.3.2.3 --- Quantification of the flavonoids and their glycosides --- p.24
Chapter 2.4 --- Results --- p.25
Chapter 2.4.1 --- Structural identification --- p.25
Chapter 2.4.1.1 --- Compound 1 --- p.25
Chapter 2.4.1.2 --- Compound 2 --- p.26
Chapter 2.4.1.3 --- Compound 3 --- p.26
Chapter 2.4.1.4 --- Compound 4 --- p.27
Chapter 2.4.1.5 --- Compound 5 --- p.27
Chapter 2.4.1.6 --- Compound 6 --- p.28
Chapter 2.4.1.7 --- Compound 7 --- p.28
Chapter 2.4.1.8 --- Compound 8 --- p.29
Chapter 2.4.2 --- Quantification of isoflavones in traditional Chinese foods --- p.29
Chapter 2.5 --- Discussion --- p.32
Chapter Chapter 3 --- Hypocholesterolemic Effects of Soymilkin Hamsters
Chapter 3.1 --- Introduction --- p.35
Chapter 3.1.1 --- Lipoproteins and their functions --- p.35
Chapter 3.1.2 --- Risk factors of cardiovascular disease --- p.36
Chapter 3.1.3 --- Hamster as an animal model of cholesterol metabolism --- p.38
Chapter 3.2 --- Objective --- p.39
Chapter 3.3 --- Materials and Methods --- p.40
Chapter 3.3.1 --- Preparation of soymilk --- p.40
Chapter 3.3.2 --- Animals --- p.40
Chapter 3.3.2.1 --- Experiment one - Hypocholesterolemic effect of soymilk in hamsters --- p.40
Chapter 3.3.2.1 --- Experiment two 一 The effect of fluid cross-over between soymilk and cow´ة s milk on serum cholesterol in hamsters --- p.41
Chapter 3.3.3 --- Serum lipid and lipoprotein determinations --- p.42
Chapter 3.3.4 --- Determination of cholesterol in organs --- p.42
Chapter 3.3.5 --- Statistics --- p.43
Chapter 3.4 --- Results --- p.46
Chapter 3.4.1 --- Experiment one-Hypocholesterolemic effect of soymilk in hamsters --- p.46
Chapter 3.4.1.1 --- Growth and food intake --- p.46
Chapter 3.4.1.2 --- "Effect of SM and CM on TG, TC and HDL-C" --- p.46
Chapter 3.4.1.3 --- Effect of SM and CM on non-HDL-C and ratio of non-HDL-C to HDL-C --- p.46
Chapter 3.4.1.4 --- Effect of SM and CM on concentration of hepatic cholesterol --- p.47
Chapter 3.4.1.5 --- "Effect of SM and CM on brain, heart and kidney cholesterol" --- p.47
Chapter 3.4.2 --- Experiment two - The effect of fluid cross-over between soymilk and cow´ةs milk on serum cholesterol in hamsters --- p.52
Chapter 3.4.2.1 --- Growth and food intake --- p.52
Chapter 3.4.2.2 --- Effect of fluid cross-over on serum TC --- p.52
Chapter 3.5 --- Discussion --- p.55
Chapter Chapter 4 --- Antioxidant Activities of Soybean Isoflavones and Their Glycosides
Chapter 4.1 --- Introduction --- p.58
Chapter 4.1.1 --- Role of low density lipoprotein oxidation in the development of atherosclerosis --- p.59
Chapter 4.1.2 --- LDL oxidation --- p.61
Chapter 4.1.3 --- Thiobarbituric acid reactive substances (TBARS) as an index of LDL oxidation --- p.62
Chapter 4.1.4 --- "The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power""" --- p.65
Chapter 4.1.5 --- "1,1-diphenyl-2-picrylhydrazyl (DPPH) as a measure of free radical scavenging capacity" --- p.65
Chapter 4.1.6 --- Antioxidant and LDL oxidation --- p.65
Chapter 4.2 --- Objective --- p.67
Chapter 4.3 --- Materials and Methods --- p.68
Chapter 4.3.1 --- Preparation of samples --- p.68
Chapter 4.3.2 --- Isolation of LDL from human serum --- p.68
Chapter 4.3.3 --- LDL oxidation --- p.69
Chapter 4.3.4 --- TBARS assay --- p.69
Chapter 4.3.5 --- FRAP assay --- p.70
Chapter 4.3.6 --- DPPH assay --- p.71
Chapter 4.3.7 --- Statistics --- p.72
Chapter 4.4 --- Results --- p.73
Chapter 4.4.1 --- Effects of seven individual soybean isoflavones and their glycosides on LDL oxidation --- p.73
Chapter 4.4.2 --- The antioxidant power of individual soybean isoflavones and their glycosides in the FRAP assay --- p.73
Chapter 4.4.3 --- Activity of individual soybean isoflavones and their glycosides as radical scavenging antioxidants --- p.74
Chapter 4.5 --- Discussion --- p.78
Chapter Chapter 5 --- Hypocholesterolemic Effects of Soybean Isoflavones in Ovariectomized Golden Syrian Hamsters
Chapter 5.1 --- Introduction --- p.83
Chapter 5.1.1 --- Coronary heart disease in women --- p.83
Chapter 5.1.2 --- Menopause as a risk factor in CHD --- p.84
Chapter 5.1.3 --- Dietary soy in treating postmenopausal hypercholesterolemia --- p.85
Chapter 5.2 --- Objective --- p.87
Chapter 5.3 --- Materials and Methods --- p.88
Chapter 5.3.1 --- Preparation of soymilk --- p.88
Chapter 5.3.2 --- Preparation of soybean extract --- p.88
Chapter 5.3.3 --- Animals --- p.89
Chapter 5.3.4 --- Serum lipid determinations --- p.90
Chapter 5.3.5 --- Determination of tissue cholesterol content --- p.90
Chapter 5.3.6 --- Extraction of neutral and acidic sterols from fecal samples --- p.90
Chapter 5.3.6.1 --- Determination of neutral sterols --- p.91
Chapter 5.3.6.2 --- Determination of acidic sterols --- p.92
Chapter 5.3.6.3 --- GLC analysis of neutral and acidic sterols --- p.92
Chapter 5.3.7 --- Statistics --- p.93
Chapter 5.4 --- Results --- p.96
Chapter 5.4.1 --- Growth and food intake --- p.96
Chapter 5.4.2 --- Effect of ovariectomy on serum TC --- p.96
Chapter 5.4.3 --- "Effect of soymilk and soybean extract on serum TC,TG and HDL-C" --- p.96
Chapter 5.4.4 --- Effect of soymilk and soybean extract on non-HDL-C and ratio of non- HDL-C to HDL-C --- p.97
Chapter 5.4.5 --- Effect of soymilk and soybean extract on concentration of hepatic cholesterol --- p.97
Chapter 5.4.6 --- Effect of soymilk and soybean extract on heart and kidney cholesterol --- p.97
Chapter 5.4.7 --- Effect of soymilk and soybean extract on fecal neutral and acidic sterols --- p.103
Chapter 5.5 --- Discussion --- p.106
Chapter Chapter 6 --- Conclusion --- p.110
References --- p.113

Cheung Lai Ming.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.
Includes bibliographical references (leaves 153-161).
Abstracts in English and Chinese.
THESIS COMMITTEE --- p.i
ACKNOWLEDGEMENTS --- p.ii
ABSTRACT --- p.iii
ABSTRACT (Chinese version) --- p.v
CONTENTS --- p.vi
LIST OF TABLES --- p.xi
LIST OF FIGURES --- p.xiii
LIST OF ABBREVIATIONS --- p.xv
Chapter CHAPTER ONE: --- INTRODUCTION --- p.1
Chapter 1.1 --- Free radical --- p.2
Chapter 1.1.1 --- Definition --- p.2
Chapter 1.1.2 --- Reaction mechanism --- p.3
Chapter 1.1.3 --- Sources of oxygen reactive species --- p.4
Chapter 1.1.3.1 --- Enzymes --- p.4
Chapter 1.1.3.2 --- The auto-oxidation of small molecules --- p.4
Chapter 1.1.3.3 --- Haem proteins --- p.5
Chapter 1.1.3.4 --- Endoplasmic reticulum sources --- p.5
Chapter 1.1.3.5 --- Mitochondrial sources --- p.5
Chapter 1.1.3.6 --- Nucleus --- p.6
Chapter 1.1.4 --- Lipid peroxidation --- p.6
Chapter 1.1.4.1 --- Initiation of lipid peroxidation --- p.7
Chapter 1.1.4.2 --- Propagation of lipid peroxidation --- p.8
Chapter 1.1.4.3 --- Products of lipid peroxidation --- p.9
Chapter 1.1.5 --- Human diseases associated with free radicals --- p.10
Chapter 1.2 --- Antioxidants --- p.12
Chapter 1.2.1 --- Definition --- p.12
Chapter 1.2.2 --- Defence against free radical damage --- p.13
Chapter 1.2.2.1 --- Catalytic free radical removal --- p.13
Chapter 1.2.2.2 --- Free radical scavenging --- p.14
Chapter 1.2.2.3 --- Removal of catalytic iron and copper ions --- p.14
Chapter 1.2.3 --- Synthetic vs. natural antioxidant --- p.15
Chapter 1.2.3.1 --- Synthetic antioxidants --- p.15
Chapter 1.2.3.2 --- Natural antioxidants --- p.16
Chapter 1.3 --- Measurement of antioxidant activity --- p.17
Chapter 1.3.1 --- Loss of substrate --- p.17
Chapter 1.3.1.1 --- Beta-carotene bleaching method --- p.17
Chapter 1.3.2 --- Measurement of free radical scavenging --- p.17
Chapter 1.3.2.1 --- "Scavenging of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH´Ø)" --- p.17
Chapter 1.3.2.2 --- Superoxide scavenging --- p.18
Chapter 1.3.2.3 --- Hydrogen peroxide scavenging --- p.18
Chapter 1.3.2.4 --- Hydroxyl radical scavenging --- p.19
Chapter 1.3.2.5 --- Peroxyl radical --- p.19
Chapter 1.3.3 --- Measurement of end product --- p.21
Chapter 1.3.3.1 --- Diene conjugation --- p.21
Chapter 1.3.3.2 --- Light emission --- p.21
Chapter 1.3.3.3 --- The thiobarbituric acid (TBA) test --- p.22
Chapter 1.3.4 --- Low-density lipoprotein oxidation --- p.22
Chapter 1.4 --- Phenolic antioxidant --- p.24
Chapter 1.4.1 --- Chemistry --- p.24
Chapter 1.4.2 --- Mechanism of action of phenolic antioxidants --- p.25
Chapter 1.4.3 --- Isolation and characterization --- p.25
Chapter 1.4.3.1 --- Extraction --- p.25
Chapter 1.4.3.2 --- Analysis of phenolic compounds --- p.27
Chapter 1.4.3.2.1 --- Colorimetric method --- p.27
Chapter 1.4.3.2.2 --- Enzymatic method --- p.28
Chapter 1.4.3.2.3 --- Paper chromatography --- p.28
Chapter 1.4.3.2.4 --- Thin-layer chromatography --- p.29
Chapter 1.4.3.2.5 --- UV-Vis absorption spectroscopy --- p.29
Chapter 1.4.3.2.6 --- High-performance liquid chromatography --- p.30
Chapter 1.4.4 --- Natural sources of phenolic antioxidants --- p.31
Chapter 1.4.4.1 --- Olive oil --- p.31
Chapter 1.4.4.2 --- Berry --- p.32
Chapter 1.4.4.3 --- Cherry --- p.32
Chapter 1.4.4.4 --- Red wine --- p.32
Chapter 1.4.4.5 --- Herb --- p.33
Chapter 1.4.4.6 --- Vegetables --- p.33
Chapter 1.5 --- Mushroom Sample --- p.34
Chapter 1.5.1 --- Pleurotus tuber-regium --- p.34
Chapter 1.5.2 --- Lentinus edodes --- p.34
Chapter 1.5.3 --- Volvariella volvacea --- p.35
Chapter 1.5.4 --- Antioxidants in fungi or mushroom --- p.37
Chapter 1.5.5 --- Phenolic compounds in mushrooms --- p.39
Chapter 1.6 --- Objectives --- p.42
Chapter CHAPTER TWO: --- MATERIALS AND METHODS --- p.43
Chapter 2.1 --- Sample Collection --- p.43
Chapter 2.2 --- Sample Preparation --- p.43
Chapter 2.3 --- Moisture Content --- p.43
Chapter 2.4 --- Solvent Extraction --- p.44
Chapter 2.4.1 --- Scheme I (Aqueous extraction only) --- p.44
Chapter 2.4.2 --- Scheme II (Methanol and water extraction) --- p.45
Chapter 2.4.3 --- Scheme III (Differential solvent extraction) --- p.46
Chapter 2.4.4 --- Scheme IV (Scaled-up extraction) --- p.47
Chapter 2.5 --- Antioxidant activity assays --- p.50
Chapter 2.5.1 --- Beta-carotene bleaching method --- p.50
Chapter 2.5.2 --- "Scavenging activity on 1,1 -diphenyl-2-picrylhydrazyl radicals" --- p.51
Chapter 2.5.3 --- Assay for erythrocyte hemolysis --- p.51
Chapter 2.5.4 --- Assay of lipid peroxidation using rat brain --- p.52
Chapter 2.5.5 --- LDL oxidation (TBARS) --- p.53
Chapter 2.5.5.1 --- LDL Isolation --- p.53
Chapter 2.5.5.2 --- Calculation of density --- p.54
Chapter 2.5.5.3 --- Lowry Method for Protein Determination --- p.55
Chapter 2.5.5.4 --- Reagents for TBARS assay --- p.55
Chapter 2.5.5.5 --- TBARS formation --- p.56
Chapter 2.6 --- Determination of total polyphenolic compounds --- p.56
Chapter 2.7 --- Fractionation --- p.57
Chapter 2.7.1 --- Fractionation of the methanol crude extracts obtained under reflux by solvent --- p.57
Chapter 2.7.2 --- Fractionation of boiling water crude extracts by ultrafiltration --- p.57
Chapter 2.8 --- Crude Protein Content (Kjeldahl method) --- p.58
Chapter 2.9 --- Total carbohydrate content --- p.59
Chapter 2.10 --- Thin-layer chromatography --- p.59
Chapter 2.11 --- High performance liquid chromatography --- p.60
Chapter 2.11.1 --- Analysis of methanol fractions --- p.60
Chapter 2.11.2 --- Analysis of water fractions --- p.61
Chapter 2.12 --- Liquid chromatography-Mass spectrometry --- p.61
Chapter 2.12.1 --- Liquid chromatography --- p.61
Chapter 2.12.2 --- Mass spectrometric analysis --- p.62
Chapter 2.13 --- Data analysis --- p.62
Chapter CHAPTER THREE: --- RESULTS AND DISCUSSION --- p.63
Chapter 3.1 --- Mushroom sample --- p.63
Chapter 3.2 --- Extraction scheme I --- p.65
Chapter 3.2.1 --- Antioxidant activity --- p.65
Chapter 3.2.1.1 --- Effect of extraction temperature --- p.65
Chapter 3.2.1.2 --- Effect of concentration of extracts --- p.66
Chapter 3.3 --- Extraction scheme II --- p.69
Chapter 3.3.1 --- Antioxidant activity --- p.69
Chapter 3.3.1.1 --- Effect of extraction temperature --- p.69
Chapter 3.3.1.2 --- Effect of concentration of extracts --- p.72
Chapter 3.3.1.3 --- Effect of solvent --- p.72
Chapter 3.4 --- Extraction scheme III --- p.75
Chapter 3.4.1 --- Extraction yield --- p.75
Chapter 3.4.2 --- Total phenolic content --- p.76
Chapter 3.4.3 --- Antioxidant activity --- p.80
Chapter 3.4.3.1 --- Beta-carotene bleaching method --- p.80
Chapter 3.4.3.1.1 --- Effect of extract concentration --- p.80
Chapter 3.4.3.1.2 --- Relation between total phenolic content and antioxidant activity --- p.82
Chapter 3.4.3.2 --- "Scavenging activity of 1,1 -diphenyl-2-picrylhydrazyl (DPPH) radical" --- p.85
Chapter 3.4.3.3 --- Assay for erythrocyte hemolysis --- p.88
Chapter 3.5 --- Extraction scheme IV --- p.91
Chapter 3.5.1 --- Yield and Fractionation --- p.91
Chapter 3.5.2 --- Chemical characterization of fractions --- p.93
Chapter 3.5.2.1 --- Protein content --- p.93
Chapter 3.5.2.2 --- Total carbohydrate content --- p.93
Chapter 3.5.2.3 --- Total phenolic content --- p.94
Chapter 3.5.3 --- Antioxidant activity --- p.99
Chapter 3.5.3.1 --- Assay for lipid peroxidation of rat brain --- p.99
Chapter 3.5.3.2 --- LDL oxidation --- p.118
Chapter 3.5.4 --- Identification of antioxidant by chromatographic methods --- p.126
Chapter 3.5.4.1 --- Thin-layer chromatography --- p.126
Chapter 3.5.4.2 --- High-performance liquid chromatography --- p.132
Chapter 3.5.4.3 --- Liquid chromatography-Mass spectrometry --- p.142
Chapter CHAPTER FOUR: --- CONCLUSION --- p.148
REFERENCES --- p.153
RELATED PUBLICATION --- p.161

Lo Kit Man.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2003.
Includes bibliographical references (leaves 193-208).
Abstracts in English and Chinese.
THESIS COMMITTEE --- p.i
ACKNOWLEDGEMENTS --- p.ii
ABSTRACT --- p.iii
ABSTRACT (Chinese version) --- p.v
CONTENT --- p.vii
LIST OF TABLES --- p.xiii
LIST OF FIGURES --- p.xviii
LIST OF ABBREVIATIONS --- p.xx
Chapter CHAPTER 1: --- INTRODUCTION --- p.1
Chapter 1.1 --- An introduction of natural antioxidants --- p.1
Chapter 1.1.1 --- Definition of antioxidants --- p.1
Chapter 1.1.2 --- Application of natural antioxidants in foods --- p.3
Chapter 1.1.2.1 --- Oxidation of foods --- p.3
Chapter 1.1.2.1.1 --- Autoxidation of food --- p.3
Chapter 1.1.2.1.2 --- Photo-oxidation of food --- p.4
Chapter 1.1.3 --- Free radicals and antioxidants --- p.6
Chapter 1.1.3.1 --- Free radicals and reactive oxygen species --- p.6
Chapter 1.1.3.1.1 --- Superoxide anion radical --- p.8
Chapter 1.1.3.1.2 --- Hydrogen peroxide --- p.9
Chapter 1.1.3.1.3 --- Hydroxyl radical --- p.10
Chapter 1.1.3.1.4 --- Peroxyl radical --- p.12
Chapter 1.1.3.1.5 --- Lipid peroxidation of cell membranes --- p.13
Chapter 1.1.3.1.6 --- Oxidation of LDL and atherosclerosis --- p.16
Chapter 1.1.4 --- Natural antioxidants and their mechanisms --- p.18
Chapter 1.1.4.1 --- Carotenoids --- p.18
Chapter 1.1.4.2 --- Phenolic compounds --- p.20
Chapter 1.1.4.2.1 --- Flavonoids --- p.21
Chapter 1.1.4.2.2 --- Phenolic acids --- p.22
Chapter 1.1.4.3 --- Sterols --- p.24
Chapter 1.1.4.4 --- Vitamins --- p.25
Chapter 1.2 --- Antioxidant in mushrooms --- p.27
Chapter 1.2.1 --- Antioxidant properties of mushrooms --- p.27
Chapter 1.2.2 --- Characterization of mushroom phenolic antioxidants --- p.30
Chapter 1.2.3 --- Biosynthesis of phenolic compounds in mushrooms or fungi --- p.33
Chapter 1.3 --- Assays for evaluation of antioxidants --- p.35
Chapter 1.3.1 --- Beta-carotene bleaching method --- p.35
Chapter 1.3.2 --- Scavenging activity of DPPH radical --- p.36
Chapter 1.3.3 --- Erythrocyte hemolysis --- p.36
Chapter 1.3.4 --- Scavenging activity of ABTS ´Ø + radical cation --- p.37
Chapter 1.3.5 --- Scavenging activity of hydroxyl radical --- p.37
Chapter 1.3.6 --- Assay for lipid peroxidation of rat brain homogenate --- p.38
Chapter 1.3.7 --- Inhibition of low-density lipoproteins (LDLs) oxidation --- p.39
Chapter 1.4 --- Analysis of phenolic antioxidants --- p.41
Chapter 1.4.1 --- Extraction of phenolic compounds --- p.41
Chapter 1.4.2 --- Determination of total phenolic content --- p.43
Chapter 1.4.3 --- Chromatographic fractionation of phenolic compounds --- p.44
Chapter 1.4.4 --- Characterization of phenolic compounds --- p.45
Chapter 1.4.4.1 --- Thin-layer chromatography (TLC) --- p.45
Chapter 1.4.4.2 --- High performance liquid chromatography (HPLC) --- p.46
Chapter 1.4.4.3 --- Liquid chromatography-Mass spectrometry (LC-MS) --- p.47
Chapter 1.5 --- Objectives --- p.49
Chapter CHAPTER 2: --- MATERIALS AND METHODS --- p.50
Chapter 2.1 --- Sample preparation --- p.50
Chapter 2.2 --- Sample extraction. --- p.51
Chapter 2.2.1 --- Small-scale methanol and water extraction --- p.51
Chapter 2.2.2 --- Large-scale methanol and water extraction and fractionation --- p.54
Chapter 2.2.2.1 --- Large-scale methanol and water extraction --- p.54
Chapter 2.2.2.2 --- Fractionation of crude extracts --- p.55
Chapter 2.2.2.2.1 --- Fractionation of methanol crude extract --- p.55
Chapter 2.2.2.2.2 --- Fractionation of water crude extract --- p.55
Chapter 2.3 --- Fractionation by column chromatography --- p.58
Chapter 2.4 --- Assays for measuring antioxidant activity --- p.60
Chapter 2.4.1 --- Beta-carotene bleaching method --- p.60
Chapter 2.4.2 --- Scavenging activity of DPPH radical --- p.62
Chapter 2.4.3 --- Erythrocyte hemolysis --- p.63
Chapter 2.4.4 --- Scavenging activity of ABTS ´Ø + radical cation --- p.64
Chapter 2.4.5 --- Scavenging activity of hydroxyl radical --- p.65
Chapter 2.4.6 --- Assay for lipid peroxidation of rat brain homogenate --- p.66
Chapter 2.4.7 --- Inhibition of human low-density lipoproteins (LDLs) oxidation --- p.67
Chapter 2.4.7.1 --- Isolation of human LDLs --- p.67
Chapter 2.4.7.2 --- Calculation of density --- p.68
Chapter 2.4.7.3 --- Lowry's method for determination of protein content --- p.69
Chapter 2.4.7.4 --- Preparation of reagents --- p.69
Chapter 2.4.7.5 --- Determination of thiobarbituric acid reactive substance (TBARS) --- p.70
Chapter 2.5 --- Total phenolic content --- p.70
Chapter 2.6 --- Total carbohydrate content --- p.71
Chapter 2.7 --- Determination of protein content- the Biuret method --- p.71
Chapter 2.8 --- Thin-layer chromatography (TLC) --- p.72
Chapter 2.9 --- High performance liquid chromatography (HPLC) --- p.73
Chapter 2.9.1 --- Analysis of subfractions of methanol crude extract --- p.73
Chapter 2.9.2 --- Analysis of fractionated subfractions and subfractions of Pevf and Aa mushrooms --- p.74
Chapter 2.10 --- Liquid chromatography- Mass spectrometry (LC-MS) --- p.74
Chapter 2.10.1 --- Liquid chromatography --- p.74
Chapter 2.10.2 --- Mass spectrometry --- p.75
Chapter 2.11 --- Data analysis --- p.75
Chapter CHAPTER 3: --- RESULTS AND DISCUSSION --- p.77
Chapter 3.1 --- Small-scale extraction scheme --- p.77
Chapter 3.1.1 --- Extraction yield --- p.77
Chapter 3.1.2 --- Assays for measuring antioxidant activity --- p.80
Chapter 3.1.2.1 --- Beta-carotene bleaching method --- p.80
Chapter 3.1.2.2 --- Scavenging activity of DPPH radical --- p.91
Chapter 3.1.2.3 --- Erythrocyte hemolysis --- p.98
Chapter 3.1.2.4 --- Summary for small-scale extraction --- p.105
Chapter 3.2 --- Large-scale extraction and fractionation scheme --- p.107
Chapter 3.2.1 --- Extraction yield for crude extracts and subfractions --- p.107
Chapter 3.2.2 --- Antioxidant activity of subfractions and crude extracts of Aa and Pevf mushrooms --- p.111
Chapter 3.2.2.1 --- Scavenging activity of ABTS ´Ø + radical cation --- p.111
Chapter 3.2.2.2 --- Scavenging activity of hydroxyl radical --- p.114
Chapter 3.2.2.3 --- Assay for lipid peroxidation of rat brain homogenate --- p.125
Chapter 3.2.2.4 --- Summary for large-scale extraction --- p.135
Chapter 3.2.3 --- Chemical characterization of the crude extracts and their sub fractions of Aa and Pevf mushrooms --- p.13 8
Chapter 3.2.3.1 --- Total phenolic content --- p.139
Chapter 3.2.3.1.1 --- Total phenolic content of crude extract and their sub fractions of Aa and Pevf mushrooms --- p.139
Chapter 3.2.3.1.2 --- Correlation between total phenolic content and antioxidant activity --- p.140
Chapter 3.2.3.2 --- Total carbohydrate content --- p.144
Chapter 3.2.3.2.1 --- Total carbohydrate content of water crude extract and their sub fractions of Aa and Pevf mushrooms --- p.144
Chapter 3.2.3.2.2 --- Correlation between total carbohydrate content and antioxidant activity --- p.144
Chapter 3.2.3.3 --- Determination of protein content- the Biuret method --- p.146
Chapter 3.2.3.3.1 --- Protein content of water crude extract and their sub fractions of Aa and Pevf mushrooms --- p.146
Chapter 3.2.3.3.2 --- Correlation between protein content and antioxidant activity --- p.147
Chapter 3.2.3.4 --- Summary of correlation between chemical components and antioxidant activity --- p.148
Chapter 3.3 --- Column fractionation of ethyl acetate and butanol subfractions of Aa mushroom --- p.151
Chapter 3.3.1 --- Rf value in TLC and yield of fractionated subfractions --- p.151
Chapter 3.3.2 --- Total phenolic content of fractionated subfractions --- p.153
Chapter 3.3.3 --- Antioxidant activity of fractionated subfractions --- p.155
Chapter 3.3.3.1 --- Scavenging activity of ABTS ´Ø + radical cation --- p.155
Chapter 3.3.3.2 --- Scavenging activity of DPPH radical --- p.158
Chapter 3.3.3.3 --- Inhibition of human low-density lipoprotein (LDL) oxidation --- p.163
Chapter 3.4 --- Chromatographic characterization of the subfractions of methanol crude extract of Aa and Pevf mushrooms --- p.167
Chapter 3.4.1 --- Thin-layer chromatography (TLC) --- p.167
Chapter 3.4.2 --- High performance liquid chromatography (HPLC) --- p.172
Chapter 3.5 --- Chromatographic and spectrometric characterization of the fractionated subfractions of the ethyl acetate and butanol subfractions of Aa --- p.177
Chapter 3.5.1 --- High performance liquid chromatography (HPLC) --- p.177
Chapter 3.5.2 --- Liquid chromatography- Mass spectrometry (LC-MS) --- p.186
Chapter CHAPTER 4: --- CONCLUSION --- p.189
REFERENCES --- p.193
RELATED PUBLICATION --- p.209

The present study determined whether dietary supplementation with reduced glutathione (GSH), glutathione ester (GSHE) or N-acetylcysteine (NAC) increased the mitochondrial level of GSH, the major antioxidant inside cells, in rat kidney cortex and medulla. Nine month-old female Lewis rats were given daily intraperitoneal injections of isotonic saline (n=6), or saline containing GSH (250mg or 0.81mmol/Kg of body wt; n=7), GSHE (12mg or 0.03mmol/Kg; n=8), or NAC (200mg or 1.22mmol/Kg; n=8) for four weeks. At the end of the injection period, the rats were anesthetized and the kidneys removed. The kidneys were separated into cortical and medullary sections, weighed, and homogenized. The sections were separated into cytosolic and mitochondrial fractions by differential centrifugation. The GSH levels were determined by a colorimetric assay. Cortical and medullary mitochondrial GSH levels were significantly increased by all three supplements. Cytosolic GSH levels were also significantly increased in both cortical and medullary sections. Thus, dietary supplementation can significantly increase the mitochondrial pool of GSH in the rat kidney.
Department of Physiology and Health Science

During extreme exercise, athletes experience increased inflammation that is similar to the acute phase response. Endurance athletes, distance runners in particular, are also more susceptible to compromised iron stores. This study evaluated inflammation, immune function and iron status in athletes completing a 50K ultramarathon. Twenty-two well-trained distance runners, 11 males and 11 females, were randomized in a double blind manner into--1) those who consumed 300 mg vitamin E and 1000 mg vitamin C (500 mg twice daily) or 2) placebos--for six weeks before and one week following a 50K ultramarathon race. Blood samples were obtained on 13 separate occasions throughout the study: before supplementation, during supplementation, the day before the race, pre-race, mid-race, immediately post-race, 2 hours following the race, and daily for six days following the race. Plasma levels of ascorbic acid and ��-tocopherol were measured by HPLC with electrochemical detection. Inflammatory cytokines, interleukin-6 (IL-6), tumor necrosis factor-�� (TNF-��), and interleukin-1�� (IL-1��) were measured using standard clinical assays. Each subject recorded immune function in an activity log and incidence of illness was tabulated as number of days ill. Ferritin was measured by enzyme immunoassay. Hemoglobin, hematocrit, and total-iron binding capacity (TIBC) and serum total iron were analyzed by standard procedures. Plasma concentrations of ascorbic acid and ��-tocopherol increased significantly in supplemented subjects (p<0.0001). Although the ultramarathon race elicited an inflammatory response, antioxidant supplementation did not alter the responses of IL-6 and TNF-��, which both increased from pre-race to mid-race, post- and post-2 h (Scheffe post-hoc analysis, p<0.0001) and returned to pre-race concentrations by 1 day after the race. Male supplemented subjects had lower IL-1�� concentrations compared to females consuming the supplement or to males consuming the placebo (ANCOVA, gender/time/treatment interaction; p<0.01) at mid-race (p<0.05 females, p<0.005 males), post 1 and 2 days (all p<0.002). Males had significantly higher ferritin levels than the female subjects (ANOVA, p<0.0001); supplementation resulted in lower ferritin concentrations at post-5 days (p<0.02, ANCOVA treatment time interaction, p<0.005). Supplementation did not reduce the days illness among those consuming antioxidants compared to those consuming the placebos. Ferritin not only increases during inflammation, it also is a measure of iron stores. Females had significantly lower levels of iron than the male subjects for each of the iron parameters measured (hemoglobin and hematocrit both p<0.0001, ferritin p<0.001, TIBC p<0.02) excluding serum total iron. The ferritin concentrations measured in the women were indicative of depleted iron stores (<12 ��g/l), and antioxidant supplementation increased hematocrit levels in the female subjects (p<0.05). This investigation indicates that female distance runners need to be aware of an increased susceptibility to iron depletion compared to their male counterparts. Antioxidant supplementation improved hematocrit levels (p<0.05) among female runners and may improve iron status among females with depleted stores. Although other investigations have suggested that antioxidant vitamins decrease exercise induced inflammation, no profound benefit of supplementation was found in this investigation though a response similar to the acute phase response was elicited by the ultramarathon race. Improvements in IL-i and ferritin in response to antioxidant supplementation may indicate that the supplementation was beneficial, but more research is needed to draw definitive conclusions.
Graduation date: 2003

Lau Wing-Sze.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (leaves 131-149).
Abstracts in English and Chinese.
Acknowledgements --- p.i
Abstract --- p.ii
Abstract (Chinese version) --- p.iv
Table of Contents --- p.vi
List of Tables --- p.x
List of Figures --- p.xii
List of Abbreviations --- p.xiv
Chapter Chapter One: --- An overview of Vigna sinensis seeds
Chapter 1.1 --- Introduction --- p.1
Chapter 1.2 --- Food and functional food --- p.2
Chapter 1.3 --- Edible legumes as an important food --- p.4
Chapter 1.4 --- Nutritional an extra-nutritional values of V. sinensis seeds --- p.5
Chapter Chapter Two: --- Purification of phenolic antioxidants from V. sinensis seeds
Chapter 2.1 --- Introduction --- p.11
Chapter 2.1.1 --- Reactive oxygen species and antioxidants --- p.12
Chapter 2.1.2 --- Phenolic flavonoids --- p.15
Chapter 2.2 --- Materials and Methods
Chapter 2.2.1 --- Chemicals and reagents --- p.24
Chapter 2.2.2 --- Plant material --- p.25
Chapter 2.2.3 --- Optimization and extraction of V. sinensis seeds constituents --- p.25
Chapter 2.2.4 --- Chromatographic separation of phenolic components --- p.26
Chapter 2.2.5 --- Determination of phenolic contents --- p.27
Chapter 2.2.6 --- Determination of free radical scavenging ability using trolox equivalent antioxidant capacity (TEAC) assay --- p.28
Chapter 2.2.7 --- Statistical analysis --- p.29
Chapter 2.3 --- Results and Discussion
Chapter 2.3.1 --- Optimization on the extraction of V. sinensis seeds --- p.30
Chapter 2.3.2 --- Extraction and fractionation of V. sinensis seeds constituents --- p.31
Chapter 2.3.3 --- Yield of various V sinensis seed fractions --- p.31
Chapter 2.3.4 --- Phenolic contents in various V. sinensis seed fractions --- p.32
Chapter 2.3.5 --- Free radical scavenging abilities of various V sinensis seed fractions --- p.33
Chapter Chapter Three: --- Effect of V. sinensis seed extract on high fat and cholesterol - feeding mice
Chapter 3.1 --- Introduction --- p.41
Chapter 3.1.1 --- Cholesterol in bloodstream circulation --- p.42
Chapter 3.1.2 --- "Relationship between LDL oxidation, atherosclerosis and coronary heart disease" --- p.43
Chapter 3.1.3 --- Diet supplements with beneficial effects on preventing coronary heart disease --- p.44
Chapter 3.2 --- Materials and Methods --- p.47
Chapter 3.2.1 --- Chemicals and reagents --- p.47
Chapter 3.2.2 --- Preparation of diets --- p.48
Chapter 3.2.3 --- Animals --- p.48
Chapter 3.2.4 --- Feeding experiments --- p.49
Chapter 3.2.5 --- Post-feeding analysis --- p.50
Chapter 3.2.5.1 --- Caecal content and health indices --- p.50
Chapter 3.2.5.2 --- Serum triglycerides --- p.51
Chapter 3.2.5.3 --- Serum total cholesterol --- p.52
Chapter 3.2.5.4 --- High-density lipoprotein (HDL) cholesterol --- p.53
Chapter 3.2.5.5 --- Low-density lipoprotein (LDL) cholesterol --- p.54
Chapter 3.2.5.6 --- Hepatic lipid and cholesterol --- p.55
Chapter 3.2.6 --- Statistical analysis --- p.55
Chapter 3.3 --- Results and Discussion --- p.56
Chapter 3.3.1 --- Food intakes and body weights of animals --- p.56
Chapter 3.3.2 --- Caecal contents and health indices --- p.56
Chapter 3.3.3 --- Effects of V sinensis seed extract on serum and hepatic levels of triglycerides and cholesterol --- p.57
Chapter Chapter Four: --- Antiproliferative activities of V. sinensis seed extracts
Chapter 4.1 --- Introduction --- p.66
Chapter 4.1.1 --- Cancer and antioxidant --- p.67
Chapter 4.1.2 --- Dietary cancer prevention agents --- p.68
Chapter 4.2 --- Materials and Methods --- p.71
Chapter 4.2.1 --- Chemicals and reagents --- p.71
Chapter 4.2.2 --- Cell lines --- p.71
Chapter 4.2.3 --- Maintenance of cell lines --- p.72
Chapter 4.2.4 --- Antiproliferation assays --- p.73
Chapter 4.2.4.1 --- MTT assay --- p.73
Chapter 4.2.4.2 --- BrdU assay --- p.73
Chapter 4.2.5 --- Cytotoxic activity determined by lactate dehydrogenase (LDH) assay --- p.77
Chapter 4.2.6 --- Time-course assay --- p.79
Chapter 4.2.7 --- Determination of IC50 --- p.79
Chapter 4.2.8 --- Statistical analysis --- p.79
Chapter 4.3 --- Results and Discussion --- p.80
Chapter 4.3.1 --- Antiproliferative activities of V. sinensis seed extracts on HepG2 cells --- p.80
Chapter 4.3.2 --- Cytotoxic activities of V. sinensis seed extracts on HepG2 cells --- p.82
Chapter 4.3.3 --- Antiproliferative activities of phenolic fraction on MCF-7cells --- p.83
Chapter 4.3.4 --- Cytotoxic activity of phenolic fraction on MCF-7 cells --- p.84
Chapter 4.3.5 --- Time-course study of antiproliferative activities of phenolic fraction on cancer cells --- p.85
Chapter 4.3.6 --- Effect of phenolic fraction on normal cells --- p.86
Chapter Chapter Five: --- Antioxidant and antiproliferative activities of selected content flavonoids from V. sinensis seeds
Chapter 5.1 --- Introduction --- p.93
Chapter 5.1.1 --- Cell cycle progression and regulation --- p.94
Chapter 5.1.2 --- Bioavailability of plant flavonoids --- p.96
Chapter 5.1.3 --- Characterization of flavonoids in V. sinensis seed --- p.98
Chapter 5.2 --- Materials and Methods --- p.102
Chapter 5.2.1 --- Chemicals and reagents --- p.102
Chapter 5.2.2 --- Determination of free radical scavenging ability of identified flavonoids from V sinensis seeds using trolox equivalent antioxidant capacity (TEAC) assay --- p.103
Chapter 5.2.3 --- Antiproliferation assays --- p.104
Chapter 5.2.4 --- Cytotoxicity assay --- p.104
Chapter 5.2.5 --- Time-course assay --- p.104
Chapter 5.2.6 --- Determination of cell cycle distribution by flow cytometry --- p.105
Chapter 5.2.7 --- Statistical analysis --- p.106
Chapter 5.3 --- Results and Discussion --- p.107
Chapter 5.3.1 --- Free radical scavenging abilities of identified flavonoids from V sinensis seeds --- p.107
Chapter 5.3.2 --- Antiproliferative activities of selected flavonoids on cancer cells --- p.109
Chapter 5.3.3 --- Cytotoxic activities of selected flavonoids on cancer cells --- p.111
Chapter 5.3.4 --- Time -course study of antiproliferative activities on cancer cells --- p.112
Chapter 5.3.5 --- Cytotoxic activities of selected flavonoids on normal cells --- p.114
Chapter 5.3.6 --- Determination of the effects of cyanidin on cancer cells by analyzing cell cycle pattern --- p.115
Chapter Chapter Six: --- Conclusion --- p.128
References --- p.131

Ho, Ka Yan.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2007.
Includes bibliographical references (leaves 111-124).
Abstracts in English and Chinese.
Thesis committee --- p.i
Acknowledgements --- p.ii
Abstract --- p.iii
摘要 --- p.v
List of Tables --- p.vii
List of Figures --- p.viii
Abbreviations --- p.x
Content --- p.xiii
Chapter Chapter 1: --- Introduction --- p.1
Chapter 1.1 --- Reactive oxygen species (ROS) --- p.1
Chapter 1.1.1 --- Definition and examples --- p.1
Chapter 1.1.2 --- Generation of ROS in biological systems --- p.2
Chapter 1.1.3 --- Features of specif ic ROS --- p.3
Chapter 1.1.3.1 --- Superoxide anion --- p.3
Chapter 1.1.3.2 --- Peroxyl radical --- p.4
Chapter 1.1.3.3 --- Hydrogen peroxide --- p.4
Chapter 1.1.3.4 --- Hydroxyl radical --- p.5
Chapter 1.1.4 --- Damaging effects of ROS on biomolecules --- p.5
Chapter 1.1.4.1 --- Lipid peroxidation --- p.6
Chapter 1.1.4.2 --- DNA damage --- p.8
Chapter 1.1.4.3 --- Protein oxidation --- p.9
Chapter 1.2 --- Antioxidants --- p.11
Chapter 1.2.1 --- Introduction --- p.11
Chapter 1.2.2 --- Mode of action --- p.11
Chapter 1.2.3 --- Endogenous Antioxidants --- p.12
Chapter 1.2.3.1 --- Antioxidant enzymes --- p.12
Chapter 1.2.3.2 --- Antioxidant compounds --- p.15
Chapter 1.2.4 --- Exogenous antioxidants --- p.16
Chapter 1.3 --- Oxidative stress --- p.17
Chapter 1.3.1 --- Balance between ROS and antioxidants --- p.17
Chapter 1.3.2 --- Diseases associated with oxidative stress --- p.18
Chapter 1.4 --- Previous studies on edible mushroom antioxidants --- p.19
Chapter 1.4.1 --- Previous studies on Agrocybe aegerita --- p.20
Chapter 1.5 --- Cell culture models for antioxidant research --- p.21
Chapter 1.6 --- Objectives --- p.23
Chapter Chapter 2 --- Materials and Methods --- p.24
Chapter 2.1 --- Materials --- p.24
Chapter 2.1.1 --- Mushroom fruiting bodies --- p.24
Chapter 2.1.2 --- Cell lines and their subcultures --- p.24
Chapter 2.2 --- Principle of Methods and Procedures --- p.26
Chapter 2.2.1 --- Sample preparation and extraction --- p.26
Chapter 2.2.2 --- Chemical assays for in vitro antioxidative properties of mushroom extracts --- p.28
Chapter 2.2.2.1 --- ABTS + scavenging activity --- p.28
Chapter 2.2.2.2 --- Hydroxyl radical scavenging activity --- p.30
Chapter 2.2.2.3 --- Hydrogen peroxide scavenging activity --- p.32
Chapter 2.2.3 --- Total phenolic content --- p.34
Chapter 2.2.4 --- Cytotoxicity of hydrogen peroxide --- p.36
Chapter 2.2.5 --- Cytoprotectivity of mushroom extracts --- p.36
Chapter 2.2.6 --- "Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay" --- p.37
Chapter 2.2.7 --- Lactate dehydrogenase (LDH) assay --- p.39
Chapter 2.2.8 --- Total cellular protein loss --- p.40
Chapter 2.2.9 --- Comet assay (Single cell gel electrophresis assay) --- p.41
Chapter 2.2.10 --- Thiobarbituric Acid Reactive Substances (TBARS) assay ..… --- p.44
Chapter 2.2.11 --- Preparation of cell lysate for evaluating cellular antioxidant defense system --- p.45
Chapter 2.2.12 --- Total Glutathione level --- p.46
Chapter 2.2.13 --- Enzyme activity --- p.49
Chapter 2.2.13.1 --- Catalase (CAT) --- p.49
Chapter 2.2.13.2 --- Glutathione peroxidases (GPx) --- p.51
Chapter 2.2.13.3 --- Glutathione Reductase (GR) --- p.53
Chapter 2.2.13.4 --- Superoxide dismutase (SOD) --- p.54
Chapter 2.2.14 --- Determination of protein --- p.56
Chapter 2.2.15 --- Statistical analysis --- p.56
Chapter Chapter 3 --- Results and discussions --- p.57
Chapter 3.1 --- Extraction yield --- p.57
Chapter 3.2 --- Chemical assays for in vitro antioxidative properties of mushroom extracts --- p.60
Chapter 3.2.1 --- ABTS + scavenging activity --- p.60
Chapter 3.2.2 --- Hydroxyl radicals scavenging activity --- p.61
Chapter 3.2.3 --- Hydrogen peroxide scavenging activity --- p.64
Chapter 3.3 --- Total phenolic content --- p.67
Chapter 3.4 --- Cytotoxicity of hydrogen peroxide --- p.69
Chapter 3.4.1 --- "Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay" --- p.71
Chapter 3.4.2 --- Lactate dehydrogenase (LDH) assay --- p.72
Chapter 3.4.3 --- Total cellular protein loss --- p.73
Chapter 3.4.4 --- Residual hydrogen peroxide level --- p.76
Chapter 3.4.5 --- Lipid peroxidation --- p.77
Chapter 3.4.6 --- DNA damage --- p.79
Chapter 3.5 --- Cytotoxicity of extracts --- p.85
Chapter 3.6 --- Protection of H2()2-induced oxidative damage in HDFa cells --- p.88
Chapter 3.6.1 --- Protective effect of mushroom water extracts --- p.88
Chapter 3.6.2 --- Protective effect of CfAa on H2()2-incluced damage to HDFa --- p.93
Chapter 3.6.3 --- Protective effect of CfAa on DNA damage in HDFa cells --- p.96
Chapter 3.7 --- Modulation of cellular antioxidant defense system by CfAa --- p.99
Chapter 3.7.1 --- Intracellular total glutathione --- p.100
Chapter 3.7.2 --- Enzyme activities --- p.102
Chapter 3.8 --- Speculation on the possible components in CfAa --- p.108
Chapter Chapter 4 --- Conclusion and further works --- p.109
References --- p.111

Ng Yuk Fan.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.
Includes bibliographical references (leaves 145-162).
Abstracts in English and Chinese.
Thesis Committee: --- p.i
Acknowledgements --- p.ii
Abstract --- p.iii
摘要 --- p.v
Content --- p.vii
List of Tables --- p.xiii
List of Figures --- p.xvi
Abbreviations --- p.xviii
Chapter Chapter 1: --- Introduction --- p.1
Chapter 1.1 --- Antioxidants --- p.1
Chapter 1.1.1 --- Definition and mode of actions of antioxidants --- p.1
Chapter 1.1.2 --- Synthetic antioxidants --- p.2
Chapter 1.1.3 --- Natural antioxidants --- p.3
Chapter 1.2 --- Changes of antioxidant activity in food processing --- p.4
Chapter 1.2.1 --- Blanching --- p.4
Chapter 1.2.2 --- Drying --- p.5
Chapter 1.2.3 --- Microwave and Infrared energy --- p.7
Chapter 1.2.4 --- Freezing --- p.8
Chapter 1.3 --- Lipid oxidation and antioxidant --- p.8
Chapter 1.3.1 --- Free radicals --- p.8
Chapter 1.3.1.1 --- Superoxide --- p.10
Chapter 1.3.1.2 --- Hydrogen peroxide --- p.11
Chapter 1.3.1.3 --- Hydroxyl radical --- p.13
Chapter 1.3.2 --- Mechanism of lipid oxidation --- p.14
Chapter 1.3.3 --- Oxidation of low-density-liporoproteins (LDLs) and coronary heart disease --- p.15
Chapter 1.3.4 --- Role of antioxidants in inhibiting lipid oxidation --- p.16
Chapter 1.4 --- Hypocholesterolemic and antioxidant activity of phenolics --- p.19
Chapter 1.5 --- Medicinal properties of mushrooms --- p.21
Chapter 1.5.1 --- Background information of mushrooms --- p.21
Chapter 1.5.2 --- Phenolics in mushrooms --- p.22
Chapter 1.5.3 --- Hypocholesterolemic effect in mushroom --- p.23
Chapter 1.5.4 --- Previous studies in Agrocybe aegerita --- p.25
Chapter 1.6 --- Animal model for hypocholesteroliemic study --- p.27
Chapter 1.6.1 --- General requirements --- p.27
Chapter 1.6.2 --- Hamster model --- p.27
Chapter 1.7 --- Principles of assays that involved in antioxidant activity --- p.30
Chapter 1.7.1 --- ABTS + radical cation scavenging activity --- p.30
Chapter 1.7.2 --- Beta carotene bleaching method --- p.31
Chapter 1.7.3 --- Ferric reducing antioxidant power (FRAP) --- p.31
Chapter 1.7.4 --- Scavenging activity of hydroxyl radical --- p.32
Chapter 1.7.5 --- Inhibition of low-density lipoproteins (LDLs) oxidation --- p.33
Chapter 1.7.6 --- Total phenolic content determination --- p.33
Chapter 1.8 --- Principles of assays in hypocholesterolemic study --- p.34
Chapter 1.8.1 --- HDL-Cholesterol determination --- p.34
Chapter 1.8.2 --- Total cholesterol determination --- p.34
Chapter 1.8.3 --- Determination of plasma total triglyceride --- p.35
Chapter 1.9 --- Objectives --- p.36
Chapter Chapter 2: --- Materials and Methods --- p.37
Chapter 2.1 --- Sample preparation --- p.37
Chapter 2.2 --- Proximate Analysis of FAa and DAa --- p.38
Chapter 2.2.1 --- Determination of crude protein --- p.38
Chapter 2.2.2 --- Determination of ash --- p.39
Chapter 2.2.3 --- Total dietary fiber --- p.39
Chapter 2.2.4 --- Determination of fat --- p.41
Chapter 2.2.5 --- Moisture content --- p.42
Chapter 2.3 --- Sample extraction --- p.42
Chapter 2.3.1 --- Small-scale extraction --- p.42
Chapter 2.3.2 --- Large-scale extraction --- p.43
Chapter 2.4 --- Total phenolic content of DAa and FAa extract --- p.44
Chapter 2.5 --- Chemical assays for in vitro antioxidative properties determination --- p.45
Chapter 2.5.1 --- Hydroxyl free radical scavenging activity --- p.45
Chapter 2.5.2 --- Beta-carotene bleaching method --- p.46
Chapter 2.5.3 --- Inhibition of human low-density-lipoproteins (LDLs) oxidation --- p.47
Chapter 2.5.4 --- Scavenging activity of ABTS+radical cation --- p.50
Chapter 2.6 --- In vivo tests for antioxidative and hypocholesterolemic effect of DAa --- p.51
Chapter 2.6.1 --- Feeding experiments --- p.51
Chapter 2.6.2 --- Collection of plasma --- p.52
Chapter 2.6.3 --- Liver sample preparation --- p.52
Chapter 2.6.4 --- Determination of in vivo antioxidative effect --- p.54
Chapter 2.6.4.1 --- FRPA assay --- p.54
Chapter 2.6.4.2 --- ABTS + radical cation scavenging activity --- p.55
Chapter 2.6.5 --- Determination of plasma lipid profiles --- p.55
Chapter 2.6.5.1 --- Plasma total cholesterol (TC) --- p.55
Chapter 2.6.5.2 --- Plasma total triglyceride (TG) --- p.56
Chapter 2.6.5.3 --- Plasma high density lipoprotein cholesterol (HDL-C) determination --- p.57
Chapter 2.6.5.4 --- Hepatic cholesterol determination by gas chromatography analysis --- p.57
Chapter 2.7 --- Statistical analysis --- p.59
Chapter Chapter 3: --- Results and discussion --- p.61
Chapter 3.1 --- Proximate analysis --- p.61
Chapter 3.2 --- Small-scale extraction scheme --- p.63
Chapter 3.2.1 --- Extraction yield --- p.63
Chapter 3.2.2 --- Antioxidant assays --- p.65
Chapter 3.2.2.1 --- Hydroxyl free radical scavenging activity --- p.65
Chapter 3.2.2.2 --- Beta-carotene bleaching method --- p.68
Chapter 3.2.2.3 --- The formation of TBARS in human LDL oxidation --- p.75
Chapter 3.2.2.4 --- Total phenolic content (TPC) in DAa and FAa ethanolic and water extracts --- p.81
Chapter 3.2.2.5 --- Correlation between total phenolic content and antioxidant activity of mushroom extracts --- p.84
Chapter 3.2.2.6 --- Comparison of antioxidant activity and TPC in DAa and FAa ethanolic and water extracts in the small-scale extraction scheme --- p.88
Chapter 3.3 --- Large-scale extraction scheme --- p.91
Chapter 3.3.1 --- Extraction yield --- p.91
Chapter 3.3.2 --- Antioxidant assays --- p.91
Chapter 3.3.2.1 --- Hydroxyl free radical scavenging activity --- p.91
Chapter 3.3.2.2 --- Beta-carotene bleaching method --- p.94
Chapter 3.3.2.3 --- ABTS + radical cation scavenging activity --- p.96
Chapter 3.3.2.4 --- Formation of TBARS in human LDL oxidation in the DAa_E_l and Daa_W_1 --- p.97
Chapter 3.3.2.5 --- Total phenolic content (TPC) of DAa_E_l and DAa_W_l --- p.97
Chapter 3.3.2.6 --- Correlation between total phenolic content and antioxidant activity --- p.101
Chapter 3.3.2.7 --- Summary of large-scale extraction scheme --- p.103
Chapter 3.4 --- In vivo antioxidant activity and hypocholesterolemic effect of DAa studied by animal model --- p.104
Chapter 3.4.1 --- Effect of DAa´ؤE_1 and DAa_W_l on body weight and food intake --- p.105
Chapter 3.4.2 --- Effect of DAa一E´ؤ1 and DAa_W_l on plasma total cholesterol (TC) in hamsters --- p.108
Chapter 3.4.3 --- Effect of DAa´ؤE_1 and DAa W l on plasma total triglycerides (TG) in hamsters --- p.114
Chapter 3.4.4 --- Effect of DAa_E_l and DAa_W_l on plasma high-density-lipoprotein cholesterol (HDL-C) in hamsters --- p.119
Chapter 3.4.5 --- Effect of DAa_E_l and DAa一W_1 on hepatic cholesterol (HC) profile in hamsters --- p.124
Chapter 3.4.6 --- Effect of DAa_E_l and DAa W l on ferric reducing antioxidant power (FRAP) in hamsters (FRAP) --- p.128
Chapter 3.4.7 --- Effect of DAa_E_l and DAa_W_l on ABTS + cation radical scavenging activity --- p.131
Chapter 3.4.8 --- The antioxidant activity and hypocholesterolemic effect of DAa extracts --- p.134
Chapter 3.4.9 --- Summary of in vivo antioxidant activity and hypocholesterolemic effect of DAa studied by animal model --- p.140
Chapter Chapter 4: --- Conclusions --- p.142
References --- p.145

Wong, Wai Yin.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2007.
Includes bibliographical references (leaves 92-108).
Abstracts in English and Chinese.
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Atherosclerosis --- p.1
Chapter 1.1.1 --- Pathogenesis of Atherosclerosis --- p.2
Chapter 1.1.2 --- Atherosclerosis and Cardiovascular Disease --- p.4
Chapter 1.2 --- Cardiovascular Disease (CVD) --- p.5
Chapter 1.2.1 --- Term Definition --- p.5
Chapter 1.2.2 --- Risk Factors --- p.6
Chapter 1.2.3 --- Current Western Medications --- p.7
Chapter 1.3 --- Reactive Oxygen Species (ROS) --- p.8
Chapter 1.3.1 --- Impact of ROS --- p.8
Chapter 1.3.2 --- "Superoxide Anion Radical, Hydrogen Peroxide, Hydroxyl Radical, Nitric Oxide" --- p.9
Chapter 1.3.3 --- ROS Production by NAD(P)H Oxidases --- p.11
Chapter 1.3.4 --- ROS Production by Mitochondria --- p.12
Chapter 1.3.5 --- Lipid Peroxidation --- p.13
Chapter 1.3.6 --- Other Sources of ROS --- p.15
Chapter 1.4 --- Antioxidants --- p.16
Chapter 1.4.1 --- Superoxide Dismutase (SOD) --- p.16
Chapter 1.4.2 --- Catalase (CAT) --- p.17
Chapter 1.4.3 --- Glutathinoe Peroxidase (GPx) --- p.17
Chapter 1.4.4 --- Glutathione-S-Transferase (GST) --- p.18
Chapter 1.4.5 --- Vitamin E --- p.18
Chapter 1.4.6 --- Vitamin C --- p.19
Chapter 1.5 --- Ageing --- p.19
Chapter 1.6 --- Antioxidants and CVD --- p.21
Chapter 1.7 --- Traditional Chinese Medicine (TCM) --- p.22
Chapter 1.7.1 --- Danshen --- p.23
Chapter 1.7.2 --- Gegen --- p.25
Chapter 1.7.3 --- Danshen-Gegen Compound Formula (DG) --- p.26
Chapter 1.8 --- Aim of Study --- p.27
Chapter Chapter 2 --- In vitro Foam Cells Formation --- p.29
Chapter 2.1 --- Materials and Methods --- p.29
Chapter 2.1.1 --- Materials --- p.29
Chapter 2.1.2 --- Methods --- p.30
Chapter 2.1.2.1 --- Herbal Preparation by Hot Water Extraction --- p.30
Chapter 2.1.2.2 --- Resident Peritoneal Macrophages Preparation --- p.31
Chapter 2.1.2.3 --- "Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl Tetrazolium Bromide (MTT) Assay" --- p.31
Chapter 2.1.2.4 --- DG Effect on in vitro Foam Cells Formation --- p.32
Chapter 2.2 --- Results and Discussion --- p.32
Chapter 2.3 --- Summary --- p.39
Chapter Chapter 3 --- In vivo Antioxidant Level --- p.40
Chapter 3.1 --- DG Effect on in vivo Antioxidant Levels on Young-adult Wistar Rats --- p.40
Chapter 3.1.1 --- Materials and Methods --- p.40
Chapter 3.1.1.1 --- Herbal Preparation by Hot Water Extraction --- p.40
Chapter 3.1.1.2 --- Assay Kits --- p.41
Chapter 3.1.1.3 --- Antibodies for Protein Expression Determination in Organs --- p.41
Chapter 3.1.1.4 --- Animals and Experimental Design --- p.41
Chapter 3.1.1.5 --- Plasma Antioxidants --- p.42
Chapter 3.1.1.6 --- Lipid Peroxidation and Protein Expression in Organs --- p.46
Chapter 3.1.1.7 --- Statistics --- p.52
Chapter 3.1.2 --- Results and Discussion --- p.53
Chapter 3.2 --- DG Effect on in vivo Antioxidant Levels on Middle-aged Wistar Rats --- p.74
Chapter 3.2.1 --- Materials and Methods --- p.75
Chapter 3.2.2 --- Results and Discussion --- p.75
Chapter 3.3 --- Summary --- p.87
Chapter Chapter 4 --- Conclusion and Future Work --- p.90
Chapter 4.1 --- Conclusion --- p.90
Chapter 4.2 --- Future work --- p.90
Reference --- p.92
Related Publication --- p.109

Wong Yuen Fan.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2000.
Includes bibliographical references (leaves 134-150).
Abstracts in English and Chinese.
ACKNOWLEDGMENTS --- p.i
ABSTRACT --- p.ii
LIST OF ABBREAIATIONS --- p.vii
TABLE OF CONTENTS --- p.ix
Chapter Chapter 1 --- General introduction
Chapter 1.1 --- History of Kudingcha --- p.1
Chapter 1.2 --- Classification of Kudingcha --- p.1
Chapter 1.3 --- Composition of Kudingcha --- p.3
Chapter 1.4 --- Introduction to phenylethanoid glycosides --- p.4
Chapter 1.4.1 --- Isolation and purification of phenylethanoid glycosides --- p.4
Chapter 1.4.2 --- Taxonomy of phenylethanoid glycosides --- p.5
Chapter 1.4.3 --- Structure of phenylethanoid glycosides --- p.5
Chapter 1.4.4 --- Biosynthesis of phenylethanoid glycosides --- p.6
Chapter 1.4.5 --- Pharmacological effects of phenylethanoid glycosides --- p.9
Chapter 1.4.5.1 --- Anticarcinogenic activity --- p.10
Chapter 1.4.5.2 --- Inhibitory activity of protein kinase C --- p.10
Chapter 1.4.5.3 --- Immunosuppressive activity --- p.11
Chapter 1.4.5.4 --- DNA repairing activity --- p.11
Chapter 1.4.5.5 --- Antibacterial and antiviral activities --- p.11
Chapter 1.4.5.6 --- Antiinflammatory and antinociceptive activities --- p.12
Chapter 1.4.5.7 --- Hepatoprotective activity --- p.12
Chapter 1.4.5.8 --- Inhibitory activity of xanthine oxidase --- p.13
Chapter 1.4.5.9 --- Antioxidative and scavenging activities --- p.13
Chapter Chapter 2 --- Isolation and purification of phenylethaonid glycosides in Kudingcha
Chapter 2.1 --- Introduction --- p.15
Chapter 2.2 --- Objectives --- p.16
Chapter 2.3 --- Materials and Methods --- p.17
Chapter 2.3.1 --- Extraction and isolation --- p.17
Chapter 2.3.2 --- High performance liquid chromatograph (HPLC)analysis --- p.19
Chapter 2.3.2.1 --- "Acteoside, ligupurpuroside A and osmanthuside B" --- p.19
Chapter 2.3.2.2 --- cis-Ligupurpuroside B and trans-ligupurpuroside B --- p.19
Chapter 2.3.3 --- Isolation and purification of isoacteoside --- p.19
Chapter 2.4 --- Results --- p.24
Chapter 2.4.1 --- A cteoside --- p.24
Chapter 2.4.2 --- Osmanthuside B --- p.24
Chapter 2.4.3 --- Ligupurpuroside A --- p.24
Chapter 2.4.4 --- trans-Ligupurpuroside B --- p.25
Chapter 2.4.5 --- cis-Ligupurpuroside B --- p.25
Chapter 2.4.6 --- Isoacteoside --- p.25
Chapter 2.4.6.1 --- Thermal stability --- p.25
Chapter 2.5 --- Discussions --- p.27
Chapter 2.5.1 --- Acteoside --- p.27
Chapter 2.5.2 --- Osmanthuside B --- p.27
Chapter 2.5.3 --- Ligupurpuroside A --- p.28
Chapter 2.5.4 --- trans-Ligupurpuroside B --- p.29
Chapter 2.5.5 --- cis-Ligupurpuroside B --- p.29
Chapter 2.5.6 --- Isoacteoside --- p.30
Chapter Chapter 3 --- Inhibitory effect of phenylethanoid glycosides isolated from Kudingcha on Cu2+-mediated LDL oxidation in vitro
Chapter 3.1 --- Introduction --- p.36
Chapter 3.2 --- Mechanisms of lipoprotein oxidation in vivo --- p.36
Chapter 3.2.1 --- Oxidants underlying LDL oxidation --- p.36
Chapter 3.2.2 --- Oxidative modification of LDL --- p.37
Chapter 3.2.3 --- Role of oxidatively modified LDL in atherogenesis --- p.38
Chapter 3.2.4 --- Antioxidants and atherosclerotic heart disease --- p.40
Chapter 3.2.5 --- Measuring the thiobarbituric acid-reactive substances (TBARS) formation as an index to monitor LDL oxidation --- p.41
Chapter 3.2.6 --- Effect of flavonoids on Cu2+-mediated human LDL oxidation --- p.41
Chapter 3.3 --- Objectives --- p.43
Chapter 3.4 --- Materials and methods --- p.44
Chapter 3.4.1 --- LDL isolation --- p.44
Chapter 3.4.2 --- LDL oxidation --- p.44
Chapter 3.4.3 --- Thiobarbituric acid-reactive substances (TBARS) assay --- p.45
Chapter 3.4.4 --- Interactions of phenylethanoid glycosides isolated from Kudingcha with Cu2+ in human LDL oxidation --- p.45
Chapter 3.4.5 --- Statistics --- p.46
Chapter 3.5 --- Results --- p.47
Chapter 3.5.1 --- Protective effect of the major phenylethanoid glycosides isolated from Kudingcha on LDL oxidation --- p.47
Chapter 3.5.2 --- Varying protective effect of individual major Kudingcha phenylethanoid glycosides --- p.47
Chapter 3.5.3 --- Interactions of Kudingcha phenylethanoid glycosides with Cu2+in human LDL oxidation --- p.51
Chapter 3.5 --- Discussions --- p.55
Chapter Chapter 4 --- Inhibitory effects of Kudingcha phenylethanoid glycosides on a-tocopherol oxidation in vitro
Chapter 4.1 --- Introduction --- p.58
Chapter 4.1.1 --- LDL oxidation and atherosclerosis --- p.58
Chapter 4.1.2 --- Role of vitamin E in LDL lipid peroxidation --- p.59
Chapter 4.1.3 --- Interaction of tocopherol interactions with other antioxidants and synergists --- p.61
Chapter 4.2 --- Objectives --- p.62
Chapter 4.3 --- Materials and Methods --- p.63
Chapter 4.3.1 --- Depletion of a-tocopherol in LDL --- p.63
Chapter 4.3.2 --- Regeneration of a-tocopherol in LDL --- p.63
Chapter 4.3.3 --- HPLC analysis of a-tocopherol in LDL --- p.64
Chapter 4.3.4 --- Statistics --- p.64
Chapter 4.4 --- Results --- p.66
Chapter 4.4.1 --- Protective effects of Kudingcha phenylethanoid glycosides on a-tocopherol depletion --- p.66
Chapter 4.4.2 --- Regeneration of a-tocopherol by acteoside --- p.70
Chapter 4.5 --- Discussions --- p.72
Chapter Chapter 5 --- Relaxing effects of Kudingcha extract and purified acteoside in rat aortic rings
Chapter 5.1 --- Introduction --- p.75
Chapter 5.1.1 --- Mechanisms of calcium mobilization --- p.76
Chapter 5.1.1.1 --- Voltage-dependent calcium channel --- p.76
Chapter 5.1.1.2 --- Thromboxane A2 Receptor-mediated calcium channel --- p.77
Chapter 5.1.1.3 --- Protein kinase C in signal transudation --- p.77
Chapter 5.1.2 --- Contractile proteins and regulation of contraction of vascular smooth muscle --- p.78
Chapter 5.2 --- Objectives --- p.82
Chapter 5.3 --- Materials and Methods --- p.83
Chapter 5.3.1 --- Arterial ring preparation --- p.83
Chapter 5.3.2 --- Vascular action of Kudingcha extract and acteoside --- p.85
Chapter 5.3.2.1 --- Relaxant responses of Kudingcha extract and acteoside on U46619 -induced contraction --- p.85
Chapter 5.3.2.2 --- Relaxant responses of Kudingcha extract and acteoside on high K+ and CaCl2-induced contraction --- p.85
Chapter 5.3.2.3 --- Relaxant responses of Kudingcha extract and acteoside on protein kinase C- mediated contraction --- p.86
Chapter 5.3.2.4 --- Effect of acteoside on acetylcholine-induced relaxation --- p.87
Chapter 5.3.3 --- Statistics --- p.87
Chapter 5.4 --- Results --- p.88
Chapter 5.4.1 --- Effects of Kudingcha extract and acteoside on U46619-induced contraction --- p.88
Chapter 5.4.2 --- Effects of Kudingcha extract and acteoside on high K+-induced contraction --- p.94
Chapter 5.4.3 --- Effect of Kudingcha extract and acteoside on protein kinase C-mediated contraction --- p.98
Chapter 5.4.4 --- Effect of acteoside on acetylcholine-induced relaxation --- p.100
Chapter 5.5 --- Discussions --- p.103
Chapter Chapter 6 --- Effect of Kudingcha on lipid contents of hamsters and New Zealand Rabbits
Chapter 6.1 --- Introduction --- p.106
Chapter 6.1.1 --- Factors related to CHD --- p.106
Chapter 6.1.2 --- Animal model --- p.107
Chapter 6.2 --- Objectives --- p.108
Chapter 6.3 --- Materials and Methods --- p.109
Chapter 6.3.1 --- Rabbit --- p.109
Chapter 6.3.1.1 --- Measurement of atheroma formation --- p.112
Chapter 6.3.2 --- Hamster --- p.114
Chapter 6.3.3 --- Serum lipid determinations --- p.116
Chapter 6.3.4 --- Determination of hepatic cholesterol content --- p.116
Chapter 6.3.5 --- Statistics --- p.117
Chapter 6.4 --- Results --- p.119
Chapter 6.4.1 --- Growth and Food intake --- p.119
Chapter 6.4.2 --- "Effect of Kudingcha supplementation on Serum TG, TC and HDL-C" --- p.119
Chapter 6.4.3 --- Effect of Kudingcha supplementation on hepatic cholesterol contents --- p.124
Chapter 6.4.4 --- Effect of Kudingcha supplementation on atheroma formation --- p.124
Chapter 6.5 --- Discussions --- p.129
Chapter Chapter 7 --- Conclusions --- p.131
References --- p.134

Experiments were conducted to investigate the role of free radicals in exercise induced cardiac adaptations and to determine if statin administration would adversely affect cardiac adaptations to exercise. In the first experiment myocardial antioxidant enzymes, cardiac function and cardiac hypertrophy were assessed following a chronic exercise protocol previously used by our lab. MPG effectively reduced myocardial oxidative stress and activation of the signaling proteins Akt and S6 following an exercise bout. Skeletal muscle mitochondria content increased to similar levels in E and E+MPG. Similar increases (P<0.05) in both exercised groups were observed for heart wt and heart wt to body wt ratio. Cardiac function at the high workload improved in E vs S as indicated by higher (P<0.05) peak systolic pressure (SP), cardiac output (CO), coronary flow, COxSP and mechanical efficiency (COxSP/VO2). MPG prevented these exercise-induced functional improvements. This study provides evidence that free radicals do not play a role in the development of exercise-induced cardiac hypertrophy, however, they are involved in functional cardiac adaptations, which may be mediated through the PI3K/Akt pathway. In the second experiment a similar exercise protocol was used to determine if statins which have been shown to prevent pathological forms of cardiac hypertrophy, would be detrimental to exercise induced cardiac adaptations. In addition to the sedentary and exercise groups sedentary+statin and exercise+statin groups were assessed. Hearts were isolated and perfused and assessed for function at low and high workloads. Exercise treatment resulted in cardiac hypertrophy in absolute and relative terms to a similar extent in statin-treated and untreated exercised rats. Additionally it resulted in significant functional increases for SP, CO, COxSP, VO₂, and EFF in both exercised groups. In conclusion, these studies provide evidence that exercise in the cold is a valid model for physiological cardiac hypertrophy and that pathological and physiological cardiac hypertrophy signal through different pathways due to the fact that two well established treatments (mpg and statins) that prevent pathological cardiac hypertrophy did not affect exercise induced cardiac hypertrophy.
text

博士
國立海洋大學
食品科學系
91
Abstract The sorghum distillery residue (SDR) has been an underutilized by-product with an estimated production of 150 ton/day in Kinmen, Taiwan. The objective of this study was to test the physiological effects of SDR, in order to utilize it as a nutraceutical ingredient in feed for cultured fish. The SDR contain 20% crude protein and 25% starch. The grey mullet fed 5% wet - SDR (W-SDR) for 90 days, and 10% dehulled wet — sorghum distillery residue (DW-SDR) for 60 days respectively, the body weight and the specific growth rate (SGR) showed no significant differences from those fed control diet. After cultivation the grey mullet (12.4±0.6 g) were fed either 20% wet-SDR or dehulled wet-SDR for 60 days, SGR averaged 0.82±0.10 % day and 0.98±0.06 % day respectively while those fed the control diet averaged 1.07 ±0.04 % day. The corresponding FCR was 3.93±0.14 or 3.14±0.13 being significantly higher than the control (2.57±0.08). The mortality of control, DW-SDR and W-SDR were 12.5±2.5, 7.5±2.5 and 15.5±5.0 respectively. In the winter trial, the grey mullet fed a diet consisting of 10% DW-SDR for 60 days (the water temperature range 17~21℃), the body weight of grey mullet from 288 ± 53g to 321 ± 61g. The SGR1 was 0.18% day. The SGR1 of control group was 0.12 % day. In the period of 60 to 75 day, the water temperature from 17℃ dropped to 14℃, the SGR2 of grey mullet fed DW-SDR diet decreased to 0. While the SGR2 of the control group reduced to —0.09 % day. The difference in SGR2 of the DW-SDR and control group became more noticeable at lower temp. dietary replacement of soybean meal and rice bran with SDR produced grey mullet more resistant to cold temperature and better growth as compared to the control. In winter trial, the grey mullet fed 10% dehulled wet-SDR had a blood viscosity of 1.9±0.69 mPas and a hematocrit value of 33 ± 3 %, being apparently lower than that of the control group 3.6±0.88 mPas and 41 ± 1 %. The blood of the control group started to clot in 6 h and showed hemolysis, while the mullet fed dehulled wet-SDR did not show blood clotting, maintained normal fluidity and integrity of erythrocyte membrane. In vitro study, RBC hemolysis of cultured grey mulle induced by H2O2 that was significantly inhibited by the SDR extract. The erythrocytes maintained elliptic or quasi-circular, in which nucleus appeared at the cell center, and cell membrane was intact. The control group showed disintegrated cell membranes and emptied cell center. Hot air-dried, wet, and dehulled wet-SDR showed 63%, 90% and 97% inhibition of the hemoglobin-catalyzed oxidation of linoleic acid in contrast to soybean (13%) and rice bran (78%). Feeds formulated either with 20% wet-SDR or dehulled wet-SDR showed an antioxidant activity of 68% being higher than the control diet (53%). Feeding these diets to cultured grey mullet resulted in a lower hydroperoxide content in gill tissue, shown by a chemiluminesence intensity of 1806 or 1409 mV as opposed to 2666 mV for fish fed a control diet. An in vitro digestibility test was developed to simulate the in vivo digestion of grey mullet. The protein digestion was 47% lower than that of the control. Inhibition of trypsin activity correlated with the amount of dietary SDR extract (r = 0.98). Based on Lineweaver-Burk plot, the inhibition of SDR on trypsin was uncompetitive - noncompetitive mixed type with changes in both Vmax and Km. W-SDR (dry basis) was found to contain 3.3 mg/g of tannin. This inhibitory effect and the tannin content in SDR decreased significantly (P<0.05) when the SDR extract was pretreated with polyethylene glycol (PEG), a tannin binding agent. A detannin treatment with PEG was tested to prevent the inhibition of dietary SDR on feed protein digestion and growth of cultured grey mullet in vivo. This treatment may provide an example to utilize other plant protein sources rich in tannin as fish feed ingredient.

Oxidative stress is the major driving force behind the aging process and many age-related diseases. However, direct experimental evidence of whether antioxidants, such as ascorbate (AA) and lipoic acid (LA) can slow the progression of aging process and/or reduce risks of developing degenerative disease is largely absent. This suggests a better understanding of the precise mechanism of how dietary micronutrient affect parameters of involved in cellular redox balance and aging are warranted. In this dissertation, young and old rats were used as our model to understand potential pro-oxidant events that contribute to increases in oxidative stress in various tissues and how antioxidants such as ascorbate and lipoic acid influence these events. Our major findings are that the age-related impairment of mitochondria and increased deposition of iron contribute significantly to heighten levels of oxidative stress, as evidenced by the resultant increases in the rates of oxidant appearance and in the levels of oxidative damage to DNA, lipids and proteins. We find that AA and LA strongly protected against transition metal-ion dependent increases in oxidative stress. AA effectively inhibited transition metal-mediated lipide peroxidation in human plasma. LA in its reduced form effectively binds iron and copper in a redox inactive manner and reversed chronically elevated levels of iron in the brain without removing enzyme bound transition metal ions. LA also significantly attenuated the age-related increase in oxidative stress associated with mitochondrial decay in the heart, as evidenced by the improvements in AA levels and glutathione redox status. The declines in tissue GSH levels in aged rats were strongly associated with the diminished γ-GCL activity (in parallel with decreased expression of the catalytic and modulatory subunits), and lowered Nrf2 expression and binding to ARE sequence in rat liver. Remarkably, all these events were effectively reversed by the administration of LA, modulating the parameters to return to the observed in young animals. The implications of this work open new avenues not only for further understanding of the aging process but also for possible strategies in its modulation by the micronutrients.
Graduation date: 2004

Lai, Tsz Ching.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2011.
Includes bibliographical references (leaves 121-136).
Abstracts in English and Chinese.
Acknowledgements
Abstract
摘要
Content
List of tables
List of figures
List of abbreviations
Chapter Chapter 1: --- Introduction --- p.1
Chapter 1.1 --- Introduction of food market trends in Hong Kong and mushroom productivity in the world --- p.1
Chapter 1.1.1 --- Agrocybe aegerita --- p.1
Chapter 1.1.2 --- Pleurotus spp --- p.2
Chapter 1.1.3 --- Pholiota nameko --- p.3
Chapter 1.2 --- Objectives --- p.5
Chapter Chapter 2: --- Chemical assays for in vitro antioxidative properties of mushroom extracts --- p.6
Chapter 2.1 --- Introduction --- p.6
Chapter 2.1.1 --- Reactive oxygen species (ROS) --- p.6
Chapter 2.1.1.1 --- Definition of ROS --- p.6
Chapter 2.1.1.2 --- Sources of ROS --- p.6
Chapter 2.1.1.2.1 --- Endogenous sources of ROS --- p.6
Chapter 2.1.1.2.2 --- Exogenous sources of ROS --- p.8
Chapter 2.1.1.3 --- Damaging effects of ROS --- p.8
Chapter 2.1.2 --- Antioxidants --- p.10
Chapter 2.1.2.1 --- Mechanism of action --- p.10
Chapter 2.1.2.2 --- Sources of antioxidants --- p.11
Chapter 2.1.2.2.1 --- Dietary antioxidants --- p.11
Chapter 2.1.2.2.2 --- Antioxidants in edible mushrooms --- p.12
Chapter 2.1.2.2.3 --- Phenolic compounds in mushrooms --- p.13
Chapter 2.2 --- Materials and Methods --- p.16
Chapter 2.2.1 --- Materials --- p.16
Chapter 2.2.1.1 --- Mushroom fruiting bodies --- p.16
Chapter 2.2.2 --- Principles of Methods and Experimental Protocols --- p.17
Chapter 2.2.2.1 --- Sample preparation --- p.17
Chapter 2.2.2.2 --- Evaluation of antioxidant capacity --- p.18
Chapter 2.2.2.2.1 --- DPPH radical scavenging activity --- p.18
Chapter 2.2.2.2.2 --- Superoxide anion scavenging activity --- p.19
Chapter 2.2.2.2.3 --- Hydroxyl radical scavenging activity --- p.20
Chapter 2.2.2.2.4 --- Hydrogen peroxide scavenging activity --- p.22
Chapter 2.2.2.3 --- Determination of phenolic compounds --- p.24
Chapter 2.2.2.3.1 --- Total phenolic content --- p.24
Chapter 2.2.2.3.2 --- Identification of phenolic acids --- p.25
Chapter 2.2.3 --- Statistical analysis --- p.27
Chapter 2.3 --- Results and Discussion --- p.28
Chapter 2.3.1 --- Extraction yield --- p.28
Chapter 2.3.2 --- Evaluation of antioxidant capacity --- p.29
Chapter 2.3.2.1 --- DPPH radical scavenging activity --- p.29
Chapter 2.3.2.2 --- Superoxide anion scavenging activity --- p.31
Chapter 2.3.2.3 --- Hydroxyl radical scavenging activity --- p.33
Chapter 2.3.2.4 --- Hydrogen peroxide scavenging activity --- p.35
Chapter 2.3.2.5 --- Comparison of the effective concentrations (EC50) of mushroom water extracts in different antioxidant assays --- p.37
Chapter 2.3.3 --- Determination of phenolic compounds --- p.38
Chapter 2.3.3.1 --- Total phenolic content --- p.38
Chapter 2.3.3.2 --- Identification of phenolic acids --- p.39
Chapter 2.4 --- Summary --- p.45
Chapter Chapter 3: --- Anti-angiogenic properties of the Aa water extract --- p.46
Chapter 3.1 --- Introduction --- p.46
Chapter 3.1.1 --- Angiogenesis --- p.46
Chapter 3.1.1.1 --- Process of angiogenesis --- p.46
Chapter 3.1.1.2 --- Regulations of angiogenesis --- p.47
Chapter 3.1.1.2.1 --- Fibroblast growth factor (bFGF) --- p.47
Chapter 3.1.1.2.2 --- Vascular endothelial growth factor (VEGF) --- p.48
Chapter 3.1.2 --- Tumor angiogenesis --- p.49
Chapter 3.1.2.1 --- ROS generation in tumor cells --- p.50
Chapter 3.1.2.2 --- Hydrogen peroxide and VEGF --- p.51
Chapter 3.1.2.3 --- Previous studies on tumor angiogenesis --- p.52
Chapter 3.1.2.3.1 --- ROS and endothelial cells proliferation --- p.52
Chapter 3.1.2.3.2 --- VEGF and endothelial cells functions --- p.53
Chapter 3.1.3 --- Use of antioxidants in cancer treatment --- p.53
Chapter 3.1.3.1 --- Antioxidant use of cancer therapy --- p.53
Chapter 3.1.3.2 --- Antioxidant and endothelial cells functions --- p.54
Chapter 3.1.3.3 --- Anti-angiogenic effects of polyphenols --- p.56
Chapter 3.1.3.3.1 --- Phenolic acids --- p.56
Chapter 3.1.3.3.2 --- Tea catechin --- p.57
Chapter 3.1.3.3.3 --- Resveratrol --- p.57
Chapter 3.1.3.3.4 --- Genistein --- p.58
Chapter 3.2 --- Principles of Methods and Experimental Protocols --- p.60
Chapter 3.2.1 --- Sample preparation --- p.60
Chapter 3.2.2 --- Toxicity of the Aa water extract --- p.60
Chapter 3.2.2.1 --- Limulus amebocyte lysate (LAL) test --- p.60
Chapter 3.2.2.2 --- Toxicity towards normal cells --- p.61
Chapter 3.2.2.2.1 --- Cell line and its subculture --- p.61
Chapter 3.2.2.2.2 --- Colorimetric (MTT) assay --- p.62
Chapter 3.2.3 --- Effect of the Aa water extract on cancer cells --- p.63
Chapter 3.2.3.1 --- Cell line and its subculture --- p.63
Chapter 3.2.3.2 --- Redox status --- p.63
Chapter 3.2.3.3 --- VEGF secretion --- p.65
Chapter 3.2.4 --- In vitro cell culture anti-angioenesis analysis --- p.66
Chapter 3.2.4.1 --- Cell line and its subculture --- p.66
Chapter 3.2.4.2 --- Endothelial cells proliferation --- p.67
Chapter 3.2.4.3 --- Endothelial cells migration --- p.68
Chapter 3.2.4.3.1 --- Wound healing assay --- p.68
Chapter 3.2.4.3.2 --- Transwell culture insert assay --- p.69
Chapter 3.2.4.4 --- Endothelial cells tubule formation --- p.71
Chapter 3.2.5 --- In vitro organ culture anti-angiogenesis analysis --- p.72
Chapter 3.2.5.1 --- Aortic ring assay --- p.72
Chapter 3.2.6 --- Statistical analysis --- p.74
Chapter 3.3 --- Results and Discussions --- p.75
Chapter 3.3.1 --- Toxicity of the Aa water extract --- p.75
Chapter 3.3.1.1 --- Limulus amebocyte lysate (LAL) test --- p.75
Chapter 3.3.1.2 --- Toxicity towards normal cells --- p.75
Chapter 3.3.2 --- Effect of the Aa water extract on cancer cells --- p.77
Chapter 3.3.2.1 --- Redox status --- p.77
Chapter 3.3.2.2 --- VEGF secretion --- p.79
Chapter 3.3.2.3 --- Relationship between intracellular ROS and VEGF secretion detected --- p.80
Chapter 3.3.3 --- Effect of the Aa water extract on angiogenesis --- p.82
Chapter 3.3.3.1 --- Endothelial cells proliferation --- p.82
Chapter 3.3.3.2 --- Endothelial cells migration --- p.84
Chapter 3.3.3.2.1 --- Wound healing assay --- p.84
Chapter 3.3.3.2.2 --- Transwell culture insert assay --- p.87
Chapter 3.3.3.3 --- Endothelial cells tubule formation --- p.90
Chapter 3.3.3.4 --- Aortic ring assay --- p.97
Chapter 3.3.4 --- Effect of phenolic acids on endothelial cells --- p.101
Chapter 3.3.4.1 --- Endothelial cells proliferation --- p.101
Chapter 3.3.4.2 --- Endothelial cells migration --- p.102
Chapter 3.3.4.2.1 --- Wound healing assay --- p.102
Chapter 3.3.4.2.2 --- Transwell culture insert assay --- p.105
Chapter 3.3.4.3 --- Endothelial cells tubule formation --- p.106
Chapter 3.3.4.4 --- Aortic ring assay --- p.112
Chapter 3.4 --- Summary --- p.116
Chapter Chapter 4 --- Conclusions and future works --- p.118
References --- p.121

碩士
國立臺灣海洋大學
水產養殖學系
103
This study was to find out the dietary effects of four natural antioxidant supplements on rearing performance, antioxidant capacity, immune response, physiological reaction and resistance to hypoxia stress of white shrimp (Litopenaeus vannamei). Four kinds of natural antioxidants used are astaxanthin (AX), curcumin (CU), Salvia extract (SA) and sesame extracts (SE). Each supplement was incorporated into a basal diet with the same diphenylpicryihydrazyl (DPPH) antioxidant capacity. These 4 diets and a control diet(C) without supplement were fed to the shrimp for 10 weeks, and each diet had 3 replicates. Rearing performance such as survival, growth and feed consumption was monitored. After rearing, the resulting shrimp were subjected to hypoxia stress and the antioxidant capacity, immune response, physiological reactions and oxygen consumption rate were monitored. The results showed no difference in survival among the treatments and the shrimp fed CU had the best growth. Compared to the control, adding antioxidants improved the antioxidant capacity such as total antioxidant capacity (TAS), Glutathioine peroxidase (GPx), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), superoxide dismutase enzymes (Superoxide dismutase, SOD) and increased Total haemocyte counts (THC). Curcumin show at TAS, GPx, ALT, AST and terms of THC, AX compared to, Salvia, sesame and control group had the best antioxidant capacity, but it is slightly inferior in SOD sesame and Salvia. When under hypoxia stress, the glucose (Glucose, GLU) and lactic acid (Lactate, LAC) of all groups all increased, but triglycerides (Triglycerides, TRI) had no change. When under hypoxia stress, oxygen consumption rates (Oxygen consumption rate, OCR) of the groups supplemented with antioxidants were lower than the control group, but were of no difference among themselves. In conclusion, these four dietary antioxidants enhanced the antioxidant capacity and immune response of white shrimp but had no effects on physiological reactions. Furthermore, CU is able to promote the growth of white shrimp.

碩士
國立中興大學
食品暨應用生物科技學系所
101
Lentinula edodes contains many physiologically active components, including ergrsterol, ergothioine, lentinan and eritadenine. In addition, its extracts are used to be a source for a potential anti-inflammatory, cholesterol reducing, and anti-tumor activity. According to previews studies, pulsed light or continuous UV exposure method can convert ergosterol to ergocalciferol (known as vitamin D2) and increase vitamin D formation in mushrooms. There are many mushrooms , such as Lentinula edodes, Flammulina velutipes, Pleurotus eryngii and Lyophyllum shimeji, Lentinula edodes contain high level ergosterol in Taiwan. Comparing with continuous UVB light method, pulsed light method has some advantages, such as shorter period of time and high peak energy. Therefore, it is worth to evaluate the change of physiological active substances during the irradiating. The purposes of this study were to explore the effect under different shot (0, 20, 40, and 60 pulses, and the radiant eneygy was 0.131 J/cm2 per pulse) of pulsed light irradiation of vitamin D converted to ergosterol , to analysis the physiological activity substances, including, ergothioneine and total phenols, and to evaluate the anti-inflammatory reaction, and antioxidant properties of Lentinula edodes 921. According to the results, powder exposed to 20, 40, and 60 shots of pulsed light, could significantly increased vitamin D2 content dose-dependently. The content of ergosterol would be decreased slightly under different irritation condition, 20, 40, and 60 shots, which were 5522.6, 5414.0, 5260.5, and 5117.8 μg/g. And the content of ergothioneine had slightly increased , which were 560.0, 570.1, 613.6, and 593.3 μg/g. Total phenols content significantly increased, contents were 6.91, 7.07, 7.24, and 7.42 mg/g. The anti-inflammation activity evaluation, we investigated the extracts of Lentinula edodes effect on macrophage celluar-RAW264.7, induced by LPS and produce NO, TNF-a and IL-6 substances. There were four kinds of extracts used in this study, included no irritated aqueous extract (NW), irradiated aqueous extract (IW) , no ethyl acetate extract (NEA) , irritation ethyl acetate extract (IEA). Overall the results of four extracts demonstrated the IW in the concentration of 500 μg/mL, had an excellent inhibition on macrophage to produce NO, stimulated by LPS, and the inhibition ratio was 74.3%.When the concentrations were 1000 and 750 μg/mL, IW had an excellent inhibition on LPS-induced TNF-a production at 24 and 48 hours. The inhibition were 13.43% and 24.91%.When the concentrations was 1000 μg/mL, NW had an excellent inhibition on LPS-induced IL-6 production at 24 hours.The inhibition was 68.05%. When the concentrations was 1000 μg/mL, IW had an excellent inhibition on LPS-induced IL-6 production at 48 hours. The inhibition was 89.04%. According to the results mentioned before, we found that the aqueous layer extracts exhibited better effect than ethyl acetate extracts. Therefore, aqueous layer extract were used in the following experiment. At 20 mg/mL NW and IW, the reducing power were 0.99 and 1.03, whereas, scavenging abilities of aqueous layer extract on DPPH were 45.69% and 51.96%. On the chelating ferrous ions, there was no significant difference between NW and IW. Overall, powder exposed 20 shots of pulsed light could significantly increase vitamin D2 content, which needed 6.7 seconds. In addition, it can also significantly enhanced the content of vitamin D2 and total phenols, and there were no influence on the content of ergosterol and ergothioneine after irritation.The anti-inflammation of irritated aqueous layer extract are better. Accoding to the previous results, we supported that the composition of aqueous layer extracts has been changed after irritation, but the mechanisms is unknown. Therefore, using NMR spectroscopy to clarify the changes of chemical structure on irritated aqueous layer extract could be proceeded in the future.