Forty-eight rat pups were handled and isolated from postnatal days 3 through 13 in order to determine whether this manipulation would alter the postnatal development of the hippocampus. Half of these animals were then reared in enriched environments from weaning until maturity to determine whether enrichment would ameliorate the expected deficits in learning ability. Beginning at 90 days of age, all animals were tested on a T-maze, rotating bar and both place and cued versions of a water maze task. The study failed to find gross deficits in learning as a result of the handling/isolation procedure, although emotional differences between groups was evident, as were sex differences. Apparently more questions have been raised than answered by this study, and possible directions for future research are discussed.

The effects of exercise training and iron intake on iron metabolism in adult female rats were investigated. Adult female Sprague-Dawley rats were assigned to either an exercise (E) or sedentary (S) group and fed either a diet containing 9 ppm (9) of dietary iron (low iron level), or 40 ppm (40) of iron (a level slightly above the National Research Council recommendations). The exercise animals were subjected to a program of swimming, 5 days/week, over a 6- week period. Total food intake and final body weight were similar between the E and S groups. In both 40-E and 9-E animals, concentration of serum iron was significantly (P<0.05) lower while total iron binding capacity was significantly elevated, when compared to sedentary counterparts. Saturation of transferrin was significantly reduced in the 9-E group. Liver and spleen weights did not differ but significant increases in cardiac weights were noted in both E groups. Gastrocnemius muscle weights were similar in both E groups and 9-S, but significantly lower in the 40-S group. In organ tissues, liver iron concentration was significantly reduced in the 9-E animals, while spleen iron level was highest in the 40-E group. Cardiac iron concentration was significantly reduced in both E and low iron diet groups while levels of iron in gastrocnemius muscle did not differ among experimental groups. In both groups of exercised rats, bone marrow iron was significantly lower when compared to sedentary animals. In response to exercise training, an increase in skeletal muscle citrate synthase activity was observed in both E groups. This study suggests that exercise affects various parameters of iron metabolism. Regardless of iron intake, physical training appeared to alter distribution of iron stores, that may be associated with alterations of hematological iron transport and iron-containing proteins. The Combination of a low iron intake and intense exercise training appeared to enhance early characteristics of a latent iron deficiency. / Ph. D.

Thesis submitted in fulfilment of the requirements for the degree Doctor of Technology: Biomedical Technology in the Faculty of Health and Wellness Sciences at the Cape Peninsula University of Technology 2014 / According to the World Health Organization, cancer is the leading cause of death in the developed world, while it is the second leading cause of death in the developing world. In particular, liver cancer is the fifth most commonly diagnosed cancer in men, however, it is the second most frequent cause of death, responsible for an estimated 700,000 deaths annually. General limited access to health services, including treatment and the overall management of cancer in developing countries often contribute to the increased mortality rates when compared to developed countries. For centuries, medicinal plants have been used to prevent, and to a certain extent, treat cancer as a readily available and affordable alternative. In many instances, the curative or preventative claims still remain anecdotal. However, increasing evidence suggest that polyphenolic components of plants possess antioxidant activities, which are credited with curative/beneficial properties of medicinal plants. The curative properties could either be related to the primary compounds present in the plant itself, or the bio-activation products of plant components affecting hepatic drug metabolising and antioxidant enzymes systems related to carcinogen metabolism and maintaining oxidative homeostasis, respectively. Similarly, chronic consumption of medicinal plants could also result in hepatotoxicity, either caused by the primary plant components or bio-activation products. Due to these observations it is paramount to understand the mechanisms involved in the metabolism of plant components to critically assess beneficial versus potential harmful properties associated with chronic consumption. The focus of the current study was aimed at elucidating the bio-activity of four multipurpose indigenous plants to Southern Africa, i.e. Adansonia digitata, Agathosma betulina, Siphonochilus aethiopicus and Myrothamnus flabellifolius. Traditionally, A. digitata has been used as an immunostimulant, anti-inflammatory and analgesic agent, while also as an antipyretic agent in the treatment of diarrhoea and dysentery. Similarly, traditional medicinal uses of A. betulina include treatment cholera, haematuria, calculus, kidney diseases, as well as infections of the bladder, urethra, and prostate among others. S. aethiopicus was traditionally employed to treat infections associated with pains and fevers, whereas M. flabellifolius served as treatment of conditions ranging from respiratory ailments, backache, kidney problems, haemorrhoids, chest pain, and asthma. In the first part of this study, the polyphenolic contents and antioxidant capacities of the four plants were characterised. The emphasis was placed on using different solvents, namely water, ethanol and acetone for the extraction of the plant material and different methodologies to assess the antioxidant contents and -capacities of the various extracts as both these factors can influence the outcome. When considering the antioxidant contents, total polyphenols, flavanols, and flavonols of the different solvent extracts prepared from the four plants were determined, whereas three different assays were used for the antioxidant capacities, i.e. oxygen radical absorbance capacity (ORAC), trolox equivalent antioxidant capacity (TEAC) and ferric-reducing antioxidant power (FRAP) assays. The A. digitata acetone extract had the highest (7.121 mg gallic acid equivalent (GAE)/milligram (mg) soluble solids), whereas the water extract of the same plant had the lowest total phenolic content (0.008 mg GAE/mg soluble solids). In general, the acetone extracts demonstrated the highest total polyphenol, flavanol, and flavonol contents, followed by the ethanol extracts, with the water extracts having the lowest contents. M. flabellifolius was the only distinct deviation from this rule, where the water extract demonstrated the highest total polyphenol content. Considering antioxidant capacities, the acetone extracts provided the highest antioxidant capacities for all plants when assessed using the TEAC (8.56-32.68 milimole (mmole) trolox equivalent (TE)/mg soluble solids) and FRAP (5.69-37.39 mmole ascorbic acid equivalent/mg soluble solids) antioxidant assays, with the exception of M. flabellifolius where the water extract demonstrated the highest activity (22.73 mmole ascorbic acid equivalent/mg soluble solids). Antioxidant capacity determinations with TEAC and FRAP assays followed similar patterns, which were different from capacities determined by the ORAC (0.46-533.54 mmoleTE/mg of soluble solids) assay. Corroborating the antioxidant content findings, the acetone extracts also demonstrated the highest antioxidant capacities (140.41-533.54 mmoleTE/mg of soluble solids), followed by ethanol (94.62-151.29 mmoleTE/mg of soluble solids) and water (0.46-134.02 mmoleTE/mg of soluble solids). Only M. flabellifolius (TEAC and FRAP) and S. aethiopicus (FRAP) deviated from this trend. Correlations between the polyphenolic contents and antioxidant capacities indicated that acetone and ethanol were more effective in extracting polyphenolic compounds than water, while also providing extracts with superior antioxidant activities. Furthermore, ORAC assay was the antioxidant capacity determining assay of choice for the aqueous plant extracts, whereas the TEAC and FRAP assays were more suitable when determining the antioxidant capacities of the acetone and ethanol plant extracts. These results confirm the notion that no single assay can comprehensively determine the antioxidant activities of plant extracts and that a battery of assays should be used, as the various antioxidant capacity determination techniques use different substrates with different targets for measurement. The second part of this study comprised an in vivo experimental animal model to assess the potential toxicity, antioxidant status and modulation of the hepatic phase 2 drug metabolising enzymes following chronic consumption of the various plant extracts in male Fisher rats. Rats consumed aqueous extracts of the various plants (2% and 5% (w/v)) as the sole source of drinking fluid for 90 days, and the serum chemical pathology parameters for monitoring liver and kidney function conducted. These included alkaline phosphatase (ALP), aspartate transaminase (AST), alanine transaminase (ALT), total iron (Fe), and creatinine (CREA). Parameters for blood and hepatic redox status included total polyphenols, ORAC, reduced glutathione (GSH), oxidised glutathione (GSSG), their ratio (GSH:GSSG), conjugated dienes (CD) and thiobarbituric acid reactive substances (TBARS). Assessment of the phase 2 hepatic xenobiotic metabolising enzymes included glutathione S-transferase (GST)  and activity in the cytosolic fraction and, UDP-glucuronosyltransferase (UDP-GT) activity in liver microsomes. When considering the liver and kidney function none of the plant extracts induced any significant toxicity, while 2% A. digitata significantly increased serum Fe. When considering the redox status, the whole blood and liver samples yielded similar results, with significant decreases in oxidised glutathione (GSSG) in rats consuming the 2% M. flabellifolius (82.76 mole/L) and 5% A. digitata (90.42 mole/L) with a resultant significant increase in the glutathione redox status (GSH:GSSG ratio of 5.69 and 5.64, respectively) when compared to rats consuming water (4.77). The GSH:GSSG ratio was also significantly increased by consumption of 2% A. betulina (8.45) and 5% S. aethiopicus (5.99). The consumption of all plant extracts, except 5% A. betulina and M. flabellifolius, significantly increased lipid peroxidation in the plasma CDs assay. These results indicated an increased antioxidant capacity in the liver with/without an associated reduced cellular oxidative stress status, which could be interpreted as a reduced susceptibility to oxidative damage. When considering the phase 2 hepatic enzymes, none of the plant extracts caused any significant changes in GST, GST or UDP-GT activities. The third part investigated the chemoprotective properties against cancer promotion in the liver utilising diethylnitrosamine (DEN) as cancer initiator and maize culture material of Fusarium verticillioides, containing the fumonisin B mycotoxins, as promoters in male Fischer rats. The rats consumed 2% (w/v) aqueous extracts of A. digitata, A. betulina, and S. aethiopicus over 28 days after cancer initiation and liver sections subjected to glutathione-S-transferase placental form positive GSTP+ staining and pre-cancerous liver foci categorised according to size. In addition, blood and liver analyses were done as described in the chronic feeding study above. Consumption of the A. digitata and, to a certain extent, S. aethiopicus extracts, altered the oxidative stress status in the liver as indicated by the increased lipid peroxidation, as determined by significantly increased liver CDs and the decreased GSH:GSSG ratio in the blood. This can be related to a subchronic toxicity due to the high total polyphenol intake as mentioned above. These underlying sub chronic toxic effects of A. digitata and S. aethiopicus are likely to be responsible for the observed inhibitory effect on the proliferation of GSTP+ minifoci in the liver. Hepatic phase 2 metabolising enzyme activities were not significantly altered by A. digitata and S. aethiopicus consumption, while GST activity was significantly increased by A. betulina treatment. Based on the findings of the current study, aqueous extracts of A. digitata, A. betulina, and S. aethiopicus may serve as hepatoprotectors with a potential to modulate liver carcinogenesis, specifically cancer promotion. To our knowledge, no other studies have attempted to describe the possible chemoprevention mechanisms of these indigenous medicinal plants. Assessments of phase 1 hepatic enzymes and other antioxidant enzymes are suggested for future studies to further describe biochemical and molecular mechanisms associated with consumption of these extracts. Additionally, identifying main compounds present in the plant extracts could culminate in development of drugs and novel nutraceuticals. It is also recommended that increasing concentrations of the plant extracts and/or the ethanol extracts to be used in future studies to better describe dose-responses of the different plants in liver carcinogenesis.

To study the effect of exercise and protein intake during pregnancy on maternal and fetal zinc status in the rat, one hundred and seventy-nine pregnant Sprague-Dawley rats were divided into four groups; sedentary-standard protein diet, sedentary-high protein diet, exercising-standard protein diet and exercising-high protein diet. The standard protein diet contained 7.22% protein, while the high protein diet contained 24.77% protein; all other nutrients were supplied in amounts required for normal parturition of the laboratory rat. After acclimatization, the exercising dams, regardless of diet, were made to swim continuously for one and one-half hours/day until sacrifice. The four major groups were further subdivided into 28 groups, designated by three-day intervals according to gestational day--days 3, 6, 9, 12, 15, 18, and 21. Uterine tissues were retained for zinc content analysis; fetal and placental tissues were separated from uterine tissue for days 15 through 21 only. The concentration of uterine zinc was affected solely by gestation; absolute placental tissue zinc values were lowest in the sedentary-high and exercising-low protein groups, while the exercising-high protein group possessed the greatest zinc value. No significant difference was detected in fetal zinc concentrations. Fetal tissue from exercising dams weighed significantly less (p<0.05) than fetal tissue from the sedentary dams; and sedentary-high protein dams produced significantly more (p<0.05) fetuses than the exercising-high protein dams. Both protein intake and exercising during pregnancy significantly affect normal parturition and zinc metabolism in the rat. / M.S.

The effect of two levels of dietary protein and exercise on iron metabolism in pregnant Sprague-Dawley rats was studied. Animals were assigned to the following diet and exercise groups on the first day of gestation: high protein sedentary (HS), high protein exercise (HEx), low protein sedentary (LS), low protien exercise (LEx). Animals in the exercise groups swam continuously for 75 minuites the first day and 90 minutes daily thereafter, throughout the study. Hemoglobin, hematocrit, liver iron concentration and spleen iron concentration were measured at day 0, 3, 6, 9, 12, 15, 18, and 21 of gestation. Mean hemoglobin, hematocrit and liver iron concentration values were lower at day 21 than at day 0 of gestation. Mean hematocrit and hemoglobin for the LEx group were the lowest for days 9 through 15. At day 15 the mean hematocrit for the LEx group was significantly (p < 0.05) lower than that of the other groups. The HEx group had the highest hematocrit and hemoglobin values at day 21 of gestation; hemoglobin was significantly (p < 0.05) higher. No difference in mean spleen iron concentration from day 0 to day 21 was found, however, the low protein groups had higher spleen iron values early in pregnancy that the high protein groups. The mean spleen iron concentration of the LEx group was significantly (p < 0.05) higher than those of the HS and HEx groups at day 6. A trend for higher liver iron concentration values of the low protein groups than high protein groups was also observed. The LEx group had a significantly (p <0.05) higher mean liver iron concentration at day 18 than the other groups. Both protein nutriture and exercise appear to affect iron metabolism in pregnant rats. / M.S.