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Doctor of Philosophy / Department of Human Nutrition / Brian Lindshield / Fortified blended foods (FBFs), in particular, corn-soybean blend (CSB), are food aid commodities widely used in infant and young children supplementary feeding programs. A United States Agency for International Development (USAID) Food Aid Quality Review report recommended developing novel FBFs using local alternative commodities such as sorghum and improving the nutritional quality of FBFs using extrusion processing. Extruded sorghum-cowpea, sorghum-soy and corn-soy FBFs were developed and compared with the non-extruded FBFs corn-soy blend 13 (CSB13) and corn-soy blend plus (CSB+) using the in-vitro digestion/Caco-2 cell model. Dry FBFs’ iron and vitamin A content ranged from 8.0 to 31.8 mg/100g and 0.54 to 1.67 mg/100g, respectively. Following in-vitro digestion, bioavailable iron and vitamin A levels were determined by measuring Caco-2 cell ferritin and vitamin A levels in response to 12-hour and 4-hour treatments, respectively, with aqueous fractions collected from digested FBFs. Most extruded FBFs’ aqueous fraction iron levels were 2- to 7-fold higher (p<0.05) than CSB13 and CSB+. However, Caco-2 cell ferritin and vitamin A levels were not significantly different among FBFs. These results suggest that consumption of newly developed extruded sorghum-cowpea, sorghum-soy and corn-soy FBFs will result in bioavailable iron and vitamin A levels comparable to traditional non-extruded CSB13 and CSB+. Thus, extruded sorghum-cowpea FBF may be a suitable alternative to corn-soybean based FBFs. Saw palmetto supplements are one of the most commonly consumed products by men with prostate cancer and/or benign prostatic hyperplasia (BPH). Some studies have found significant improvements in BPH with saw palmetto supplementation, whereas others found no benefits. The variation in the efficacy in these trials may be a result of differences in the putative active components, fatty acids and phytosterols, of the saw palmetto supplements. We quantified fatty acids and phytosterols in 20 commercially available liquid, powder, dried berry, and tincture saw palmetto supplements. Liquid saw palmetto supplements contained significantly higher (p<0.05) concentrations of total fatty acids (908.5 mg/g), individual fatty acids, total phytosterols (2.04 mg/g), and individual phytosterols, than the other supplement categories. Our findings suggest that liquid saw palmetto supplements may be the best choice for individuals who want to take a saw palmetto supplement.

A crucial step in the drug discovery and development process is the assessment of membrane permeability properties of new chemical entities and researchers are constantly searching for cost-effective, high through-put models with as high as possible predictive value. In addition, a thorough understanding of the membrane permeability pathways and metabolism mechanisms are required when evaluating drug disposition and pharmacokinetics. Various in vitro methods/techniques are available to measure the rate of permeation of compounds across epithelial cell membranes to estimate oral drug absorption in humans. The aim of this study is to compare three in vitro models (i.e. excised rat intestinal tissue, excised pig intestinal tissue and Caco-2 human cell cultures) in terms of drug permeability characteristics by means of different techniques including the Ussing type Sweetana-Grass diffusion chamber apparatus, everted sac glass apparatus and the Transwell® plate apparatus. The transport of abacavir sulphate was determined in two directions (i.e. apical-to-basolateral or AP - BL and basolateral-to-apical or BL - AP) across excised rat intestinal tissue, excised pig intestinal tissue and Caco-2 cell monolayers. The test solution was applied to the donor side and samples (200 μl) were drawn from the acceptor side at 20 min intervals for a period of 2 h. The concentration of abacavir in the samples was then measured by means of a validated high performance liquid chromatography (HPLC) method. The transepithelial electrical resistance (TEER) was measured before and after each transport experiment to give an indication of the integrity of the cell membranes. The apparent permeability coefficient (Papp) and efflux ratio (ER) values were calculated and used to compare the different methods and techniques in terms of drug permeation characteristics. All three of the in vitro methods, in all of the techniques employed, showed higher transport of abacavir in the BL - AP direction than in the AP - BL direction. This indicates that all three in vitro methods had intact active efflux transporters over the entire study period. The excised rat intestinal method showed similar drug permeability characteristics in both techniques compared to that of the Caco-2 cell monolayers. In contrast, the excised pig intestinal method only showed similar drug permeability characteristics in the Sweetana-Grass diffusion apparatus when compared to the Caco-2 cell monolayers. This phenomenon can possibly be explained by the relatively large surface area of the pig tissue used in the everted sac technique where the role of physiological and other factors take effect. These factors may include the thickness of the membrane and mucus layer as well as variables such as diet, age, gender and size of the pigs obtained from the abattoir that cannot be controlled. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2015

A crucial step in the drug discovery and development process is the assessment of membrane permeability properties of new chemical entities and researchers are constantly searching for cost-effective, high through-put models with as high as possible predictive value. In addition, a thorough understanding of the membrane permeability pathways and metabolism mechanisms are required when evaluating drug disposition and pharmacokinetics. Various in vitro methods/techniques are available to measure the rate of permeation of compounds across epithelial cell membranes to estimate oral drug absorption in humans. The aim of this study is to compare three in vitro models (i.e. excised rat intestinal tissue, excised pig intestinal tissue and Caco-2 human cell cultures) in terms of drug permeability characteristics by means of different techniques including the Ussing type Sweetana-Grass diffusion chamber apparatus, everted sac glass apparatus and the Transwell® plate apparatus. The transport of abacavir sulphate was determined in two directions (i.e. apical-to-basolateral or AP - BL and basolateral-to-apical or BL - AP) across excised rat intestinal tissue, excised pig intestinal tissue and Caco-2 cell monolayers. The test solution was applied to the donor side and samples (200 μl) were drawn from the acceptor side at 20 min intervals for a period of 2 h. The concentration of abacavir in the samples was then measured by means of a validated high performance liquid chromatography (HPLC) method. The transepithelial electrical resistance (TEER) was measured before and after each transport experiment to give an indication of the integrity of the cell membranes. The apparent permeability coefficient (Papp) and efflux ratio (ER) values were calculated and used to compare the different methods and techniques in terms of drug permeation characteristics. All three of the in vitro methods, in all of the techniques employed, showed higher transport of abacavir in the BL - AP direction than in the AP - BL direction. This indicates that all three in vitro methods had intact active efflux transporters over the entire study period. The excised rat intestinal method showed similar drug permeability characteristics in both techniques compared to that of the Caco-2 cell monolayers. In contrast, the excised pig intestinal method only showed similar drug permeability characteristics in the Sweetana-Grass diffusion apparatus when compared to the Caco-2 cell monolayers. This phenomenon can possibly be explained by the relatively large surface area of the pig tissue used in the everted sac technique where the role of physiological and other factors take effect. These factors may include the thickness of the membrane and mucus layer as well as variables such as diet, age, gender and size of the pigs obtained from the abattoir that cannot be controlled. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2015

Field pea (Pisum sativum L.) seeds have high nutritional value but also contain phytate which can inhibit the absorption and utilization of nutrients. Phytate is the main storage form of phosphorus in the seeds but chelates Fe, Zn and some other micronutrients and is not well digested by monogastrics. Peas with pigmented seed coats contain polyphenols which also have anti-nutritional properties. To increase the nutritional value of field pea seeds, two low phytate lines (1-150-81 and 1-2347-144) containing higher inorganic phosphorus concentration (IN-P) and lower phytate-phosphorus concentration (PA-P) than the normal phytate varieties were developed from CDC Bronco in previous research. The objectives of this research were 1) to determine the effect of genotype and environment on iron bioavailability in a set of five pea varieties differing in phytate concentration and iron concentration using in vitro digestion/Caco-2 cell culture bioassay; 2) to determine the effect of seed coats on iron bioavailability by testing whole seeds compared to dehulled seeds in varieties differing in seed coat pigmentation using in vitro digestion/Caco-2 cell culture bioassay; 3) to determine the inheritance of iron bioavailability in field pea by evaluating recombinant inbred lines differing in phytate concentration using in vitro digestion/Caco-2 cell culture bioassay; 4) to determine the effects of pea with the low phytate trait on body weight and hemoglobin concentration of chickens. Iron concentration (FECON) did not differ significantly between normal and low phytate varieties. Iron bioavailability (FEBIO) of the two low-phytate lines was 1.4 to 1.9 times higher than that of the three normal phytate varieties, and growing environment also had a significant effect on FEBIO. Peas with pigmented seed coats contained 7 times lower FEBIO than peas with non-pigmented seed coat. The removal of the seed coat increased the FEBIO in peas with pigmented seed coat 5 to 6 times. From previous research on PR-15 recombinant inbred lines (RILs) which were developed from a cross between low phytate line 1-2347-144 and a normal phytate variety CDC Meadow, it was found that PA-P was controlled by a single gene. FEBIO, in this study, was also found to follow a bimodal frequency distribution, characteristic of single gene control, and it was highly correlated with PA-P in the PR-15 lines. In vivo studies were used to evaluate iron absorption of chickens fed with low and normal phytate pea diets. The diets containing the low-phytate pea lines had no significant effect on chicken body weight and hemoglobin level, compared with the diets containing normal phytate pea varieties. An unexpected high FECON was discovered in the diets that was traced to the ingredients of limestone and dicalcium-phosphate which likely affected the experimental results.

Nutriomics is the study of the whole range of nutritional components (nutriome) in foods. In order to further understand the molecular basis for the positive health benefits of fruits identified from epidemiology, the mass balance through the human digestion and absorption should be studied. The components of the nutriome studied in this research were sugars, carotenoids, phenolics and organic acids, all important for defining dietary – human health relationships and linking to evidence obtained from epidemiological studies. An attempt to approach a realistic human mimetic digestion and absorption model has been carried out in this study using a static in-vitro model of the human digestive system. Two major novelities in this model compared to other in-vitro models are (i) the use of particles of solid fruit products that mimic the products of human chewing and (ii) a cell-based (Caco-2) in-vitro intestinal absorption model. Hence, imitative bioavailability, i.e. releasing nutrients and potential levels of target compounds reaching the portal circulatory system could be assessed. The fruits studied were tomato, mango, papaya; each as fresh, dried and juiced forms. In-vivo chewing suggested 0.5 cm size modes for dried products and 1.5 cm for fresh products. The agglomerates that were obtained from the chewing of dried products disaggregated during in-vitro digestions in the presence of acids (gastric simulation) or sodium bicarbonate at pH 6 (small intestinal simulation). The extent of this disaggregation followed the order: tomato > mango > papaya. Although all fresh samples contained separated cells, their responses to a 5 mm texture analysis probe (mimicking teeth cusps) varied depending on fruit products. All matrices were hardened by drying, becoming more brittle and breaking easier to produce smaller size modes. Variation between individual participants in the size of their chewed particles was lower for fresh products and high for dried products. The in-vitro digestion and absorption model developed had simulated particle sizes of approximately 0.5 cm3 for dried products or 1.5 cm3 (thickness varied with the products) for fresh products in a 9:1 ratio mix with blended samples, and were digested in-vitro using the following steps: 1. ‘Chewing’: pH 6.9; 37 C, 10 min, in a shaking-water bath (55 rpm) with human alpha-amylase (100 U/L). 2. ‘Gastric’ digestion: pH 2; 37 C, 60 min, in a shaking-water (55 rpm) with porcine pepsin (40 µg/L). 3. ‘Intestinal’ digestion: pH 6; 37 C, 60 min, in a shaking-water bath (55 rpm) with porcine pancreatin and bile extract (1.4 µg/L and 8.6 µg/L, respectively). 4. Caco-2 cell monolayer in-vitro passages: aged 22 days post confluent monolayers in a 24 transwell-insert well plate seeded at 105 cells, pH 7.4 with renewal of apical and basolateral solutions every 30 min for bioavailability estimations. In this study, two models of basolateral – apical solution renewals were carried out: both apical and basolateral were renewed (model A) and basolateral only was renewed (model B). To study metabolites produced by Caco-2 cells, the bioassays were carried out for 22 h without renewals of apical and basolateral solutions (model C). An overview of nutriomics analysis of in-vitro digestions of mango, papaya and tomato based on principal component analysis (PCA) suggested: (1) fruit types led to variable nutriome releases: in-vitro digestions affected tomato >mango >papaya; (2) processing varied nutriome releases from fruit products with juicing tended to release more nutriome components, whereas drying and unprocessed (fresh) did not show noticeably different patterns; (3) gastric and simultaneous gastric-intestinal digestions were similar in nutriome releases whereas contributions of intestinal digestion alone were negligible for water soluble nutriome components; and overall (4) during in-vitro digestions there were no interactions among releasing nutriome from the fruit products studied (independent nutriome releasing processes). Phenolic components showed molecular changes during in-vitro digestion and processing, due to, heating effects, pH or enzymic degradations. Caco-2 bioassays using model compounds showed a range of monolayer responses as follows: (1) mannitol, lycopene and catechin were strictly retained in the apical solution; (2) sugars, caffeine and atenolol were translocated in the apical-to-basolateral direction as intact molecules; (3) Beta-carotene partially disappeared from the apical solution without basolateral release. Models A – C consistently confirmed these responses. Low recoveries provided evidence for cellular metabolisms of (particularly) phenolic and carotenoid molecules by the Caco-2 cell monolayers.

Tocopheryl Polyethylene Glycol Succinate 1000 (TPGS 1000) can inhibit P-glycoprotein (P-gp); TPGS 1000 was not originally designed to inhibit an efflux pump. Recent work from our laboratories demonstrated that TPGS activity has a rational PEG chain length dependency. In other recent work, inhibition mechanism was investigated and appears to be specific to the ATPase providing P-gp energy. Based on these observations, we commenced rational surface-active design. The current work summarizes new materials tested in a validated Caco-2 cell monolayer model; rhodamine 123 (10 μM) was used as the P-gp substrate. These results demonstrate that one may logically construct non-ionic surfactants with enhanced propensity to inhibit in vitro efflux. One new surfactant based inhibitor, Tocopheryl Polypropylene Glycol Succinate 1000 (TPPG 1000), approached cyclosporine (CsA) in its in vitro efflux inhibitory potency. Subsequently, TPPG 1000 was tested for its ability to enhance the bioavailability of raloxifene - an established P-gp substrate - in fasted male rats. Animals dosed with raloxifene and TPPG 1000 experienced an increase in raloxifene oral bioavailability versus a control group which received no inhibitor. These preliminary results demonstrate that one may prepare TPGS analogs that possess enhanced inhibitory potency in vitro and in vivo.

Los cereales son empleados como dieta complementaria a la lactancia materna a partir del 4º mes ya que suministra nutrientes esenciales (especialmente hidratos de carbono, proteínas, minerales y vitaminas (particularmente tiamina). Los alimentos infantiles elaborados a partir de harinas procedentes de cereales pueden presentar compromiso en la biodisponibilidad de determinados minerales, por ello, es de gran importancia tratar de establecer los tratamientos tecnológicos necesarios para que la utilización de estos nutrientes esenciales sea la máxima. Se ha comprobado que el método más sencillo y eficaz de conseguir la eliminación del ácido fítico, es la adición de fitasa exógena. / Dietary minerals intake is of interest of human beings in general, but particularly for infant and young children in the first year of life, when growth is accelerated. Insuficient mineral intake in this period, mainly of iron, calcium and zinc, is responsible for diseases such as anaemia, rickets, osteoporosis or inmune diseases. Cereals are introduced to infants at the age of four to six months to supplement breastmilk and follow-on formula, since this is a period of rapid growth and development. Phytic acid (myoinositol hexa-phosphoric acid, IP6) is the major phosphorus storage compound of most seeds and cereal grains, and it has a strong ability to chelate multivalent metal ions, specially iron, zinc, and calcium.Based on this knowledge, complete phytate degradation by means of technological treatments is desirable, in order to overcome its negative effects on mineral bioavailability. Food processing-such as cooking, bread-making and fermentation-is known to reduce phytic acid content. However, the bioavailability of minerals can be considerably increased by dephytinization adding an exogenous phytase.

M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Obesity has steadily increased and represents a major public health problem worldwide, reducing quality of life and causing a range of health problems. Obesity has emerged as the fifth leading risk of global deaths. Annually, 2.8 million adults die as a result of being overweight or obese. The increase of obesity remains inexplicable in terms of genetic susceptibility to obesity. The genetic loci identified by genome-wide association studies (GWASs) explains about 2% of the heritability for obesity. Perhaps other factors such as epigenetics may be involved in the increase of obesity and may offer solutions for the management of obesity. Epigenetics is defined as a heritable change in gene expression without altering the genome sequences. It may help in providing a logical explanation between the genome and environment which shapes obesity risk and may help to explain the "missing heritability". Epigenetics may affect two mechanisms, namely: i) DNA methylation,and ii) histone modifications. DNA methylation might give scientists a link to the rise in obesity.The study aimed to investigate the effect of sugars used as sweeteners in sugar-sweetened beverages (SSB) on DNA methylation in a Caco-2 cell line in vitro. Four major objectives were pursued in the study which were to:(1) stimulate the Caco-2 cells with varying concentrations of sugar sweeteners and assess the morphological changes of the cells; (2) evaluate the cytotoxicity of different concentrations of the sugar sweetener on the Caco-2 cell line using the Alamar blue and LDH assay; (3) obtain genomic DNA from the treated Caco-2 cell line and perform bisulfite conversion and rest; and (4) amplify the WT1, MEG3, TNFRSF9, ATP10A, and CD44 obesity-associated genes and ascertain their degree of methylation. Caco-2 cells were stimulated with sugar sweeteners at varying concentrations (low, medium and high) for an incubation period of 62 days,and images of the cells were captured for morphological characterisation. The incubation condition entailed cells plated in a 12 or 96 well plate, incubated in a humidified 5% CO2 incubator at 37 °C and there is nutrient renewal every three days.Alamar blue, a cell proliferation colourimetric assay and lactate dehydrogenase assays (LDH), a homogenous membrane fluorimetric assay were used for the cytotoxicity studies. The results of the characterisation showed that different concentrations of sugar sweeteners affected the morphology of the cells as the incubation period progressed. The cytotoxicity results of both LDH and Alamar blue depicted low concentration of sweeteners that had low-to-moderate toxicity and the medium and high concentration of the sweeteners had a moderate to high toxicity on the Caco-2 cells. DNA from the Caco-2 cells was extracted. Techniques used to study DNA methylation such as bisulfite conversion, PCR amplification and restriction enzymes that have differential sensitivity to 5-methyl-cytosine were performed. The quality of DNA extracted was good. The bisulfite conversion was conducted andno amplification was observed, as a contingency plan Normal PCR was performed to amplify the CpG islands, and there was amplification. In conclusion, the study showed that a low concentration of a sugar sweetener (fructose: glucose) used in beverages had low toxicity to the Caco-2 cell line and prolonged exposure of the low concentration might have an adverse effect on the cells' morphology. At medium concentrations, the sugar sweetener used in beverages had medium toxicity to Caco-2 cells; prolonged exposure may lead to morphological changes. These findings indicated that control of dietary glucose intake is an important strategy in combating the development of obesity and type-2 diabetes. DNA methylation could not be established.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Martin Huličiak Supervisor: PharmDr. Lukáš Červený, Ph.D Title of diploma thesis: Study of interactions of antiviral drugs with intestinal drug efflux ABC transporters P-gp, MRP2 and BCRP are efflux transporters, members of the family of ATP binding cassette (ABC) transporters. These transporters are located on the apical membrane of the intestinal epithelium, where they may limit absorption of orally administered drugs. Study of drug interactions with/on intestinal efflux transporters is necessary to provide safe and effective treatment. The Caco-2 cell line is FDA recommended in vitro model of intestinal barrier and it is used for bidirectional testing of substrates and inhibitors of ABC transporters in preclinical research. However, this methodology has several shortcomings, so the need of introduction of new experimental models is increasing and the ex vivo method based on human or rat intestine is a promising option. Precision-cut intestinal slices (PCIS) represent a mini-model of the organ and contain all types of cells of the tissue. We used both in vitro model using Caco-2 cell monolayers for drug transport study and in our lab established ex vivo method of PCIS for accumulation study...