Ovarian cancer (OvCa) is the most fatal gynecological malignancy, with over 21,290 new cases diagnosed in 2015. The 5 year survival rate for patients diagnosed at Stage IV is 17% in the United States. It has been reported that estrogen (E2) can promote metastasis of ovarian tumor cells through its nuclear receptor (ER). E2 is a potent mitogen and can stimulate the growth and invasion of ovarian cancer cells. We have previously shown that flaxseed diet decreases the severity and incidence of OvCa in hens, the only animal model that develops OvCa spontaneously and the disease is pathologically and histologically similar to the human disease.. While it is well established that the n-3 polyunsaturated fatty acids from flaxseed are anti-inflammatory the phytoestrogenic properties of its lignan, secoisolaricirescinol diglucoside (SDG) have not been fully explored. SDG metabolites, enterolactone (ED) and enterodiol (EL) can decrease ER signaling by competing for binding sites with E2, potentially decreasing expression of E2 target genes involved in proliferation and survival. Our goal was to analyze the effect of flaxseed diet on E2 signaling and metabolism in the normal and cancerous ovarian tissues. We hypothesized that due to the phytoestrogenic properties of the flax lignan, flaxseed diet can affect the downstream signaling of ER there by altering the expression of its target genes. In the 3 year old pre-neoplastic hen ovaries, 15% flaxseed supplemented diet was most effective in decreasing the expression of ER and its target genes like IGFBP4, IGFBP5, IRS1 that are a part of the IGF signaling pathway and are also implicated in a significant number of malignancies. Whole flaxseed diet also decreased the expression of the anti-apoptotic protein, BCL2L1 possibly by reducing the activation of the NFkB pathway. Ovarian tumors from 4 year old hens appeared to over-express estrogen receptor along the glandular area of the tumor but not the stroma. In the 3 year old pre-neoplastic hen ovaries, whole flaxseed and its components, defatted flax meal and flax oil decreased AKT2 mRNA expression but did not affect its activation. Another major regulator of intracellular signaling, ERK 1/2 MAPK appeared to be upregulated in tumors from flaxseed fed hens. Whole flaxseed diet was also significant in altering the metabolism of E2. This was suggested by the increased 2-hydroxyestrone/16-hydroxyestrone (2OHE/16OHE) ratio (a marker for reduced risk of cancer), in the serum from normal hens that were fed 15% flaxseed. It was also demonstrated that whole flaxseed and its components led to a significant decrease in CYP1B1 expression, an enzyme frequently upregulated in cancers, in the hen ovarian tumor tissues. Levels of the pro-apoptotic and anti-proliferative metabolite, 2-methoxyestradiol were significantly increased with whole flaxseed diet in the serum from 3 year old (pre-neoplastic hens) as well as 4 year old hens (cancerous and normal hens). These results demonstrated that whole flaxseed had a more significant effect in decreasing the expression of the cancer implicated end-points and increasing the levels of protective metabolites in comparison to either of its components, individually. Mechanistic studies with the in vitro model using the BG1FR ovarian cancer cells indicated that that 2-methoxyestradiol could induced apoptosis with a parallel increase in p38 activation. Since we observe a similar correlation between 2-methoxyestradiol and p38 in vivo, we believe that flaxseed diet maintains an anti-proliferative, anti-inflammatory and pro-apoptotic ovarian microenvironment by increasing 2-methoxyestradiol levels.

Flaxseed is an excellent source of bioactive compounds, alpha-linolenic acid (ALA) and secoisolariciresinol diglucoside (SDG), which when added to food products enhance their functionality. However, when flaxseed is added to foods, overall acceptability and sensory properties can be affected, which may negatively affect the role of functional food in providing health benefits. This study was designed to determine the effects of adding milled flaxseeds (20g or 30g) and storage (1 month and 6 months) at - 200C in banana and cinnamon muffins on the sensory properties of muffins using both consumer acceptability evaluation and descriptive analysis, physical and chemical measurements, and ALA and SDG concentrations. Results were correlated using partial least square (PLS) to provide an explicit demonstration of association between overall acceptability of muffins and results from various measurements conducted. It was revealed that the addition of flaxseed reduced overall acceptability due to the enhancement of flax aroma and flavor, oil aroma and flavor, sour aroma and taste, brown color, and firmness., while storage had no impact. The negative influence of these sensory attributes was addressed by the addition of flavorings. Cinnamon flavoring reduced the presence of oil aroma and flavor, and sour aroma and taste while firmness decreased when pureed banana was added. But the intensities of flax aroma and flavor, and brown color were still pronounced in muffins even after the addition of the two flavorings. ALA and SDG concentrations were markedly increased in muffins when flaxseed was added. Storage did not significantly affect the levels of ALA in all muffins but prolonged storage increased SDG level, which can be associated with its role in enhancing extraction proficiency of SDG from the muffin matrix. Although results revealed that acceptability of muffins was significantly higher among clinical trial participants, the mean liking values had moderate deviation between consumers and clinical trial participants. This indicated that muffins fortified with flaxseeds can be acceptable by consumers with diverse wants. / May 2016

The overall goal of this research was to develop a plant protein-based microcapsule capable of carrying, protecting and delivering flaxseed oil within the food and gastrointestinal environment. Specifically, the research aimed to: a) screen a variety of plant proteins and pre-treatment conditions based on their emulsifying properties for use as a wall material; b) develop and optimize encapsulation protocols for entrapping flaxseed oil; and c) study the oxidative stability and delivery of entrapped oils from capsules under different environmental and simulated gastrointestinal conditions. In Chapter 3 and 4, the emulsifying and physicochemical properties of legume and oilseed protein isolates, respectively produced from isoelectric precipitation and salt extraction were investigated. Findings in Chapter 3 indicated that both the legume source and method of production showed significant effects on the emulsifying and physicochemical properties of chickpea (ChPI), faba bean (FbPI), lentil (LPI), pea (PPI), and soy (SPI) protein isolates. The emulsion capacity (EC) values ranged between 476-542 g oil/g protein with LPI showing the highest capacity. Isoelectric-precipitated ChPI and LPI displayed higher emulsion activity index (EAI) (~46.2 m2/g), (emulsion stability index) ESI (~84.9 min) and (creaming stability) CS (98.6%), which were comparable to those of SPI. In Chapter 4, findings indicated that both protein source and method of production had significant effects on the physicochemical and emulsifying properties of canola (CaPI) and flaxseed protein isolates (FlPI). CaPI showed significantly higher EC (~515.6 g oil/g protein) than FlPI (~498.9 g oil/g protein). EAI for FlPI was found to be higher (~40.1 m2/g) than CaPI (~25.1 m2/g) however, ESI values of CaPI and FlPI were similar. Creaming stability of emulsions stabilized by CaPI and FlPI ranged between 86.1 and 96.6%. CaPI and FlPI were shown to have emulsion forming properties; however their stability was low. In Chapter 5, ChPI and LPI-stabilized emulsions were optimized based on pH, protein concentration and oil content for their ability to form and stabilize oil-in-water emulsions using response surface methodology. Droplet charge was shown to be only affected by pH, while droplet size and creaming index were affected by protein concentration, oil content and pH. Optimum conditions for minimal creaming (no serum separation after 24 h), small droplet size (<2 μm), and high net droplet charge (absolute zeta potential (ZP) value >40 mV) were identified as: 4.1% protein, 40.0% oil, and pH 3.0 or 8.0, regardless of the plant protein used for emulsion preparation. Flaxseed oil was microencapsulated by freeze (Chapter 6) or spray (Chapter 7) drying employing ChPI or LPI and maltodextrin. Effects of emulsion formulation (oil, protein and maltodextrin levels) and protein source (ChPI vs. LPI) on the physicochemical characteristics, oxidative stability, and release properties of the resulting capsules were investigated. Optimized capsule designs were found to have high encapsulation efficiencies, low surface oil, and afforded protection against oxidation over a 25 d room temperature storage study relative to free oil. Microcapsules were also able to deliver 84.2% of the encapsulated oil in the simulated gastrointestinal environments.

Basic and clinical research documents the benefits of dietary milled flaxseed (MFX), a rich source of alpha-linolenic acid (ALA) and lignans, in the attenuation of risk factors key to regulating cardiovascular disease (CVD) progression. ALA has antihypertensive properties and the lignan metabolites, enterodiol (END) and enterolactone (ENL), have antioxidative potential. The effectiveness of these bioactives to reduce risk factors of CVD may be dependent upon their plasma concentrations. To study this, we first designed and validated a method using supported liquid extraction and gas chromatography/mass spectrometry to isolate and quantify enterolignans in plasma. Applying this technique, we examined MFX doses of 10-40 g/d administered to healthy, younger adults (18-49 years of age) for 4 weeks. Ten g/d was sufficient to significantly increase circulating ALA (1.5 fold) and enterolignans (5-31 fold). There was no significant dose-dependent response. In another investigation, younger (18-29 years of age) and older (45-69 years of age) healthy adults were studied to determine if age influenced enterolignan metabolism. CVD is associated with advanced age but older people may not be able to obtain lignan metabolites from dietary MFX. Following 4 weeks of MFX consumption, both age groups increased plasma total enterolignans (END + ENL) with no between-group differences. This suggested that older and younger adults metabolize MFX lignans equally. A final study assessed MFX bioactives in plasma of peripheral artery disease patients >40 years of age. Plasma enterolignans increased 10-50 fold and ALA 1-2 fold after only one month of MFX ingestion. Dietary MFX also attenuated total (11%) and LDL (15%) cholesterol in these patients after 1-6 months of administered MFX compared to placebo. The attenuation in cholesterol was due to the high fiber content of flaxseed, and not to ALA and enterolignans, despite their marked increase in circulation. MFX did not interfere with cholesterol-lowering medications but instead decreased cholesterol levels beyond the effects of medications alone. To conclude, dietary supplementation with MFX resulted in an increase in plasma enterolignan and ALA concentrations in healthy younger and older adults and in patients with pre-existing CVD. The cholesterol-lowering benefits of MFX were additional to cholesterol-lowering drugs and likely attributed to MFX fiber. / May 2016

Oral consumption of flaxseed improves serum lipid parameters, and the flaxseed lignan, secoisolariciresinol diglucoside (SDG) may mediate these effects. SDGs therapeutic potential cannot be fully realized until its mechanism of action and pharmacokinetics are more completely characterized.</p>This research aimed to assess SDGs effects in dietary models of hypertriglyceridemia, hypercholesterolemia, and obesity. Furthermore, this thesis work provided preliminary pharmacokinetic data on SDGs aglycone form, secoisolariciresinol (SECO). Dietary manipulations were used to induce hyperlipidemic states in female Wistar or male Sprague-Dawley rats. Groups of 10 rats were randomly assigned to one of three (obesity and cholesterol diets) or four (fructose) treatment groups: 1) Normal diet with 0.0 µmol SDG/kg body weight; 2) Dietary manipulation with 0.0 µmol SDG/kg; 3) Dietary manipulation with 4.4 µmol SDG/kg; and 4) 10% fructose in water with 8.8 µmol SDG/kg. Lignan or vehicle (saline) was administered daily by oral gavage for four weeks (2 weeks for male Sprague-Dawleys). After four (or two) weeks of SDG administration, body and liver weights were recorded, serum lipids were measured using enzymatic kits, hepatic fat accumulation was determined by histochemical analysis and hepatic mRNA expression of triglyceride pathway targets was evaluated using real-time RT-PCR. A 10% fructose in water model was effective for the induction of hypertriglyceridemia in male Sprague-Dawley rats but ineffective in female Wistar rats of similar age. Neither 4.4 nor 8.8 µmol SDG/kg improved serum and hepatic triglyceride parameters in male Sprague-Dawley rats on a 10% fructose in water diet. It is suspected that gender and strain are important factors for this model of hypertriglyceridemia. Dietary manipulations for the induction of hypercholesterolemia and obesity in female Wistar rats were not effective following 4 weeks administration of a 1% cholesterol diet and a 45% fat diet respectively. Since previous studies were able to successfully induce hypercholesterolemia in the same model, it is suspected that the differences in age of the animals accounted for the inconsistent results. Strain, gender and age of animals were identified as important considerations when trying to induce hyperlipidemic states through dietary manipulations.</p>SDG (4.4 µmol/kg) dosed daily for four weeks caused no gross morphological organ changes or alterations in blood chemistry or hematology parameters. Following an intravenous bolus (10 mg/kg), secoisolariciresinol (SECO) disposition was consistent with two-compartment pharmacokinetics, with distribution and elimination half-lives at 26 seconds and 5 minutes, respectively. No SECO was detected in the plasma following an oral bolus (10 mg/kg). Further investigation into SDGs hypolipidemic effects are required to elucidate its mechanism of action. A complete pharmacokinetic study is warranted to fully understand SDGs safety and efficacy.

Flaxseed (FS) is a rich source of lignans and n-3 polyunsaturated fatty acids (PUFA), compounds that may help preserve normal bone cell function during aging. Understanding the effect of FS alone or combined with lower doses of estrogen therapy on bone and other estrogen-responsive tissues (e.g. uterus) is of particular interest to postmenopausal women who combine dietary bioactives (e.g. FS) with pharmacological agents (e.g. estrogen monotherapy) to attenuate postmenopausal bone loss. The overall objective of my research was to determine the effects and mechanisms of FS, alone or combined with lower doses of estrogen therapy, on bone and uterus health by measuring a comprehensive set of outcomes in the ovariectomized rat model of postmenopausal osteoporosis. The results demonstrated that FS enhances the protective effect of low-dose estrogen therapy (LD) on vertebral bone mineral density (BMD), three-dimensional microarchitecture and strength in ovariectomized rats. Moreover, FS exerts a stronger effect on bone outcomes when combined with LD than when combined with ultra-low-dose estrogen therapy (ULD). These studies also showed that FS feeding results in higher lignans and n-3 PUFAs in vertebrae, tibias and femurs. Histological analyses at the lumbar vertebra (LV) showed that there were no differences in TRAP-5β, CTX, or OPG/RANKL ratio between the FS+LD and LD groups. FS+LD did however result in lower protein expression of osteocalcin, a marker of bone formation and overall bone turnover, and higher expression of OPG compared to the negative control (NEG), while LD did not. While these findings suggest that FS+LD results in greater attenuation of deterioration of bone tissue compared to LD due to a reduction in bone turnover, significant differences between FS+LD and LD were not observed. Elucidating these specific mechanisms of action require further investigation. In the uterus, FS+LD did not induce greater cell proliferation or differences in qualitative indices of uterine morphology compared to LD. These findings suggest that there may be no increase in the risk of endometrial hyperplasia and carcinoma with FS+LD compared to LD. These findings may lead to the development of strategies that combine food bioactives and current pharmacological agents to more effectively normalize bone turnover during aging.

Flaxseed (FS) is a rich source of lignans and n-3 polyunsaturated fatty acids (PUFA), compounds that may help preserve normal bone cell function during aging. Understanding the effect of FS alone or combined with lower doses of estrogen therapy on bone and other estrogen-responsive tissues (e.g. uterus) is of particular interest to postmenopausal women who combine dietary bioactives (e.g. FS) with pharmacological agents (e.g. estrogen monotherapy) to attenuate postmenopausal bone loss. The overall objective of my research was to determine the effects and mechanisms of FS, alone or combined with lower doses of estrogen therapy, on bone and uterus health by measuring a comprehensive set of outcomes in the ovariectomized rat model of postmenopausal osteoporosis. The results demonstrated that FS enhances the protective effect of low-dose estrogen therapy (LD) on vertebral bone mineral density (BMD), three-dimensional microarchitecture and strength in ovariectomized rats. Moreover, FS exerts a stronger effect on bone outcomes when combined with LD than when combined with ultra-low-dose estrogen therapy (ULD). These studies also showed that FS feeding results in higher lignans and n-3 PUFAs in vertebrae, tibias and femurs. Histological analyses at the lumbar vertebra (LV) showed that there were no differences in TRAP-5β, CTX, or OPG/RANKL ratio between the FS+LD and LD groups. FS+LD did however result in lower protein expression of osteocalcin, a marker of bone formation and overall bone turnover, and higher expression of OPG compared to the negative control (NEG), while LD did not. While these findings suggest that FS+LD results in greater attenuation of deterioration of bone tissue compared to LD due to a reduction in bone turnover, significant differences between FS+LD and LD were not observed. Elucidating these specific mechanisms of action require further investigation. In the uterus, FS+LD did not induce greater cell proliferation or differences in qualitative indices of uterine morphology compared to LD. These findings suggest that there may be no increase in the risk of endometrial hyperplasia and carcinoma with FS+LD compared to LD. These findings may lead to the development of strategies that combine food bioactives and current pharmacological agents to more effectively normalize bone turnover during aging.

Oral consumption of flaxseed improves serum lipid parameters, and the flaxseed lignan, secoisolariciresinol diglucoside (SDG) may mediate these effects. SDGs therapeutic potential cannot be fully realized until its mechanism of action and pharmacokinetics are more completely characterized.</p>This research aimed to assess SDGs effects in dietary models of hypertriglyceridemia, hypercholesterolemia, and obesity. Furthermore, this thesis work provided preliminary pharmacokinetic data on SDGs aglycone form, secoisolariciresinol (SECO). Dietary manipulations were used to induce hyperlipidemic states in female Wistar or male Sprague-Dawley rats. Groups of 10 rats were randomly assigned to one of three (obesity and cholesterol diets) or four (fructose) treatment groups: 1) Normal diet with 0.0 µmol SDG/kg body weight; 2) Dietary manipulation with 0.0 µmol SDG/kg; 3) Dietary manipulation with 4.4 µmol SDG/kg; and 4) 10% fructose in water with 8.8 µmol SDG/kg. Lignan or vehicle (saline) was administered daily by oral gavage for four weeks (2 weeks for male Sprague-Dawleys). After four (or two) weeks of SDG administration, body and liver weights were recorded, serum lipids were measured using enzymatic kits, hepatic fat accumulation was determined by histochemical analysis and hepatic mRNA expression of triglyceride pathway targets was evaluated using real-time RT-PCR. A 10% fructose in water model was effective for the induction of hypertriglyceridemia in male Sprague-Dawley rats but ineffective in female Wistar rats of similar age. Neither 4.4 nor 8.8 µmol SDG/kg improved serum and hepatic triglyceride parameters in male Sprague-Dawley rats on a 10% fructose in water diet. It is suspected that gender and strain are important factors for this model of hypertriglyceridemia. Dietary manipulations for the induction of hypercholesterolemia and obesity in female Wistar rats were not effective following 4 weeks administration of a 1% cholesterol diet and a 45% fat diet respectively. Since previous studies were able to successfully induce hypercholesterolemia in the same model, it is suspected that the differences in age of the animals accounted for the inconsistent results. Strain, gender and age of animals were identified as important considerations when trying to induce hyperlipidemic states through dietary manipulations.</p>SDG (4.4 µmol/kg) dosed daily for four weeks caused no gross morphological organ changes or alterations in blood chemistry or hematology parameters. Following an intravenous bolus (10 mg/kg), secoisolariciresinol (SECO) disposition was consistent with two-compartment pharmacokinetics, with distribution and elimination half-lives at 26 seconds and 5 minutes, respectively. No SECO was detected in the plasma following an oral bolus (10 mg/kg). Further investigation into SDGs hypolipidemic effects are required to elucidate its mechanism of action. A complete pharmacokinetic study is warranted to fully understand SDGs safety and efficacy.

It is becoming increasingly evident that the development of atherosclerotic coronary heart disease (CHD) can largely be regulated by lifestyle and dietary choices. The type of fatty acids regularly consumed may promote or prevent atherogenesis. Flaxseed, the richest plant source of the omega-3 fatty acid alpha-linolenic acid (ALA) is thought to protect against atherosclerotic disease. However, the mechanism(s) by which flaxseed exerts these anti-atherogenic effects requires further investigation. Alternatively, there are dietary fatty acids that are thought to induce significant deleterious effects upon our cardiovascular health. Epidemiological evidence associates dietary trans fatty acids (TFAs) with atherosclerotic CHD. This evidence has largely focused on the main source of TFAs in the North American diet, industrially hydrogenated vegetable shortening (iTFAs). It is assumed that TFAs stimulate atherosclerosis but the only studies to date have shown no effect of TFAs on atherosclerosis. Even less is known of the impact of naturally occurring TFAs from dairy and meat products of ruminant animals (rTFAs) on atherosclerotic disease. We investigated the effects of flaxseed supplementation on atherosclerosis and vascular function in two animal models, the hypercholesterolemic rabbit and the cholesterol fed, low density lipoprotein receptor (LDLr-/-) deficient mouse. New Zealand White rabbits and LDLr-/- mice were fed a diet containing flaxseed in the absence or presence of dietary cholesterol for a period of 6 to 24 weeks. We found that dietary flaxseed inhibits the atherogenic effects of a high cholesterol diet in both animal models. The anti-atherogenic effect was achieved in the mouse model through a capacity to lower circulating cholesterol levels and at a cellular level by inhibiting cell proliferation and inflammation. This reduction is also associated with an improved vascular relaxation response as demonstrated in the rabbit model. We also investigated the effects of consuming TFAs from two sources, industrially hydrogenated iTFAs rich in elaidic TFA (C18:1t-9) or naturally-occurring ruminant rTFAs rich in vaccenic TFA (C18:1t-11), on atherosclerotic development in the LDLr-/- mouse in the presence or absence of elevated dietary cholesterol. Our results demonstrate that consuming iTFAs dose dependently initiates atherosclerotic development but not beyond the effects of dietary cholesterol alone. However, consuming rTFAs rich in vaccenic acid protects against hyperlipidemia and atherosclerosis in the presence or absence of dietary cholesterol. The effects of combining dietary flaxseed and iTFAs in the diet were also examined in this model. Adding whole ground flaxseed or flaxseed oil (ALA) to diets containing low and high doses of iTFAs completely prevented atherosclerotic development in the absence of dietary cholesterol. Flaxseed was also able to partially prevent atherosclerosis caused by iTFAs and cholesterol. Our results suggest that the omega-3 ALA fatty acid rich content of flaxseed is mainly responsible for the anti-atherogenic effects of flaxseed. Our results highlight potential mechanisms for the beneficial effects of dietary flaxseed and the mixed effects of TFAs on cardiovascular health and underscore the need for further basic and clinical investigations.

Dietary flaxseed has cardioprotective effects that may be achieved through its rich content of the omega-3 fatty acid, alpha linolenic acid (ALA). We investigated the effects of dietary flaxseed both with and without an atherogenic cholesterol-enriched diet to determine the effects of dietary flaxseed on the expression of the adipose cytokines leptin and adiponectin. Rabbits were fed one of four diets: a regular (RG) diet, or a regular diet with added 0.5% cholesterol (CH), or 10% ground flaxseed (FX), or both (CF) for 8 weeks. Levels of leptin and adiponectin expression were assessed by RT-PCR in visceral adipose tissue. Consumption of flaxseed significantly increased plasma and adipose levels of ALA. Leptin, but not adiponectin, mRNA expression was lower in CH animals and was elevated in CF animals. Changes in leptin expression were strongly and positively correlated with adipose ALA levels and inversely correlated with levels of en face atherosclerosis. Our data demonstrate that the type of fat in the diet as well as its caloric content can specifically influence leptin expression. The findings support the hypothesis that the beneficial cardiovascular effects associated with flaxseed consumption may be related to a change in leptin expression.