This thesis evaluated supplementation with two components of flaxseed during exercise training on inflammation in older adults.<P>Experiment 1: This experiment assessed secoisolariciresinol diglucoside (SDG) supplementation during aerobic exercise training on inflammation in older adults. Methods: One hundred subjects aged 50y or older were randomized to receive either SDG or placebo before completing a 6-month walking program. Fasting concentrations of interleukin-6 and tumor necrosis factor-á, glucose, triacylglycerol (TAG), high density lipoprotein (HDL), low density lipoprotein, and total cholesterol as well as leukocyte cell count were measured every two months while body composition, resting blood pressure, and a composite Z-score of six metabolic syndrome risk factors were assessed at baseline and 6 months. Results: Men on placebo increased metabolic syndrome composite Z-score (p<0.05). TAG increased (p=0.017) in men on placebo relative to men on SDG and men on SDG decreased (p=0.045) DBP relative to men on placebo. Conclusions: SDG had no effect on inflammation while it is effective in attenuating risk factors associated with metabolic syndrome in older males but not females.<p>Experiment 2: This experiment evaluated alpha-linolenic acid (ALA) supplementation during strength exercise training on inflammation in older adults. Methods: Fifty-one healthy older adults (65.4±0.8y) were randomized to receive ALA or a placebo before completing a 12 wk strength training program. Subjects were evaluated at baseline and 12 weeks for TNF-á and IL-6, muscle strength, body composition, and muscle thickness. Results: Males supplementing with ALA decreased IL-6 concentration (p=0.003). The female placebo and male ALA group had a significant increase in knee flexor thickness (p<0.05). Chest and leg press strength, lean tissue mass, and muscle thickness significantly increased, while percent fat and total body mass decreased with training (p<0.05), with no difference between ALA and placebo. Conclusions: ALA lowers IL-6 in older men, but has minimal effect on muscle mass and strength during resistance training.<p>General Conclusion: A composite score of metabolic syndrome is attenuated in males supplementing with SDG. ALA reduces IL-6 in older men. Older men, but not older women, derive specific health benefits from increased consumption of components of flaxseed consumed during an exercise program.

The rising use of natural products creates an imperative need for an enhanced awareness of the safety of current and new products making their way into the marketplace. An important example is natural products containing lignans as the principal active component. Despite their structural similarity the lignan of creosote bush can cause hepato- and renal toxicity while the lignans of flaxseed have no reported serious toxicity. This dissertation aimed to investigate the oxidative metabolism of such lignans to determine whether reversible, competitive interactions and/or bioactivation may explain the differences in their apparent toxicity.<p> The first objective was to study the metabolism and bioactivation of nordihydroguaiaretic acid (creosote bush) and secoisolariciresinol (flaxseed). Nordihydroguaiaretic acid metabolism in rat liver microsomes led to the production of three glutathione adducts formed via ortho¬-quinone reactive intermediates. This metabolism was independent of NADPH and thus attributed to autoxidation. Secoisolariciresinol metabolism yielded lariciresinol and no glutathione adducts suggesting an absence of bioactivation to reactive quinone intermediates.<p> The second objective was to study the autoxidation of nordihydroguaiaretic acid. The major autoxidation product was a unique, stable schisandrin-like cyclolignan which was the result of nordihydroguaiaretic acid cyclization. The half-life of nordihydroguaiaretic acid in aqueous solution, pH 7.4, 37ºC is 3.14 hours suggesting the cyclolignan may be responsible for some of the biological effects of nordihydroguaiaretic acid.<p> The third objective was to study the inhibition of cytochrome P450 isoforms 1A2, 2B, 2C11 and 3A by lignans derived from creosote bush and flaxseed. None of the lignans caused irreversible inhibition. Both creosote bush and flaxseed lignans caused reversible inhibition of P450 enzyme activity that involved competitive or mixed-type inhibition, however the inhibition was present at nonphysiologically relevant concentrations. Activation of cytochrome P450 isoforms was also observed at low lignan concentrations. The results suggest that P450-mediated bioactivation or reversible inhibition cannot explain the differences in toxicity noted between the lignans of creosote bush and flaxseed.<p> This work suggests a minimal risk for drug-lignan interactions at P450 enzymes. Further studies are warranted to determine the presence and biological and toxicological role of the nordihydroguaiaretic acid cyclolignan in herbal preparations.

The rising use of natural products creates an imperative need for an enhanced awareness of the safety of current and new products making their way into the marketplace. An important example is natural products containing lignans as the principal active component. Despite their structural similarity the lignan of creosote bush can cause hepato- and renal toxicity while the lignans of flaxseed have no reported serious toxicity. This dissertation aimed to investigate the oxidative metabolism of such lignans to determine whether reversible, competitive interactions and/or bioactivation may explain the differences in their apparent toxicity.<p> The first objective was to study the metabolism and bioactivation of nordihydroguaiaretic acid (creosote bush) and secoisolariciresinol (flaxseed). Nordihydroguaiaretic acid metabolism in rat liver microsomes led to the production of three glutathione adducts formed via ortho¬-quinone reactive intermediates. This metabolism was independent of NADPH and thus attributed to autoxidation. Secoisolariciresinol metabolism yielded lariciresinol and no glutathione adducts suggesting an absence of bioactivation to reactive quinone intermediates.<p> The second objective was to study the autoxidation of nordihydroguaiaretic acid. The major autoxidation product was a unique, stable schisandrin-like cyclolignan which was the result of nordihydroguaiaretic acid cyclization. The half-life of nordihydroguaiaretic acid in aqueous solution, pH 7.4, 37ºC is 3.14 hours suggesting the cyclolignan may be responsible for some of the biological effects of nordihydroguaiaretic acid.<p> The third objective was to study the inhibition of cytochrome P450 isoforms 1A2, 2B, 2C11 and 3A by lignans derived from creosote bush and flaxseed. None of the lignans caused irreversible inhibition. Both creosote bush and flaxseed lignans caused reversible inhibition of P450 enzyme activity that involved competitive or mixed-type inhibition, however the inhibition was present at nonphysiologically relevant concentrations. Activation of cytochrome P450 isoforms was also observed at low lignan concentrations. The results suggest that P450-mediated bioactivation or reversible inhibition cannot explain the differences in toxicity noted between the lignans of creosote bush and flaxseed.<p> This work suggests a minimal risk for drug-lignan interactions at P450 enzymes. Further studies are warranted to determine the presence and biological and toxicological role of the nordihydroguaiaretic acid cyclolignan in herbal preparations.

Doctor of Philosophy / Department of Human Nutrition / Weiqun Wang / Wheat lignans are phenylpropane dimers linked by β-β bonds with a 1, 4-diarylbutane structure. They are biosynthesized in the cell cytoplasm through action of enzymes of the phenylpropanoid pathway. Pinoresinol lariciresinol reductase (PLR) catalyzes the final steps of biosynthesis of wheat lignans. In epidemiological and clinical investigations, studies show that high plasma lignan amounts correlate with reduced risks of breast, colon, and prostate cancers. However, in some of the studies, the results are not consistent. More consistent results are observed when animal and cell culture models are used. Our previous studies in the Wang lab demonstrated that treatment of human colon cancer cells, SW480 with lignans results in a dose and time dependent inhibition of cancer cell growth. In the first paper, we investigated direct experimental cancer preventative characteristics of a wheat lignan, secoisolariciresinol diglucoside (SDG) vs. its metabolite enterolactone in human colon cancer SW480 cells. Treatment of cancer cells with 0-40 µM SDG or enterolactone resulted into inhibition of cancer cell growth as observed by reduction of cell numbers. The reduction appeared related to induction of S-phase cell cycle arrest rather than cytotoxic effect. Further analysis revealed that SDG was more stable in cell culture medium than enterolactone. HPLC-MS/ESI showed that enterolactone is the principle metabolite in cancer cells but undetectable SDG or its metabolites were in the cells treated with SDG. In the second paper, we investigated over expression of the PLR gene and enhancement of lignan levels in transgenic wheat. We transformed wheat cultivars (‘Bobwhite’, ‘Madison’, and ‘Fielder’ respectively) with the Forsythia intermedia PLR gene under the regulatory control of the maize ubiquitin promoter. Of the total 217 transgenic wheat lines, we successfully obtained 7 transformants with the inserted ubiquitin PLR gene as screened by PCR. Real-time PCR further indicated 109-117% PLR over expression over the transgenic control in 3 transformants of the 7 at T0 generation. In addition, the levels of SDG, as determined by HPLC was found to be significantly elevated in one of the 3 positive transgenic plants. To the best of our knowledge, this is the first study reported that genetically engineered wheat with over expressed PLR enzyme enhancing phytochemical lignan has been successfully achieved.