Faculty of Health Sciences School of Pharmacology 9410866v kermifrum@yahoo.com / ABSTRACT Thirty plant species traditionally used to treat skin pathologies were chosen from the readily available ethnobotanical literature. Four plants (aqueous or methanol extracts) displayed promising 5-lipoxygenase inhibitory activity with IC50 values below 61 ppm. These included Aloe greatheadii, Melianthus comosus, Pentanisia prunelloides and Warburgia solutaris. Essential oils generally displayed superior 5- lipoxygenase inhibitory activity with IC50 values between 22 and 75 ppm. These included the essential oils of Ballota africana, Helichrysum odoratissimum, Heteropyxis natalensis and Lippia javanica. A large proportion of the plants exhibited dose-dependent DPPH anti-oxidant activity with IC50 values between 5 and 94 ppm for the most active. These included Halleria lucida, Croton sylvaticus, Melianthus comosus, Lippia javanica and Pentanisia prunelloides. Aqueous extracts of Melianthus comosus exhibited the most potent anti-inflammatory and anti-oxidant activity. The methanol extract of the leaves of Halleria lucida was subjected to activity guided fractionation and two anti-oxidant molecules were isolated, namely luteolin-5-Oglucoside and verbascoside (acteoside). Isobologram construction resulted in a concentration-dependent additive and antagonistic interaction being recognised between the two isolated compounds. Warburgia salutaris displayed promising 5-lipoxygenase inhibitory activity. Two isolated compounds, mukadiaal and warburganal were found to partially contribute to the anti-inflammatory activity of the plant. The essential oils of Helichrysum odoratissimum, Heteropyxis natalensis and Lippia javanica were subjected to gas chromatography and major compounds contributing to possible anti-inflammatory effects identified. These included β-caryophyllene, 1,8-cineole, limonene and α- humulene. Enantiomers and racemic mixtures of limonene displayed significantly different 5-lipoxygenase inhibitory activity suggesting stereoselectivity of the enzyme-catalysed reaction. The monoterpene 1,8-cineole appeared to cause partial potentiation of the anti-inflammatory activity displayed by limonene. These results provide some in vitro scientific rationale for their traditional use as dermatological agents.

Many studies highlighted the biological potential of vitamin E, especially tocotrienols (T3), a vitamin E subfamily, particularly in the field of cardiovascular diseases and chronic inflammation. A pharmacophore based virtual screening of these substances against various antiinflammatory targets showed that this class could be considered as potential inhibitors of 5- lipoxygenase, a key enzyme in the biosynthesis of chemoattractant and vasoactive leukotrienes. Consequently, this screening was confirmed by in vitro assays. However, usual natural sources of T3 provide complex mixtures involving particularly challenging purification processes. Thus, this work aims at designing and optimizing efficient semisynthesis towards pharmacologically relevant T3 derivatives were developed from δ- tocotrienol, the main T3 isolated from Bixa orellana seeds, a renewable and easily available vegetal source from tropical regions, analyzed mainly by HPLC chromatography. Verification of the most effective reaction conditions of semisynthesis and synthesis another potential inhibitors of 5-LOX based on tocotrienols' structure are the following aims of the work. During this study, the semisynthesis based on δ-tocotrienol was completely optimized and 3 new T3 derivatives were synthesized and fully characterized....

Most plant oxylipins, a large class of diverse oxygenated polyunsaturated fatty acids and their derivatives, are produced through the lipoxygenase (LOX) pathway. Recent progress in dicots has highlighted the biological roles of oxylipins in plant defense responses to pathogens and pests. In contrast, the physiological function of LOXs and their metabolites in monocots is poorly understood. We cloned and characterized three maize LOXs ZmLOX10 ZmLOX11 and ZmLOX12. Both ZmLOX10 and ZmLOX11 apeared to be 13-LOX, whereas ZmLOX12 is a unique 9-LOX. Whereas leaf was the preferential site of ZmLOX10 expression, ZmLOX11 was strongly expressed in silks. Induction of these ZmLOX10 and ZmLOX12 by wounding and defense-related compounds suggested their role in plant resistance mechanisms against pests and pathogens. Abscisic acid, however, was the only inducer of ZmLOX11 in leaves. Higher increase in ZmLOX10 transcripts in maize infected by fungus Cochliobolus carbonum implicated this gene in resistance responses to necrotrophic pathogens. In addition, ZmLOX10 was shown to be the first reported LOX to be regulated by a circadian clock. It was found that ZmLOX10 was also inducible by low temperatures. Phenotypical studies of wild type and mutant near isogenic lines showed that expression of ZmLOX12, specific to underground organs, was required for pathogenesis of F. verticillioides on maize mesocotyls.

Most plant oxylipins, a large class of diverse oxygenated polyunsaturated fatty acids and their derivatives, are produced through the lipoxygenase (LOX) pathway. Recent progress in dicots has highlighted the biological roles of oxylipins in plant defense responses to pathogens and pests. In contrast, the physiological function of LOXs and their metabolites in monocots is poorly understood. We cloned and characterized three maize LOXs ZmLOX10 ZmLOX11 and ZmLOX12. Both ZmLOX10 and ZmLOX11 apeared to be 13-LOX, whereas ZmLOX12 is a unique 9-LOX. Whereas leaf was the preferential site of ZmLOX10 expression, ZmLOX11 was strongly expressed in silks. Induction of these ZmLOX10 and ZmLOX12 by wounding and defense-related compounds suggested their role in plant resistance mechanisms against pests and pathogens. Abscisic acid, however, was the only inducer of ZmLOX11 in leaves. Higher increase in ZmLOX10 transcripts in maize infected by fungus Cochliobolus carbonum implicated this gene in resistance responses to necrotrophic pathogens. In addition, ZmLOX10 was shown to be the first reported LOX to be regulated by a circadian clock. It was found that ZmLOX10 was also inducible by low temperatures. Phenotypical studies of wild type and mutant near isogenic lines showed that expression of ZmLOX12, specific to underground organs, was required for pathogenesis of F. verticillioides on maize mesocotyls.

Maize production is limited agronomically by the availability of water and nutrients during the growing season. Of these two limiting factors, water availability is predicted to increase in importance as climate change and the growing urban landscape continue to stress limited supplies of freshwater. Historically, efforts to breed maize for water-limited environments have been extensive; especially in the areas of root architecture and flowering physiology. As progress has been made and new traits have been discovered and selected for, the different responses to drought stress at specific developmental stages of the maize plant have been selected as a whole when drought tolerance is evaluated. Herein we attempt to define the characteristics of the maize drought response during different developmental stages of the maize plant that can be altered through plant breeding. Towards breeding for drought tolerance, 400 inbred lines from a diversity panel were amplified and sequenced at the ZmLOX4 and ZmLOX5 loci in an effort to characterize their linkage disequilibrium and genetic diversity. Understanding these characteristics is essential for an association mapping study that accompanies this project, searching for novel and natural allelic diversity to improve drought tolerance and aflatoxin resistance in maize. This study is among the first to investigate genetic diversity at important gene paralogs ZmLOX4 and ZmLOX5 believed to be highly conserved among all Eukaryotes. We show very little genetic diversity and very low linkage disequilibrium in these genes, but also identified one natural variant line with knocked out ZmLOX5, a variant line missing ZmLOX5, and five line variants with a duplication of ZmLOX5. Tajima's D test suggests that both ZmLOX4 and ZmLOX5 have both been under neutral selection. Further investigation of haplotype data revealed that ZmLOX12, a member of the ZmLOX family, showed strong LD that extends much further than expected in maize. Linkage disequilibrium patterns at these loci of interest are crucial to quantify for future candidate gene association mapping studies. Knockout and copy number variants of ZmLOX5, while not a surprising find, are under further investigation for crop improvement.

Chapter 2 In solution, py(mi)ridinols 1.33, 1.34 and 1.35 are 2-, 5- and 28-fold more reactive antioxidants, respectively, than α-TOH (the most potent lipid-soluble antioxidant in nature). In order to develop a highly-reactive fluorescent indicator of lipid peroxidation in cells, we sought to couple these antioxidants with boron-dipyrro- methene (BODIPY) dyes, such that the resulting conjugates will display a significant fluorecence enhancement upon oxidation. This chapter details efforts towards the synthesis of these compounds. Chapter 3 Lipoxygenases are a family of important enzymes that catalyze the dioxygenation of arachidonic acid to yield a variety of potent lipid mediators that have been implicated in the pathogenesis of numerous degenerative conditions. We have undertaken a preliminary study of the effect of replacing the unsaturation in the related polyunsaturated lipid linoleic acid with cyclopropane rings on both the oxidizability of the lipid, as well as lipoxygenase’s ability to utilize it as a substrate. We anticipate that these analogs will be useful in co-crystallization studies with the enzyme that will provide unique insight into substrate acquisition, binding and the necessary conformation for catalysis. / Thesis (Master, Chemistry) -- Queen's University, 2011-11-10 16:15:05.643

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Dehulled black beans were micronized at 90⁰C, 100⁰C, 110⁰C, 120⁰C, 130⁰C and 140⁰C; milled to flour and tested for lipoxygenase activity. Non micronized black bean flour was higher in lipoxygenase activity than flours at ≥120ºC (p=≤0.05). Micronized (100⁰C, 110⁰C, 120⁰C) and non micronized black bean flour was added to low fat beef burgers (6%). C18:3 was significantly higher in the black bean flour samples (raw and cooked). Whole wheat flour had the highest amount of C18:2 in all samples (p= ≤0.05). The all beef control was significantly higher in Newton value, drip loss, cook loss and percent shrinkage compared to burgers with binders (p= ≤0.05). Ninety-three participants participated in an consumer sensory panel. Results showed higher acceptability of micronized burgers compared to all beef or whole wheat flour controls. This study demonstrated incorporation of black bean flour into low fat beef burgers can improve their physical, chemical and sensory properties.

Soybean production and consumption has increased in recent decades. However, trypsin inhibitor activity and “beany” flavour are two drawbacks limiting the utilization of soybean. In the present study, micronization, ethanol washing, and enzymatic hydrolysis (alone or in combination) were used to treat soybean. Micronization at 100 °C and 135 °C decreased the activity of both trypsin inhibitors (53% and 80% respectively), and lipoxygenase (51% and 99%, respectively). Ethanol increased the trypsin inhibitor activity while alcalase hydrolysis decreased its activity. Different treatment combinations affected trypsin inhibitor activity, with micronization having a major influence. “Beany” flavour related volatiles (hexanal, (E)- 2-hexenal, 1-hexanol, heptanal, (E)-2-octenal, (E)-2-nonenal, (E,E)-2,4-nonadienal, 2,4-decadienal, (E,E)-2,4-decadienal, 1-octen-3-ol, 2-pentylfuran and 3-octen-2-one) were significantly decreased with micronization. Ethanol effects varied with different volatiles. Soybean micronized at 135°C and washed with 65% ethanol was recommended for soybean processing due to its low trypsin inhibitor activity and low “beany” related volatile content.

Dehulled black beans were micronized at 90⁰C, 100⁰C, 110⁰C, 120⁰C, 130⁰C and 140⁰C; milled to flour and tested for lipoxygenase activity. Non micronized black bean flour was higher in lipoxygenase activity than flours at ≥120ºC (p=≤0.05). Micronized (100⁰C, 110⁰C, 120⁰C) and non micronized black bean flour was added to low fat beef burgers (6%). C18:3 was significantly higher in the black bean flour samples (raw and cooked). Whole wheat flour had the highest amount of C18:2 in all samples (p= ≤0.05). The all beef control was significantly higher in Newton value, drip loss, cook loss and percent shrinkage compared to burgers with binders (p= ≤0.05). Ninety-three participants participated in an consumer sensory panel. Results showed higher acceptability of micronized burgers compared to all beef or whole wheat flour controls. This study demonstrated incorporation of black bean flour into low fat beef burgers can improve their physical, chemical and sensory properties.