Orientador: Eutimio Gustavo Fernández Núñez / Banca: Regildo Mário Gonçalves da Silva / Banca: Nelson Barbosa Machado Neto / Resumo: O estresse oxidativo e as inflamações estão associados a muitas doenças como diabetes, câncer, doenças cardiovasculares e neurodegenerativas, incluindo a aterosclerose, causando preocupação em todo o mundo. Como alternativa, as plantas podem desempenhar um papel chave no combate a essas desordens devido suas propriedades farmacológicas que são relacionadas a uma ampla gama de compostos com atividade antioxidante, entre eles, os compostos polifenólicos. Nesse contexto as pimentas podem ser uma alternativa para a obtenção desses compostos. Porém, a concentração desses metabólitos secundários é baixa e afetada por diversos fatores abióticos. Como alternativa para contornar esse problema foram utilizadas metodologias relacionadas com as culturas in vitro de células e de tecidos vegetais. O presente trabalho teve como objetivo padronizar a obtenção de explantes (Capítulo 1) a serem utilizados em uma cultura de calos otimizada através de uma abordagem sistêmica (Capítulo 2). No capítulo 1, por meio de um delineamento experimental fatorial completoadois níveis (24) determinou-se numericamente o efeito de quatro fatores [O tipo de solução para condicionamento osmótico (água e solução aquosa de KNO3 a 1%), meio germinativo (ágar e ágar + ácido giberélico a 1,88 mg.L-1), tempo (15 e 30 dias) e dois genótipos (Pitanga e Cambuci)] nas taxas de surgimento de cotilédones e na germinação. As respostas dos genótipos a solução de condicionamento osmótico e ao meio de germinação foram diferent... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The oxidative stress and inflammations are associated with many diseases such as diabetes, cancer, cardiovascular and neurodegenerative diseases including atherosclerosis causing concern worldwide. As an alternative, the plants can play a key role due to their pharmacological properties against these disorders; these attributes are most of the times associated with the presence of polyphenolic compounds. In this context, peppers can be an alternative to obtain these compounds. However, the presence of secondary metabolites is low and affected by several abiotic factors. As an alternative to circumvent this problem, methodologies related to in vitro cultures of plant cells and tissues were applied. The objective of the present work was to standardize the extraction of explants (Chapter 1) to be used in a callus culture optimized through a systemic approach (Chapter 2). In Chapter 1, through a two-level full factorial experimental design (24 ) was numerically determined the effect of four factors [The solution type for osmotic conditioning (water and 1% KNO3 aqueous solution), germination medium (agar and agar + gibberellic acid at 1.88 mg.L -1, GA3), post-sowing time (15 and 30 days) and two genotypes (Pitanga and Cambuci)] in cotyledon emergence and germination rates. The genotypes responses to the osmotic conditioning solution and to the germination medium were different. The optimal combination of treatments for in vitro germination and development of seedlings for Pitanga and Cambuci was water + agar and water + agar-GA3, respectively. In Chapter 2, aiming to optimize systemically the Cambuci pepper callus culture stage for higher biomass and secondary metabolites productionwere evaluated explant's origin, plant growth regulator type and their concentrations through a multilevel factorial design. The values obtained in the optimized conditions were: callus... (Complete abstract click electronic access below) / Mestre

Chiang Yee-Ting. / Thesis submitted in: December 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 116-130). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / List of Abbreviations --- p.iv / List of Tables --- p.v / List of Figures --- p.vi / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- "Free radical, oxidative stress and antioxidants" --- p.2 / Chapter 1.1.1 --- Free radicals and reactive oxygen species (ROS) --- p.2 / Chapter 1.1.2 --- Oxidative stress and human diseases --- p.3 / Chapter 1.1.3 --- Dietary antioxidants --- p.5 / Chapter 1.1.4 --- Synthetic antioxidants --- p.5 / Chapter 1.2 --- Flavonoids ´ؤ polyphenolic compounds in plants --- p.8 / Chapter 1.2.1 --- Sources and biosynthesis of flavonoids --- p.8 / Chapter 1.2.2 --- Classification and dietary occurrence of flavonoids --- p.11 / Chapter 1.2.3 --- Functions of flavonoids in plants --- p.15 / Chapter 1.2.4 --- Effects of flavonoids in mammals --- p.16 / Chapter 1.2.5 --- Therapeutic application of flavonoids --- p.17 / Chapter 1.2.6 --- Absorption and metabolism of flavonoids --- p.26 / Chapter 1.3 --- Plant of interest --- p.28 / Chapter 1.4 --- Method used to characterize flaovnoid antioxidants in Vigna sinensis seeds --- p.30 / Chapter 1.5 --- Method used to evaluate the antioxidant activity --- p.31 / Chapter 1.5.1 --- p-carotene bleaching method --- p.31 / Chapter 1.5.2 --- "a,a-diphenyl- --- p.32 / Chapter 1.5.3 --- Single cell gel electrophoresis assay (Comet assay) --- p.32 / Chapter 1.6 --- Research objectives --- p.34 / Chapter 2 --- Materials and Methods --- p.35 / Chapter 2.1 --- Plant materials and chemicals --- p.35 / Chapter 2.2 --- Sample preparation --- p.36 / Chapter 2.2.1 --- Methanolic extraction method --- p.36 / Chapter 2.2.2 --- Acidic methanolic extraction method --- p.36 / Chapter 2.2.3 --- Optimization of extraction time --- p.37 / Chapter 2.3 --- Standards preparation --- p.37 / Chapter 2.4 --- Characterization of flavonoid antioxidants in V. sinensis seed extracts --- p.38 / Chapter 2.5 --- Evaluation of antioxidant activity --- p.39 / Chapter 2.6 --- Determination of free radical scavenging ability --- p.41 / Chapter 2.7 --- Evaluation of the protective effects on DNA damage --- p.42 / Chapter 2.7.1 --- Preparation of reagents --- p.42 / Chapter 2.7.2 --- Blood sample --- p.43 / Chapter 2.7.3 --- Hydrogen peroxide treatment --- p.43 / Chapter 2.7.3.1 --- Co-incubation system --- p.43 / Chapter 2.7.3.2 --- Pre-incubation system --- p.43 / Chapter 2.7.4 --- Establishment of optimal assay conditions --- p.44 / Chapter 2.7.4.1 --- Hydrogen peroxide concentration --- p.44 / Chapter 2.7.4.2 --- Sample volume --- p.44 / Chapter 2.7.4.3 --- Incubation time --- p.44 / Chapter 2.7.4.4 --- Hydrogen peroxide treatment time --- p.44 / Chapter 2.7.5 --- Ethidium bromide-acridine orange cell viability determination --- p.45 / Chapter 2.7.6 --- Slide preparation --- p.45 / Chapter 2.7.7 --- Comet assay --- p.45 / Chapter 2.7.8 --- Quantification of DNA damage --- p.47 / Chapter 2.7.9 --- Statistical analysis --- p.47 / Chapter 3 --- Results / Chapter 3.1 --- Comparison on the free radical scavenging abilities on two different V. sinensis seed extracts --- p.48 / Chapter 3.1.1 --- Optimal extraction time of methanolic extraction method --- p.48 / Chapter 3.1.2 --- Optimal extraction time of acidic methanolic extraction method --- p.48 / Chapter 3.1.3 --- pH values of two different V. sinensis seed extracts --- p.49 / Chapter 3.1.4 --- Free radical scavenging abilities of the two different V. sinensis seed extracts --- p.49 / Chapter 3.2 --- Determination of the stability of the V. sinensis seed extracts --- p.50 / Chapter 3.2.1 --- Effects of storage on the free radical scavenging ability of methanolic V. sinensis seed extract --- p.50 / Chapter 3.2.2 --- Effects of storage on the free radical scavenging ability of acidic V. sinensis seed extract --- p.50 / Chapter 3.2.3 --- Effect of storage on the antioxidant activity of methanolic V.sinensis seed extract --- p.51 / Chapter 3.2.4 --- Effect of storage on the antioxidant activity of acidic V. sinensis seed extract --- p.52 / Chapter 3.3 --- Identification of the flavonoid antioxidants in the acidic V. sinensis seed extract --- p.53 / Chapter 3.4 --- Evaluation of free radical scavenging abilitiesof identified flavonoids using the DPPH radical scavenging method --- p.54 / Chapter 3.5 --- Evaluation of antioxidant activities of the identified flavonoids using the β-carotene bleaching assay --- p.56 / Chapter 3.6 --- Evaluation of protective effects on DNA damage using the Comet assay --- p.57 / Chapter 3.6.1 --- Optimal comet assay conditions --- p.57 / Chapter 3.6.1.1 --- Hydrogen peroxide concentration --- p.57 / Chapter 3.6.1.2 --- Sample volume --- p.58 / Chapter 3.6.1.3 --- Incubation time with the seed extract in the co-incubation system --- p.58 / Chapter 3.6.1.4 --- Hydrogen peroxide treatment time --- p.58 / Chapter 3.6.1.5 --- Incubation time with the seed extract in the pre-incubation system --- p.59 / Chapter 3.6.2 --- Protective effects of the V. sinensis seed extracts and phenolic compounds --- p.59 / Chapter 3.6.2.1 --- Protective effects in pre-incubation system --- p.59 / Chapter 3.6.2.2 --- Protective effects in co-incubation system --- p.60 / Chapter 3.6.3 --- Protective effects of the identified flavonoids in acidic V.sinensis seed extracts and phenolic compounds --- p.60 / Chapter 3.6.3.1 --- Protective effects in pre-incubation system --- p.60 / Chapter 3.6.3.1.1 --- At 0.5 mM concentration --- p.60 / Chapter 3.6.3.1.2 --- At 1 mM concentration --- p.61 / Chapter 3.6.3.2 --- Protective effects in co-incubation system --- p.62 / Chapter 3.6.3.2.1 --- At 0.5 mM concentration --- p.62 / Chapter 3.6.3.2.2 --- At 1 mM concentration --- p.62 / Chapter 4 --- Discussion --- p.100 / Chapter 4.1 --- Comparison on the two different extraction methods --- p.100 / Chapter 4.1.1 --- Methanolic extraction and acidic methanolic extraction --- p.100 / Chapter 4.1.2 --- Free radical scavenging abilities on the two different V sinensis seed extracts --- p.100 / Chapter 4.2 --- Stabilities of two different V. sinensis seed extracts --- p.101 / Chapter 4.2.1 --- Change in antioxidant activity during storage --- p.101 / Chapter 4.2.2 --- Comparison on the stabilities of the extracts assayed under different conditions --- p.102 / Chapter 4.3 --- Identification of flavonoid antioxidants in the acidic methanolic V sinensis seed extract --- p.103 / Chapter 4.4 --- Antioxidant activities of the individual identified flavonoid antioxidants --- p.104 / Chapter 4.4.1 --- Antioxidant activities of the identified flavonoid antioxidants and the selected phenolic compounds in hydrophilic assay system --- p.106 / Chapter 4.4.2 --- Antioxidant activities of the identified flavonoid antioxidants and the selected phenolic compounds in lipophilic assay system --- p.107 / Chapter 4.5 --- Evaluation of protective effects on DNA damage using Comet assay --- p.109 / Chapter 4.5.1 --- Optimal conditions in Comet assay --- p.109 / Chapter 4.5.1.1 --- Effect of hydrogen peroxide concentration --- p.109 / Chapter 4.5.1.2 --- Effect of sample volume --- p.109 / Chapter 4.5.1.3 --- Effect of hydrogen treatment time --- p.110 / Chapter 4.5.1.4 --- Pre-incubation and co-incubation systems --- p.110 / Chapter 4.5.2 --- Protective effects of two different V. sinensis seed extracts and six phenolic compounds --- p.111 / Chapter 4.5.3 --- Protective effects of the identified flavonoids and the phenolic compounds --- p.112 / Chapter 4.6 --- Health beneficial properties of V. sinensis seeds --- p.113 / Chapter 5 --- Conclusion --- p.114 / References --- p.116

Polysulfides, produced from olefin sulfurization, are common additives to various petroleum-derived products such as engine oils, hydraulic fluids, rubbers and polymeric materials in an effort to protect these materials from oxidative degradation. The mechanisms by which organosulfur compounds act as antioxidants are often described as ‘secondary antioxidants’ since it is believed that they slow the rate of radical initiation by decomposing hydroperoxides. Organosulfurs also figure in various natural products from the genus Allium, of which garlic is a prominent species. Our group previously shown that thiosulfinates (RS(O)SR), key components of garlic, are potent radical-trapping antioxidants (RTAs) through the intermediacy of sulfenic acids (RSOH). In an effort to shed light on the antioxidant mechanisms of other organosulfur constituents of garlic and sulfurized olefins, in Chapter 2, we reported on the antioxidant activity of trisulfide-1-oxides (RS(O)SSR) at 37 °C. In that study, we found these moieties to be effective RTAs, equal in potency to hindered phenols, the industry standard. Trisulfide-1-oxides were shown to react with peroxyl radicals via a concerted bimolecular homolytic substitution reaction, forming a perthiyl radical as product. In Chapter 3, inspired by the findings at 37 °C, we investigated the RTA activity of polysulfides at elevated temperatures, from 100 °C to 160 °C, and found that tetrasulfides were surprisingly potent RTAs at and above 100 °C. Paralleling the reactivity of trisulfide-1-oxides, tetrasulfides were found to react with peroxyl radicals via a concerted bimolecular homolytic substitution reaction, also forming a perthiyl radical as an initial product. Perthiyl radicals are formed in multiple reactions described in the abovementioned studies and although they were shown to rapidly recombine, their apparent stability, as well as preliminary computational investigations into the S-H bond strength in hydropersulfides (RSSH), prompted us to investigate hydrogen-atom transfers from hydropersulfides. As reported in Chapter 4, we found that hydropersulfides (RSSH) are very potent hydrogen-atom donors, making them highly effective RTAs in organic and aqueous media, even besting thiols in many H-atom transfer reactions with alkyl, alkoxyl and peroxyl radicals. The one-electron oxidation exhibited by hydropersulfides parallels the one we previously reported for sulfenic acids (RSOH). Previous successful studies on the one-electron oxidation of sulfenic acids prompted us to make use of the persistence imparted by a triptycene backbone to study two-electron processes, namely - thiol oxidation by hydrogen peroxide (H2O2). Indeed, the reaction of thiols with H2O2 is central to many processes essential to life, from protein folding to redox signaling. However, kinetic and mechanistic characterization of their subsequent reactions had not been reported due to the instability of the sulfenic acid intermediate under study. Gratifyingly, as reported in Chapter 5, using our triptycene model with an appended fluorine atom, we were able to investigate the complete series of consecutive reactions with hydrogen peroxide, from thiol to sulfenic acid to sulfinic acid to sulfonic acid, which has hitherto not been possible, and found that the reaction is specific base-catalyzed. Sulfinic acids (RSO2H), the primary product of sulfenic acid oxidation, have been used increasingly in recent years in many synthetic applications such as sulfonylation reactions. As described in Chapter 6, using a similar approach to previous studies, we synthesized a triptycene sulfinic acid and demonstrated that the triptycene backbone is also effective a rendering the sulfinic acid and sulfonyl radical (RSO2•) persistent enough to study H-atom transfer reactions. We showed that sulfinic acids are good H-atom donors to alkyl and alkoxyl radicals but they are not effective RTAs due to the propensity of sulfinic acids to autoxidize through the reaction of sulfonyl radicals with oxygen.

A wide variety of sorghum genotypes with a pigmented pericarp were grown in varying environments and were analyzed for total phenols, condensed tannins, flavan-4-ols, and in vitro antioxidant activity. In addition, sorghum flavonoids were separated, characterized, and quantified using HPLC-PDA and LC-MS. Total phenols and in vitro antioxidant activity increased when sorghums had a pigmented testa causing the presence of condensed tannins. Flavan-4-ol levels were highest in sorghums with a black pericarp (5.8-16.1 abs/mL/g), followed by those with a red pericarp (1.1-9.2 abs/mL/g). Sorghums with a black pericarp had the highest 3-deoxyanthocyanin levels (308-1885 µg/g) and these were increased when the grain had minimal weathering and was darkest in color. Sorghums with a lemon-yellow pericarp had the highest flavanone levels (260-3586 µg/g) with eriodictyol being the main flavanone. Flavanone levels were increased when the grain was bright yellow with minimum weathering and were high compared to those found in common sources (238-574 µg/g, fresh wts.). No flavonoids were predominant in sorghums with a red pericarp. Flavonoid composition varied when all sorghums were grouped by secondary plant color. Sorghums with tan secondary plant color, including those with a white pericarp, had higher levels of flavones (50-932 µg/g) than those with red/purple secondary plant color (0-172 µg/g). On the other hand, 3-deoxyanthocyanin levels were higher in red/purple plant sorghums (14-1885 µg/g) than in tan plant sorghums (0-24 µg/g). Among red/purple plant sorghums, lemon-yellow pericarp sorghums had the highest levels of flavones (51-172 µg/g). Environment and weathering had an effect on flavonoid levels. The 3-deoxyanthocyanins were reduced for sorghums grown in a dry environment (i.e. Lubbock, TX) and flavonoid levels were increased in grains with minimum weathering or molding. This study reports that all sorghums, including those with a white pericarp, have flavonoids and their levels and compositions are affected by the genotype. This information will be helpful for plant breeders, food scientists, and the pharmaceutical/nutraceutical industries in selecting sorghums with desired healthy components.

A wide variety of sorghum genotypes with a pigmented pericarp were grown in varying environments and were analyzed for total phenols, condensed tannins, flavan-4-ols, and in vitro antioxidant activity. In addition, sorghum flavonoids were separated, characterized, and quantified using HPLC-PDA and LC-MS. Total phenols and in vitro antioxidant activity increased when sorghums had a pigmented testa causing the presence of condensed tannins. Flavan-4-ol levels were highest in sorghums with a black pericarp (5.8-16.1 abs/mL/g), followed by those with a red pericarp (1.1-9.2 abs/mL/g). Sorghums with a black pericarp had the highest 3-deoxyanthocyanin levels (308-1885 µg/g) and these were increased when the grain had minimal weathering and was darkest in color. Sorghums with a lemon-yellow pericarp had the highest flavanone levels (260-3586 µg/g) with eriodictyol being the main flavanone. Flavanone levels were increased when the grain was bright yellow with minimum weathering and were high compared to those found in common sources (238-574 µg/g, fresh wts.). No flavonoids were predominant in sorghums with a red pericarp. Flavonoid composition varied when all sorghums were grouped by secondary plant color. Sorghums with tan secondary plant color, including those with a white pericarp, had higher levels of flavones (50-932 µg/g) than those with red/purple secondary plant color (0-172 µg/g). On the other hand, 3-deoxyanthocyanin levels were higher in red/purple plant sorghums (14-1885 µg/g) than in tan plant sorghums (0-24 µg/g). Among red/purple plant sorghums, lemon-yellow pericarp sorghums had the highest levels of flavones (51-172 µg/g). Environment and weathering had an effect on flavonoid levels. The 3-deoxyanthocyanins were reduced for sorghums grown in a dry environment (i.e. Lubbock, TX) and flavonoid levels were increased in grains with minimum weathering or molding. This study reports that all sorghums, including those with a white pericarp, have flavonoids and their levels and compositions are affected by the genotype. This information will be helpful for plant breeders, food scientists, and the pharmaceutical/nutraceutical industries in selecting sorghums with desired healthy components.

Meat lipid oxidation causes negative quality effects, especially in further processed products. Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and rosemary extract (RM) are common antioxidants, but plant-based tannin compounds have been shown to be effective antioxidants. The objective was to evaluate antioxidant effectiveness, pH, color, and sensory effects of several tannin sources. In study 1, 19 percent fat ground beef was mixed with no treatment (control), BHA/BHT (0.02percent), RM (0.2percent), or 0.25percent or 0.5percent powders of Chardonnay grapeseed (CG), Norton grapeseed (NG), CitruSmart (CS), or dried cherry (CP). Raw and cooked patties were aerobically stored for 0 to 5 days at 4 degrees C. Cooked patties were analyzed using the TBARS (thiobarbituric acid reactive substances) method. Raw patties were analyzed for pH, instrumental color, subjective color, and ingredient specks. Study 2 was similar, except the treatments were 0.25percent and 0.5percent powders of CG, chestnut (CN), mimosa (M), and quebracho (Q) tannins, as well as 0.5percent NG, 0.02percent BHA/BHT, 0.2percent RM, and a control. In study 1, only the NG and CG pre-cooked patties had similar or lower TBARS values compared to RM and BHA/BHT patties. All tannin treatments, except 0.25percent CG and 0.25percent CS, lowered pH compared to the control. Patties with NG and 0.5percent CG were darker, and tannin-treated patties were not redder than the control. Patties with CG and 0.5percent NG were less yellow. Subjectively, tannin-treated patties did not have less discoloration during storage, and the CG and NG patties had numerous visible ingredient specks. In study 2, CN, M, Q, and 0.5percent CG treated patties had low TBARS during storage. pH was slightly lower in CG and CN patties than the control. All tannin-treated patties were darker than the control patties, except patties containing 0.25percent CG and Q, and control patties had the highest red values. Tannin-treatments patties, except CN and Q patties, were less yellow. Subjectively, only 0.25percent M patties had less discoloration than control patties, while 0.5percent CN patties were more discolored. Norton and Chardonnay grapeseed flour and chesnut, mimosa, and quebracho powders would be recommended for pre-cooked ground beef patties based on their antioxidant effectiveness in these studies.

The effects of sorghum bran addition on table tortillas and tortilla chip properties were evaluated. Texture, phenol content, antioxidant activity, and sensory characteristics were evaluated. Texture was measured by objective and subjective tests. Products were analyzed for phenols following the Folin-Ciocalteu procedure and for antioxidant potential following the ABTS (2,2'-azinobis (3- ethylbenzothiazoline-6-sulfonic acid) method. Sensory properties were evaluated using a nine point hedonic scale. Bran from two specialty sorghums: sumac (high tannin) and black (high anthocyanins) was added at 0, 5, and 10% to table tortillas and tortilla chips. For table tortillas the interaction of sorghum bran with an antistaling formula containing guar gum, carboxymethylcellulose and maltogenic alpha-amylase was assessed. Tortillas containing sorghum bran had a more friable structure than the control. This detrimental effect was overcome by the antistaling formula. Additives made fluffier tortillas with improved texture and appearance. Tortillas containing sorghum bran and the antistaling formula were acceptable to panelists. At 5% sorghum bran inclusion, there was no significant difference in sensory attributes from the control aside from appearance. Tortillas containing sorghum bran had a dark natural color comparable to that of blue corn tortillas. Tortilla chip texture was not significantly affected by addition of bran to the formula. As in table tortillas, addition of sorghum bran produced minor changes in the texture and flavor of the product, but a significant change in appearance acceptability. Tortilla chips had a dark color, comparable to the one of blue corn tortilla chips. Sumac bran yielded larger amounts of phenols and antioxidant activity than black bran. Levels of phenols and antioxidant potential increased with increased bran. Although processing caused a measurable loss of sorghum bran antioxidants, table tortilla and tortilla chips were still a significant source of phenols and antioxidant activity. The addition of sorghum bran produced tortillas and tortilla chips with increased levels of dietary fiber and antioxidants, without adversely affecting other sensory properties.

Thesis (MSc)--University of Stellenbosch, 2008. / Bibliography. Also available via the Internet.

Thesis (Ph.D.)--University of Delaware, 2009. / Principal faculty advisor: Brian J. Bahnson, Dept. of Chemistry & Biochemistry. Includes bibliographical references.

Thesis (M.S.)--Rutgers University, 2008. / "Graduate Program in Pharmaceutical Science." Includes bibliographical references (p. 41-42).