Dissertations / Theses on the topic 'Phenolic phytochemicals'

Fruits and vegetables are known to play an important role in human health due to the range of phytochemicals they contain. Twenty-one peach genotypes and 45 plum genotypes with different flesh and skin color were analyzed for their antioxidant content and antioxidant activity. Anthocyanin content, phenolic content and antioxidant activity were higher in red-flesh than in light-colored flesh peaches. Carotenoid content was higher in yellow-flesh peaches. Among the peaches, the antioxidant activity was well correlated with phenolic content. The anthocyanin content among the plums increased with the red color intensity. Red-flesh plums generally had higher phenolic content than the other plums. Antioxidant activity was higher in red-flesh genotypes; however, it was strongly correlated only with the phenolic content in light-colored flesh plums. Extracts from selected genotypes of peaches and plums and their fractions were evaluated against two breast cancer cell lines (MDA-MB-435 and MCF-7) and one non-cancerous breast line (MCF-10A). The cells were cultured in the presence of peach and plum extracts and their fractions at various concentrations (0-500 µg/ml) and the cell viability and antiproliferation activity was evaluated by MTT assay and Coulter Counter. There was a dose-dependent reduction on cell viability of estrogen-negative MDA-MB-435 breast cancer cells. Only weak activity against MCF-7 was observed at high extract concentrations. There was no activity against MCF-10A after 24 h treatment. Fraction I, which consists of mainly phenolic acids such as chlorogenic acid and a caffeic acid derivative, reduces MDA-MB-435 breast cancer cell viability with the lowest IC50. The second most effective fraction was Fraction II which contained anthocyanins. Fraction III (flavonols) and Fraction IV (polymerized compounds) had no effect on the cell lines. Phenolic acids present in fraction I induced apoptosis in MDA-MB-435 estrogen receptor-negative cell line. Fraction I did not induced apoptosis in MCF-10A, a noncancerous cell line even at higher concentrations than the ones tested in MDA-MB- 435. Apoptosis induced by Fraction I was caspase 3 and PARP independent. After treatment with 50 µg of chlorogenic acid equivalent/ml there was an activation of p- ERK.

The rise in the incidence of neurodegenerative diseases leads to an increased demand for the development of alternative therapeutic strategies. Phytochemicals have proved beneficial effects for human health, exerting protective roles towards several diseases including neurodegenerative disorders. Thus, the search for new bioactivities is a field in constant growth. The beet and fruits belonging to Opuntia spp. are rich in betalains and phenolic compounds, which confer several benefits to human health such as regulators of antioxidant and anti-inflammatory responses as well as potential protective effect for chronic diseases.
info:eu-repo/semantics/publishedVersion

Two dietary polyphenols, delphinidin and resveratrol, were selected for this study; delphinidin, an anthocyanin flavonoid, was found to elicit cardioprotective properties from a pilot study and resveratrol, a stilbene flavonoid, one of the most studied of the polyphenols and was chosen as a worthy comparator. In vitro studies using cultured endothelial cells exposed to a variety of reactive oxygen species (ROS) were used to determine if delphinidin and resveratrol could protect these cells from oxidative damage. It was found that physiological concentrations of delphinidin protected these cells from hydroxyl-radical damage. However, resveratrol failed to show any protective effects. Oxidative stress was induced in porcine coronary artery using diethyldithiocarbamate (DETCA), an inhibitor of the superoxide dismutase (SOD). Sensitivity to nitric oxide (NO) was preserved in endothelium-denuded vessels that were pre-treated with delphinidin, but not with resveratrol. However, studies where a balloon catheter was used to induce injury to the endothelium, delphinidin pre-treatment did not preserve sensitivity to NO in these vessels. Taken together, these results suggest that delphinidin, but not resveratrol, has potential to protect the endothelium from oxidative stress by preserving/protecting NO bioavailability and by acting directly as an antioxidant or indirectly upregulating antioxidant systems.

Aims: Cancer is the first cause of death in the Peruvian population; searching alternative treatments of medicinal plants constitute a promissory field to find new anticancer drugs. The main objective in this study was to evaluate the phytochemical screening, total phenolic content, antioxidant and cytotoxic activity of ethanol extract of Chromolaena laevigata (C. laevigata) on human tumor cell lines. Study Design: The fresh leaves of C. laevigata were soaked with ethanol followed by phytochemical screening using standard methods. Place and Duration of Study: Laboratory of Applied Chemistry, Faculty of Pharmacy and Biochemistry, Universidad Nacional San Luis Gonzaga de Ica, Ica, Peru; Laboratory “Abraham Vaisberg Wolach”, Universidad Peruana Cayetano Heredia, Lima, Peru. Methodology: Phytochemical screening was assessed by using chemical reactives. Total phenolic content (TPC) was developed using Folin Ciocalteu reactive and the antioxidant activity was determined against DPPH and ABTS radicals by spectrophotometry. The cytotoxic activity was determined on human tumor cell lines followed as: MCF-7, H-460, HT-29, M-14, K-562 and DU-145. Results: Phytochemical study confirmed flavonoids and phenolic compounds in ethanol extract. TPC resulted 45.21 ± 3.5 mg of gallic acid equivalent/g of dried extract. The highest antioxidant extract for DPPH and ABTS radical scavenging tests were IC50 = 11.66 ± 1.0 μg/mL, IC50= 12.45 ± 0.50 μg/mL respectively. Ethanolic extracts (μg/mL) showed a low cytotoxicity on human tumor cell lines (CI50 > 20 μg/mL) for DU-145, HT-29, MCF-7 and M-14. Whereas, for H-460, and K562 tumor cell lines showed high cytotoxicity. Conclusion: In our findings, C. laevigata demonstrated a high antioxidant and total phenolic content. The ethanol extract exhibited better cytotoxic effect compared with 5-FU. Hence, This medicinal plant could be effective to prevent chronical diseases as cancer and oxidative stress disorders.

Thesis (Ph.D.) - University of Glasgow, 2008.
Ph.D. thesis submitted to the Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, 2008. Includes bibliographical references. Print version also available.

PhD (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal Universtiy of Technology
The genus Tulbaghia has been used in traditional medicine to treat various ailments such as fever, earache, tuberculosis and esophageal cancer. However, there is limited scientific evidence to support its use. Therefore the objectives of this study were to perform phytochemical analysis, investigate the antioxidant, antimicrobial, anticancer, immunomodulatory activities and toxicity of crude acetone and water extracts from selected Tulbaghia species. Standard methods were used for preliminary phytochemical analysis. The total phenolic content of the plant extracts was determined using the folin ciocalteu method whereas the total flavonoids were determined by using the aluminium chloride colorimetric method. DPPH and ABTS assays were used to evaluate the antioxidant activity. The antimicrobial activity was assessed by agar well diffusion, microtiter dilution and time kill assays. For anticancer studies, the antiproliferative activity of the extracts was evaluated using the MTT assay on Hkesc-1 and KB cells. Morphological changes of the cancer cells treated with extracts were examined using light microscopy. Induction of apoptosis was assessed using fluorescence microscopy and acridine orange/ethidium bromide staining. Flow cytometry analysis was conducted to examine the multicaspase activity and cell cycle arrest. For immunomodulatory activity, the Greiss reagent and Luminex cytokine assays were used to determine the effect of the extracts on NO production and the concentration of the cytokines in the treated cells, respectively. Toxicity of selected Tulbaghia species was examined by investigating the effect of the extracts on the metabolic activity and cell membrane integrity on the treated RAW264.7 cells using the MTT and LDH assays, respectively. The zebrafish assay was used to evaluate the embryotoxicity and teratogenic effects of crude acetone and water extracts of T. violacea at 24 h intervals for 96 h post fertilisation (hpf). The percentage mortality, hatchability and heart rate were examined. Phytochemical screening of eight Tulbaghia species demonstrated the presence of flavonoids, glycosides, tannins, terpenoids, saponins and steroids. The amount of total phenol and flavonoid content varied in different plant extracts ranging from 4.50 to 11.10 milligrams gallic acid equivalent per gram (mg GAE/g) of fresh material and 3.04 to 9.65 milligrams quercetin equivalent per gram (mg QE/g) of fresh material respectively. The IC50 values based on DPPH and ABTS for T. alliacea (0.06 and 0.06 mg/mL) and T. violacea (0.08 and 0.03 mg/mL) were generally lower showing potential antioxidant activities. For antimicrobial activity, the acetone extracts of T. acutiloba, T. alliacea, T. leucantha, T. ludwigiana, T. natalensis and T. simmleri showed moderate antimicrobial activity against all test organisms while the water extracts showed moderate to no activity. One species, T. cernua, showed poor activity against all the tested microbes. The acetone and water extracts of T. violacea showed the greatest antibacterial and antifungal activity against all the tested microorganisms with minimum inhibitory concentration ranging from 0.1 mg/mL to 3.13 mg/mL. The acetone extracts of T. violacea also exhibited both bacteriostatic/fungistatic and bactericidal/fungicidal activity depending on the incubation time and concentration of the extract. The bactericidal/fungicidal activity was observed at x2 MIC. The results for anticancer activity showed that treatment of Hkesc-1 cells with acetone and water crude extracts had anti-proliferative activity with IC50 values of 0.4 mg/mL and 1.625 mg/mL, respectively while KB had 0.2 mg/mL and 1 mg/mL, respectively. Morphological changes such as blebbing, cell shrinkage and rounding were observed in the treated cells suggesting that apoptosis was taking place. AOEB staining showed that the level of apoptosis was dependent on the concentration of the extracts. The activation of multicaspase activity in both Hkesc-1 and KB treated cells was also concentration dependent leading to cell death by apoptosis and the induction of cell cycle arrest at the G2/M phase. Immunomodulatory activity results indicated that cell viability was above 80% when concentrations of 50 µg/mL or less of both acetone and water crude was used. Treatment with the acetone extract had no significant effect (p>0.05) on the LPS induced NO production in RAW264.7 cells except at 50 µg/mL where significant inhibition was observed. The water extract had no significant effect (p>0.05) on NO production at all the concentrations. Treatment of LPS–induced RAW264.7 cells with acetone extract stimulated the production of IL-1α, IL-6 and TNF-α, but had no significant effect (p > 0.05) on IL-1β. On the other hand, treatment with the water extracts stimulated the production of IL-1α, IL-6 but had no significant effect (p>0.05) on TNF-α and IL-1β. Treatment of LPS-induced RAW264.7 cells with the acetone extract had very little stimulatory effect on IL-4, IL-5 and IL-13 and no significant effect on IL-10 whereas for the water extract a significant stimulatory effect was only observed for IL-4 after 48 h of treatment. High concentrations (>10000 pg/mL) of MCP-1, MIP1-α, MIP1-β, MIP-2, GCSF, GM-CSF, RANTES and IP-10 were also observed in acetone and water extract treated RAW264.7 cells. For toxicity studies, acetone and aqueous crude leaf extracts from T. alliacea, T. simmleri, and T. violacea had a significant inhibitory (p<0.05) effect on the RAW264.7 cells after 48h treatment. Acetone extracts from T. alliacea, T. simmleri and T. violacea resulted in IC50 values of 0.48 mg/mL, 0.72 mg/mL and 0.1 mg/mL, respectively. Treatment with water extracts showed minimal toxic effect indicated by higher IC50 values of 0.95 mg/mL, 2.49 mg/mL and 0.3 mg/mL for T. alliacea, T. simmleri and T. violacea, respectively. The LDH release by macrophages after 24 h treatment with acetone extracts was observed to be concentration dependent while treatment with water extracts did not induce LDH release. The zebra fish assay showed a lethal dose (LD50) for the T. violacea acetone crude extract of 20 μg/mL whereas that for water extract was 85 μg/mL. The observed teratogenic effects included scoliosis, edema of the pericardial cavity, retarded yolk resorption, hook-like/bent tail and shorter body length. In conclusion, the results from this study indicate that the extracts from the eight Tulbaghia species examined contain phytochemicals that may have the antioxidant, antimicrobial, anticancer and immunomodulatory properties. Extracts from T. violacea were observed to be the most potent. This study thus supports the use of T. violacea in treating bacterial and fungal infections in traditional medicine. The results of this study also confirm the anticancer potential of T. violacea. The immunomodulatory activity of the acetone and water extracts from T. violacea indicated a dominantly pro-inflammatory activity. Traditional medicine prepared form T. violacea may be of benefit to individuals with weak immune systems. The toxicity of selected Tulbaghia species was observed to be concentration, extract and time dependent. Therefore, traditional medicine prepared from Tulbaghia extracts should be taken with caution preferably in small doses over a short period of time. Future studies will focus on the identification of the bioactive compound(s) responsible for the antimicrobial, anticancer and immunomodulatory activities.

Perilla L. are significant for multi-pharmacological effect. The aim of this work is to study Perilla L. growth and develop¬ment tendencies, productivity; composition of biologically active compounds and their variations during the vegetation period, and the biological effect of the extracts; to select perspective plants for cultivation in Lithuania and for production of medicinal preparations. The objectives of the Study: To investigate and determine growth dynamics of Perilla L. species and varieties during the vegetation period and to assess the influence of climate conditions on the vegetation rhythmic. To assess the quantity dynamics of Perilla L. species and varieties herbal medicinal raw material and to compare the productivity of Perilla L. species and varieties. To determine the quantity of the essential oil in Perilla L. species and varieties and to identify the chemotypes of the plants. To determine the qualitative composition and variation dynamics of the phenolic acids in Perilla L. species and varieties. To determine the composition and variation dynamics of the flavone complex in Perilla L. species and varieties. To determine the composition and variation dynamics of antho¬cyanidines in Perilla L. species and varieties. To assess the radical scavenging activity of the extracts of Perilla L. species and varieties raw materials. To research the effect of Perilla L. extracts on the oxidative phosphorylation in the rat heart mitochondria.
Perilla L. genties vienmečiai vaistiniai augalai yra augaliniai imunomoduliatoriai, pasižymintys daugeliu farmakologinių poveikių. Darbo tikslas: Ištirti Vidurio Lietuvoje auginamų Perilla L. rūšių ir varietetų augimo ir vystymosi dėsningumus, biologiškai aktyvių junginių sudėtį ir jų įvai¬ravimą vegetacijos metu bei ekstraktų biologinį poveikį; atrinkti perspek¬tyvius augalus auginimui Lietuvoje. Uždaviniai: Ištirti ir nustatyti Perilla L. augimo dinamiką vegetacijos metu ir įvertinti klimatinių veiksnių įtaką augalų vegetacijai. Įvertinti Perilla L. vaistinės augalinės žaliavos kiekio dinamiką vege¬tacijos metu ir palyginti Perilla L. rūšių ir varietetų produk¬tyvumą. Nustatyti Perilla L. rūšių ir varietetų eterinio aliejaus kiekį auga¬luose vegetacijos metu ir identifikuoti augalų chemotipus. Nustatyti Perilla L. rūšių ir varietetų fenolinių rūgščių kokybinę sudėtį ir jų kitimo dėsningumus vegetacijos metu. Nustatyti Perilla L. rūšių ir varietetų flavonų komplekso sudėtį ir kitimo dinamiką vegetacijos metu. Nustatyti Perilla L. rūšių ir varietetų antocianidinų sudėtį bei kitimo dinamiką vegetacijos metu. Įvertinti Perilla L. rūšių ir varietetų žaliavų ekstraktų antiradikalinį aktyvumą. Ištirti Perilla L. rūšių ir varietetų ekstraktų poveikį žiurkės širdies mitochondrijų oksidaciniam fosforilinimui. Tyrimų rezultatai ir poveikiu pasižyminčių junginių identifikavimas atveria perspektyvas ateities tyrimams, kurie reikalingi kuriant preparatus iš perilių augalinių žaliavų.

The aim of this research was to investigate the phytochemistry of two species Sanicula europaea and Teucrium davaeanum which are traditionally used in treatment of wounds. Four compounds were isolated from the 80% methanolic extract of S. europaea; bis-(2-ethylhexyl) phthalate (1), palmitic acid (2), rosmarinic acid (3), saniculoside N (4). Compounds 1 and 2 were isolated for the first time from this species. The structure elucidation of the isolated compounds was on the basis of 1D, 2D NMR spectroscopy and mass spectrometry measurements. Two compounds were isolated from the crude glycosides extract of T.davaeanum; 6 is a phenylethanoid glycoside and 8 is an iridoid glycoside, from the data available these may be new compounds for which the names davaeanuside A and davaeanuside B are proposed respectively." The total polyphenol content of S. europaea L, T. davaeanum leaves-flowers and T. davaeanum stem were found to be 5.0, 1.20 and 0.65 mg per 100 mg dried plant material respectively. A study of the antioxidant activity of the 50 % ethanol extracts of S. europaea and T. davaeanum showed that on a mg/mg basis S. europaea and T. davaeanum have approximately 5%, 8 % antioxidant capacity of Trolox respectively. A study of the cytotoxic activity of davaeanuside A (6), iridoid glycoside (7), davaeanuside B (8) and saponin compound (10) isolated from the crude glycosides extract of T. davaeanum revealed that saponin compound (10) inhibited the growth of Hela cells by 50 % at 50 μg/ml, P< 0.001, but the other compounds did not show activities against the tested cell lines at 100 μg/ml. The results of this work provide some basis for the traditional use of these species in the treatment of wounds.

CAPES; Fundação Araucária
A espécie Moringa oleifera (Moringaceae) é uma planta que possui ampla aplicação industrial, alto valor nutricional e que, além disso, também exibe diversas atividades biológicas. Utilizadas na medicina popular, as folhas de M. oleifera já demonstraram possuir grande variedade de moléculas bioativas, inclusive compostos fenólicos, os quais são, possivelmente, os responsáveis pelo potencial antioxidante desta parte da planta. Apesar do crescente interesse sobre a espécie e, especificamente, sobre o seu potencial fitoquímico, são escassos os trabalhos que relatam o isolamento e a identificação dos compostos bioativos presentes nas suas folhas, principalmente em exemplares cultivados no Brasil. Sendo assim, os objetivos deste trabalho foram comparar dois métodos de extração de compostos bioativos e, na sequência, isolar bioguiadamente compostos com atividade antioxidante das folhas de M. oleifera coletadas no município de Itajaí (Santa Catarina, Brasil). O monitoramento bioguiado foi realizado com ensaios in vitro de determinação da atividade antioxidante: capacidade de redução do reagente Folin-Ciocalteau, FRAP, sequestro dos radicais DPPH e ABTS, além do ORAC. A técnica de CLAE-DAD foi utilizada para a caracterização química e acompanhamento das etapas do isolamento. A principal diferença prática entre os métodos de extração avaliados foi o preparo de um extrato hidroalcoólico inicial, no processo de extração 1. A partir dos resultados de determinação da atividade antioxidante, interpretados com o auxílio de ferramentas estatísticas (teste de Tukey e teste t pareado), foi possível perceber que o potencial das folhas de M. oleifera sofreu variações em função da forma de extração e dos solventes utilizados. Em geral, as frações produzidas a partir do processo de extração 1 apresentaram maior atividade antioxidante e perfis cromatográficos com sinais mais intensos. Com base nestes resultados, a fração obtida com acetato de etila, no processo de extração 1, foi selecionada para dar sequência ao isolamento bioguiado. A purificação desta fração em coluna aberta preenchida com sílica gel gerou 61 subfrações, as quais, após análise de CCD, foram agrupadas em 18. A avaliação da atividade antioxidante das subfrações agrupadas mostrou que cinco apresentavam grande potencial. Contudo, em função do rendimento, apenas três puderam dar sequência ao isolamento. Nesta etapa, uma análise adicional foi realizada: a determinação da atividade antioxidante por CLAE on-line com o ABTS•+, que permitiu definir quais dos compostos presentes nas três subfrações possuíam maior potencial e, por isso, seriam isolados. Desta forma, cinco compostos foram isolados pela técnica de CLAE semipreparativa, sendo que dois foram testados frente ao ensaio de sequestro do DPPH•. Os valores de EC50 obtidos, 30,34 e 38,72 μg/mL, estão próximos aos encontrados na literatura para substâncias isoladas de outras matrizes naturais. A técnica de RMN permitiu identificar um flavonol glicosilado. Os resultados deste trabalho mostraram que as folhas de M. oleifera coletadas em Itajaí são fonte de compostos fenólicos com potencial antioxidante e, por isso, promissoras para aplicação nas indústrias de cosméticos, alimentos e farmacêutica.
Moringa oleifera (Moringaceae) is a plant that has wide industrial application, high nutritional value and, also, exhibits several biological activities. Used in folk medicine, M. oleifera leaves have already been shown to possess a wide variety of bioactive molecules, including phenolic componds, which are possibly responsible for the antioxidant potential of this part of the plant. Despite the growing interest in this species and, specifically, in its phytochemical potential, there are few studies about the isolation and identification of bioactive compounds present in M. oleifera leaves, especially in specimens grown in Brazil. Therefore, the aims of this work were to compare two methods for extracting bioactive componds and, than, to isolate compounds with antioxidant activity of M. oleifera leaves collected in Itajaí (Santa Catarina, Brazil) by a bioguided study. The bioguided monitoring was carried out with in vitro assays to determine the antioxidant activity: Folin-Ciocalteau reagent reduction capacity, FRAP assay, DPPH and ABTS radical scavenging methods and, also, the ORAC assay. HPLC-DAD technique was used for chemical characterization and monitoring of the isolation stages. The main practical difference between the evaluated extraction methods was the preparation of an initial hydroalcoholic extract, in the extraction process 1. From the results of the antioxidant activity determination, interpreted with the aid of statistical tools (Tukey’s test and paired t-test), it was possible to see that the potential of M. oleifera leaves varied depending on the extraction form and on the solvents used. In general, the fractions prepared from the extraction process 1 showed higher antioxidant activity and chromatographic profiles with more intense signals. Based on these results, the fraction obtained with ethyl acetate, in the extraction process 1, was selected for the bioguided isolation. The purification of this fraction on an open column of silica gel generated 61 subfractions, which, after TLC analysis, were grouped in 18. The evaluation of the antioxidant activity of grouped subfractions showed that five of them presented great potential. However, depending on the yield, only three could follow the isolation. In this step, an additional analysis was performed: the determination of the antioxidant activity by an on-line HPLC method with the ABTS•+. This technique allowed defining which of the compounds presented in each subfraction had higher potential and, therefore, would be isolated. In this way, five compounds were isolated by semipreparative HPLC, two of them were tested by the DPPH• scavenging assay. The obtained EC50 values, 30.34 and 38.72 μg/mL, are close to those found in literature for substances isolated from other natural matrices. The NMR technique allowed identifying a flavonol glucoside. The results of this work showed that M. oleifera leaves collected in Itajaí are source of phenolic compounds with antioxidant potential and, therefore, are promising for the application in cosmetics, food and pharmaceutical industries.

Ethanol extracts of “jenipapo” (Genipa americana Linnaeus), “umbu" (Spondia tuberosa Arruda), “siriguela” (Spondia purpurea Linnaeus) and “mangaba” (Hancornia speciosa Gomes) were prepared from separate pulp, seeds and peel; except mangaba which are used pulp and peel. The investigation of antioxidant capacity of the ethanol extracts were carried out by the methods of determining the content of ascorbic acid and total phenolics; scavenging 2, 2 -diphenyl-1-picrilhydrazyl and 2, 2’- azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical, antioxidant capacity of reduction of iron and copper and lipid peroxidation using a biomimetic membrane system, and measurement enzymatic of catalase and superoxide dismutase. It was also analyzed the acetylcholinesterase inhibitory activity, cytotoxic effect on corneal epithelial cells of sheep, as well as phytochemicals assays and identification of phenolic compounds and organic acids present in the extracts by UPLC - MS. The highest values of total phenolic content were obtained with peel and seed extracts and to ascorbic acid in the seed of siriguela, showing better antioxidant activities of seeds and peel extracts of siriguela and umbu. Lipid peroxidation assays indicated that genipap pulp is a promising antioxidant. Genipap pulp and siriguela seed ethanol extracts presented an acetylcholinesterase inhibition zone similar to that of the positive control, carbachol. The investigation of phenols and organic acid contents revealed the presence of quercetin (48,38 to 3,88 μg.g-1), quinic acid (43,28 to 41,88 μg.g-1) and citric acid (3,78 to 0,43 μg.g-1) in several extracts and chlorogenic acid with the highest amount found in siriguela seeds (356,93 μg.g-1). These data suggest new uses for these foods as potential antioxidant supplements for the food, pharmaceutical and cosmetic industries.
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
A partir das cascas, polpas e sementes do jenipapo (Genipa americana Linnaeus), umbu (Spondia tuberosa Arruda), siriguela (Spondia purpurea Linnaeus) e mangaba (Hancornia speciosa Gomes) foram preparados extratos etanólicos, com exceção da mangaba onde o extrato foi preparado utilizando casca e polpa. Os extratos foram submetidos à investigação da capacidade antioxidante através dos métodos de determinação do conteúdo de ácido ascórbico e fenóis totais, sequestro do radical 2,2-difenil-1-picril-hidrazil e 2,2´- azinobis (3-etilbenzotiazolina-6-ácido sulfônico), capacidade antioxidante de redução do ferro e cobre, lipoperoxidação utilizando um sistema de membranas biomimético e mensuração enzimática da catalase e superóxido dismutase. Também foi avaliada a atividade inibitória da enzima acetilcolinesterase, o efeito citotóxico em células epiteliais da córnea de ovelhas, além da realização de ensaios fitoquímicos e a identificação de compostos fenólicos e ácidos orgânicos presentes nos extratos. Os maiores teores de fenóis totais foram obtidos nos extratos das cascas e sementes e no referente ao ácido ascórbico na semente da siriguela, apresentando melhor atividade antioxidante os extratos das sementes e cascas da siriguela e do umbu. Porém, no ensaio de peroxidação lipídica, o extrato etanólico da polpa de jenipapo demonstrou ser um antioxidante promissor. Os extratos etanólicos da polpa do jenipapo e da semente da siriguela apresentaram uma zona de inibição da enzima acetilcolinesterase semelhantes ao controle positivo, carbacol. Na investigação dos compostos fenólicos e ácidos orgânicos por UPLC-MS verificou-se a presença nos extratos de quercetina (48,38 a 3,88 μg.g-1), ácido quínico (43,28 a 41,88 μg.g-1), ácido cítrico (3,78 a 0,43 μg.g-1) em vários extratos. E ácido clorogênico, na semente da siriguela (356,93 μg.g-1). Os resultados obtidos desses extratos sugerem novas formas de utilização desses alimentos como potenciais suplementos antioxidantes na indústria alimentícia, farmacêutica e cosmética.

The chronic diseases such as diabetes mellitus and cardiovascular disease are among the leading causes of death globally. These are strongly associated with obesity as a result of change in dietary pattern towards high calories and reduced physical activity, which now affects both developing and developed countries. Excessive cellular energy from high calorie foods results in incomplete reduction of oxygen resulting in oxidative stress leading to reactive oxygen species (ROS). The harmful effects of ROS can be balanced by the combination of non-enzymatic antioxidants and antioxidant enzymes. Hence, the maintenance of balance between oxidants and antioxidants through well-designed diet that can modulate cellular protection through critical energy and reductant pathways coupled to antioxidant enzymes is essential. Therefore, the aim of this dissertation was to develop strategies for designing diets enriched in phenolic phytochemicals for chemoprevention of diseases. This was achieved through understanding the critical metabolic pathways contributing to cellular protection against oxidation dysfunction-linked chronic diseases. Based on structure-function rationale, the potential antioxidant, anti-hyperglycemia and anti-hypertension functionality of plant foods such as clonal lines of Lamiaceae family, Rhodiola, wine, tea, vegetables (peppers, eggplant and pumpkin) and grains (corn and legume) were investigated. Results indicated that specific phenolic profiles had high anti-hyperglycemia and anti-hypertension potential which generally corresponded to total phenolic content and free radical scavenging-linked antioxidant activity. Dietary chemoprevention of bacterial pathogens such as Helicobacter pylori and Staphylococcus aureus was investigated through inhibition of critical control points that breakdown cellular energy and antioxidant pathways. Results indicated that lemon balm with the highest gallic acid and caffeic acid contents had potent anti-S. aureus activity. L-Lactic acid, a major food ingredient, had high anti-H. pylori activity through inhibition of proline dehydrogenase and catalase. Overall cellular protection through modulation of energy and reductant stimulating metabolic pathways was investigated in model eukaryotes. Rhodiola crenulata with high anti-hyperglycermia activity, induced apoptosis in V14a breast cancer cell line. Further, phenolic phytochemicals from R. crenulata protected Saccharomyces cerevisiae from UV-induced oxidative stress and delayed death. The cellular protection with phenolic phytochemicals was linked to modulation of proline-linked oxidative phosphorylation coupled to pentose phosphate pathway (PPP).

Research Doctorate - Doctor of Philosophy (PhD)
The poorest prognostic outcome for pancreatic cancer (PC) patients, among all gastrointestinal malignancies, can be attributed to the molecular heterogeneity and lack of specific therapeutic strategies. The emergence of resistance against the common chemotherapeutic drug gemcitabine has also been widely reported. Several studies have demonstrated improved efficacy using gemcitabine in conjunction with plant polyphenolics and antioxidants for PC treatment. This suggests that plant secondary metabolites should be investigated further in a search for adjuncts to current PC treatments. Moreover, plant-derived bioactive compounds have played a key role in the development of anticancer drugs over many decades. Eucalypts dominate the Australian landscape with over 800 distinct species. Eucalypt-derived phytochemicals have been associated with a wide range of bioactivity, both in traditional indigenous Australian bush medicine and in the scientific literature. However, a few eucalypt species and their essential oils have to date been exploited for their anticancer properties. An extensive review (Chapter 1) confirmed that more research was required to gain an improved understanding of the anticancer potential of Australian eucalypt phytochemicals with activity specific to PC. Therefore, the research reported herein was designed to address two main aspects, namely; (1) determining the optimal extraction conditions for phenolics and antioxidants from eucalypts, and (2) assessing their antiproliferative activity against PC cells including the delineation of potential molecular mechanisms of action responsible for this activity. Conventional extraction with water was employed to prepare crude extracts from eight different eucalypt species and was shown to be the most efficient method for extracting phenolics and antioxidants when compared to microwave-assisted (MAE) and ultrasound-assisted extractions (UAE) (Chapter 2). Crude extracts derived from Angophora floribunda, Angophora hispida and Eucalyptus microcorys were demonstrated to possess the most potent phytochemical profile, exhibiting statistically similar cytotoxicities against MIA PaCa-2 cells as discussed in Chapter 3. In addition, E. microcorys crude extracts exerted significantly greater cytotoxicity against glioblastoma, neuroblastoma and lung cancer cells than the other extracts. In MIA PaCa-2 cells, E. microcorys crude extracts induced caspase 3/7-mediated apoptosis. Therefore, the aqueous E. microcorys extract was subjected to further investigation to obtain a greater depth of understanding of their bioactivity. Chapter 4 focuses on the significant antioxidant, antifungal and antibacterial properties of aqueous E. microcorys extract. Subsequent bioassay-guided fractionation of E. microcorys aqueous crude extract using semi-preparative Reversed-Phase (RP) HPLC revealed that fraction-1 was significantly more efficacious in terms of its antioxidant and antiproliferative activity against MIA PaCa-2 cells in comparison to other four fractions, as stated in Chapter 5. Flow cytometry analyses validated that the cytotoxicity was mediated by induction of apoptosis and abrogation of the cell cycle in the G2/M phase. Western blot analysis showed that the active fraction significantly downregulated the antiapoptotic protein B-cell lymphoma 2 (Bcl-2) and upregulated the proapoptotic proteins Bcl-2 homologous antagonist killer (Bak) and Bcl-2-associated protein (Bax) and cleaved Poly (ADP-ribose) polymerase (PARP) in MIA PaCa-2 cells. Combination treatment of the active fraction with gemcitabine increased apoptosis and cell cycle abrogation of MIA PaCa-2 greater than either mono treatment, indicating a potential additive/synergistic effect against the PC cells. Untargeted metabolomics using High performance/pressure liquid chromatography/electrospray ionisation/mass spectroscopy/mass spectroscopy (HPLC-ESI/MS/MS) revealed the tentative identities of the phytochemicals in the active fraction to be mostly phenolic compounds, of which several have previously been described to possess antipancreatic cancer activity. The findings presented in this thesis provide further scientific evidence of the antipancreatic cancer activity of extracts from Australian eucalypts. This is the first report to optimise the MAE and UAE techniques and parameters for extracting phenolic compounds and antioxidants from Eucalyptus robusta and establish the antiproliferative activity of species belonging to all three main genera of Australian eucalypts against the PC cells. Bioassay-guided fractionation of E. microcorys aqueous crude extract, investigation of bioactive compounds in the most potent fraction by liquid chromatography-mass spectroscopy (LC-MS) based-metabolomics and studies to obtain a mechanistic explanation of antiproliferative activity against PC cells are other key contributions of this project.

The rapidly growing world population, increasing severity of climate change, and constantly evolving environmental pressures have drawn into question whether current agricultural practices can meet the growing food demands healthily, equitably and sustainably. This has resulted in the rising popularity of natural biostimulants, particularly seaweed extracts, to increase crop productivity in an eco-friendly and safe manner. To better understand the complex modes of action underpinning the well-reported benefits of seaweed biostimulants to crops, their phytochemical composition requires further characterisation. Hydroxybenzoic acids, a subclass of phenolic acids, are an important class of phytochemicals and the aim of this study was to characterise their profile in commercial seaweed biostimulants. This work used modern analytical technologies to investigate salicylic acid and other benzoic acid derivatives in a commercial seaweed biostimulant, and then assessed the biological activity of the monohydroxybenzoic acids using plant growth assays. Qualitative HPLC-ESI-MS/MS methods were developed for the analysis of hydroxybenzoic acids and related derivatives. The various benzoic acid derivatives investigated include monohydroxybenzoic acids, dihydroxybenzoic acids, trihydroxybenzoic acids, methoxylated hydroxybenzoic acids, methoxylated benzoic acids, and an amino substituted benzoic acid. The HPLC-ESI-MS/MS methods for the analysis of the various derivatives were then employed to investigate the presence of these compounds in the commercial seaweed biostimulant. The compounds found to be present were the monohydroxybenzoic acids, 2,3- and 3,4-dihydroxybenzoic acid, syringic acid, and anthranilic acid. A HPLC-ESI-MS/MS method for the analysis of the monohydroxybenzoic acids was optimised and partially validated for the quantification of salicylic acid and its isomers in a commercial seaweed biostimulant. Sample preparation employed acidified acetonitrile partitioning of the seaweed biostimulant before mixed-mode solid-phase extraction. The three isomers were successfully separated using a reversed-phase biphenyl stationary phase with a methanol/water mobile phase acidified with formic acid. The MS/MS detection employed the characteristic MRM transition of m/z 137  93 of the monohydroxybenzoic acids. The concentrations of 2-, 3- and 4-hydroxybenzoic acid in a commercial seaweed biostimulant were found to be 137, 3409, and 1748 μg/L, respectively. Tomato seedling plant growth bioassays were conducted to investigate the biological effects of salicylic acid and its isomers on plant growth. Fresh and dry root and shoot weight data along with longest root length data were assessed to evaluate the biological effects of the various treatments on tomato seedling growth. It was found that a significant increase in root growth was observed when the commercial seaweed biostimulant was fortified with a combination of the three monohydroxybenzoic acids, using dosages that correlate to the concentrations determined in the seaweed biostimulant in this study.

Pecan kernels from six cultivars were analyzed for phenolic content and antioxidant properties. In addition, kernels from two cultivars were irradiated with 0, 1.5 and 3.0 kGy using E-Beam irradiation and stored in accelerated conditions (40 °C and 55% R.H.). Changes in phytochemical profile and antioxidant properties were monitored for 134 days. Cultivars differed greatly in their phytochemical content. Total extractable phenolic content (TP) ranged from 62 to 106 milligrams of chlorogenic acid equivalents per gram of defatted kernel. Antioxidant capacity (AC) measured by the DPPH free radical had a strong correlation with TP. Shells from each cultivar were 6, 4.5 and 18 times greater for TP, AC and condensed tannin content (CT). Gallic and ellagic acids, epicatechin and catechin were identified in hydrolyzed extracts of all cultivars. Prior to hydrolysis, no compounds were positively identified. Fatty acid profile of kernel oil had a strong inverse correlation between oleic and linoleic oil. Kernels from the same cultivar but different location differed in their fatty acid composition but had similar TP. Irradiation of â Kanzaâ and â Desirableâ kernels with 1.5 and 3.0 kGy had no detrimental effects on AC and TP by the end of experiments. Phenolic profile was similar for all treatments. Tocopherol content decreased with irradiation treatments, but no further degradation was observed throughout storage. Peroxide values increased slightly after 98 and 134 days of storage for â Desirableâ kernels, with slight differences between controls and irradiated samples. Color of kernels decreased in lightness and yellowness and increased in redness with no differences between irradiated samples and controls. For the first time the effect of pecan cultivar and E-Beam irradiation was assessed in phytochemical and antioxidant attributes of pecan kernels. Additionally, irradiation with E-Beam had no significant detrimental effects in phytochemical composition and only a slight increase in peroxide value, indicating potential as pecan kernel sanitization.

The total phenolic content (TPC) of common vegetables grown in Ontario was determined by the Folin-Ciocalteu method, and it was found that the broccoli inflorescence had an exceptionally high TPC on average value, followed by cabbage, onion, potato and carrot. The TPC values of darkpurple potatoes and carrots were higher than the common potatoes and carrots. Positive correlations between the TPC and TAA were observed with varied degrees in all vegetables. Choice of cultivar and production practices can be used to increase TPC and TAA in a wide range of vegetables. Insecticide application did not influence the TPC and TAA of broccoli leaves and flowers. Higher N rate decreased the TPC and TAA of cabbage cultivar ‘Huron’ and of carrot. Fungicide and biofungicide applications did not influence TAA in carrots. Fertilizer applications did not influence the TAA of onions, but there was a decrease in TPC. The rate of MAP (mono ammonium phosphate 52% P2O5) affected the TAA of onions, but the influence was inconsistent between two antioxidant assays. High temperature with possibly high rainfall capacity occurred in the year increased the TPC and TAA of most studied vegetable crops.
Ontario Ministry of Agriculture, Food and Rural Affairs/University of Guelph Sustainable Production Systems Program

The relationship between phenolic compounds and sensory attributes has been studied using pure solutions and more recently in baked products made using commercial blends of white or red wheat varieties. However, research is lacking that investigates the relationship between phytochemical content of pure varieties of light red or medium red wheat and the perceived sensory attributes in the context of baked product matrix. Darker red wheat is believed to contain higher amounts of phenolic phytochemicals which has been speculated to be the reason for off-flavours in baked products, thus having a negative impact on consumer acceptance of wholegrain baked products made using red wheat. Compared to baked products made using light red wheat, the medium red wheat products were perceived to be more intense in sensory attributes such as bitterness and astringency, among other properties. A number of non-volatile and volatile phytochemicals in low and intermediate moisture baked products were found to be correlated with the sensory attributes perceived by trained panellists. The results of this research will be useful to wheat breeders, processors and fellow researchers in improving their understanding of samples they are working with and integrate new ideas into their research as it provides a) an easy technique to classify wheat grains and, b) a database to further explore the relationship between phytochemistry and flavour of baked products.
OMAFRA

O conceito de sustentabilidade resume-se à noção de desenvolvimento económico, social e ambiental, de forma a não provocar danos significativos ao ambiente e aos recursos naturais existentes. Nos dias de hoje, o crescimento da população é uma das questões mais preocupantes, sendo indispensável repensar o problema dos desperdícios originados pelas indústrias agroalimentares. Anualmente, estas indústrias desperdiçam toneladas de resíduos que pouca ou nenhuma utilização têm. Contudo, grande parte destes desperdícios alimentares contém quantidades consideráveis de compostos bioatividade, cuja atividade pode ter diversas aplicações, sendo de destacar a produção de nutracêuticos. As indústrias agroalimentares produtoras e/ou transformadoras de azeite, vinho, café e castanha geram uma grande quantidade de resíduos alimentares potencialmente atrativos para outras indústrias, que os podem reutilizar com vista a aumentar a sua produtividade e lucro. Tendo em conta este problema, este trabalho de conclusão de ciclo tenta mostrar a importância do reaproveitamento dos resíduos alimentares com maior expressão a nível económico, ambiental e social. Dados as características químicas e os efeitos benéficos que estes resíduos alimentares podem causar à saúde humana, a sua reutilização para a formação de novos produtos pode ser vantajosa para as indústrias farmacêuticas, cosméticas e alimentares.
Sustainability is summed up as the concept of economic, social and environmental development in way that doesn’t cause significant damage to the environment and existing natural resources. Nowadays, population growth is one of the most concerning factors and it is imperative to rethink the issue of the waste that originates from the agrifood sector. Every year, these industries produce tons of waste with very little utility. However, most of the food waste has considerable amounts of bioactive compounds, whose activity can be used to produce nutraceuticals. The agri-food and/or manufacturing industries that produce olive oil, wine, coffee and chestnut produce an enormous amount of food waste that are potentially attractive to other industries that will reuse them with the goal of increasing their profit and productivity. Considering this issue, this work of conclusion of period tries to show the importance of reusing food waste with bigger economical, environmental and social impact. Given the chemical characteristics and the beneficial effects this waste can cause to human health, its reuse to create new products can be an advantage for the pharmaceutical, cosmetic and food industries.

Phytochemicals are defined as biologically active, non-nutrient (i.e. not essential for the maintenance of life), plant compounds. They are present in all fruits, vegetables, grains, and other plant foods, and have been shown to be beneficial to human health. Flavonols are a group of phytochemicals present in a variety of different vegetables including broccoli, Brussels sprouts, cabbage, cauliflower, kale and bok choy. Flavonols have been shown to exhibit powerful antioxidant activity as well as possess potential protective properties against certain cancers. The aim of this research was to measure the levels of the major flavonols in bok choy and to assess the antiproliferative activity of crude bok choy extracts, selected fractions, and individual flavonol compounds on human colon cancer cells (HT-29) in vitro. The flavonol composition of three bok choy cultivars (Sumo, Karate, and Miyako) was determined after acid hydrolysis by HPLC-PDA/ESI-MSn . Kaempferol (85.5 – 122 mg/100 g DW), isorhamnetin (38.3 – 66.7 mg/100 g DW), and quercetin (10 – 20.6 mg/100 g DW) were the main flavonols present. The Miyako variety contained the highest levels of both quercetin and isorhamnetin, however, the levels of kaempferol were comparable in all three cultivars. The total flavonol aglycone content in the three bok choy cultivars did not vary significantly (183.3 mg/100 g DW, 159.9 mg/100 g DW, and 197.3 mg/100 g DW for Sumo, Karate, and Miyako respectively), therefore, no conclusions were made as to whether one bok choy cultivar may contain more health-promoting flavonols than another. Several glycoside and hydroxycinnamic acid derivatives of quercetin, isorhamnetin, and kaempferol were identified in the alkaline and hydroalcoholic bok choy extracts by HPLCPDA/ESI-MSn . Two flavonol-3-sophoroside-7-glucosides and three flavonol-3,7-diglucosides were identified in the bok choy after alkaline hydrolysis, and six complex flavonol glycosidehydroxycinnamic acid derivatives, two flavonol di-glycosides, and one flavonol mono-glucoside were identified in the hydroalcoholic extracts. Kaempferol-3-O-sophoroside-7-O-glucoside was purified by preparative HPLC and SPE and fully characterised by NMR, UV and MSn . The ability of crude bok choy extracts, selected fractions, and individual flavonol compounds to inhibit cell proliferation of the human colon adenocarcinoma cell line, HT-29, in vitro was assessed using the MTT assay. The antiproliferative effects on HT-29 cells was evident for all extracts/fractions/compounds examined, however, the crude bok choy extracts were found to be the most potent. The IC50 values for the three bok choy cultivars ranged between 1.58 – 4.01 mg/L after 72 hours of exposure. Preliminary results suggested that bok choy has the potential to positively influence human health, both from a chemopreventive perspective as well as a chemotherapeutic.