O câncer de mama caracteriza-se globalmente como a neoplasia de maior incidência e mortalidade na população feminina. Antocianinas e elagitaninos presentes em frutas como as berries destacam-se por seu promissor efeito protetor em diferentes estágios do desenvolvimento do câncer de mama. Grumixama (G; Eugenia brasiliensis Lam) é uma espécie de cereja nativa do Brasil que assim como as amora-preta (AP; Rubus spp) e silvestre (AS; Morus nigra L.) contém alto teor de antocianinas e elagitaninos. Poucos estudos focaram na ação anticâncer destas berries no câncer de mama. Portanto, o objetivo do presente estudo foi avaliar os efeitos de extratos ricos em antocianinas ou elagitaninos de G, AP e AS no crescimento e na expressão de genes e miRNAs das linhagens de células humanas de câncer de mama MCF-7 (receptor de hormônio positiva) e MDA-MB 231 (receptor de hormônio negativa). Não se observou citotoxicidade após 72 e 96 horas de tratamento com os extratos (25-200&#181g/mL) ricos em antocianinas (ASANT, APANT e GANT) ou elagitaninos (APELA e GELA), em ambas as linhagens celulares. Após 72 horas de tratamento, GANT e APANT induziram parada de ciclo celular em G0/G1 (12,5 e 50&#181g/mL, p<0,05) em células MCF-7. Após 96h, ASANT, APANT e GANT induziram parada de ciclo celular em G0/G1 (12,5 e 50&#181g/mL; p<0,05) nessas mesmas células. Entretanto, na concentração de 200&#181g/mL apenas GANT induziu parada em G0/G1 (72 e 96h; p<0,05). Em células MDA-MB 231, após 96h APANT e GANT induziram parada de ciclo celular em G0/G1 nas concentrações testadas (12,5, 50 e 200&#181g/mL, p<0,05), assim como ASANT nas concentrações de 12,5 e 50&#181g/mL (p<0,05). Em células MCF-7, após 72h APELA e GELA induziram aumento da proporção de células em subG0 (200&#181g/mL, p<0,05). Em MDA-MB 231, após 72 e 96h, APELA e GELA (200&#181g/mL) induziram aumento da proporção de células em subG0 (p<0,05) e parada em G0/G1 (p<0,05). Em células MCF-7, GANT induziu morte celular por apoptose (p<0,05) após 72 e 96h de tratamento. Entretanto, em MDA-MB 231 os extratos ricos em antocianinas não induziram morte celular. Em células MCF-7, após 96h GELA e APELA induziram principalmente necrose (p<0,05). Em MDA-MB 231, APELA e GELA induziram apoptose (p<0,05) após 72 e 96h. Em células MDA-MB 231, após 72h de tratamento foi observada inibição da proliferação celular por GELA, GANT e APELA (200&#181g/mL; p<0,05). Em células MDA-MB 231, 48h de tratamento com GELA; GANT e APELA (200&#181g/mL) aumentaram a expressão 5 genes (ESR2, FOXA1, JUN, PTGS2,VEGFA) e inibiram a expressão de 10 genes (ADAM23, ATM, BCL2, CDH1, EGF, GLI1, ID1, MKI67, SNAI2 e THBS1) correlacionados ao câncer de mama. Adicionalmente, GELA; GANT e APELA (200&#181g/mL) induziram aumento da expressão de miR- 210(p<0,05) e APELA (200&#181g/mL) reduziu a expressão de miRNA 19a/b (p<0,05) em células MDAMB 231. Coletivamente estes resultados sugerem que antocianinas de grumixama e elagitaninos de amora preta e grumixama apresentam potencial efeito protetor contra o câncer de mama. Adicionalmente, essa ação anticarcinogênica pode ser mediada por indução de morte celular, mais especificamente apoptose, redução de proliferação celular e modulação da expressão de genes e miRNAs relacionados ao câncer de mama. / Breast cancer is characterized as the neoplasia with the highest incidence and mortality rates in women worldwide. Anthocyanins and ellagitannins present in certain fruits, such as berries, stand out for their promising protective effect at different stages of breast cancer development. Grumixama (G; Eugenia brasiliensis Lam), a cherry species from Brazil, as well as blackberry (AP; Rubus spp) and mulberry (AS; Morus nigra L.) contain elevated concentrations of anthocyanins and ellagitannins. Few studies focused on the anticarcinogenic action of these berries in breast cancer development. Therefore, the aim of the present study was to evaluate the effects of extracts rich in anthocyanins or ellagitannins from G, AP and AS on cellular growth and genes and miRNAs expression in human MCF-7 (hormone receptor positive) and MDA-MB 231 (hormone receptor negative) cell lines. No cytotoxicity was observed after 72 and 96 hours of treatment with extracts rich in anthocyanin (25-200 &#181g/mL) (ASANT, APANT and GANT) or ellagitannins (APELA and GELA) in both cell lines. After 72 hours of treatment, GANT and APANT induced cell cycle arrest at G0/G1 (12.5&#181g/mL and 50&#181g/mL, p<0.05) in MCF-7 cells. After 96h, ASANT, APANT and GANT induced cycle arrest at G0/G1 (12.5 and 50&#181g/mL; p<0.05) in MCF-7 cells. However, at 200&#181g/mL, only GANT induced G0/G1 (72 and 96h; p<0.05). In MDA-MB 231 cells, after 96h APANT and GANT induced cell cycle arrest at G0/G1 with the three tested concentrations (12.5, 50 and 200&#181g/mL, p<0.05), as well as ASANT at concentrations 12,5 and 50&#181g/mL (p <0.05). In MCF-7 cells, after 72h APELA and GELA induced an increase in the proportion of cells in subG0 (200&#181g/mL, p<0.05). In MDA-MB 231, after 72 and 96h, APELA and GELA (200&#181g/mL) induced an increase in the proportion of cells in subG0 (p<0.05) and cell cycle arrest in G0/G1 (p<0.05). In MCF-7 cells, GANT induced apoptosis (p<0.05) after 72 and 96h of treatment. However, in MDA-MB 231, extracts rich in anthocyanins did not induce cell death. In MCF-7 cells, after 96h GELA and APELA induced mainly necrosis (p<0.05). In MDA-MB 231, APELA and GELA induced apoptosis (p<0.05) after 72 and 96h. In MDA-MB 231 cells, inhibition of cell proliferation by GELA, GANT and APELA (200?g/mL; p<0.05) was observed after 72h of treatment. In MDA-MB 231 cells, treatment for 48h with GELA, GANT and APELA (200&#181g) increased expression of 5 genes (ESR2, FOXA1, JUN, PTGS2, VEGFA) and inhibited expression of 10 genes (ADAM23, ATM, BCL2, CDH1, EGF, GLI1, ID1, MKI67, SNAI2 and THBS1) correlated with breast cancer. In addition, GELA; GANT and APELA (200&#181g/mL) induced increased expression of miR-210 (p<0.05) and APELA (200&#181g/mL) reduced the expression of miRNA 19a/b (p<0.05) in MDA-MB cells 231. Collectively these results suggest that anthocyanins of grumixama and ellagitannins of blackberry and grumixama have potential protective effect against breast cancer. Additionally, this anticarcinogenic action can be mediated by induction of cell death, more specifically apoptosis, reduction of cell proliferation and modulation of the expression of genes and miRNAs related to breast cancer.

Orientador: Adriana Vitorino Rossi / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-19T14:28:20Z (GMT). No. of bitstreams: 1 Coelho_AlineGuadalupe_M.pdf: 2011995 bytes, checksum: 9c482d314d20b2ffa92f0b204911224f (MD5) Previous issue date: 2011 / Resumo: Antocianinas (ACYS) são corantes naturais que conferem cor a folhas, flores e frutas, são derivados glicosilados do cátion flavílio, da classe dos flavonóides e apresentam potencial para uso como corante, além de atividade antioxidante e terapêutica. Essas características estimulam buscar formas de viabilizar a utilização desses corantes na indústria de diversos segmentos, além de fomentar pesquisas acerca de sua estabilidade. Nesse trabalho, realizou-se o estudo da degradação térmica de extratos de ACYS, obtidos de uva (Vitis vinifera L. 'Brasil') e jabuticaba (Myrciaria Cauliflora). Os padrões das ACYS majoritárias para cada fruta malvidina-3-glicosídeo e cianidina-3-glicosídeo foram utilizados como referência. Foram realizados ensaios de estabilidade, para extratos obtidos a 25, 55 e 85 °C, com pH ajustado para 3,0 e mantido natural, armazenados sob-refrigeração ou a temperatura ambiente. Além disso, foram realizados ensaios de degradação acelerada a 55 e 85 °C com monitoramento espectrofotométrico e análises dos extratos e padrões iniciais e degradados por UHPLC-MS. Os ensaios de estabilidade dos extratos foram monitorados por até 170 dias. Foi verificado o efeito da baixa temperatura de armazenamento, para a estabilidade dos extratos de uva, enquanto para os extratos de jabuticaba foram verificados também o efeito da diminuição do pH, e do aumento da concentração. Os estudos da degradação térmica indicaram o aumento da velocidade da degradação das ACYS, com o aumento da temperatura. As análises de UHPLC-MS dos extratos degradados indicaram que a temperatura de degradação não altera a rota de degradação das ACYS nas amostras. As reações de degradação dos padrões seguem ajustes de segunda ordem com energias de ativação de 95,2±0,1 e 89,34±0,05 kJ mol, e tempos de meia vida de 78 ± 2 e 4,2± 0,1 e 58,7 e 3,8±0,4 horas para degradação a 55 e 85 °C da cianidina-3-glicosídeo e malvidina-3-glicosídeo, respectivamente / Abstract: Anthocyanins (ACYS) are natural dyes that give color to leaves, flowers and fruits, they are glycosylated derivatives of flavylium cation, the class of flavonoids and have potential for use as a dye, as well as antioxidant activity and therapy. These features facilitate finding ways to encourage the use of these dyes in various industry segments, and foster research about its stability. In this work, we carried out the study of thermal degradation of ACYS extracts obtained from grape (Vitis vinifera L. 'Brazil') and jabuticaba (Myrciaria cauliflora). Standards of the ACYS (malvidin-3-glucoside and cyanidin-3-glucoside) present in major quantity in each fruit were used as a reference. Stability tests over time, for extracts obtained at 25, 55 and 85 ° C, with pH adjusted to 3.0 and maintained course, and stored under refrigeration, were performed at room temperature. Further tests were carried out in order to determine accelerated degradation at 55 and 85 °C and were analyzed through spectrophotometry. Inicial and degraded standards were analyzed by UHPLC-MS. Monitoring of the extracts was performed for up to 170 days, and the effect of storage temperature for stabilization of grape extracts was verified. In case of jabuticaba extracts, the effect of lower pH, the concentration of the extracts was also verified in addition to storage temperature. The thermal degradation studies indicated that the increased speed of degradation of ACYS with increasing temperature. The UHPLC-MS analysis of degraded extracts to indicate, that the degradation temperature does not change the route of degradation of samples. The standards degradation reaction followed fit second order adjustment with activation energies of 95.2±0.1 and 89.34±0.05 kJ mol, half-life of 78±2, and 4.2± 0.1 and 58.7±0.4 and 3.8 hours to decay to 55 and 85° C of cyanidin-3-glucoside and malvidin-3-glucoside, respectively / Mestrado / Quimica Analitica / Mestre em Química

Abstract Flavonoids are a class of secondary metabolites in plants that are involved in many important functions. Various flavonoid compounds have also been reported to be beneficial for human health. Bilberry (Vaccinium myrtillus L.) is the characteristic field layer species in boreal forests and the fruits of bilberry are rich in anthocyanin pigments, a subclass of flavonoids. In the present work, flavonoid biosynthesis was examined in different tissues of bilberry. The focus was on the developing fruits of wild type and natural color mutants of bilberry, and on effect of solar radiation on flavonoid biosynthesis in bilberry leaves. For the isolation of RNA for gene expression analysis, a method was optimized for different tissues of bilberry. The cDNA fragments of five genes from the flavonoid pathway, coding phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavanol 4-reductase and anthocyanidin synthase, were isolated from bilberry using polymerase chain reaction technique, sequenced, and labelled with dioxigenin-dUTP label. These homologous, bilberry-specific probes were used for determining the expression of the flavonoid pathway genes in bilberry fruits, flowers and leaves with a modified non-radioactive method developed in the course of the study. The anthocyanins, catechins, proanthocyanidins, flavonols and hydroxycinnamic acids in fruits, leaves and different fractions of bilberry were identified and quantified with high-performance liquid chromatography combined with a diode array detector and mass spectrometer. The results demonstrate a correlation between anthocyanin accumulation and expression of the flavonoid pathway genes during the ripening of berries. A correlation between flavonol and anthocyanin biosynthesis was detected in bilberry and also in previous literature collected from flavonol and anthocyanin analyses from other fruits. Accordingly, models for the connection between flavonol and anthocyanin synthesis in fruit species were suggested. Activation of the expression of flavonoid pathway genes and accumulation of flavonoids and hydroxycinnamic acids was detected in leaves growing under direct solar radiation, compared to the shadow leaves of the same plants. Based on the results, it is suggested that cyanidin of anthocyanins and flavonol quercetin play a predominant role in the defence against high solar radiation in Vaccinium leaves. The results give new information about the biosynthesis of flavonoids in bilberry at the gene level, in addition to the information of the composition and content of flavonoids during fruit development and in different parts of the bilberry plant. Also, new information was obtained of the roles of flavonoids in protecting plants from excess solar radiation.

Abstract The aim was to study the effects of drought and indirect drought-related stresses on anthocyanin content in bilberry (Vaccinium myrtillus L.). Anthocyanin content was also studied in relation to developmental stage (juvenile vs. mature leaves, previous vs. current-year stems). It was hypothesised that drought-stressed plants accumulate anthocyanins, but their direct role in osmotic regulation was questioned. Direct drought was created by preventing water supply. Freezing-induced dehydration was accomplished by removal of snow. Effects of summertime chilling (+2 °C during active growth, +5/0 °C during frost hardening) on water status were tested. The trace metal Nickel (Ni) was applied to the soil, as Ni may interfere with plant water relations at the rhizospheric level. Salt (Na+) was added to the soil to observe salt-induced disturbance in osmotic balance and ion homeostasis. Tissue water content (TWC) decreased and anthocyanin level increased under direct drought, especially in the mature leaves. The freezing-stressed plants contained the same TWC and anthocyanin levels in mid-winter compared to plants that overwintered below snow. The freezing-stressed plants had lower TWC and a similar anthocyanin level in early spring, and lower TWC and anthocyanin level in late spring than plants that overwintered below snow. In the summer and autumn following snow removal, current-year stems of freezing-stressed plants had the same TWC, but higher levels of anthocyanins than current-year stems of plants that overwintered below snow. New growth was thus affected by the freezing stress experienced by previous-year stems. Chilling had no effect on anthocyanins. Although TWC decreased and anthocyanin level increased from active growth to the beginning of frost hardening, no increase was observed during frost hardening. Ni did not cause drought stress in the aboveground shoots, but anthocyanin level decreased in the aboveground shoots along with Ni accumulation in the belowground stems. Na+ increased TWC in the belowground stems, but decreased TWC and anthocyanin level in the aboveground stems. It is proposed that anthocyanins do not have direct role in osmotic regulation, or in the development of freezing tolerance. It is suggested that the increase in anthocyanin level under direct drought stress is mainly due to the photoprotection of chlorophylls by anthocyanins. This is supported by two facts: (1) At increased anthocyanin level in the juvenile leaves, chlorophyll a was stabilized despite continuing drought stress, and (2) after the initial peak in the mature leaves, the accumulation of anthocyanins ceased although the drought became more severe. As chlorophyll a decreased in the mature leaves due to senescence, there was less demand for such high levels of anthocyanins.

Os frutos da pitangueira pertencem à família Myrtaceae e podem ser encontrados em três variedades da mesma espécie, reconhecidos de acordo com a cor dos frutos como amarelo, vermelho e roxo. O objetivo deste estudo é caracterizar as três variedades de frutos de pitanga, com relação aos principais carotenoides e compostos fenólicos, e seu comportamento durante o desenvolvimento. Os frutos foram obtidos da região de Campinas (IAC) e fornecidos pela EMBRAPA - (RS). Os extratos metanólico dos frutos foram caracterizados quanto ao conteúdo antocianinas monoméricas, onde somente a variedade roxa, das diferentes plantas e região, apresentou aumento de concentração de antocianinas monoméricas com o desenvolvimento (0,59 a 1,94 mg cy-3-glu eq/g b.s. e 1,43 a 8,47 cy-3-glu eq/g b.s. para o estádio intermediário e maduro, respectivamente. Além disso, a variedade roxa no estádio maduro apresentou maior concentração de fenólicos totais (6,52 a 18,42 mg de AG/g b.s.) quando comparados às outras. O ácido cítrico foi identificado, por CLAE/DAD, como o ácido orgânico majoritário, seguido pelos ácidos succínico e málico, em todas as variedades, com maior concentração no estádio verde. De acordo com a concentração de ácido ascórbico, a pitanga não é uma boa fonte de vitamina C, apresentando valor máximo de 4,0 mg/g b.s no estádio maduro. Os açúcares majoritários encontrados foram a glicose e a frutose. A antocianina majoritária, identificada por LC-ESI-MS/MS, da variedade roxa, no estádio maduro foi a cianidina-3-glicosídeo, com uma variação de 8,05 a 640,53 mg/100g b.s, já o flavonoide majoritário na variedade amarela e vermelha foram miricetina e quercetina em ambas variedades. O carotenoide majoritário identificado por CLAE/DAD, em todas variedades no estádio maduro foi o licopeno, onde a variedade vermelha se destaca pela maior concentração. Dessa forma, o perfil de flavonoides não é alterado com o desenvolvimento, mas é observado uma redução no seu conteúdo nas variedades vermelha e amarela; já a variedade roxa apresenta alteração no perfil de flavonoides, com a expressão de antocianinas no estádio maduro. Por outro lado, o perfil de carotenoides em todas as variedades não sofreu alteração. / The fruits of the pitangueira belong to the family Myrtaceae and can be found in three varieties of the same species, recognized according to the color of the fruits as yellow, red and purple. The aim of this study was characterize the fruits of three pitanga varieties, according to the carotenoids and phenolic compounds composition, and their behavior during ripening. The fruits were obtained from the Campinas region and also provided by EMBRAPA - (RS). Monomeric anthocyaninswere detected only on purple variety, presenting 0.59 to 1.94 mg cy-3-&#927;-glu eq/g d.w. and 1.43 to 8.47 cy-3-&#927;-glu eq/g d.w.) for the intermediate and mature stages, respectively. In addition, the purple variety at the mature stage had the highest total phenolics concentration (6.52 to 18.42 mg of AG/g d.b.) as compared to other varieties. The citric acid was identified as the major organic acid, followed by succinic and malic acids, in all varieties, with a higher concentration in the green stage. According to the concentration of ascorbic acid, pitanga could not be considered a good source of vitamin C, presenting the highest amount of the 4.0 mg/g d.w. at the mature stage. The major soluble sugars found were glucose and fructose. The major anthocyanin of the purple variety, identified by LC-ESI-MS/MS in the mature stage, was cyanidin-3-&#927;-glucoside, ranging from 8.05 to 640.53 mg /100g d.w., whereas the major flavonoids in the yellow and red varieties were myricetin and quercetin. The flavonoid profile was not altered with ripe developing, but a decrease in its content was observed in the red and yellow varieties; on the other hand, the purple variety presented a change in the flavonoid profile, with the expression of anthocyanins in the mature stage. The carotenoids profile in all varieties was the same. The major carotenoid identified by HPLC/DAD, at the mature stage, was lycopene, where the red variety presented the highest concentration.

This diploma thesis concerns the determination of anthocyanin pigments in the elderberry pomace (Sambucus nigra L). The theoretical part describes in general the plant specification, its chemical composition, botanical characteristics and the possibility of utilization of elderberry in food processing industry and in pharmacy. However, the main concern of this thesis are the anthocyanins. This part is closely focused on chemical structure and factors that have significant influence over their stability. There are briefly summarized the positive effects of these colourants and their use in the food industry. In the experimental part, the elderberry juice and the extracts from the rhizomes were prepared. Two different solvents, a 1: 1 water / ethanol mixture and a mixture of water and methanol in the same ratio were used as extractors. To identify and quantify the dyes in the prepared samples, a chromatographic separation system (HPLC) with a Kinetex EVO C18 column and isocratic elution of the combined mobile phases of water of acetonitrile and formic acid was developed. The analysis only took 12 minutes. Two major dyes cyanidin-3-glucoside and cyanidin-3-sambubioside were identified and quantified by HPLC. The largest amount of anthocyanins was obtained by extracting mixture methanol / water (594.6 ± 0.42 mg.100 g-1). The content of anthocyanin dyes in dry extracts and juice was also determined by the often-used pH-differential method. Both results were compared. The HPLC method provided very accurate results against the less demanding pH-differential assay method, which proved to be suitable for the indicative measurement of anthocyanin content in real samples.

Contents of this diploma thesis was introduction of method determination of anthocyanin pigments by liquid chromatography. Anthocyanin pigments was determined in various cultivars of elderberry (Sambucus nigra L.) and in selected samples of red wine. In the first part of the theory is described elderberry, its species and chemical composition. Some possibilities of processing and utilization of elderberry in medicine are mentioned here too. The next part of the theory is aimed at chemical agents in wine, characteristics of anthocyanins and their utilization in food processing industry. The methods for determination of anthocyanin pigments with focus on HPLC are also mentioned there. The main objective of the experimental part of this thesis was comparison of content of anthocyanins in various cultivars of elderberry and in selected samples of red wine. In samples of red wine was observed not only cultivars´ influence, but also influence of the crop year on the content of anthocyanin pigments. The separation of the anthocyanin pigments by high-performance liquid chromatography with diode array detection (HPLC/DAD) was performed. The highest content of anthocyanin pigments in elderberry was determined in the cultivar Haschberg. In the case of red wine, the richest source of anthocyanins was the cultivar André 2009 – Rakvice. Higher content of anthocyanins was explicitly proved in young wines rather than in older wines.

No description available.

No description available.

No description available.