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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Moraceae family consists of approximately 1000 species, distributed in 61 genera. Morus alba has small flowers, simple stems up to 33 feet tall and whole leaves, serrated or lobed, fruit September to November. M. alba is used in traditional medicine as hypoglycemic, antioxidant, anti-inflammatory and in the treatment of menopausal symptoms. Flavonoids such as quercetin, rutin and alkaloid 1- deoxinojirimicina were isolated and identified in their leaves. The physico-chemical quality control was performed with M. Alba s leaves collected in four seasons. The values found in the analysis of loss on drying and foreign matter are agreement with established for herbal drugs. The collection made in March had the highest content of total ash and acid insoluble and swelling index was higher in September. The dosages of polyphenols and flavonoids were higher in summer. The evaluation of anti-inflammatory activity of M. alba in induction of granulomatous tissue showed that animals treated with hydroethanolic extract presented 20.24 ± 6.94% inhibition of the formation of granulomatous tissue while those treated with nimesulide has 21.42 ± 6.52%, confirming significant anti-inflammatory activity. The dosages of AST, ALT and creatinine were performed with the same animals, demonstrating absence of toxicity in liver and kidney. In determining the in vitro antimicrobial activity, the fractions ethyl acetate and chloroform showed the best responses front of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Aspergillus fumigatus, Prothoteca zophii and Candida albicans. The cytotoxicity of the hydroethanolic extract by cell cultures of Chinese hamster ovary (CHO) and cells of connective tissue of mouse (NCTC) clone 929 showed, respectively, IC50 of 0.34 mg/mL and 3.24 mg/mL. / A família Moraceae compreende aproximadamente 1000 espécies, distribuídas em 61 gêneros. Morus alba possui flores pequenas, caule simples com até 10 metros de altura e folhas inteiras, serreadas ou lobadas, frutifica de setembro a novembro. M. alba é utilizada na medicina tradicional como hipoglicemiante, antioxidante, anti-inflamatória e no tratamento dos sintomas da menopausa. Flavonóides como quercetina, rutina e o alcalóide 1-deoxinojirimicina foram isolados e identificados nas suas folhas. O controle de qualidade físico-químico foi realizado com folhas de M. alba coletadas nas quatro estações. Os valores encontrados nas análises de perda por dessecação e matéria estranha estão de acordo com os estabelecidos para drogas vegetais. A coleta realizada em março apresentou o maior conteúdo de cinzas totais e insolúveis em ácido e o índice de intumescência foi mais elevado em setembro. As dosagens de polifenóis e flavonóides foram mais elevadas no verão. A avaliação da atividade anti-inflamatória de M. alba em modelo de indução de tecido granulomatoso demonstrou que os animais tratados com o extrato hidroetanólico apresentaram 20,24 ± 6,94% de inibição da formação de tecido granulomatoso enquanto que os tratados com nimesulida apresentaram 21,42 ± 6,52%, confirmando significativa atividade anti-inflamatória. As dosagens de AST, ALT e creatinina foram realizadas com os mesmos animais, demonstrando ausência de indícios de toxicidade hepática e renal. Na determinação da atividade antimicrobiana in vitro, as frações acetato de etila e clorofórmio apresentaram as melhores atividades frente à Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Aspergillus fumigatus, Prothoteca zophii e Candida albicans. A citotoxicidade do extrato hidroetanólico por culturas de células de ovário de hamster chinês (CHO) e células do tecido conectivo de camundongo (NCTC) clone 929 apresentaram, respectivamente, IC50 de 0,34 mg/mL e 3,24 mg/mL, demonstrando ação citotóxica in vitro.

Submitted by Livia Mello (liviacmello@yahoo.com.br) on 2016-10-03T12:47:48Z No. of bitstreams: 1 TesePLSB.pdf: 13905738 bytes, checksum: 1789e96e4fee04313be2237c09868fd3 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-10T19:44:40Z (GMT) No. of bitstreams: 1 TesePLSB.pdf: 13905738 bytes, checksum: 1789e96e4fee04313be2237c09868fd3 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-10T19:44:51Z (GMT) No. of bitstreams: 1 TesePLSB.pdf: 13905738 bytes, checksum: 1789e96e4fee04313be2237c09868fd3 (MD5) / Made available in DSpace on 2016-10-10T19:45:01Z (GMT). No. of bitstreams: 1 TesePLSB.pdf: 13905738 bytes, checksum: 1789e96e4fee04313be2237c09868fd3 (MD5) Previous issue date: 2015-08-13 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Mulberry leaves are commonly used by women to alleviate hot flashes, shivering and night sweats. These symptoms appear due to the irregular estrogen secretion by ovaries during climacteric and are often alleviated using natural products for non-hormonal replacement therapy (NHRT). A recurrent issue regarding the use of plant extracts as therapeutics is the lack of standardization, compromising their efficacy. In the present work, hydroethanolic extracts of Morus alba leaves with pharmaceutical potential were qualitatively and quantitatively standardized. All extracts were screened by in vitro assays for estrogenicity based in estrogen receptor cell model (ER+ MFC-7). The ones containing up to 35% ethanol (v/v) induced cell proliferation like 17--estradiol. The extracts with alcohol level between 35 and 75% behaved as partial agonists, while higher alcohol content extracts killed the cells. In ER selectivity in vitro assay based on ER- cell model (MDA-MB-231), extracts prepared with up 55% ethanol were inactive and the others killed them. Cell-based assays using non-tumoral fibroblasts showed cytotoxicity for extracts with ethanol concentration higher than 60%. Thus, extracts prepared using 0-35% ethanol were the more suitable for phytoestrogenic sources in NHRT. Ethanol solutions (50-60%) were the best ones to get an extract with antitumoral potential for triple negative breast cancer subtype. Multivariate exploratory techniques were applied to analyze the chemical profile provided by FT-ICR-MS and LC-DAD-MS. Extracts with estrogenic activity had 3-O-(6”-glycosyl), 3-O-(6”- rutinosyl) e 3-O-(6”-malonylglycosyl) derivatives of quercetin and kaempferol. These 6 coumpounds were then defined as chemical markers for the quantitative standardization of mulberry leaf extracts, expressed as total flavonoids content (TFC). The maximum TFC was below 3% (w/w) in the total agonistic-like extracts. In the partial agonist-like and highly toxic ones the average levels were 4.8% and 4,4% respectively. The influence of environmental factors over the bioactive compounds content in mulberry leaves was also investigated. Four-seasons: the highest TFC was found in the beginning of spring (4.9%) and the lowest one in autumn (2.0%). Circardian cycle: the maximum TFC was reached in early morning (2.4%), gradually reducing throughout the photoperiod. Senescence: TFC average increment at 0.53% monthly. Hydric stress: higher TCF values (2.0%) were found during drought periods. Quantitative analysis of the chemical markers was also performed in samples of mulberry-based herbal drugs. The TCF results showed a huge variability among them (0.28 to 13.88%), clearly evidencing the lack of standardization of raw materials to be used in formulations. This work highlighted that mulberry leaf hydroethanolic extracts are sources for phytoestrogens and hence have promising potential for treatment of climacteric symptoms, but the control of extraction solvent composition is the key to efficacy. Small changes on it can interfere substantially in the expected biological activity, hence the concentration of bioactive compounds must be kept under an optimal range, even in an unfavorable environmental condition. / As folhas de amoreira são popularmente utilizadas por mulheres no alívio das ondas de calor e sudoreses noturnas, sintomas decorrentes da produção irregular de estrogênio no climatério. Um problema recorrente no uso comum de produtos naturais com fins medicinais é o comprometimento da eficácia, pois não há controle de qualidade dos produtos. Neste trabalho foi realizada a padronização química qualitativa e quantitativa de extratos hidroetanólicos de folhas de Morus alba com potencial para uso no tratamento desses sintomas. Em testes in vitro de estrogenicidade com células ER+ (MCF-7) extratos com até 35% (v/v) de etanol:água estimularam o crescimento celular como o 17--estradiol. Aqueles contendo entre 35 e 75% de álcool atuaram como agonistas parciais, enquanto extratos com elevado teor alcoólico induziram a morte celular. Nos estudos de seletividade para o receptor estrógeno com células ER- (MDA-MB-231), extratos preparados com até 55% etanol foram inativos, enquanto os demais levaram à morte das células. Estudos com células de fibroblastos não tumorais (BALB/c 3T3 clone 31) mostraram citotoxicidade para extratos com percentual alcoólico superior a 60%. Assim, extratos preparados com 0-35% etanol foram os mais indicados para uso como fonte de fitoestrógenos em NHRT e aqueles com 50 - 60% foram indicados como antitumorais em câncer de mama triplo-negativo. Ferramentas quimiométricas multivariadas aplicadas na análise do perfil químico dos extratos por FT-ICR-MS e LC-DAD-MS associaram a atividade estrogênica aos derivados 3-O-(6”-glicosil), 3-O-(6”- rutinosil) e 3-O-(6”- malonilglicosil) dos flavonoides quercetina e campferol, os quais foram selecionados como marcadores químicos para a padronização quantitativa do extrato em teor de flavonoides totais. Nos extratos com perfil de agonismo total, o teor máximo encontrado foi inferior a 3% (m/m), enquanto naqueles com agonismo parcial e com elevada citotoxicidade, os teores médios foram respectivamente 4,8% e 4,4%. A influência de fatores ambientais no teor dos compostos bioativos em folhas de amoreira também foi avaliada. Estações do ano: o maior teor foi encontrado no início da primavera (4,9%) e o menor no início do outono (2,0%). Ciclo circadiano: o valor máximo foi obtido no início da manhã (2,38%), com redução gradual ao longo do fotoperíodo. Senescência: crescimento no teor total dos flavonoides a uma taxa mensal média de 0,53%. Estresse hídrico: valores mais altos (2,0%) foram obtidos em períodos de estiagem. Análises quantitativas dos marcadores também foram realizadas em amostras de medicamentos fitoterápicos comerciais de amora, mostrando grande variabilidade nos teores (0,28 a 13,88%), evidenciando a falta de padronização dos extratos nas formulações. Esse trabalho mostrou que extratos hidroetanólicos de folhas de Morus alba possuem fitoestrógenos e têm potencial aplicação em medicamentos fitoterápicos. Além disso, o controle da composição do solvente extrator é fundamental para garantir a eficácia do extrato, visto que pequenas alterações podem tanto alterar significativamente a atividade biológica esperada, como manter a concentração dos compostos bioativos em uma faixa ótima, mesmo quando a sua produção é desfavorecida por fatores ambientais. / FAPESP 2011/24137-0 / CNPq 146902/2011-4

Memoria para optar al Título Profesional de Ingeniero Agrónomo Mención: Producción Animal / El estudio se llevó a cabo en la Estación Experimental Germán Greve de la Universidad de Chile. Se evaluaron los efectos de la suplementación con morera (Morus alba) sobre la producción y composición de la leche en el último tercio de lactancia en cabras. Se utilizaron 30 cabras mestizas (F1), cruza de Anglo Nubian y Criolla que se encontraban en el último tercio de la lactancia, las que fueron asignadas a los tratamientos según edad, tipo de parto y progenie. Dado que el grupo de animales presentó cuatro factores de variación: biotipo, fecha de parto, edad y número de parto, los animales fueron agrupados en éstas variables, de modo que cada grupo quedara representado en igual magnitud. Los subgrupos resultantes se asignaron al azar en los tratamientos experimentales que fueron: a) T0, dieta en base a heno de alfalfa y b) T1, dieta basada en hojas y tallos de morera. Las cabras permanecieron en el ensayo 60 días. El forraje se suministró en forma fresca picada. Se recolectaron muestras del material ofrecido y rechazado para el análisis de los suplementos (digestibilidad aparente de la materia seca, proteína bruta, fibra detergente neutro y energía metabolizable). También se realizó un control lechero donde se evaluó: producción de leche por tratamiento, proteína y sólidos totales de la leche. Por último, se determinó ganancias de peso en los animales sometidos a los tratamientos. Se observaron diferencias significativas (P<0,05) para producción de leche, pero no hubo diferencias para el consumo de materia seca ni composición química de la leche. Los registros de producción mostraron diferencias entre tratamientos, se encontró diferencias significativas de una mejor respuesta de las cabras alimentadas con morera. Los resultados sobre el peso vivo de las cabras reflejaron que hubo diferencias estadísticamente significativas (P<0,05) en el cambio de peso al final del experimento con el tratamiento suplementario de morera. / The study was carried out in the Experimental Station German Greve University of Chile. The effects of supplementing with mulberry (Morus alba) on production and composition of milk in goats as evaluated. Thirty goats (F1), crosses of Anglian Nubian and Creole that were in the last third of lactation, were assigned to the treatments according to: strain, parturition data, age and number of parturition. The animals were grouped according to these variables, so that each group was represented. The experimental treatments were: a) T0, alfalfa hay as supplement b) T1, mulberry leaves and stems as supplement. The goats remained in the assay for 60 days. The forage was provided chopped fresh form. Samples of offered and rejected forage were collected for chemical analysis dry matter apparent digestibility, crude protein, neutral detergent fibber and metabolizable energy. Also a milk control was done to determine: milk production, protein and total solids in milk. Finally, live weight gains was determined in both treatments. Significant differences (P<0.05) for milk production was observed, but there was not differences for dry matter intake and milk chemical composition. The production registries showed differences between treatments, there were tendencies of one better answer of the goats fed with mulberry. Finally live weight showed statistically significant differences (P<0.05) in favour of the mulberry supplemented treatment.

Plant estrogens have long been associated with health benefits. The potential of tissue culture techniques for the production of several secondary metabolites has been known for many years. Tissue cultures stimulate the production or induce the biosynthesis of novel compounds not found in the mature plant. Tissue culture of Morus alba, family Moraceae, is known to contain phytoestrogens, was established on plant-hormone supplemented Murashige and Skoog (MS) medium. Petiole and the stem tissue from mature trees were the best explants for initiation and proliferation of calli. The best callus proliferation was obtained on MS medium containing 1-napthalene acetic acid (1mg/ml) and benzylaminopurine (0.5mg/ml) for M. alba. Comparison of phytoestrogens of Moraceae species from in vivo and in vitro tissue isolation were carried out. The estrogenic activities of callus extracts were assayed in an estrogen-responsive yeast system expressing the human estrogen receptor alpha. Male callus extracts had higher estrogenic activity than male and female extracts from in vivo and in vitro tissues. Isolation and characterization of phytoestrogens from above tissues were carried out using solid phase extraction, high perfomance liquid chromatography and mass spectrometry techniques. Biochanin A, an isoflavonoid, was isolated as one of the compounds in male callus extracts. Biochanin A has been known to have an antiestrogenic acitivity in mammals. Isoflavonoid compounds have been characterized as strong protein tyrosine kinase inhibitors in variety of animal cells. Isoflavones are structurally similar to estradiol, and display agonistic and antagonistic interactions with the estrogen receptor. Isoflavones possess therapeutic and preventive properties such as being used for postmenopausal osteoporosis, breast cancer, and inhibition of tumors.

<p>&nbsp;Upotrebom različitih ekstragenasa izvr&scaron;ena je ekstrakcija ploda, lista i korena belog (<em>Morus alba</em> L.) i crnog duda (<em>Morus nigra</em> L. ). Ekstrakcija etanolom je izvr&scaron;ena pri predhodno određenim optimalnim uslovima ekstrakcije (70% koncentracija etanola, temperatura 60&deg;C i odnos rastvarač:droga 20 ml/g). Ispitan je hemijski sastav ekstrakata različitih delova duda. Ispitivanja hemijskog sastava etanolnih ekstrakata obuhvatala su određivanje sadržaja ukupnih fenola, ukupnih flavonoida, ukupnih i monomernih antocijana, kvalitativni i kvantitativni sastav polifenolnih komponenata, kao i sadržaj mikro-, makro- i toksičnih elemenata. Najvedi sadržaj ukupnih fenolnih jedinjenja određen je u ekstraktu korena <em>M. nigra</em> (186 mg EHK/g), a najvedi sadržaj flavonoidnih jedinjenja u ekstraktu lista <em>M. nigra</em> (67 mg ER/g). U ekstraktima lista duda po svom sadržaju u odnosu na ukupan sadržaj polifenolnih jedinjenja ističe se kafena kiselina (od 53,34 do 62,11%), dok se u ekstraktima korena izdvaja hlorogenska kiselina (udeo vedi od 74%). U pogledu mineralnog sastava ekstrakata relativno veliki sadržaj Ca i Mg je karakterističan za obe vrste ekstrakata (vedi od 1.400 &mu;g/g). Mikroelementi u svim ispitanim uzorcima duda po opadajudem nivou, su Fe, B, Zn, Cu i Mn. U ekstraktima dobijenim superkritičnim CO₂određen je sadržaj masnih kiselina i ukupnih karotenoida. U ispitivanim ekstraktima dominante su nezasidene masne kiseline, linolna i linolenska kiselina, a značajan je udeo i zasidene masne kiseline (palmitinske kiseline).</p><p>Ispitivanje delovanja ekstrakata obuhvatalo je evaluaciju antioksidativne aktivnosti pradenjem sposobnosti neutralizacije slobodnih radikala, redukcionog potencijala i inhibicije lipidne peroksidacije. Po svim primenjenim metodama najvedu aktivnost je pokazao ekstrakt lista crnog duda. U zavr&scaron;noj fazi rada ispitana je in vitro citotoksična aktivnost ekstrakata duda, njihovim delovanjem na rast tri delijske linije: Hep2, RD i L2OB. Ekstrakti lista <em>M. nigra</em> su se pokazali kao najpotentniji inhibitori&nbsp;rasta na sve tri delijske linije (IC₅₀&lt;30 &mu;g/g). Na osnovu svih sprovedenih ispitivanja kao proizvod sa najboljim karakteristikama pokazao se ekstrakti lista <em>M. nigra</em>.</p> / <p>Using different extraction solvents extracts of fruit, leaves and roots of white (<em>Morus alba</em> L.) and black mulberry (<em>Morus nigra</em> L.) were prepared. Extraction by ethanol was carried out at the previously determined optimal extraction conditions (70% ethanol concentration, temperature 60&deg;C and the ratio of solvent: drug 20 ml/g). The chemical composition of extracts obtained from different parts of the mulberry were analysed. These analyses included the determination of total phenols, total flavonoids, monomeric and total anthocyanins, qualitative and quantitative composition of polyphenolic components, and the content of micro-, macro- and toxic elements. The highest phenolic content was determined in the extract of <em>M. nigra</em> root (186 mg ECA/ g), while the highest content of flavonoid compounds was determined in the extract of <em>M. nigra</em> leaves (67 mg ER/g). In the case of mulberry leaf extracts dominant phenolic compound, in relation to the total phenolic content, was caffeic acid (with contribution from 53.34 to 62.11%), while in the extracts of root extracts dominant phenolic compounds was chlorogenic acid (a higher proportion of 74%). In terms of mineral composition of the extracts relatively high content of Ca and Mg was characteristic of both types of extracts (greater than 1,400 mg/g). Measured content of trace elements in mulberry extracts decrease in the next order: Fe&gt; B&gt; Zn&gt; Cu&gt; Mn. In the extracts obtained by supercritical CO₂ fatty acids and total carotenoids were determined. In these extracts dominant unsaturated fatty acids were linoleic and linolenic acid. Also, significant proportion of saturated fatty acid (palmitic acid) has been detected.</p><p>Testing of extracts activity included the evaluation of antioxidant activity applying the methods based on: neutralize free radicals, reducing power potential and the inhibition of lipid peroxidation. All applied methods showed the highest activity of black mulberry leaf extract. In the final phase of work in vitro cytotoxic activity of mulberry extracts was examined. Their effect on growth of three cell lines: Hep2, RD, and L2OB were analysed. Extracts of <em>M. nigra</em> proved to be the most potent inhibitors of growth in all three cell lines (IC₅₀&lt;30 mg/g). On the basis of all conducted tests it has been proven that <em>M. nigra</em> extracts are products with the best characteristics.</p>