Thesis (Ph. D.)--University of Chicago, 1916. / "A Private Edition Distributed by the University of Chicago Libraries." "Reprinted from the Botanical gazette, vol. LXI, no. 6." Also available on the Internet.

The Hawaiian species of Bidens are morphologically and ecologically diverse taxa which have evolved from a single ancestral species. Adaptive radiation has occurred without the evolution of physiological or genetic interspecific isolating mechanisms since all species are interfertile and genetic distances among populations, based on isozyme loci, show little correlation with morphological differences or taxonomic classification. This disparity between the evolution of morphological and biochemical characters makes it of interest to determine whether or not there has been divergence in secondary metabolites in these species. Leaves and roots of 19 species and six subspecies of Hawaiian Bidens were examined for polyacetylenes. Eleven C₁₃ hydrocarbons, aromatic and thiophenyl derivatives, one C₁₄ tetrahydropyran and three C₁₇ hydrocarbons were isolated and identified. All can be derived from oleic acid. Polyacetylenes were not detected in the leaves of 13 taxa although they are found in the roots of all species. The occurrence of 2-(2-phenylethyne-1-yl)-5 acetoxymethyl thiophene in Bidens has not been previously reported. Most taxa could be distinguished by their complement of leaf and root acetylenes and no variation was found within taxa except in B. torta. There appears to be no taxonomically significant pattern to the distribution of polyacetylenes above the species level in this group. The complexity of polyacetylene inheritance was assessed using experimentally produced interspecific hybrids. Crosses between species which do not produce leaf acetylenes resulted in F₁, individuals without acetylenes. Crosses between species which produce leaf acetylenes and those which do not yielded hybrids with acetylenes not always identical to parental arrays. Progeny from parents with different sets of acetylenes expressed a combination of the major compounds found in both parents. In all cases, nonparental acetylenes in the F₁, generation were biosynthetically closely related to compounds found in the parents. Polyacetylene synthesis was not segregated in the F₂ individuals from Type B crosses. De novo biosynthesis of polyacetylenes in Bidens leaves was investigated in pulse-chase studies. ¹⁴C-labelled acetylenes were recovered from three species of Bidens administered ¹⁴C0₂ and subsequently allowed to metabolize in ¹⁴C0₂ for 12, 24 and 168 hours. Radioactive C₁₃ ene-tetrayne-ene was also isolated from the roots of all plants, indicating that translocation of ¹⁴C-labelled precursors from aerial tissues occurred. Phenylheptatriyne (PHT) was detected in two day old seedlings of B. alba, suggesting that polyacetylene biosynthesis begins during germination or soon thereafter. Quantities in the leaves continue to increase up to and beyond 24 days while amounts in the hypocotyls peak at seven days. Relative PHT values in the roots are 100 times higher than those in the aerial tissues for the first 24 days, but there is also a gradual decline in these levels beginning at two weeks and continuing beyond the experimental period. Phenylheptatriyne is absent from the roots of mature B. alba. Many polyacetylenes are toxic to biological systems in the presence of UV-A radiation. These in vitro effects have led to speculation about the putative functions of polyacetylenes in the organisms which produce them. Nineteen species of phylloplane yeasts and yeast-like fungi were isolated from species of Hawaiian Bidens with and without leaf acetylenes. Although all these organisms, members of the Sporobolomycetaceae, Cryptococcaceae and Fungi Imperfecti, were photosensitive to some polyacetylenes and resistant to others, there was no correlation between the presence or absence of leaf polyacetylenes and the distribution of these saprophytes among species of Bidens. Nevertheless, it is significant that the only pathogenic species isolated in this study, ColIetolrichum gloeosporiodes , did not colonize Bidens leaves containing C₁₃ aromatic acetylenes to which it is extremely photosensitive in vitro. / Science, Faculty of / Botany, Department of / Graduate

As análises dos voláteis por SPME/CG-EM das partes aéreas, flores e frutos de Bidens sulphurea e Bidens gardneri permitiram-nos constatar diferenças em suas composições químicas. Porém, em todas as frações analisadas os compostos majoritários foram os sequiterpenos b-cariofileno, germacreno D e biciclogermacreno. Apesar dos constituintes majoritários coincidirem nas frações analisadas das duas espécies, foi possível constatar a presença exclusiva de determinados metabólitos em cada fração. A partir das frações hexânicas, oriundas dos extratos etanólicos de B. sulphurea (partes aéreas e flores) e B. gardneri (partes aéreas), foram identificadas trinta e cinco, dezenove e vinte substâncias por CG-EM, respectivamente. Na fração hexânica das partes aéreas de B. sulphurea (BsfcEt/Hx) foram identificados, como constituintes majoritários, o óxido de cariofileno, espatulenol e _- cariofileno, enquanto que na fração hexânica de suas flores (BsflorEt/Hx) os principais constituintes identificados foram _-amirina e _-sitosterol e na fração hexânica das partes aéreas de B. gardneri (BgfcEt/Hx) foram os metabólitos _-estigmasterol e o trans-fitol. O estudo químico da espécie B. sulphurea (partes aéreas e flores) conduziu ao isolamento de um sesquiterpeno (1), 5 flavonas (2, 10, 13-15), 8 flavonóis (3-8, 11, 16), 1 aurona (9) e 2 chalconas (12, 17). Já o estudo de B. gardneri (partes aéreas) conduziu ao isolamento de 4 ácidos clorogênicos (21, 22, 27, 28), 3 poliacetilenos (18-20), 2 flavonas (25, 27), 3 flavanonas (23, 24, 30) e 2 chalconas (29, 31). Os metabólitos 3-O-_-glicopiranosil-tetradeca- 6(E),12(E)-dieno-8,10-diino-1,14-diol (18), 1-O-_-glicopiranosil-14-hidróxi-tetradeca-6(E), 12(E)-dieno-8,10-diino-3-ona (19), 4-metóxi-7-O-_-glicopiranosil-8,3-diidróxi-flavanona (23), 4-metóxi-7-O-_-(6-acetil)-glicopiranosil-8,3-diidróxi-flavanona (24) e 7-O-_-(6- trans-p-cumaroil)-glicopiranosil-8,34-triidróxi-flavanona (30) estão sendo descritos pela primeira vez na literatura. Enquanto que os flavonóides 3-O-_-xilopiranosil-quercetina (5), 3-O-a-arabinofuranosil-kaempferol (8) e 3-O-b-(6-trans-cafeoil)-galactopiranosil-quercetina (16) estão sendo relatados pela primeira vez na família Asteraceae e as substâncias 4(15)- eudesmeno-1_,6_-diol (1), 6-C-_-glicopiranosil-apigenina (2), 3-O-_-arabinofuranosilquercetina (6), 8-C-_-glicopiranosil-apigenina (13), 6-C-_-glicopiranosil-luteolina (14), 8-C- _-glicopiranosil-luteolina (15), ácido 1-metil-5-O-E-cafeoilquínico (22) e 7-O-_- glicopiranosil-apigenina (25) estão sendo relatadas pela primeira vez no gênero Bidens. Além disso, convém destacar que a partir das substâncias 2-O-_-glicopiranosil-trideca-3(E),11(E)- dieno-5,7,9-triino-1,13-diol (20), 4-metóxi-4-O-_-glicopiranosil-okanina (29) e 4-O-_-(6- trans-p-cumaroil)-glicopiranosil-okanina (31) há poucos relatos na literatura e ainda não foram descritos dados de suas propriedades biológicas. Na avaliação da atividade antialérgica das substâncias isoladas, os flavonóides causaram inibição da liberação de _-hexosaminidase de forma dose-dependente, sendo que as substâncias 11 e 31 foram as mais ativas e apresentaram CI50 de 5,1 ± 1,3 M e 5,8 ± 1,2 M, respectivamente. Dentre os extratos e frações avaliados biologicamente, observou-se que BsfcEt/Hx (maior teor de sesquiterpenos) causou um estímulo da liberação de _-hexosaminidase, enquanto que BsfcEt/Ac foi a fração mais ativa (CI50 = 1,3 ± 1,1g/mL). As análises desta última fração e de BsfcEt/DCM por CLAE-DAD-EM e CLAE-DAD-EM/EM revelaram que seus constituintes majoritários são os flavonóis 3, 4, 6 e 7. / The SPME/GC-MS analyses of aerial parts, flowers and fruits of Bidens sulphurea and Bidens gardneri showed the differences in their chemical compositions. However the sesquiterpenes _-caryophyllene, germacrene D and bicyclogermacrene were identified in all fractions analyzed as major compounds. It was observed the exclusive presence of metabolites in each fraction, in spite of major constituents were equal in fractions analyzed of two species. Thirty-five, nineteen and twenty substances were identified in the hexane fractions from ethanol extracts of B. sulphurea (aerial parts and flowers) and B. gardneri (aerial parts) by GC-MS. The major compounds were caryophyllene oxide, spathulenol and _-caryophyllene in the hexane fraction from aerial parts of B. sulphurea (BsfcEt/Hx), while _-amyrin and _- sitosterol were identified in the hexane fraction of its flowers and _-stigmasterol and transphytol were main constituents identified in the hexane fraction from aerial parts of B. gardneri. The chemical study of species B. sulphurea (aerial parts and flowers) led to the isolation of one sesquiterpene (1), five flavones (2, 10, 13-15), eight flavonols (3-8, 11, 16), one aurone (9) and two chalcones (12, 17). From B. gardneri (aerial parts), four chlorogenic acids (21, 22, 27, 28), three polyacetylenes (18-20), two flavones (25, 27), three flavanones (23, 24, 30) and two chalcones (29, 31) were isolated. The substances 3-O-_-glucopyranosyltetradeca-6(E),12(E)-dien-8,10-diin-1,14-diol(18), 1-O-_-glucopyranosyl-14-hydroxytetradeca-6(E),12(E)-dien-8,10-diin-3-one(19),4-methoxy-7-O_glucopyranosyl-8,3-dihydroxyflavanone (23), 4-methoxy-7-O-_-(6-acetyl)-glucopyranosyl-8,3-dihydroxyflavanone(24) and 7-O-_-(6-trans-p-coumaroyl)-glucopyranosyl-8,34-trihydroxyflavanone(30) are described for the first time in the literature. Whereas the flavonols 3-O-_-xylopyranosyl quercetin (5), 3-O-a-arabinofuranosyl kaempferol (8) and 3-O--(6-transcaffeoyl)-galactopyranosyl quercetin (16) are described for the first time in the Asteraceaeand 4(15)-eudesmene-1_,6_-diol (1), 6-C-_-glucopyranosyl apigenin (2), 3-O-_-arabinofuranosyl quercetin (6), 8-C-_-glucopyranosyl apigenin (13), 6-C-_-glucopyranosylluteolin (14), 8-C-_-glucopyranosyl luteolin (15), 1-methyl-5-O-E-caffeoylquinic acid (22)and 7-O-_-glucopyranosyl apigenin (25) for the first time in the genus Bidens. Moreover,there are few reports of the isolation and there are not studies of biological activities from 2-O-_-glucopyranosyl-trideca-3(E),11(E)-dien-5,7,9-triin-1,13-diol (20), 4-methoxy-4-O-_-glucopyranosyl okanin (29) and 4-O-_-(6-trans-p-coumaroyl)-glucopyranosyl okanin (31).The flavonoids showed inhibition of _-hexosaminidase released with dependent-doseresponse and the substances 11 (IC50 = 5,1 ± 1,3 M) and 31 (IC50 = 5,8 ± 1,2 M) were themost active. The BsfcEt/Hx fraction (highest concentration of sesquiterpenes) induced _-hexosaminidase released, while the BsfcEt/Ac fraction exhibited the lower IC50 (1,3 ±1,1g/mL). The flavonoids 3, 4, 6 and 7 were identified, by HPLC-DAD-MS and HPLCDAD-MS/MS, as major constituents in BsfcEt/Ac and BsfcEt/DCM fractions.

As análises dos voláteis por SPME/CG-EM das partes aéreas, flores e frutos de Bidens sulphurea e Bidens gardneri permitiram-nos constatar diferenças em suas composições químicas. Porém, em todas as frações analisadas os compostos majoritários foram os sequiterpenos b-cariofileno, germacreno D e biciclogermacreno. Apesar dos constituintes majoritários coincidirem nas frações analisadas das duas espécies, foi possível constatar a presença exclusiva de determinados metabólitos em cada fração. A partir das frações hexânicas, oriundas dos extratos etanólicos de B. sulphurea (partes aéreas e flores) e B. gardneri (partes aéreas), foram identificadas trinta e cinco, dezenove e vinte substâncias por CG-EM, respectivamente. Na fração hexânica das partes aéreas de B. sulphurea (BsfcEt/Hx) foram identificados, como constituintes majoritários, o óxido de cariofileno, espatulenol e _- cariofileno, enquanto que na fração hexânica de suas flores (BsflorEt/Hx) os principais constituintes identificados foram _-amirina e _-sitosterol e na fração hexânica das partes aéreas de B. gardneri (BgfcEt/Hx) foram os metabólitos _-estigmasterol e o trans-fitol. O estudo químico da espécie B. sulphurea (partes aéreas e flores) conduziu ao isolamento de um sesquiterpeno (1), 5 flavonas (2, 10, 13-15), 8 flavonóis (3-8, 11, 16), 1 aurona (9) e 2 chalconas (12, 17). Já o estudo de B. gardneri (partes aéreas) conduziu ao isolamento de 4 ácidos clorogênicos (21, 22, 27, 28), 3 poliacetilenos (18-20), 2 flavonas (25, 27), 3 flavanonas (23, 24, 30) e 2 chalconas (29, 31). Os metabólitos 3-O-_-glicopiranosil-tetradeca- 6(E),12(E)-dieno-8,10-diino-1,14-diol (18), 1-O-_-glicopiranosil-14-hidróxi-tetradeca-6(E), 12(E)-dieno-8,10-diino-3-ona (19), 4-metóxi-7-O-_-glicopiranosil-8,3-diidróxi-flavanona (23), 4-metóxi-7-O-_-(6-acetil)-glicopiranosil-8,3-diidróxi-flavanona (24) e 7-O-_-(6- trans-p-cumaroil)-glicopiranosil-8,34-triidróxi-flavanona (30) estão sendo descritos pela primeira vez na literatura. Enquanto que os flavonóides 3-O-_-xilopiranosil-quercetina (5), 3-O-a-arabinofuranosil-kaempferol (8) e 3-O-b-(6-trans-cafeoil)-galactopiranosil-quercetina (16) estão sendo relatados pela primeira vez na família Asteraceae e as substâncias 4(15)- eudesmeno-1_,6_-diol (1), 6-C-_-glicopiranosil-apigenina (2), 3-O-_-arabinofuranosilquercetina (6), 8-C-_-glicopiranosil-apigenina (13), 6-C-_-glicopiranosil-luteolina (14), 8-C- _-glicopiranosil-luteolina (15), ácido 1-metil-5-O-E-cafeoilquínico (22) e 7-O-_- glicopiranosil-apigenina (25) estão sendo relatadas pela primeira vez no gênero Bidens. Além disso, convém destacar que a partir das substâncias 2-O-_-glicopiranosil-trideca-3(E),11(E)- dieno-5,7,9-triino-1,13-diol (20), 4-metóxi-4-O-_-glicopiranosil-okanina (29) e 4-O-_-(6- trans-p-cumaroil)-glicopiranosil-okanina (31) há poucos relatos na literatura e ainda não foram descritos dados de suas propriedades biológicas. Na avaliação da atividade antialérgica das substâncias isoladas, os flavonóides causaram inibição da liberação de _-hexosaminidase de forma dose-dependente, sendo que as substâncias 11 e 31 foram as mais ativas e apresentaram CI50 de 5,1 ± 1,3 M e 5,8 ± 1,2 M, respectivamente. Dentre os extratos e frações avaliados biologicamente, observou-se que BsfcEt/Hx (maior teor de sesquiterpenos) causou um estímulo da liberação de _-hexosaminidase, enquanto que BsfcEt/Ac foi a fração mais ativa (CI50 = 1,3 ± 1,1g/mL). As análises desta última fração e de BsfcEt/DCM por CLAE-DAD-EM e CLAE-DAD-EM/EM revelaram que seus constituintes majoritários são os flavonóis 3, 4, 6 e 7. / The SPME/GC-MS analyses of aerial parts, flowers and fruits of Bidens sulphurea and Bidens gardneri showed the differences in their chemical compositions. However the sesquiterpenes _-caryophyllene, germacrene D and bicyclogermacrene were identified in all fractions analyzed as major compounds. It was observed the exclusive presence of metabolites in each fraction, in spite of major constituents were equal in fractions analyzed of two species. Thirty-five, nineteen and twenty substances were identified in the hexane fractions from ethanol extracts of B. sulphurea (aerial parts and flowers) and B. gardneri (aerial parts) by GC-MS. The major compounds were caryophyllene oxide, spathulenol and _-caryophyllene in the hexane fraction from aerial parts of B. sulphurea (BsfcEt/Hx), while _-amyrin and _- sitosterol were identified in the hexane fraction of its flowers and _-stigmasterol and transphytol were main constituents identified in the hexane fraction from aerial parts of B. gardneri. The chemical study of species B. sulphurea (aerial parts and flowers) led to the isolation of one sesquiterpene (1), five flavones (2, 10, 13-15), eight flavonols (3-8, 11, 16), one aurone (9) and two chalcones (12, 17). From B. gardneri (aerial parts), four chlorogenic acids (21, 22, 27, 28), three polyacetylenes (18-20), two flavones (25, 27), three flavanones (23, 24, 30) and two chalcones (29, 31) were isolated. The substances 3-O-_-glucopyranosyltetradeca-6(E),12(E)-dien-8,10-diin-1,14-diol(18), 1-O-_-glucopyranosyl-14-hydroxytetradeca-6(E),12(E)-dien-8,10-diin-3-one(19),4-methoxy-7-O_glucopyranosyl-8,3-dihydroxyflavanone (23), 4-methoxy-7-O-_-(6-acetyl)-glucopyranosyl-8,3-dihydroxyflavanone(24) and 7-O-_-(6-trans-p-coumaroyl)-glucopyranosyl-8,34-trihydroxyflavanone(30) are described for the first time in the literature. Whereas the flavonols 3-O-_-xylopyranosyl quercetin (5), 3-O-a-arabinofuranosyl kaempferol (8) and 3-O--(6-transcaffeoyl)-galactopyranosyl quercetin (16) are described for the first time in the Asteraceaeand 4(15)-eudesmene-1_,6_-diol (1), 6-C-_-glucopyranosyl apigenin (2), 3-O-_-arabinofuranosyl quercetin (6), 8-C-_-glucopyranosyl apigenin (13), 6-C-_-glucopyranosylluteolin (14), 8-C-_-glucopyranosyl luteolin (15), 1-methyl-5-O-E-caffeoylquinic acid (22)and 7-O-_-glucopyranosyl apigenin (25) for the first time in the genus Bidens. Moreover,there are few reports of the isolation and there are not studies of biological activities from 2-O-_-glucopyranosyl-trideca-3(E),11(E)-dien-5,7,9-triin-1,13-diol (20), 4-methoxy-4-O-_-glucopyranosyl okanin (29) and 4-O-_-(6-trans-p-coumaroyl)-glucopyranosyl okanin (31).The flavonoids showed inhibition of _-hexosaminidase released with dependent-doseresponse and the substances 11 (IC50 = 5,1 ± 1,3 M) and 31 (IC50 = 5,8 ± 1,2 M) were themost active. The BsfcEt/Hx fraction (highest concentration of sesquiterpenes) induced _-hexosaminidase released, while the BsfcEt/Ac fraction exhibited the lower IC50 (1,3 ±1,1g/mL). The flavonoids 3, 4, 6 and 7 were identified, by HPLC-DAD-MS and HPLCDAD-MS/MS, as major constituents in BsfcEt/Ac and BsfcEt/DCM fractions.

Thesis (M.S.)--University of Wisconsin--Madison, 1961. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 72-74).

Dois dos principais aspectos da Química de Produtos Naturais são a identificação e a determinação estrutural dos compostos orgânicos presentes em materiais de origem vegetal ou animal. Para realizar esta tarefa são empregados diversos métodos de separação e espectroscópicos. Dentre os métodos espectroscópicos, destaca-se a espectroscopia de ressonância magnética nuclear pela riqueza dos dados fornecidos. Entretanto, o desenvolvimento da área exige métodos cada vez mais eficientes e sensíveis, para permitir o estudo de amostras em quantidades cada vez menores. Neste trabalho, empregamos três estratégias distintas para aumentar a eficiência da análise de amostras de produtos naturais por RMN. A primeira estratégia busca acelerar o processo de identificação dos componentes de frações de extratos vegetais através da separação virtual dos componentes da mistura, baseada nas diferenças de seus coeficientes de difusão. Esta estratégia permitiu identificar os quatro componentes majoritários da fração em acetato de etila do extrato etanólico das partes aéreas de Bidens sulphurea (Asteraceae) através de um estudo de RMN DOSY-2D. A segunda estratégia é dedicada ao problema da baixa sensibilidade da RMN 13C, o que torna difícil, e em alguns casos, proibitiva em termos do tempo necessário, a análise de amostras muito pequenas. Dada a dificuldade de se obter diretamente um espectro com uma relação sinal/ruído adequada, uma abordagem possível se baseia em estratégias de pós-processamento de um espectro de RMN em que predomina o ruído. A estratégia utilizada neste trabalho fornece um espectro reconstruído de secções que foram previamente processadas para a eliminação do ruído pela decomposição do valor singular (SVD). Os espectros assim processados nos auxiliaram na identificação e/ou elucidação estrutural de dois alcalóides obtidos de Duguetia furfuraceae (Annonaceae) e um poliacetileno e dois flavonóides de B. sulphurea (Asteraceae). A terceira estratégia utilizada neste trabalho se baseia na ampliação dos parâmetros rotineiramente obtidos numa análise por RMN, com a finalidade de fornecer novos subsídios para a determinação da estrutura de produtos naturais. Neste sentido, a determinação das constantes de acoplamento heteronuclear forneceu dados valiosos para a identificação de dois poliacetilenos obtidos de B. gardneri. / Two major aspects of Natural Products Chemistry are the identification and structure determination of organic compounds in materials of vegetable or animal. To accomplish this task are used various methods of separation and spectroscopic. Among spectroscopic methods, we highlight the spectroscopy of nuclear magnetic resonance by the wealth of data provided. However, the development of the area requires methods increasingly efficient and sensitive to allow the study of small or diluted samples. In this work, we employ three different strategies to increase the efficiency of analysis of samples of natural products by NMR. The first strategy seeks to accelerate the process of identifying the components of fractions of plant extracts through the virtual separation of the components of the mixture based on differences in their diffusion coefficients. With this strategy we have identified four major components of the in ethyl acetate fraction of ethanol extract of aerial parts of Bidens sulphurea (Asteraceae) by 2D DOSY NMR. The second strategy is devoted to the problem of low sensitivity of 13C NMR, which makes it difficult, and in some cases prohibitive in terms of time, the analysis of very small samples. Due to the difficulty of obtaining directly a spectrum with a signal-to-noise ratio adequate, a possible approach is based on strategies for post-processing of an NMR spectrum in which noise predominates. This strategy used here provides a spectrum of reconstructed sections were first processed to remove noise by singular value decomposition (SVD). Spectra processed of this way helped us in identifying and / or structural elucidation of two alkaloids derived from Duguetia furfuraceae (Annonaceae) and a polyacetylene and two flavonoids from Bidens gardneri (Asteraceae). The third strategy used in this work is based on expansion of the parameters routinely obtained in an analysis by NMR in order to provide new subsidies for the structure determination of natural products. In this sense, the determination of heteronuclear coupling constants provided valuable data for the identification of two polyacetylenes obtained from B. gardneri.

Zugl.: Uppsala, Univ., Diss., 1984.

Senescence was examined in the economic weed Bidens pilosa, with the objectives to a) determine the effects of deflowering and defruiting on growth, chlorophyll content, photosynthesis and transpiration; b) to identify the stage of development of the head at which the flowers, seeds/fruit produce senescence signals; and c) to test for senescence activity in plant extracts made from the receptacles and leaves of Bidens pilosa. Total chlorophyll content in the controls, in association with the development of fruit, was lower in the final harvests when compared with earlier harvests in both pot and field-grown plant experiments. Deflowered Bidens pilosa plants had a higher chlorophyll concentration than both defruited and control plants in both pot and field-grown plants. Stem death of the control plants was higher than that of deflowered plants in both field and pot experiments. The present results suggest that deflowering is essential if the leaves are to be harvested commercially because it retards senescence and maintains growth. Fruit and flower heads were responsible for the reduction in leaf and stem growth after flowering in Bidens pilosa. Removing these organs slowed plant decline, suggesting that the flower head and especially the fruit are responsible for senescence. In contrast, the fruit were the main organs responsible for the decline in leaf chlorophyll concentration. In pot-grown plants in full sunlight, photosynthesis and transpiration were low in deflowered plants compared with the control and defruited plants 45 days after treatment, and it coincided with a low stomatal conductance. These results suggest that stomatal conductance played a role in lowering photosynthesis in deflowered plants. In contrast, the control plants had a higher stomatal conductance than deflowered plants 75 days after treatment, yet photosynthesis and transpiration rates were the same in both treatments. Thus stomatal conductance alone does not successfully explain differences in photosynthesis in these treatments. The dry weight of head with mature dry fruit was higher in plants grown at high light intensities than at medium or low light intensities. It coincided with a greater decline in chlorophyll concentration in the leaf nearest to the head and fruit. In contrast, photosynthesis was the same at all light intensities in the leaf nearest to the head and fruit. This suggests that high light accelerated the process of fruit maturation of the fruit which then influenced senescence in the leaf nearest to the flower head. Ethanolic and water extracts of senescent receptacles purified using paper chromatography, induced senescence of leaves in light but not in the dark. In ethanolic extracts, activity was detected in R[f]s 0.1, 0.2 and 0.3. In water extracts, activity was detected in R[f] 0.1. Senescent leaf extracts purified using column chromatography also induced senescence in light under greenhouse conditions. At high concentrations, activity was detected in fraction 10 eluted with ethyl acetate: methanol (55:45); fraction 11 eluted with ethyl acetate: methanol (50:50); fraction 12 eluted with methanol (100%) and in fraction 13 eluted with ethylacetate : isopropanol: water: acetic acid (52:28:28:4). Under growth room conditions, activity was detected in fractions 12, eluted with methanol (100%) and 13, eluted with ethyl acetate: isopropanol: water: acetic acid (52:28:28:4) in the presence of light. Fraction 1 (R[f] 0.00-0.10) from senescent receptacles, non-senescent and senescent leaves, obtained following thin layer chromatography of ethanolic extracts induced senescence under light. Fraction 1 was eluted with methanol. This fraction lacked activity when eluted with ethyl acetate. Fraction 4 (R[f] 0.25 - 0.35) from non-senescent leaf extracts, which co-chromatographed with 4-chloroindole acetic acid, gave activity in bean cuttings kept under continuous low light. Senescent leaf extracts showed no activity. Fraction 7 (R[f] 0.9 - 1.0) from non-senescent leaf extracts, also induced senescence in bean cuttings under light. The same Fraction from senescent leaf extracts lacked activity. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2000.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2013 / Made available in DSpace on 2013-12-05T22:39:08Z (GMT). No. of bitstreams: 1 317579.pdf: 2266949 bytes, checksum: c109cde9b9c0ab36f488fbf71f7b4fbb (MD5) Previous issue date: 2013 / Introdução. Bidens pilosa L. é uma planta considerada medicinal que contém poliacetilenos presumivelmente responsáveis por ações antitumorais. As quinonas, como a juglona, também compreendem uma classe de substâncias de interesse por seu potencial antitumoral. Além disto, evidências indicam que a associação de ascorbato com algumas quinonas pode potencializar a atividade antitumoral. Objetivos. Avaliar o efeito antitumoral do extrato de B.pilosa obtido pela tecnologia supercrítica, a fim de obter um extrato rico em poliacetilenos (SFE) com atividade superior em comparação ao extrato obtido por maceração hidroetanólica (HCE). Testar a administração de juglona isoladamente ou em associação ao ascorbato em células T24 pela citotoxicidade; caracterizado o mecanismo de morte celular induzido, indução de estresse oxidativo, efeito antiproliferativo e preliminarmente, anti-invasivo in vitro. Por fim, avaliar a administração de juglona e ascorbato em associação a quimioterápicos convencionais, doxorrubicina e cisplatina. Metodologia. A composição fitoquimica dos extratos foi avaliada por cromatografia em camada delgada e espectrofotometria UV-Vis. A citotoxicidade para células MCF-7 e T24 foi avaliada pelo ensaio do MTT. O potencial lesivo sobre o DNA foi avaliado in vitro pela motilidade eletroforética de DNA plasmidial e imunoeletroforese para fosforilação da histona gama-H2Ax em células T24. Foram avaliados marcadores de danos oxidativos, geração celular de EROs e conteúdo de GSH, também a fosforilação do fator de iniciação eucariótico 2 eIF2a. Foi avaliada a morfologia das células em processo de morte, sendo também verificada a ativação de caspases por espectrofluorimetria e clivagem da poli (ADP-ribose) polimerase (PARP) por imunoeletroforese. A atividade antiproliferativa foi medida pelo ensaio de formação de colônias. A inibição da motilidade celular foi investigada pelo ensaio de migração. A atividade antitumoral in vivo foi medida em camundongos Balb-c portadores de tumor ascítico de Ehrlich. Resultados. Os espectros sobre os constituintes majoritários de SFE apresentaram bandas típicas de poliacetilenos com picos de absorção na faixa do UV registrados em 208, 269 e 334 nm. A citotoxicidade in vitro foi dependente da concentração dos extratos. Às 24h, a CI50 foi 811 e 437 µg/mL, respectivamente para HCE e SFE, reduzindo para 291 µg/mL às 48h, no caso de SFE. Às 24h, a CI50 foi 28,5 µM para juglona, reduzindo para 6,3 µM, quando associada ao ascorbato 1 mM. A citotoxicidade da cisplatina chegou a dobrar devida sua associação com juglona e ascorbato. HCE causou danos no DNA em 160 µg/mL, ao passo que 40 µg/mL de SFE causaram danos equivalentes. Juglona iniciou danos sobre o DNA em 20 µM, ao passo que 5 µM associados ao ascorbato 1 mM causaram danos equivalentes. A juglona induziu à geração celular de EROs e consumo de GSH; iniciando fosforilação do eIF2a que indica estresse de retículo endoplasmático. Estes efeitos foram potencializados em até 4 vezes pela associação ao ascorbato. A juglona sozinha ou associada ao ascorbato não causou ativação de caspases; corroborando a morfologia das células T24 em processo de morte, o processo induzido provavelmente está mais relacionado à necrose. Em tratamentos subletais, a juglona diminuiu a proliferação e a motilidade de células T24, ambos os processos potencializados pela associação ao ascorbato, que induziu à morte clonogênica. Finalmente, os ensaios in vivo indicaram que ambos os extratos de B. pilosa apresentaram atividade, mas SFE causou redução superior do volume de líquido ascítico e células compactadas (4 ± 1 e 1 ± 0,4 mL, respectivamente), ao mesmo tempo em que resultou em maior aumento no tempo de sobrevida (~31%) em comparação aos animais do controle negativo. A inibição do crescimento tumoral determinada para HCE foi de cerca de 40%, ao passo que esta determinação ultrapassou 60% no caso de SFE. Conclusão. A extração com fluido de CO2 supercrítico é uma alternativa para a obtenção de um extrato de B. pilosa rico em poliacetilenos citotóxicos com atividade antitumoral superior em comparação ao extrato obtido por maceração hidroetanólica. O ascorbato potencializa a citotoxicidade, o efeito antiproliferativo e inibidor da motilidade de células T24 in vitro da juglona. Os achados deste trabalho levam a sugerir que a dose do tratamento feito com juglona pode ser ajustada, em termos de redução da mesma, em até 4 vezes, se a juglona for administrada em associação ao ascorbato.<br> / Abstract: Introduction. Bidens pilosa L. is a plant considered medicinal containing polyacetylenes expected to be responsible for antitumor actions. Quinones, such as juglone, comprise a class of substances of interest also due to their antitumor potential. Furthermore, evidences indicate that the combination of ascorbate with some quinones can result in potentiated antitumor activity. Objectives. The supercritical technology was assessed to obtain an extract from B.pilosa rich in polyacetylenes (SFE) with superior antitumor activity compared to that of the extract obtained by hydroethanol maceration (HCE). Juglone was tested administered alone and/or combined with ascorbate on T24 cells for cytotoxicity, the mechanism of cell demise was characterized, oxidative stress induction; antiproliferative and, preliminarily, anti-invasive effects. Juglone and ascorbate were evaluated for the cytotoxicity in combination with the conventional chemotherapics doxorubicin and cisplatin. Methodology. The phytochemical composition of extracts was evaluated by thin layer chromatography with UV-Vis spectroscopy. Cytotoxicity on MCF-7 and T24 cells was checked by the MTT assay. The damaging potential on DNA was assessed in vitro by the electrophoretic motility of plasmid DNA and immunoelectrophoresis for phosphorylation of histone gamma-H2Ax in T24 cells. Markers of oxidative stress, ROS generation and GSH consumption, and the phosphorylation of the eukaryotic initiation factor eIF2á were analyzed as well. The induced cell death process was monitored by caspase activation through spectrofluorimetry and poly (ADP-ribose) polymerase (PARP) cleavage by immunoelectrophoresis. The antiproliferativo action was measured by the colony forming assay. Cell motility inhibition was investigated by a migration assay. In vivo antitumor activity was measured in Ehrlich carcinoma-bearing Balb-c mice. Results. Spectra about the major constituents in SFE presented typical bands of polyacetylenes recorded in UV at 208, 269 and 334 nm. Cytotoxicity in vitro was concentration-dependent. At 24 h, IC50 was 811 and 437 ìg/mL, respectively for HCE and SFE on MCF-7 cells, falling to 291 ìg/mL at 48 h, in the case of SFE. At 24h, IC50 was 28.5 ìM for juglone on T24 cells, falling to 6.3 ìM when it was combined with ascorbate 1 mM. At 24 h, the cisplatin#s cytoxicity on T24 cells increased up to 2-fold due to its combination with juglone and ascorbate. HCE caused DNA damage at 160 ìg/mL, whereas SFE at 40 ìg/mL caused equivalent damage. Juglone triggered DNA damage at 20 ìM, whereas 5 ìM in combination with ascorbate 1 mM caused equivalent damage. Juglone caused reduction in terms of ROS generation and increased GSH consumption, triggering eiF2á phosphorylation that indicates endoplasmic reticulum stress. These actions were potentiated up to 4-fold due to the combination of juglone with ascorbate. Juglone alone or combined with ascorbate did not cause caspase activation, corroborating T24 cell morphology under demise, the induced cell death process seems to be close related to necrosis. Under sublethal treatments, juglone reduced the proliferation and the motility of T24 cells, both actions were potentiated due to the combination with ascorbate, something that finally induced to clonogenic cell death. In vivo assays indicated that both extracts from B. pilosa had activity, but SFE caused superior reduction in terms of ascitic fluid volume and packed cells (4 ± 1 e 1 ± 0.4 mL, respectively). Furthermore, SFE increased more the animals life span (~31%) compared to animals from the negative control. The tumor growth inhibition by HCE was about 40%, whereas more than 60% was reached by SFE. Conclusion. Supercritical extraction with fluid CO2 is a choice to obtain an extract from B. pilosa rich in cytotoxic polyacetylenes with superior antitumor activity in comparison to that of the extract obtained by hydroethanol maceration. Ascorbate potentiated the cytotoxicity, the antiproliferative action and the inhibition of T24 cells motility in vitro by juglone. These findings lead to suggest that the dose of the treatment done with juglone can be adjusted, in terms of reduction, up to 4-fold, if juglone is administered in combination with ascorbate.

Dois dos principais aspectos da Química de Produtos Naturais são a identificação e a determinação estrutural dos compostos orgânicos presentes em materiais de origem vegetal ou animal. Para realizar esta tarefa são empregados diversos métodos de separação e espectroscópicos. Dentre os métodos espectroscópicos, destaca-se a espectroscopia de ressonância magnética nuclear pela riqueza dos dados fornecidos. Entretanto, o desenvolvimento da área exige métodos cada vez mais eficientes e sensíveis, para permitir o estudo de amostras em quantidades cada vez menores. Neste trabalho, empregamos três estratégias distintas para aumentar a eficiência da análise de amostras de produtos naturais por RMN. A primeira estratégia busca acelerar o processo de identificação dos componentes de frações de extratos vegetais através da separação virtual dos componentes da mistura, baseada nas diferenças de seus coeficientes de difusão. Esta estratégia permitiu identificar os quatro componentes majoritários da fração em acetato de etila do extrato etanólico das partes aéreas de Bidens sulphurea (Asteraceae) através de um estudo de RMN DOSY-2D. A segunda estratégia é dedicada ao problema da baixa sensibilidade da RMN 13C, o que torna difícil, e em alguns casos, proibitiva em termos do tempo necessário, a análise de amostras muito pequenas. Dada a dificuldade de se obter diretamente um espectro com uma relação sinal/ruído adequada, uma abordagem possível se baseia em estratégias de pós-processamento de um espectro de RMN em que predomina o ruído. A estratégia utilizada neste trabalho fornece um espectro reconstruído de secções que foram previamente processadas para a eliminação do ruído pela decomposição do valor singular (SVD). Os espectros assim processados nos auxiliaram na identificação e/ou elucidação estrutural de dois alcalóides obtidos de Duguetia furfuraceae (Annonaceae) e um poliacetileno e dois flavonóides de B. sulphurea (Asteraceae). A terceira estratégia utilizada neste trabalho se baseia na ampliação dos parâmetros rotineiramente obtidos numa análise por RMN, com a finalidade de fornecer novos subsídios para a determinação da estrutura de produtos naturais. Neste sentido, a determinação das constantes de acoplamento heteronuclear forneceu dados valiosos para a identificação de dois poliacetilenos obtidos de B. gardneri. / Two major aspects of Natural Products Chemistry are the identification and structure determination of organic compounds in materials of vegetable or animal. To accomplish this task are used various methods of separation and spectroscopic. Among spectroscopic methods, we highlight the spectroscopy of nuclear magnetic resonance by the wealth of data provided. However, the development of the area requires methods increasingly efficient and sensitive to allow the study of small or diluted samples. In this work, we employ three different strategies to increase the efficiency of analysis of samples of natural products by NMR. The first strategy seeks to accelerate the process of identifying the components of fractions of plant extracts through the virtual separation of the components of the mixture based on differences in their diffusion coefficients. With this strategy we have identified four major components of the in ethyl acetate fraction of ethanol extract of aerial parts of Bidens sulphurea (Asteraceae) by 2D DOSY NMR. The second strategy is devoted to the problem of low sensitivity of 13C NMR, which makes it difficult, and in some cases prohibitive in terms of time, the analysis of very small samples. Due to the difficulty of obtaining directly a spectrum with a signal-to-noise ratio adequate, a possible approach is based on strategies for post-processing of an NMR spectrum in which noise predominates. This strategy used here provides a spectrum of reconstructed sections were first processed to remove noise by singular value decomposition (SVD). Spectra processed of this way helped us in identifying and / or structural elucidation of two alkaloids derived from Duguetia furfuraceae (Annonaceae) and a polyacetylene and two flavonoids from Bidens gardneri (Asteraceae). The third strategy used in this work is based on expansion of the parameters routinely obtained in an analysis by NMR in order to provide new subsidies for the structure determination of natural products. In this sense, the determination of heteronuclear coupling constants provided valuable data for the identification of two polyacetylenes obtained from B. gardneri.