Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Iveta Rajtmajerová Supervisor: PharmDr. Jana Pourová, Ph.D. Title of diploma thesis: The in vitro effects of selected fenolic substances on isolated rat vessels Silymarin flavonolignans are endowed with various aspects. Recently their hepatoprotective effect has been discussed widely as well as their positive influence on the cardiovascular system. This diploma thesis is concerned with the vasodilatory effects of silybin-A, silybin-B, silybin-A + B and isosilybin-A on rat aorta. The aim of this study was to test the vasodilatory effect of selected substances. The dependence of aortic relaxation on the increasing concentration of tested substances was measured. Isolated Wistar breed rat aorta was used. DRC curves were created and EC50 values were assessed from the obtained values of vessel tension. The results were evaluated subsequently. The vasodilator potential of silybin-A and isosilybin-A is comparable; hence the position of the substituents may be irrelevant in having any impact on relaxation of the vessels. Different vasodilatory effects of silybin-A and silybin-B have been observed thus, the relaxation could be stereoselective. The testing of the equimolar mixture of silybin-A and...

O objetivo deste trabalho foi investigar o comportamento fisiológico e bioquímico de plantas de citros (Citrus spp.) submetidas à aplicação do herbicida glyphosate. Para tanto, foram realizados quatro estudos. No primeiro, avaliou-se o efeito de glyphosate (doses entre 0 e 720 g e.a.ha-1, aplicadas sobre a parte aérea das plantas) nas concentrações foliares de chiquimato, aminoácidos livres totais e fenóis e na fotossíntese de plantas de citros em condições controladas. Foram testadas mudas de limão ‘Cravo’ (Citrus limonia L. Osbeck), com 4 meses de idade, e mudas de ‘Valência’ (Citrus sinensis L. Osbeck) sobre ‘Swingle’ (Poncirus trifoliata (L.) Raf x Citrus paradisi Macf), com 24 meses de idade. No segundo estudo avaliou-se, em condições de campo, a toxicidade de glyphosate em plantas submetidas à aplicação atingindo o caule (doses entre 0 e 2160 g e.a.ha-1) ou toda a parte aérea (doses entre 0 e 720 g e.a.ha-1). Os materiais vegetais testados foram plantas de laranja ‘Valência’ sobre ‘Swingle’ e limão ‘Cravo’, com 20 e 27 meses de idade, respectivamente. No terceiro avaliou-se a queda de frutos de laranjeira ‘Pera’ (Citrus sinensis L. Osbeck) em função do glyphosate. No último estudo investigou-se a possibilidade de aplicação acidental simulada de glyphosate causar intoxicação e queda de frutos em plantas de laranja ‘Pera’. No primeiro estudo verificou-se efeito de glyphosate somente nas concentrações de chiquimato e aminoácidos livres totais das mudas de limão ‘Cravo’; porém, os efeitos foram transitórios. Não houve efeito nos conteúdos de fenóis e clorofila total e na atividade fotossintética. Não se constatou qualquer efeito nas características bioquímicas e na fotossíntese das mudas de ‘Valência’, indicando significativa tolerância ao herbicida. No estudo a campo, as plantas de citros atingidas pelo glyphosate no caule não sofreram intoxicação. Apenas a aplicação nas doses de 360 e 720 g e.a.ha-1 sobre a parte aérea afetou as plantas. O principal sintoma de intoxicação consistiu na emissão de brotações deformadas, indicando efeito nas regiões meristemáticas. Os efeitos nas folhas pré-formadas foram pequenos ou inexistentes. Todas as plantas atingidas pelo herbicida se recuperaram entre seis e doze meses após a aplicação. Nos estudos para avaliar a queda de frutos observou-se que o glyphosate promove a produção de etileno, causando a queda destes quando estão em fase final de maturação. A queda ocorreu principalmente devido ao contato direto com a pulverização e não devido à translocação do herbicida. No último estudo, constatou-se que a aplicação acidental simulada foi parcialmente interceptada pelo mato, não causando efeito significativo na queda de frutos e no desenvolvimento vegetativo das plantas de citros. / The objective of this research was to evaluate physiological and biochemical responses of citrus (Citrus spp.) plants sprayed with the herbicide glyphosate. Four studies were done to attain this objective. By the first one, it were investigated the effects of glyphosate (dosages from 0 to 720 g a.e.ha-1 applied on plants canopies) on the leaf contents of shikimate, total free amino acids and phenolic compounds, and on photosynthesis of citrus plants under controlled conditions. The experiments were conducted using ‘Rangpur Lime’ (Citrus limonia L. Osbeck) rootstock, with 4 months old, and ‘Valencia’ (Citrus sinensis L. Osbeck) scion grafted on ‘Swingle’ (Poncirus trifoliata (L.) Raf x Citrus paradisi Macf) rootstock, with 24 months old. By the second trial, it was evaluated, under field conditions, the toxicity of glyphosate on citrus plants sprayed directly to the the trunk (dosages from 0 to 2160 g a.e.ha-1) or on the canopy (dosages from 0 to 720 g a.e.ha-1). The plants tested were ‘Valencia’ var. grafted on ‘Swingle’ var. and on ‘Rangpur Lime’ var., with 20 and 27 months old, respectively. By the third trial, it was evaluated the fruit dropping of ‘Pera’ var. (Citrus sinensis L. Osbeck) in relation to glyphosate spray. The last trial was conducted to evaluate if an accidental simulated spray of glyphosate can cause toxicity and fruit dropping in ‘Pera’ citrus var. The results by the first study showed effects of glyphosate only on shikimate and total free amino acids contents of ‘Rangpur Lime’ seedlings. However, these effects were temporaries. No effect was found on phenolic compound contents and on the photosynthetic characteristics evaluated. It was not postrayed any effect on biochemical and photosynthetic characteristics of the ‘Valencia’ plants, indicating significant glyphosate tolerance. By the second study, the citrus plants sprayed with glyphosate on their trunks have not showed intoxication. Only the application of glyphosate at dosages of 360 and 720 g a.i.ha-1 on the canopies affected the plants. The main toxic symptoms consisted of deformation of new flushes, indicating effect on plant meristems. The effects of glyphosate were low or none in the mature leaves. All plants sprayed by glyphosate have been recovered by from six to twelve months after the application. In the study, in order to evaluate the fruit dropping, it was found that the herbicide glyphosate induced ethylene production, causing fruit dropping when they are in final phase of ripening. Fruit dropping occurred mainly due to the direct contact of the spraying instead of the herbicide translocation. By the last study it was found that the accidental simulated spraying is partially intercepted by weeds, do not causing significant effect on fruit dropping and citrus growth.

O objetivo deste trabalho foi investigar o comportamento fisiológico e bioquímico de plantas de citros (Citrus spp.) submetidas à aplicação do herbicida glyphosate. Para tanto, foram realizados quatro estudos. No primeiro, avaliou-se o efeito de glyphosate (doses entre 0 e 720 g e.a.ha-1, aplicadas sobre a parte aérea das plantas) nas concentrações foliares de chiquimato, aminoácidos livres totais e fenóis e na fotossíntese de plantas de citros em condições controladas. Foram testadas mudas de limão ‘Cravo’ (Citrus limonia L. Osbeck), com 4 meses de idade, e mudas de ‘Valência’ (Citrus sinensis L. Osbeck) sobre ‘Swingle’ (Poncirus trifoliata (L.) Raf x Citrus paradisi Macf), com 24 meses de idade. No segundo estudo avaliou-se, em condições de campo, a toxicidade de glyphosate em plantas submetidas à aplicação atingindo o caule (doses entre 0 e 2160 g e.a.ha-1) ou toda a parte aérea (doses entre 0 e 720 g e.a.ha-1). Os materiais vegetais testados foram plantas de laranja ‘Valência’ sobre ‘Swingle’ e limão ‘Cravo’, com 20 e 27 meses de idade, respectivamente. No terceiro avaliou-se a queda de frutos de laranjeira ‘Pera’ (Citrus sinensis L. Osbeck) em função do glyphosate. No último estudo investigou-se a possibilidade de aplicação acidental simulada de glyphosate causar intoxicação e queda de frutos em plantas de laranja ‘Pera’. No primeiro estudo verificou-se efeito de glyphosate somente nas concentrações de chiquimato e aminoácidos livres totais das mudas de limão ‘Cravo’; porém, os efeitos foram transitórios. Não houve efeito nos conteúdos de fenóis e clorofila total e na atividade fotossintética. Não se constatou qualquer efeito nas características bioquímicas e na fotossíntese das mudas de ‘Valência’, indicando significativa tolerância ao herbicida. No estudo a campo, as plantas de citros atingidas pelo glyphosate no caule não sofreram intoxicação. Apenas a aplicação nas doses de 360 e 720 g e.a.ha-1 sobre a parte aérea afetou as plantas. O principal sintoma de intoxicação consistiu na emissão de brotações deformadas, indicando efeito nas regiões meristemáticas. Os efeitos nas folhas pré-formadas foram pequenos ou inexistentes. Todas as plantas atingidas pelo herbicida se recuperaram entre seis e doze meses após a aplicação. Nos estudos para avaliar a queda de frutos observou-se que o glyphosate promove a produção de etileno, causando a queda destes quando estão em fase final de maturação. A queda ocorreu principalmente devido ao contato direto com a pulverização e não devido à translocação do herbicida. No último estudo, constatou-se que a aplicação acidental simulada foi parcialmente interceptada pelo mato, não causando efeito significativo na queda de frutos e no desenvolvimento vegetativo das plantas de citros. / The objective of this research was to evaluate physiological and biochemical responses of citrus (Citrus spp.) plants sprayed with the herbicide glyphosate. Four studies were done to attain this objective. By the first one, it were investigated the effects of glyphosate (dosages from 0 to 720 g a.e.ha-1 applied on plants canopies) on the leaf contents of shikimate, total free amino acids and phenolic compounds, and on photosynthesis of citrus plants under controlled conditions. The experiments were conducted using ‘Rangpur Lime’ (Citrus limonia L. Osbeck) rootstock, with 4 months old, and ‘Valencia’ (Citrus sinensis L. Osbeck) scion grafted on ‘Swingle’ (Poncirus trifoliata (L.) Raf x Citrus paradisi Macf) rootstock, with 24 months old. By the second trial, it was evaluated, under field conditions, the toxicity of glyphosate on citrus plants sprayed directly to the the trunk (dosages from 0 to 2160 g a.e.ha-1) or on the canopy (dosages from 0 to 720 g a.e.ha-1). The plants tested were ‘Valencia’ var. grafted on ‘Swingle’ var. and on ‘Rangpur Lime’ var., with 20 and 27 months old, respectively. By the third trial, it was evaluated the fruit dropping of ‘Pera’ var. (Citrus sinensis L. Osbeck) in relation to glyphosate spray. The last trial was conducted to evaluate if an accidental simulated spray of glyphosate can cause toxicity and fruit dropping in ‘Pera’ citrus var. The results by the first study showed effects of glyphosate only on shikimate and total free amino acids contents of ‘Rangpur Lime’ seedlings. However, these effects were temporaries. No effect was found on phenolic compound contents and on the photosynthetic characteristics evaluated. It was not postrayed any effect on biochemical and photosynthetic characteristics of the ‘Valencia’ plants, indicating significant glyphosate tolerance. By the second study, the citrus plants sprayed with glyphosate on their trunks have not showed intoxication. Only the application of glyphosate at dosages of 360 and 720 g a.i.ha-1 on the canopies affected the plants. The main toxic symptoms consisted of deformation of new flushes, indicating effect on plant meristems. The effects of glyphosate were low or none in the mature leaves. All plants sprayed by glyphosate have been recovered by from six to twelve months after the application. In the study, in order to evaluate the fruit dropping, it was found that the herbicide glyphosate induced ethylene production, causing fruit dropping when they are in final phase of ripening. Fruit dropping occurred mainly due to the direct contact of the spraying instead of the herbicide translocation. By the last study it was found that the accidental simulated spraying is partially intercepted by weeds, do not causing significant effect on fruit dropping and citrus growth.