Foi estudado o efeito da lactona sesquiterpênica licnofolido sobre o \"burst\" respiratório de leucócitos polimorfonucleares inflamatórios (PMN) estimulados por forbol (PMA), pelo peptídeo quimiotático fMLP ou zimozan opsonizado (OZ). O licnofolido inibiu de forma dose-dependente a liberação de O2•- pelos PMN, sem alteração do período \"Iag\" do complexo NADPH. oxidase. O efeito foi mais acentuado quando os PMN foram estimulados diretamente pela via de proteína quinase C. A adição de ditiotreitol ou glutationa reduzida (GSH) às suspensões celulares antes da incubação com licnofolido preveniu parcialmente o efeito inibitório. O tratamento dos PMN com a lactona determinou uma queda drástica dos níveis celulares de GSH livre, sem incremento de glutationa oxidada (GSSG). A reação direta entre GSH e licnofolido foi confirmada com a detecção de um aduto glutationil-licnofolido através de identificação por espectrometria de massa (ESI-MS/MS). A S-tiolação protéica induzida pelo PMA foi reduzida em PMN tratados com Iicnofo/ido, como detectado através de determinação de incorporação de [35S], sendo que 80% desses tióis foram identificados como GSH. Uma série de proteínas S-glutationadas foi detectada através de autoradiografias, sendo que aquelas correspondentes a 38 e 24 kDa tiveram essa modificação póstraducional suprimida pelo tratamento com dose de licnofolido capaz de suprimir o \"burst\" respiratório dos PMN. Estes resultados indicam que a depleção celular de GSH causada pelo licnofolido impede a sustentação do \"burst\" respiratório pelos PMN, em correlação direta com a diminuição de S-glutationação protéica. / An investigation was made into the action of the sesquiterpene lactone lychnopholide on the respiratory burst of inflammatory polymorphonuclear leukocytes. Lychnopholide determined concentration-related inhibition of the generation of phorbol 12-myristate 13-acetate-, chemotatic peptide-, and opsonized zymozan-induced superoxide anion with no effect on the lag time of the assembly of the NADPH oxidase complex, such action was greater on the protein kinase C pathway that on both membrane receptor dependent stimuli via. Subsequent additions of D-glucose, Ca2+, Mg2+, dithiothreitol ar reduced glutathione (GSH) did not reverse the inhibitory action. The addition of both thiols prior to the lychnopholide treatment partially hindered the inhibition rate. The endogenous level of GSH in leukocytes was drastically depleted under the lychnopholide treatment, without corresponding increases occurring in the oxidized form (GSSG). A direct reaction between glutathione and lychnopholide was confirmed from a glutathionyl-lychnopholide adduct detected by electrospray mass spectrometry analysis and identified by tandem mass analysis in cellular extracts. Protein S-thiolation induced by PMA stimulation was decreased in lactone-treated PMN as detected by [35S] scintillation count, which indicated that about 80% of the thiols were glutathione. A subset of S-glutathionylated proteins was identified through gel electrophoresis, which revealed that the modification of the phorbol-triggered protein sulfhydryl in the protein bands corresponding to 38 and 24 kDa was precluded by the lychnopholide treatment correlated with respiratory burst inhibition. These results show that GSH depletion determined by lychnopholide treatment renders PMN to sustain respiratory burst, whose action is proportional to protein S-glutahionylation decrease.

A busca por novos fármacos inspirados em substâncias de origem natural é uma estratégia conhecida e há tempos utilizada. As lactonas sesquiterpênicas (LST) são um grupo de substâncias amplo e diverso com várias atividades farmacológicas descritas, e cuja característica estrutural determinante é a de um esqueleto principal contendo 15 átomos de carbono e um anel lactônico. O mecanismo de ação das LST está intrinsicamente relacionado com reações de adição do tipo Michael frente a biomoléculas, provocando alquilações. Há na literatura estudos aprofundados sobre a química medicinal das LST, entretanto pouco há sobre o metabolismo desse grupo de substâncias em ambientes fisiológicos. Neste sentido, este trabalho é focado no estudo do metabolismo e também das vias de fragmentação por eletrospray da budleína A, que possui estrutura química do tipo furanoeliangolido, e pertence à classe dos germacranolidos. Foram realizados ensaios in vitro utilizando-se os modelos de oxidação biomimética com metaloporfirina, microssomas hepáticos e metabolismo pela microbiota intestinal de ceco de porco (pig cecum model), sendo nos dois últimos testado também o correlato 4,5-dihidro-2\',3\'-epoxi-15-desoxigoyazensolido. O estudo de fragmentação também abordou a comparação entre budleína A e a centraterina - um estereoisômero que se diferencia apenas pela orientação da cadeia lateral ligada ao C-8 - em espectrômetros distintos e com suporte de métodos computacionais para cálculos de energia (Gaussian 03 em base B3LYP/6-31G(d)). Nos estudos de fragmentação observou-se a diferença de intensidade dos sinais para íons fragmento comuns às duas LST (m/z 275, 257 e 83), além da presença do íon fragmento de m/z 293 apenas para a budleína A, permitindo a diferenciação destes isômeros por meio de espectrometria de massas com ionização por eletrospray, sem a necessidade de ressonância de magnética nuclear. Os produtos de oxidação detectados na reação com metaloporfirinas acusaram a epoxidação na cadeia lateral envolvendo C-2\' e C-3\' com a formação de diastereoisômeros. No ensaio com microssomas não foram detectados produtos para a budleína A, enquanto que para a substância correlata observou-se a abertura do epóxido na cadeia lateral e adição de uma hidroxila, formando um diol vicinal. No modelo de ceco de porco observaram-se a formação de adutos de LST com o aminoácido cisteína, os quais foram posteriormente degradados pela ação da microbiota intenstinal, dando origem a diversos metabólitos compostos pela LST e partes de cisteína. Sendo assim, este trabalho lança bases para a maior compreensão do metabolismo de LST do tipo furanoeliangolido em modelos distintos e também contribui para os estudos de espectrometria de massas para este tipo de substância, além de descrever uma ferramenta analítica útil na diferenciação de dois estereoisômeros. / The search for new drugs inspired on compounds from natural products is a wellknown strategy with several successful cases. Sesquiterpene lactones (STL) are a wide and diversified group of compounds which has already many pharmacological activities reported. Their fundamental moiety includes a skeleton containing 15 carbons and a lactone ring and the mechanism of action is related to Michael addition type reactions with biomolecules, promoting alkylation. There are a high amount of studies regarding the medicinal chemistry of the STL, however there are few information about the metabolism of these compounds under physiological environments. On this way, the aims of this work are focused on the study of the metabolism as well as the fragmentation pathways of budlein A, which is a furanoheliangolide and belongs to the germacranolides class. On this work the following experiments were carried out: in vitro oxidative metabolism with metalloporphyrin and microsomes; intestinal metabolism by using the pig cecum model (microbiota). For microsomes and intestinal metabolism the compound 4,5- dihydro-2\',3\'-epoxy-15-deoxy-goyazensolide was also applied. Fragmentation studies compared the fragmentation patterns of budlein A and centratherin - which is a stereoisomer with -orientation for the side chain bonded at C-8 - by using different spectrometers being supported by computational methods for energies calculations (Gaussian 03 at level B3LYP/6-31G(d)). For the fragmentation studies it was observed the difference of signal intensities for fragment ions which are common for both STL (m/z 275, 257 e 83), moreover the ion m/z 293 was detected only for budlein A, allowing the differentiation between these isomers by electrospray ionization mass spectrometry instead nuclear magnetic resonance. The reactions with metalloporphyrin yielded two diastereoisomers of the STL with an epoxide at the side chain between C-2\'and C-3\'. On the microsome assay any product was detected for budlein A, while for the correlated compound the epoxide ring was opened and a hydroxyl was added at C-3\', forming a vicinal diol. On the pig cecum model it was observed the formation of adducts due to the reaction of the STL and the amino acid cysteine. These adducts were later degraded by the action of the microbiota yielding different metabolites composed by STL and residues of cysteine. Thus, this work may contribute to the improvement of the knowledge about the metabolism of STL furanoheliangolide type in different models as well as for the studies regarding the mass spectrometry of this type of compound. The development of a useful analytical tool for the differentiation of two isomers was also an important achievement.

A presente tese relata o estudo químico de duas espécies vegetais pertencentes à família Asteraceae: Mikania sp. nov; e Wunderlichia crulsiana. Da espécie Mikania sp. nov. foram estudados os extratos diclorometânicos das folhas e dos galhos, tendo sido isolados e identificados sete ácidos diterpênicos; dos óleos voláteis das folhas e dos galhos foram identificadas vinte substâncias entre monoterpenos e sesquiterpenos. Da espécie Wunderlichia crulsiana foram estudados os extratos diclorometânicos das flores e dos galhos. Das flores foram isolados e identificados palmitatos e acetatos de triterpenoílas. Dos galhos foram isolados e identificados acetatos de triterpenoílas e triterpenonas e identificados por CG triterpenóis acetilados e palmitatos de triterpenoílas hidrolisados e posteriormente acetilados. Deste extrato foram também isolados e identificados três lactonas sesquiterpênicas e um sesquiterpeno. A identificação das substâncias foi realizada através de RMN de 1H, de 13C (BBD e DEPT 135º), CG/EM e co-injeção de padrões. Os extratos brutos de ambas as plantas apresentaram discreta atividade, quando submetidos a um ensaio antifúngico. As frações, contendo os triterpenóis, acetatos e palmitatos de triterpenoflas e as lactonas sesquiterpênicas, foram testadas quanto à atividade anti-infíamatória mostrando-se bastante ativas. Tanto os extratos brutos quanto as fraqões supracitadas não se mostraram ativos em um ensaio antitumoral. Estes resultados indicam a seletividade da atividade dos extratos e frações, possivelmente não contendo substâncias citotóxicas. / This work describes the chemical study of two plants which belong to the family Asteraceae: Mikania sp. nov. and Wunderlichia crulsiana. The dichlorometane extracts from leaves and stems of Mikania sp. nov. we studied and seven diterpenic acids were isolated and identified. The volatile oil from leaves and stems of this plant were also studied and twenty substances among monoterpenes and sesquiterpenes were identified. From Wunderlichia crulsiana we analysed the dichlorometane extracts from flowers and stems. From flowers we isolated and identified triterpenes esterified with palmitic acid and acetic acid. From stems we isolated and identified triterpenes esterified with acetic acid and 3-oxo-triterpenes. By GC, we identified hydroxylated triterpenes that were acetylated, and triterpenes esterified with palmitic acid that were hydrolysed and acetylated. From stems we also isolated and identified three sesquiterpene lactones and a sesquiterpene. The compounds were identified by PMR, CMR (BBD and DEIT 135º), GC/MS and co-injection with authentic samples The extracts of both plants showed low activity when submitted to bioassay with Cladosporium sphaerospermum. The fractions which contain the hydroxylated triterpenes, triterpenes esterified with acetic and palmitic acids and sesquiterpene lactones were submitted to anti-inflammatory bioassay and showed 42%, 29%, 29% and 47% of activity, respectively. The above mentioned extracts and the fractions did not show significant activity on the Saccharomyces cerevisae bioassay, so there were not citotoxic substances in them.

Foi estudado o efeito da lactona sesquiterpênica licnofolido sobre o \"burst\" respiratório de leucócitos polimorfonucleares inflamatórios (PMN) estimulados por forbol (PMA), pelo peptídeo quimiotático fMLP ou zimozan opsonizado (OZ). O licnofolido inibiu de forma dose-dependente a liberação de O2•- pelos PMN, sem alteração do período \"Iag\" do complexo NADPH. oxidase. O efeito foi mais acentuado quando os PMN foram estimulados diretamente pela via de proteína quinase C. A adição de ditiotreitol ou glutationa reduzida (GSH) às suspensões celulares antes da incubação com licnofolido preveniu parcialmente o efeito inibitório. O tratamento dos PMN com a lactona determinou uma queda drástica dos níveis celulares de GSH livre, sem incremento de glutationa oxidada (GSSG). A reação direta entre GSH e licnofolido foi confirmada com a detecção de um aduto glutationil-licnofolido através de identificação por espectrometria de massa (ESI-MS/MS). A S-tiolação protéica induzida pelo PMA foi reduzida em PMN tratados com Iicnofo/ido, como detectado através de determinação de incorporação de [35S], sendo que 80% desses tióis foram identificados como GSH. Uma série de proteínas S-glutationadas foi detectada através de autoradiografias, sendo que aquelas correspondentes a 38 e 24 kDa tiveram essa modificação póstraducional suprimida pelo tratamento com dose de licnofolido capaz de suprimir o \"burst\" respiratório dos PMN. Estes resultados indicam que a depleção celular de GSH causada pelo licnofolido impede a sustentação do \"burst\" respiratório pelos PMN, em correlação direta com a diminuição de S-glutationação protéica. / An investigation was made into the action of the sesquiterpene lactone lychnopholide on the respiratory burst of inflammatory polymorphonuclear leukocytes. Lychnopholide determined concentration-related inhibition of the generation of phorbol 12-myristate 13-acetate-, chemotatic peptide-, and opsonized zymozan-induced superoxide anion with no effect on the lag time of the assembly of the NADPH oxidase complex, such action was greater on the protein kinase C pathway that on both membrane receptor dependent stimuli via. Subsequent additions of D-glucose, Ca2+, Mg2+, dithiothreitol ar reduced glutathione (GSH) did not reverse the inhibitory action. The addition of both thiols prior to the lychnopholide treatment partially hindered the inhibition rate. The endogenous level of GSH in leukocytes was drastically depleted under the lychnopholide treatment, without corresponding increases occurring in the oxidized form (GSSG). A direct reaction between glutathione and lychnopholide was confirmed from a glutathionyl-lychnopholide adduct detected by electrospray mass spectrometry analysis and identified by tandem mass analysis in cellular extracts. Protein S-thiolation induced by PMA stimulation was decreased in lactone-treated PMN as detected by [35S] scintillation count, which indicated that about 80% of the thiols were glutathione. A subset of S-glutathionylated proteins was identified through gel electrophoresis, which revealed that the modification of the phorbol-triggered protein sulfhydryl in the protein bands corresponding to 38 and 24 kDa was precluded by the lychnopholide treatment correlated with respiratory burst inhibition. These results show that GSH depletion determined by lychnopholide treatment renders PMN to sustain respiratory burst, whose action is proportional to protein S-glutahionylation decrease.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O diterpeno, lactona do Ãcido hawtriwaico (LAHT) isolado dos capÃtulos florais de Egletes viscosa Less. (Asteraceae) foi avaliado nos modelos de edema de orelha e nocicepÃÃo induzida por capsaicina em camundongos. A LAHT (12,5; 25 e 50 mg/kg, v.o.) atenuou significativamente a resposta ao edema de orelha induzido pela aplicaÃÃo tÃpica de capsaicina (250 Âg), de maneira dose dependente (45,7; 86,9 e 100 % respectivamente). A resposta ao edema de orelha induzido pela capsaicina foi tambÃm significativamente inibida em 74,8 % pelo vermelho de rutÃnio (VR; 3mg/kg, s.c.), um antagonista nÃo competitivo do receptor da capsaicina (TRPV1). A LAHT (50 mg/kg, v.o.) nÃo modificou a resposta ao edema de pata induzido por composto 48/80 (10 Âg), histamina (10 Âg) ou serotonina (10 Âg), demonstrando que a LAHT nÃo bloqueia a desgranulaÃÃo de cÃlulas mastocitÃrias ou os receptores de histamina e serotonina. No entanto, o edema de pata induzido por substÃncia P (SP) foi significativamente suprimido pela LAHT (25 e 50 mg/kg, v.o.), em 29,4 e 53,3 % respectivamente, bem como o aumento da permeabilidade capilar induzido pela injeÃÃo intraperitoneal de Ãcido acÃtico. No modelo de nocicepÃÃo, a LAHT (12,5; 25 e 50 mg/kg, v.o.) suprimiu de forma significativa o comportamento nociceptivo de lamber a pata induzido pela injeÃÃo intraplantar de 1,6 Âg de capsaicina (27,1; 32,3 e 52 % respectivamente). A resposta à nocicepÃÃo induzida pela capsaicina foi significativamente inibida pelo VR (3 mg/kg, s.c.) em 64,9 %. O efeito antinociceptivo da LAHT (50 mg/kg, v.o.) nÃo foi afetado pelo prÃ-tratamento por naloxona, mas foi significativamente antagonizado pela teofilina e glibenclamida, respectivos bloqueadores de adenosina e canais de KATP dependentes. A LAHT (50 mg/kg, v.o.) nÃo alterou o tempo de sono, nÃo prejudicou a atividade locomotora e nÃo alterou a coordenaÃÃo motora dos camundongos como evidenciado nos testes do tempo de sono induzido por pentobarbital, do campo aberto e rota-rod respectivamente. Esses dados sugerem que a LAHT inibe a inflamaÃÃo neurogÃnica aguda, possivelmente pela inibiÃÃo da liberaÃÃo de SP ou bloqueio de seus receptores, e a nocicepÃÃo possivelmente pelo envolvimento de adenosina endÃgena e canais de KATP dependentes / The diterpene, 12-acetoxy-hawtriwaic acid lactone (AHAL) isolated from the flower buds of Egletes viscosa Less. (Asteraceae), a popular medicinal plant largely encountered in Cearà State was evaluated in mice for its anti-inflammatory and antinociceptive potential, using capsaicin-induced ear edema and hindpaw nociception as experimental models. AHAL (12.5, 25 and 50 mg/kg, p.o.) significantly attenuated the ear edema response to topically applied capsaicin (250Âg), in a dose-related manner (45.7, 86.9 and 100 % respectively). This response to capsaicin was also greatly inhibited by ruthenium red (3 mg/kg, s.c.), a non-competitive capsaicin receptor (TRPV1) antagonist by 74.8%. The anti-edema effect of AHAL (50 mg/kg, p.o.) seems unrelated either to inhibition of mast cell degranulation or to antagonism at histamine and serotonin receptor since AHAL did not modify the paw edema response-induced by intraplantar injections of compound 48/80 (10 Âg), histamine (10 Âg) or serotonin (10 Âg). However the substance P (SP) induced hindpaw edema was significantily inhibited by AHAL (25 and 50 mg/kg, p.o.) by 29.4 and 53.3 % respectively, as well as the increase in capillary permeability induced by intra-peritoneal acetic acid. In the hindpaw nociceptive model, AHAL suppressed the nocifensive paw-licking behavior induced by intraplantar injection of capsaicin (1.6 Âg). This response to capsaicin was also greatly inhibited by ruthenium red (3 mg/kg, s.c.) by 64.9 %. The antinociceptive effect of AHAL (50 mg/kg, p.o.) was unaffected by naloxone pre-treatment but was significantly antagonized by theophylline and glibenclamide, the respective blockers of adenosine receptor and KATP-sensitive channels. AHAL (50 mg/kg, p.o.) did not impair the ambulation or motor coordination of mice in open-field and rota-rod tests. These data allow us to conclude that AHAL possesses anti-inflammatory and antinociceptive properties. It possibly inhibits acute neurogenic inflammation by the inhibition of SP release or its receptor blockade and the nociception through mechanisms that involve the endogenous adenosine and opening of ATP-sensitive potassium channels

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Os terpenos Ãcido centipÃdico e lactona do Ãcido hawtiwaico (LAH), isolados dos capÃtulos florais de Egletes viscosa Less, popularmente conhecida como macela ou macela da terra, foram avaliados em modelos de dermatite de contato irritativa induzida pelo 13-acetato-12-o-tetradecanoil-forbol (TPA) e de dermatite de contato alÃrgica induzida por oxazolona (OXA) em camundongos. O Ãcido centipÃdico e a LAH, por via tÃpica (0,125; 0,25 e 0,5mg/orelha), reduziram significativamente o edema de orelha induzido por TPA (2,5Âg/orelha) em 45,5; 55,5; 61,1% e 33,3; 42,2 e 63,3%, respectivamente. Quando administrados por via oral, (12,5; 25 e 50mg/kg), reduziram o edema em 46,6; 59,3; 67,9% e 58,7; 59,7; 71%, respectivamente quando comparado ao controle veÃculo. A dexametasona na dose tÃpica de 0,05mg/orelha e na dose oral de 1mg/kg reduziu o edema de orelha em 90 e 73,1%, respectivamente. O Ãcido centipÃdico e a LAH, na dose de 0,5mg/orelha, via tÃpica reduziu de a atividade da mieloperoxidase (MPO) em 90,1 e 94,6%, respectivamente, enquanto a dexametasona (0,05mg/orelha) reduziu em 96,2% a atividade da MPO quando comparado ao controle veÃculo. Os terpenos na dose tÃpica de 0,5mg/orelha foram capazes de reduzir os nÃveis teciduais de TNF-α, o mesmo sendo observado com a dexametasona. A aÃÃo antiinflamatÃria dos terpenos no modelo da dermatite pelo TPA foram confirmados pelos achados histolÃgicos. O modelo de dermatite de contato induzida por oxazolona (OXA, 1%/orelha), provocou uma hiperplasia epidÃrmica onde o INF-γ teve papel crucial. Os terpenos Ãcido centipÃdico (0,25 e 0,5) e a LAH (0,5mg/orelha), por via tÃpica, diminuÃram, a hiperplasia epidÃrmica induzida por oxazolona (1%/orelha)em todos os perÃodos de observaÃÃo (4Â - 19Â dia). O Ãcido centipÃdico e a LAH na dose de 0,5mg/orelha diminuÃram significativamente os nÃveis teciduais de INF-γ (92,2 e 99,4%, respectivamente) quando comparados ao controle veÃculo. Quando administrado por via oral, o Ãcido centipÃdico (12,5; 25 e 50mg/kg) reduziu de forma significativa a hiperplasia epidÃrmica do 7Â ao 19Â dia de observaÃÃo. A dexamentasona na dose tÃpica (0,05/mg) e na dose oral (1mg/kg) reduziu a hiperplasia epidÃrmica em 100 e 75%, respectivamente. Os resultados encontrados, comprovados pelo estudo histolÃgico, demonstram o efeito antiinflamatÃrio desses terpenos em modelo de dermatite de contato alÃrgica crÃnica onde a hiperplasia epidÃrmica e o aumento nos nÃveis teciduais de INF-γ sÃo caracterÃsticas importantes, como no caso da psorÃase / The terpens centipedic acid (CA) and lactone of hawtriwaic acid (LAH) isolated from the flowers chapters of Egletes viscosa Less, popularly known as macela or macela da terra, were evaluated on 13-acetate-12-o-tetradecanoil-forbol (TPA) induced irritative contact dermatitis model and on oxazolone induced allergic contact dermatitis in mice. The centipedic acid and LAH on the following dosage of 0,125; 0,25 and 0,5 mg/ear using the skin surface as a via reduced in a significative way the ear oedema induced by application of TPA (2,5 μg/ear). The values of reduction are the following respectively: 45,5; 55,1; 61,1% and 33,3; 42,2 and 63,3%. When the oral administration was used the doses of 12,5; 25 and 50 mg/kg had showed a significative oedema reduction of 46,6; 59,3; 67,9% and 58,7; 59,7; 71%, respectively. Dexametasone through skin surface administration on dosage of 0,05 mg/ear and oral dosage of 1mg/kg have reduced in a significative way the ear oedema in 90 and 73,1%, respectively. The centipedic acid and LAH using the dosage of 0,05 mg/ear through the skin surface had reduced the mieloperoxydase activity in 90,1 and 94,6%, respectively, while dexametasone (0,05 mg/ear) had reduced in 96,2% when compared to the group treated only with the vehicle. The terpens on the topic dosage of 0,05 mg/ear were capable to reduce the tecidual levels of TNF-α as well as dexametasone. The antiinflamatory action of the terpens on TPA induced dermatitis model was confirmed through histological analysis of the tissue. The oxazolone induced contact dermatitis model (OXA 1%/ear) had promoted an epidermic hyperplasia in which INF-γ have a central role. The products under evaluation centipedic acid (0,25 and 0,5 mg/ear) and LAH (0,5 mg/ear) through skin surface reduced in a significative way the epidermic hyperplasia Throughout the observation period (4Â- 19Â day). Centipedic acid and LAH on the following dosage 0,5/mg reduced in a significative way the tecidual levels of INF-γ (92,2 and 99,4%, respectively) after 19 days of treatment. When using oral administration the centipedic acid (12,5; 25 e 50 mg/kg) reduced the epidermic hyperplasia from seventh through tenth ninth day. Dexametasone through skin surface administration on dosage of 0,05 mg/ear and oral dosage of 1mg/kg have reduced the epidermic hyperplasia in 100 and 75%, respectively The data that were found plus the histological analysis have showed the antiinflammatory effect, mainly through topic administration, of these terpens on allergic contact dermatitis models in which epidermic hyperplasia and increased tecidual levels of INF-γ are important features, such as psoriasis

A presente tese relata o estudo químico de duas espécies vegetais pertencentes à família Asteraceae: Mikania sp. nov; e Wunderlichia crulsiana. Da espécie Mikania sp. nov. foram estudados os extratos diclorometânicos das folhas e dos galhos, tendo sido isolados e identificados sete ácidos diterpênicos; dos óleos voláteis das folhas e dos galhos foram identificadas vinte substâncias entre monoterpenos e sesquiterpenos. Da espécie Wunderlichia crulsiana foram estudados os extratos diclorometânicos das flores e dos galhos. Das flores foram isolados e identificados palmitatos e acetatos de triterpenoílas. Dos galhos foram isolados e identificados acetatos de triterpenoílas e triterpenonas e identificados por CG triterpenóis acetilados e palmitatos de triterpenoílas hidrolisados e posteriormente acetilados. Deste extrato foram também isolados e identificados três lactonas sesquiterpênicas e um sesquiterpeno. A identificação das substâncias foi realizada através de RMN de 1H, de 13C (BBD e DEPT 135º), CG/EM e co-injeção de padrões. Os extratos brutos de ambas as plantas apresentaram discreta atividade, quando submetidos a um ensaio antifúngico. As frações, contendo os triterpenóis, acetatos e palmitatos de triterpenoflas e as lactonas sesquiterpênicas, foram testadas quanto à atividade anti-infíamatória mostrando-se bastante ativas. Tanto os extratos brutos quanto as fraqões supracitadas não se mostraram ativos em um ensaio antitumoral. Estes resultados indicam a seletividade da atividade dos extratos e frações, possivelmente não contendo substâncias citotóxicas. / This work describes the chemical study of two plants which belong to the family Asteraceae: Mikania sp. nov. and Wunderlichia crulsiana. The dichlorometane extracts from leaves and stems of Mikania sp. nov. we studied and seven diterpenic acids were isolated and identified. The volatile oil from leaves and stems of this plant were also studied and twenty substances among monoterpenes and sesquiterpenes were identified. From Wunderlichia crulsiana we analysed the dichlorometane extracts from flowers and stems. From flowers we isolated and identified triterpenes esterified with palmitic acid and acetic acid. From stems we isolated and identified triterpenes esterified with acetic acid and 3-oxo-triterpenes. By GC, we identified hydroxylated triterpenes that were acetylated, and triterpenes esterified with palmitic acid that were hydrolysed and acetylated. From stems we also isolated and identified three sesquiterpene lactones and a sesquiterpene. The compounds were identified by PMR, CMR (BBD and DEIT 135º), GC/MS and co-injection with authentic samples The extracts of both plants showed low activity when submitted to bioassay with Cladosporium sphaerospermum. The fractions which contain the hydroxylated triterpenes, triterpenes esterified with acetic and palmitic acids and sesquiterpene lactones were submitted to anti-inflammatory bioassay and showed 42%, 29%, 29% and 47% of activity, respectively. The above mentioned extracts and the fractions did not show significant activity on the Saccharomyces cerevisae bioassay, so there were not citotoxic substances in them.

A busca por novos fármacos inspirados em substâncias de origem natural é uma estratégia conhecida e há tempos utilizada. As lactonas sesquiterpênicas (LST) são um grupo de substâncias amplo e diverso com várias atividades farmacológicas descritas, e cuja característica estrutural determinante é a de um esqueleto principal contendo 15 átomos de carbono e um anel lactônico. O mecanismo de ação das LST está intrinsicamente relacionado com reações de adição do tipo Michael frente a biomoléculas, provocando alquilações. Há na literatura estudos aprofundados sobre a química medicinal das LST, entretanto pouco há sobre o metabolismo desse grupo de substâncias em ambientes fisiológicos. Neste sentido, este trabalho é focado no estudo do metabolismo e também das vias de fragmentação por eletrospray da budleína A, que possui estrutura química do tipo furanoeliangolido, e pertence à classe dos germacranolidos. Foram realizados ensaios in vitro utilizando-se os modelos de oxidação biomimética com metaloporfirina, microssomas hepáticos e metabolismo pela microbiota intestinal de ceco de porco (pig cecum model), sendo nos dois últimos testado também o correlato 4,5-dihidro-2\',3\'-epoxi-15-desoxigoyazensolido. O estudo de fragmentação também abordou a comparação entre budleína A e a centraterina - um estereoisômero que se diferencia apenas pela orientação da cadeia lateral ligada ao C-8 - em espectrômetros distintos e com suporte de métodos computacionais para cálculos de energia (Gaussian 03 em base B3LYP/6-31G(d)). Nos estudos de fragmentação observou-se a diferença de intensidade dos sinais para íons fragmento comuns às duas LST (m/z 275, 257 e 83), além da presença do íon fragmento de m/z 293 apenas para a budleína A, permitindo a diferenciação destes isômeros por meio de espectrometria de massas com ionização por eletrospray, sem a necessidade de ressonância de magnética nuclear. Os produtos de oxidação detectados na reação com metaloporfirinas acusaram a epoxidação na cadeia lateral envolvendo C-2\' e C-3\' com a formação de diastereoisômeros. No ensaio com microssomas não foram detectados produtos para a budleína A, enquanto que para a substância correlata observou-se a abertura do epóxido na cadeia lateral e adição de uma hidroxila, formando um diol vicinal. No modelo de ceco de porco observaram-se a formação de adutos de LST com o aminoácido cisteína, os quais foram posteriormente degradados pela ação da microbiota intenstinal, dando origem a diversos metabólitos compostos pela LST e partes de cisteína. Sendo assim, este trabalho lança bases para a maior compreensão do metabolismo de LST do tipo furanoeliangolido em modelos distintos e também contribui para os estudos de espectrometria de massas para este tipo de substância, além de descrever uma ferramenta analítica útil na diferenciação de dois estereoisômeros. / The search for new drugs inspired on compounds from natural products is a wellknown strategy with several successful cases. Sesquiterpene lactones (STL) are a wide and diversified group of compounds which has already many pharmacological activities reported. Their fundamental moiety includes a skeleton containing 15 carbons and a lactone ring and the mechanism of action is related to Michael addition type reactions with biomolecules, promoting alkylation. There are a high amount of studies regarding the medicinal chemistry of the STL, however there are few information about the metabolism of these compounds under physiological environments. On this way, the aims of this work are focused on the study of the metabolism as well as the fragmentation pathways of budlein A, which is a furanoheliangolide and belongs to the germacranolides class. On this work the following experiments were carried out: in vitro oxidative metabolism with metalloporphyrin and microsomes; intestinal metabolism by using the pig cecum model (microbiota). For microsomes and intestinal metabolism the compound 4,5- dihydro-2\',3\'-epoxy-15-deoxy-goyazensolide was also applied. Fragmentation studies compared the fragmentation patterns of budlein A and centratherin - which is a stereoisomer with -orientation for the side chain bonded at C-8 - by using different spectrometers being supported by computational methods for energies calculations (Gaussian 03 at level B3LYP/6-31G(d)). For the fragmentation studies it was observed the difference of signal intensities for fragment ions which are common for both STL (m/z 275, 257 e 83), moreover the ion m/z 293 was detected only for budlein A, allowing the differentiation between these isomers by electrospray ionization mass spectrometry instead nuclear magnetic resonance. The reactions with metalloporphyrin yielded two diastereoisomers of the STL with an epoxide at the side chain between C-2\'and C-3\'. On the microsome assay any product was detected for budlein A, while for the correlated compound the epoxide ring was opened and a hydroxyl was added at C-3\', forming a vicinal diol. On the pig cecum model it was observed the formation of adducts due to the reaction of the STL and the amino acid cysteine. These adducts were later degraded by the action of the microbiota yielding different metabolites composed by STL and residues of cysteine. Thus, this work may contribute to the improvement of the knowledge about the metabolism of STL furanoheliangolide type in different models as well as for the studies regarding the mass spectrometry of this type of compound. The development of a useful analytical tool for the differentiation of two isomers was also an important achievement.

Orientador: Luiz Carlos Dias / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-12T11:13:03Z (GMT). No. of bitstreams: 1 Melgar_GliseidaZelayaran_D.pdf: 8992583 bytes, checksum: 4cacbcdc703a19bccdf5bea13368488a (MD5) Previous issue date: 2008 / Resumo: Em 1995 Miao e colaboradores relataram o isolamento da estavamicina (1), um novo produto natural, membro da família dos pirrolocetoindanos, a partir de uma cultura líquida de Streptomyses sp., isolada de uma amostra de terra coletada na Índia. A estavamicina contém uma interessante subestrutura hexahidroindeno de fusão de anel trans com 5 centros estereogênicos, uma cadeia lateral que contém outros 2 centros estereogênicos, um álcool dialílico, três duplas ligações e um resíduo de carboxilato de sódio. Apresenta atividade inibidora moderada contra a ligação do fator de transcrição EBV BZLF1 com o DNA com um valor de IC50 = 50 mM.O fragmento C11-C26 (206), contendo o grupo pirrol e 5 centros estereogênicos da estavamicina, foi preparado a partir do (R)-3-hidroxi-2-metilpropionato de metila, após uma sequência de reações, que envolveu 14 etapas (rota linear mais longa) e um rendimento global de 7%. As principais características incluem a preparação de uma imida a,b-insaturada utilizando a reação de Horner-Wadsworth-Emmons, a reação de olefinação de Takai, o acoplamento cruzado de Stille, seguido da cicloadição intramolecular de Diels-Alder que fornece dois adutos bicíclicos, sendo o majoritário correspondente ao produto desejado (para os casos de R = (R)-Bn e R = H). A última etapa foi a preparação da lactona tricíclica seguida da abertura utilizando o 2-lítio-N-MEM-pirrol.O fragmento C1-C6 (235) foi preparado a partir do 3-hidroxi-pentanodioato de dietila, após uma seqüência de reações, que envolveu 7 etapas e um rendimento global de 11,5%. As principais características incluem a elegante reação de transesterificação por quebra de simetria, redução do ácido com borana seguida por oxidação de swern e olefinação utilizando o procedimento modificado de Stork-Wittig.Em conseqüência, a rota de obtenção dos fragmentos C1-C6 e C11-C26 aqui descrita é, em princípio, prontamente aplicável para a preparação da estavamicina e análogos que eventualmente pudessem apresentar atividade farmacológica destacada. / Abstract: Epstein-Barr virus (EBV) is a human herpes virus that infects lymphocytes and epithelial cells. Is has been estimated that this virus infects a large part of the world¿s population. In 1995, stawamycin (1), a new natural product from the pyrroloketoindane family was isolated by Miao et. al from a liquid culture of Streptomyces sp, and displayed moderate inhibitory activity against the binding of the EBV BZLF1 transcription factor to DNA with IC50 = 50 mM in a DNA binding assay. Stawamycin has a trisubstituted trans-fused bicyclo[4.3.0]nonane substructure containing five stereogenic centres and a side chain that contains two stereogenic centres at C3 and C9 (absolute configuration not determined), a doubly allylic alcohol and a sodium carboxylate residue. To determine the relative configurations between C3 and C9, to establish the absolute configuration of stawamycin, and to provide material for further biological studies as well as access to novel analogues, we initiated a study towards the synthesis of this very interesting compound. We wish to describe here our successful efforts towards the preparation of the C1¿C6 as well as the C11¿C26 carbocyclic fragment of stawamycin. The bicyclo[4.3.0]nonane (C11¿C26) fragment of stawamycin has been prepared by a sequence involving 14 steps (7% overall yield) from methyl (R)-(-)-3-hydroxy-2- methylpropionate. Key steps are a Pd-catalysed Stille coupling reaction between a vinyl iodide and a vinyl stannane followed by an intramolecular Diels¿Alder cycloaddition reaction to give the desired adduct as the major isomer. The best result was obtained with the use of a triene bearing an achiral oxazolidinone in the presence of Et2AlCl to promote the IMDA cycloaddition reaction. The last step was an preparation of the tricyclic lactone followed by the opening by means of the 2-lítio-N-MEM-pirrol.The (C1¿C6) fragment of stawamycin has been prepared from diethyl-3- hydroxypentanedioate by a sequence which involved a symmetry breaking reaction of a cyclic anhydride, followed by the formation of a Z-vinyliodide employing a Stork-Wittig procedure. / Doutorado / Quimica Organica / Doutor em Ciências

Kinetic inactivation of factor XIIIa and MTG were performed in a pressure range from 0.1 to 400 MPa at 40°C within a time from 0 to 60 min in a TRIS-acetate buffer at pH 6.0. The inactivation of both enzymes at these conditions followed a first order reaction model. The high inactivation rate constant of 26.6 x10-3/min-1 for factor XIIIa at low pressure (50 MP) indicated that this enzyme is much easier to inactivate than MTG, which achieved an inactivation rate constant value of 9.7 x10-3/min at higher pressure (200 MPa). An inactivation volume of –10.17±0.5 cm3/mol confirmed that MTG is very stable under high pressure. The stability of MTG under high pressure and thermal treatment was related to its conformational changes. Enzyme inactivation was accompanied by secondary and tertiary structure changes until an irreversible protein precipitation is achieved. The tertiary structure, represented by circular dichroism spectra in the aromatic region showed differences among native and MTG samples treated under high pressure, as well as at elevated temperature. Tyrosine bands, indicating protein unfolding, increased proportionally with increasing pressure treatment above 400 MPa. Nevertheless, compared to pressure, a maximal enhancement could be observed after thermal treatment at 0.1 MPa at 80°C. That demonstrated the exposure of hydrophobic groups to the protein surface with a concomitant protein unfolding. The spectra in the far ultraviolet region showed that increasing high pressure and high temperature lead to alterations in the secondary structure. The mathematical algorithms CONTIN used to calculate secondary structures stated that the 24.5% of alpha-helix of native MTG decreased to 17.2% after a treatment at 400 MPa at 40°C for 60 min and to 6.5% after a treatment at 0.1 MPa at 80°C for 2 min. However, beta-strand structures remained relatively stable after these several treatments. MTG is arranged in a way that the active site is located between beta-strand domains that are surrounded by alpha-helices, the results of this investigation suggested that MTG activity is related with the relative stability of alpha-helix and the outstanding stability of the central beta-strand structure. The irreversible precipitated protein observed at 600 MPa at 40°C for 60 min and 0.1 MPa at 80°C for 2 min was caused principally by the formation of disulfides bonds, because high pressure and high thermal treatment lead to the exposition of the Cys64 residue towards the solvent with the subsequent ability to react with neighbouring cysteine residues. Furthermore, the reaction between protein and reducing sugars resulted in the formation of Maillard products. Furosine, as an indicator of the early stages of Maillard reaction was measured. Concentration values of 261.0 mg/g protein from samples treated at 600 MPa and 40°C and 238.5 mg/g protein from samples treated at and 0.1 MPa and 80°C for 2 min were obtained. Pentosidine a subsequent product observed in the advanced Maillard reaction was also present. Concentrations of 13.7 and 6.7 mg/g protein were obtained in the samples treated at 600 MPa and 40°C for 60 min and 0.1 MPa and 80°C for 2 min, respectively. Kinetic inactivation studies of MTG in a pressure range from 0.1 to 600 MPa at 10, 30, 40, and 50°C within a long time range from 0 to 140 h were performed in order to study MTG stability under the simultaneous effect of pressure and temperature. The inactivation kinetic showed a first and very fast step and a second very slow step suggesting irreversible inactivation behaviour. Activation energy and entropy difference decreased with increasing pressure. Thereby, the inactivation rate constants of enzyme were less temperature dependent at high pressure. The effect of pressure and temperature on MTG inactivation had a synergistic behaviour. At temperatures of 10, 30, and 40°C, increasing pressure leads to increasing inactivation rate constants. However at 50°C a tendency change occurred. Negative activation volumes of –16.2±0.5, -13.6±0.1, -11.2±0.3 cm3/mol were obtained for 10, 30 and 40°C respectively and for treatment at 50°C a positive value of about +3.0±2.0 cm3/mol in a pressure range from 0.1 to 300 and a negative volume of –11.0±0.4 cm3/mol MPa from 300 to 600 MPa were calculated. A pressure/temperature diagram from inactivation rate constants was performed to represent MTG stability. The diagram shows that in a pressure and temperature range from 0.1 to 550 MPa and 10 to 40°C, pressure induces MTG stabilization against heat denaturation. At 50°C in range from 0.1 to 300 MPa, pressure induces also enzyme stabilization again heat denaturation, but at the same temperature and above 300 MPa the enzyme was inactivated. After MTG stability analysis, reaction kinetics from MTG with individual caseins in a TRIS-acetate buffer pH 6.0 were performed under atmospheric pressure (0.1 MPa) and high pressure (400 MPa) at 40°C. The reaction was monitored by gel permeation chromatography under in three assumptions: 1) The initial velocity kinetics was obtained from a non-progressive enzymatic reactions with the products. 2) The substrate concentration exceeded enzyme concentration. 3) The sum of the individual catalytic constants of the reactive glutamine residues inside caseins are represented by a single MTG-monomeric casein complex. Enzyme reaction kinetics of MTG with the individual caseins carried out at 0.1 MPa at 40°C showed Michaelis-Menten-Henri behaviour with maximal velocities of 2.7 x 10-3, 0.8 x 10-3, and 1.3 x 10-3 mmol/L∙min and Km values of 59 x 10-3, 64 x 10-3 and 50 x 10-3 mmol/L of beta-, alpha-s1-, and whole-casein, respectively. This suggested that MTG achieved a maximal velocity with ß-casein, but had the best affinity with acid casein followed by beta- casein and finally alpha-s1-casein. Enzyme reaction kinetics of beta-casein carried out at 400 MPa and 40°C also showed a Michaelis-Menten-Henri behaviour with a similar maximal velocity of 2.6 x 10-3 mmol/L×min, but the Km value of 144 x 10-3 mmol/L showing kinetical similarity to a non-competitive inhibition. The reaction of MTG with alpha-s1-casein under high pressure did not fit in to Henri-Michaelis-Menten kinetics. Kinetic parameters showed that the affinity of MTG to beta- and alpha-s1-casein under atmospheric pressure is higher than the affinity of MTG to these caseins under high pressure. This loss of affinity can be explained by a constant number of reactive glutamine residues of casein, although the protein is unfolding at high pressure, a decrease of enzyme activity of MTG to 74% after treatment at 400 MPa at 40°C for 15 min and self association of casein under thermal and high pressure treatment. Fur technological application, the formation of acid milk gels was studied under the influence of MTG within its range of pH stability. Simultaneous addition of MTG and different concentrations of glucono-delta-lactone (Gdl) to casein solutions (5% w/v) at 40°C was analysed. Gels firmness was accessed by oscillation rheometry and gel permeation chromatography. Oscillation rheometry data showed that the time of gelation decreased with an increasing Gdl concentration added to the system, however higher concentrations of Gdl caused the formation of weaker gels. Addition of 1 g Gdl/g protein without MTG caused gelation within 5 min and a storage module value G´ of 48.9 Pa. With the simultaneous addition of 1 g Gdl/g protein and 6 U MTG/ g protein the gelation time was 4 min and the reached storage modulus was 63.7 Pa. However, the addition of 0.21 g Gdl/g protein and 6 U/g protein MTG increase the gelation time to about 69 min, but, a higher module value G´ of 111.0 Pa was achieved. Addition of high Gdl concentration caused a rapid drop of pH below 5 leading to a fast enzyme inactivation. However addition of very low Gdl concentrations was also not optimal. The simultaneous influence of MTG and Gdl concentration on the gelation time and elastic properties was evaluated by a central composite rotatable design (CCRD). The resulting quadratic storage modulus model showed that, MTG concentration had a significant influence on storage modulus G´ and, that the firmness of the gels increase in direct proportion with MTG activity with the existence of a optimum Gdl concentration, whereas the resulting linear model of the gelation time stated that Gdl concentration has a significant influence on the gelation time, while it is independent of the MTG activity. A maximal firmness of 136 ± 2 Pa was reached between a range of 0.24 - 0.27 g Gdl/g protein and 5.8 U MTG/g within a time from 49 to 59 min. Gel permeation chromatography analysis demonstrated that acid gels induced by Gdl were formed by reversible cross-linking like electrostatic interactions and hydrogen bonds as well as disulfide bonds caused by temperature treatment. Whereas, the addition of MTG proved the formation of non-reversible cross-linking like oligomers based on Ne-(g-glutamyl)- lysine, which gave more firmness and stabilization on the casein gel network.