Phytol aus dem Chlorophyllabbau ist limitierend für die Tocopherol (Vitamin E)-Synthese Als Bestandteil von Chlorophyll ist Phytol das am häufigsten vorkommende Isoprenoid in der Biosphäre. Große Mengen an Chlorophyll werden jährlich degradiert und dabei wird Phytol freigesetzt, über dessen Verbleib jedoch wenig bekannt ist. Es sollte der Nachweis erbracht werden, dass im Zuge des Chlorophyllabbaus hydrolysiertes Phytol Eingang in die Synthese anderer Phytylderivate findet. Während der Gehalt an Tocopherol, Chlorophyll und Fettsäurephytylester entwicklungs- bzw. seneszenzabhängig ist, bleibt der Gehalt an Phyllochinon etwa gleich. Auch in Samen ist der Gehalt von Tocopherol, Chlorophyll und Fettsäurephytylester entwicklungsabhängig. Es wurde gefolgert, dass nur die Synthesen von Tocopherol und Fettsäurephytylester während des Chlorophyllabbaus stimuliert werden. Daher sollten Mutanten analysiert werden, welche im Chlorophyllabbau inhibiert sind. Da Chlorophyllase den ersten Schritt des Chlorophyllabbaus katalysiert, wurden zwei unabhängige T-DNA-Insertionsmutanten für Chlorophyllase1 (CHL1) und eine T-DNA-Insertionsmutante für Chlorophyllase2 (CHL2) identifiziert und eine chl1-1chl2-Doppelmutante erzeugt. Die Analyse der Chlorophyllidanteile ergab eine im Vergleich zum Wildtyp starke Reduktion in den chl1-Mutanten, während der Chlorophyllidanteil von chl2 ähnlich hoch dem Wildtyp ist. Der Chlorophyllidanteil sich entwickelnder chl1-1chl2-Pflanzen nahm in der Seneszenz zu. Die Chlorophyllasemutanten zeigten kein verändertes Seneszenzverhalten im Vergleich zu den Wildtypen. Ferner konnte in den chl1-Linien nur geringfügig weniger Tocopherol und Fettsäurephytylester als in den Wildtypen nachgewiesen werden. Auch der Tocopherolgehalt der Samen war in den Chlorophyllasemutanten unverändert zu den Wildtypen. Aufgrund dessen wurde gefolgert, dass neben den Chorophyllasen CHL1 und CHL2 weitere Chlorophyllhydrolasen in Samen und Blättern von Arabidopsis existieren. Daher wurde auf andere Mutanten zurückgegriffen, in denen der Chlorophyllabbau stark inhibiert ist und die Seneszenz nach Dunkelinkubation im Vergleich zum Wildtyp deutlich verzögert ist. Eine deutliche Korrelation zwischen vermindertem Chlorophyllabbau und Gehalt an Tocopherol und Fettsäurephytylester konnte in den staygreen-Mutanten pao1 und zwei unabhängigen SGR (staygreen)-RNAi-Linien nachgewiesen werden. Damit konnte eindeutig gezeigt werden, dass die Synthese von Tocopherol und der Fettsäurephytylester durch die Chlorophyllhydrolyse induziert wird. Es wurde gefolgert, dass vor allem unter Seneszenz- bzw. Stressbedingungen dieser alternative Syntheseweg von Phytol eine Rolle spielt. Dennoch kommt der Phytylsynthese durch die de novo-Isoprenoidsynthese auch eine Bedeutung zu. Nach Behandlung von stickstoffmangelgestressten Wildtyppflanzen mit dem Inhibitor Fosmidomycin, welcher die plastidäre de novo-Isoprenoidsynthese hemmt, war der Tocopherolgehalt gegenüber stickstoffmangelgestressten Kontrollpflanzen stark reduziert. Ferner konnte eine T-DNA-Insertionsmutante der Geranylgeranylreduktase (GGR) identifiziert werden. Diese Mutante kann nur auf Nährmedium überleben, hat nur wenige grüne Blätter und bildet keine Samen. Es konnte kein Phyllochinon, Chlorophyll und keine Fettsäurephytylester, jedoch geringe Mengen Tocopherol nachgewiesen werden. Der Resttocopherolgehalt wird auf die Nebenaktivität einer anderen Reduktase zurückgeführt. Weiterhin wurde nur das Geranylgeranylderivat des Chlorophylls identifiziert. Diese Ergebnisse erlauben den Schluss, dass die phytylgruppenübertragenen Enzyme der Tocopherol-, Phyllochinon- und Fettsäurephytylestersynthese eine hohe Substratspezifität für die Phytylgruppe aufweisen. Nach Fütterung von Phytol konnte in ggr Tocopherol und Chlorophyll bestimmt werden. Aufgrund dessen kann gefolgert werden, dass Chlorophyllsynthetase aus Arabidopsis sowohl Geranylgeranyl-, als auch Phytylpyrophosphat als Substrat nutzen kann und damit ein breiteres Substratspektrum aufweist. / Phytol from chlorophyll degradation is limiting for tocopherol (vitamin E)-synthesis As a part of the chlorophyll molecule phytol belongs to the most abundant isoprenoid of the biosphere. Huge amounts of chlorophyll are degraded annually. During this process phytol is released, but only little is known about the fate of phytol. The goal of the project was to provide evidence that during chlorophyll degradation released phytol enters the pathway of the synthesis of further phytyl derivatives. While the content of tocopherol, chlorophyll and fatty acid phytyl esters are growth and stress related the content of phylloquinone does not change during development or under stress conditions. Also in seeds the content of these phytyl derivates are dependent on development. Hence only tocopherol and fatty acid phytyl ester synthesis are induced during chlorophyll degradation. Therefore mutants were analysed that are inhibited in chlorophyll degradation. Chorophyllase catalyses the first step during chlorophyll degradation. Two independent T-DNA insertion mutants of Chlorophyllase1 (CHL1) and one for Chlorophyllase2 (CHL2) were identified. Furthermore chl1-1 and chl2 were crossed to produce the chl1-1chl2 double mutant. The mutation resulted in a strong reduction of the chlorophyllide fraction in chl1 mutants while the chlorophyllide fraction of chl2 was similar to wild type. The chlorophyllide fraction in developing chl1-1chl2 plants increased during senescence. For all chlorophyllase mutants no retardation of senescence was observed. Compared to wild type only marginal reductions in tocopherol and fatty acid phytyl ester contents could be observed for the chl1 mutants. The seed tocopherol content of the chlorophyllase mutants was similar to wild type. Therefore, it was concluded that in leaves and seeds of Arabidopsis besides CHL1 and CHL2 further chlorophyll hydrolases exist that induce chlorophyll degradation. Thus, staygreen mutants exhibiting strongly inhibited chlorophyll degradation were analysed. Compared to wild type the staygreen mutants pao1 and two independent SGR (staygreen)-RNAi-lines show a strong retardation of senescence under dark incubation. A clear correlation between reduced chlorophyll degradation and tocopherol and fatty acid phytyl ester content could be demonstrated. With this it was possible to verify that tocopherol and fatty acid phytyl ester synthesis are induced by chlorophyll hydrolysis. This alternative pathway seems to play an important role in particular under stress and senescence conditions. Nevertheless, after application of Fosmidomycin, an inhibitor of the plastidic de novo isoprenoid synthesis pathway, to nitrogen starved wild type plants the tocopherol content was strongly reduced compared to nitrogen starved control plants. Therefore, also the plastidic de novo isoprenoid synthesis plays a significant role for tocopherol synthesis. Moreover, a T-DNA insertion mutant for Geranylgeranyl reductase (ggr) was identified and isolated. This mutant can survive only on nutrition medium, contains only a few green leaves and produces no seeds. There was no phylloquinone, chlorophyll and fatty acid phytyl ester detectable, but minor amounts of tocopherol. The residual amounts of tocopherol were attributed to side activities of another reductase. Obviously, the phytyl transferring enzymes of tocopherol, phylloquinone and fatty acid phytyl ester synthesis exhibit a strong substrate specificity of the phytyl group. After feeding phytol to ggr tocopherol and chlorophyll were detectable in this mutant. Therefore, it was concluded that chlorophyll synthetase from Arabidopsis can use geranylgeranyl pyrophosphate as well as phytyl pyrophosphate as substrates.

Nosso grupo tem caracterizando diversos produtos da via de isoprenóides em Plasmodium falciparum que é promissora para o desenvolvimento de novas drogas. Dentre os compostos isoprenicos identificados estão as vitaminas K1 e E que apresentam uma cadeia isoprenica fitil. Em plantas o fitol é originado da degradação da clorofila. mas, como ainda não existem evidências da presença de clorofila no parasito, sugerimos que este possa ser formado pela degradação dessas vitaminas. Parte do fitol seria ligado a ácidos graxos utilizado como constituinte de membrana, e parte sofreria fosforilações, formando as vitaminas E e K1 como uma segunda via de biossíntese desse composto, assim como ocorre em Arabidopsis thaliana. Analises por HPLC e TLC utilizando o marcador [3H]GGPP, confirmaram a presença de fitol nos estágios intraeritrocíticos. Além disso, mostramos que o [3H]-fitol pode ser convertido em fitil-P e fitil-PP por uma atividade fitol quinase utilizando nucleotídeos como doador fosfato. / Our group has characterized differents products from isoprenoid pathway in Plasmodium falciparum which is promising for the development of new drugs. Among the isoprenics compounds identified there are the vitamins K1 and E which have a isoprenic phytyl side chain . In plants the phytol is obtained from the chlorophyll degradation. Until now theres no evidence that support its presence in the paraste. Our hypothesis that is phytol can be produced by the degradation of these vitamins E and K1. In the parasite phytol would fulfill to functions: used as a constituent of membrane, bounded to fatty acids and; be converted through phosphorylations vitamins E and K1 as a second biosynthetic pathway as well as in Arabidopsis thaliana. Analisys through and HPLC and TLC with radioactive [3H]GGPP confirmed the presence of phytol in intraerythrocytic stages. In addition, we showed that [3H]-phytol is converted into phytyl-P and phytyl-PP through a phytol kinasey using nucleotides as Phosphate donor.

Tocoferóis, compostos com atividade de vitamina E (VTE), são antioxidantes lipofílicos sintetizados exclusivamente por organismos fotossintetizantes. A produção desses compostos ocorre a partir da ligação de um grupo cromanol a uma cadeia isoprênica, esta originada a partir de duas vias metabólicas possíveis: pela síntese de novo ou pela ativação do fitol liberado durante a quebra da clorofila, esta dependente de fitol quinase (VTE5). Conhecer os mecanismos responsáveis pela síntese e pelo acúmulo de vitamina VTE em plantas cultivadas é de grande interesse devido sua importância para a fisiologia vegetal e para a saúde humana. Frutos de tomate e seus derivados constituem fonte significativa de VTE na dieta humana. Para além da importância nutricional, o tomateiro emerge como um interessante modelo de estudo dos mecanismos regulatórios subjacentes à biossíntese de tocoferóis, visto que seu fruto combina uma ativa síntese de novo de isoprenóides juntamente com a degradação de clorofila durante o amadurecimento. Em estudo anterior, loci para caracteres quantitativos (QTL) para tocoferol em frutos foram identificados a partir da determinação dos níveis das isoformas α, β, μ e δ em uma população de linhagens introgredidas (ILs) de Solanum pennellii. Genes candidatos dentro dos intervalos dos QTL foram propostos, incluindo alguns relacionados à defitilação da clorofila e ao metabolismo fitol, CLOROFILASE (CLH) e um homólogo à VTE5, nomeado FARNESOL QUINASE (FOLK). Nesse contexto, o presente trabalho apresenta contribuições para o entendimento da regulação da biossíntese de VTE em tomateiro. Para tanto, adotaram-se diferentes abordagens, as quais incluem: a caracterização inicial da regulação transcricional dos genes envolvidos na biossíntese de tocoferóis ao longo do desenvolvimento de tomateiro; a exploração dos determinantes genéticos envolvidos no QTL para tocoferol a partir da análise do perfil transcricional de ILs; a análise integrada das mudanças do metabolismo de tocoferóis e outros isoprenóides em mutantes de tomateiro deficientes no amadurecimento e na degradação de clorofila; e, por fim, o estudo detalhado do metabolismo do fitol por meio da caracterização funcional dos genes codificantes para VTE5, FOLK e CLH(1). Os resultados obtidos fornecem valiosas informações sobre os mecanismos que controlam o acúmulo de VTE, além de expor inúmeras conexões entre o metabolismo de tocoferol e outras vias metabólicas que, em última análise, impactam a fisiologia de tomateiro / Tocopherols, compounds with vitamin E activity, are lipid-soluble antioxidants exclusively synthesized by photosynthetic organisms. The formation of tocopherol involves the condensation of a chromanol group with an isoprenoid chain, derived from two possible metabolic pathways: from the de novo biosynthesis or from chlorophyll phytol tail recycling, which depends on phytol kinase (VTE5) activity. Understanding the mechanisms underlying synthesis and accumulation of vitamin E in crops is of great interest because of its implications for plant physiology and human health. Tomato fruit and its derivatives constitute a significant dietary source of VTE for humans. Beyond the nutritional value, tomato emerges as an interesting study model of the regulatory mechanisms underlying tocopherol biosynthesis, since fruit couples an active de novo synthesis of isoprenoids together with chlorophyll degradation along ripening. In a previous work, quantitative trait loci (QTL) for VTE content were identified in ripe fruits by tocopherol determination of α, β, μ and δ isoform levels in a population of Solanum pennellii introgression lines (IL). Candidate genes within QTL intervals were proposed, including some related to chlorophyll dephytylation and phytol metabolism, a CHLOROPHYLLASE (CLH) and a VTE5 homolog, named FARNESOL KINASE (FOLK). In this context, this work presents contributions to understanding the regulation of VTE biosynthesis in tomato. For this, different approachs were taken including: an initial characterization of transcriptional regulation of the genes involved in tocopherol biosynthesis along tomato development; the exploitation of genetic determinants involved in QTL for tocopherol from the transcriptional profile analyses of ILs; the integrated analyses of tocopherols metabolism changes and other isoprenoids in ripening impaired and chlorophyll degraded tomato mutants; and, finally, the detailed study of phytol metabolism by means of functional characterization of the genes encoding for VTE5, FOLK and CLH(1). Our results provide valuable insights into the mechanisms that control the VTE accumulation and also expose several cross-talks between the tocopherol metabolism and other metabolic pathways that, ultimately, impact on tomato physiology

Made available in DSpace on 2015-05-14T12:59:29Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1832337 bytes, checksum: ca9e6d049955c1cebd5d5990ce433130 (MD5) Previous issue date: 2011-11-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / This study was conducted in order to draw a profile of pharmacological activity of phytol, a alcohol diterpene. The study was conducted in three steps: (1) evaluation of acute toxicity, (2) evaluation the activity of phytol on the CNS, focusing on the analgesic activity, and (3) evaluation of antioxidant activity. In the first two stages, the study was conducted in vivo and in third, the study was in vitro. The results were expressed as mean ± standard error of mean (S.E.M.), were considered significant when p <0.05. In the first step, the phytol showed low toxicity, causing no changes in biochemical and hematological parameters of the animals, have been determined the LD50 in 1153.39 (944.56 1.408.40) mg/kg. In the following tests, the phytol was administered intraperitoneally (i.p.) at doses of 25, 50, 100 and 200 mg/kg. The phytol showed profile of depressant drugs CNS, without compromising the motor coordination of animals. The antinociceptive activity of phytol was investigated by chemical models (test of abdominal contortions induced by acetic acid and formalin test) and thermal (hot plate test) of nociception in mice. In all tests, the phytol showed a highly significant antinociceptive effect at both the central and peripheral. However, the effect of phytol was not reversed by the antagonists naloxona (opioid system) and glibenclamida (K +ATP channels), demonstrating that at least directly, the phytol does not act by these mechanisms. In the antioxidant activity in vitro of phytol through three methodologies, one for evaluating the effect of phytol on lipid peroxidation in the TBARS test, and other two to investigate if the phytol acted as substance scavenging of free radicals to hydroxyl radical (OH) and nitric oxide (NO). In all tests, the phytol showed strong antioxidant activity, which can be attributed to their structural feature, since phytol is a unsaturated alcohol of branched-chain, and antioxidant properties may be related to the hydroxyl group (OH) present in its molecule. Probably the phytol, by reacting with a free radical, donates hydrogen atoms with an unpaired electron (H.), converting free radicals into less reactive species. Since several evidences show the involvement of these reactive species inthe mechanism of pain, the antioxidant activity of phytol may be contributing to its antinociceptive effect. / O presente estudo foi desenvolvido com a finalidade de traçar um perfil da atividade farmacológica do fitol, um diterpeno álcool. O estudo foi realizado em três etapas: (1) avaliação da toxicidade aguda, (2) avaliação da atividade do fitol sobre o SNC, com enfoque na atividade analgésica, e (3) avaliação da atividade antioxidante. Nas duas primeiras etapas, o estudo foi realizado in vivo e na terceira, o estudo foi in vitro. Os resultados foram expressos em média ± erro padrão da média (E.P.M.), sendo considerados significativos quando apresentaram p < 0,05. Na primeira etapa, o fitol apresentou baixa toxicidade, sem causar alterações nos parâmetros bioquímicos e hematológicos dos animais, apresentando uma DL50 de 1.153,39 (944,56 1.408,40) mg/kg. Nos testes seguintes, o fitol foi administrado intraperitonealmente (i.p.) nas doses de 25, 50, 100 e 200 mg/kg.O fitol apresentou perfil de droga depressora do SNC, sem comprometer a coordenação motora dos animais. A atividade antinociceptiva do fitol foi investigada através de modelos químicos (teste das contorções abdominais induzidas pelo ácido acético e o teste da formalina) e térmico (teste da placa quente) de nocicepção em camundongos. Em todos os testes, o fitol apresentou um efeito antinociceptivo significativo, tanto em nível central como periférico. Contudo, o efeito antinociceptivo do fitol não foi revertido pelos antagonistas naloxona (via opióide) e glibenclamida (canais de K+ATP), indicando que o fitol não atua por esses mecanismos, pelo menos diretamente. Na avaliação da atividade antioxidante in vitro do fitol, foram empregadas três metodologias, sendo uma para avaliar o efeito do fitol sobre a peroxidação lipídica, no teste de TBARS, e as outras duas para investigar se agia como substância seqüestradora de radicais livres, para o radical hidroxila (OH) e o óxido nítrico (NO). Em todos os testes, o fitol demonstrou forte atividade antioxidante, a qual pode ser atribuída a sua característica estrutural, uma vez que o fitol é um álcool insaturado de cadeia ramificada, e a propriedade antioxidante pode estar relacionada com o grupo hidroxila (OH) presente na sua molécula. Provavelmente, o fitol, ao reagir com um radical livre, doa átomos de hidrogênio com um elétron desemparelhado (H.),convertendo os radicais livres em espécies menos reativas. Como várias evidências mostram a participação destas espécies reativas no mecanismo da dor, a atividade antioxidante do fitol pode estar contribuindo com o seu efeito antinociceptivo.

Nosso grupo tem caracterizando diversos produtos da via de isoprenóides em Plasmodium falciparum que é promissora para o desenvolvimento de novas drogas. Dentre os compostos isoprenicos identificados estão as vitaminas K1 e E que apresentam uma cadeia isoprenica fitil. Em plantas o fitol é originado da degradação da clorofila. mas, como ainda não existem evidências da presença de clorofila no parasito, sugerimos que este possa ser formado pela degradação dessas vitaminas. Parte do fitol seria ligado a ácidos graxos utilizado como constituinte de membrana, e parte sofreria fosforilações, formando as vitaminas E e K1 como uma segunda via de biossíntese desse composto, assim como ocorre em Arabidopsis thaliana. Analises por HPLC e TLC utilizando o marcador [3H]GGPP, confirmaram a presença de fitol nos estágios intraeritrocíticos. Além disso, mostramos que o [3H]-fitol pode ser convertido em fitil-P e fitil-PP por uma atividade fitol quinase utilizando nucleotídeos como doador fosfato. / Our group has characterized differents products from isoprenoid pathway in Plasmodium falciparum which is promising for the development of new drugs. Among the isoprenics compounds identified there are the vitamins K1 and E which have a isoprenic phytyl side chain . In plants the phytol is obtained from the chlorophyll degradation. Until now theres no evidence that support its presence in the paraste. Our hypothesis that is phytol can be produced by the degradation of these vitamins E and K1. In the parasite phytol would fulfill to functions: used as a constituent of membrane, bounded to fatty acids and; be converted through phosphorylations vitamins E and K1 as a second biosynthetic pathway as well as in Arabidopsis thaliana. Analisys through and HPLC and TLC with radioactive [3H]GGPP confirmed the presence of phytol in intraerythrocytic stages. In addition, we showed that [3H]-phytol is converted into phytyl-P and phytyl-PP through a phytol kinasey using nucleotides as Phosphate donor.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-04-27T15:03:55Z No. of bitstreams: 1 josianemellodasilva.pdf: 3247422 bytes, checksum: 485b2562114e70ff7d059e0faa2e1b97 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-12T15:49:44Z (GMT) No. of bitstreams: 1 josianemellodasilva.pdf: 3247422 bytes, checksum: 485b2562114e70ff7d059e0faa2e1b97 (MD5) / Made available in DSpace on 2017-05-12T15:49:44Z (GMT). No. of bitstreams: 1 josianemellodasilva.pdf: 3247422 bytes, checksum: 485b2562114e70ff7d059e0faa2e1b97 (MD5) Previous issue date: 2015-03-13 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A espécie arbórea e nativa Lacistema pubescens Mart. (Lacistemataceae) apresenta-se distribuída em vários estados brasileiros e é conhecida popularmente, conforme a região em que se encontra, como espeto-vermelho, canela-vermelha, sabonete e cafezinho. Esse trabalho descreve o estudo fitoquímico e as atividades citotóxicas frente a linhagens tumorais e não tumorais, leishmanicida e anti-inflamatória tópica da partição hexânica das folhas de L. pubescens. A partição em hexano (PHEX) foi submetida a um fracionamento cromatográfico e originou 17 frações (FH1 a FH17). Na avaliação da atividade citotóxica, as frações FH7, FH9, FH13 e FH16 foram as mais ativas para células tumorais. As frações FH7 e FH9F3 (obtida após fracionamento de FH9) tiveram o valor de CI50 determinado, sendo FH9F3 a fração que apresentou a maior resposta frente à linhagem HL60 e Jurkat. O fracionamento de PHEX foi também biomonitorado para a atividade leishmanicida. As frações obtidas da PHEX mais seletivas para as formas promastigotas foram FH7, FH9 e FH13. A PHEX e FH7 tiveram sua atividade antiamastigota determinada sobre L. amazonensis. Com relação à atividade anti-inflamatória observou-se que a PHEX, administrada topicamente, apresentou resultados significativos no modelo do edema de orelha induzido pelo óleo de cróton, fenol, histamina, ácido araquidônico e etil fenil propionato (EPP), em camundongos. Estes resultados foram confirmados através de análises histopatológicas e ensaio enzimático. Uma formulação farmacêutica com PHEX (proPHEX) foi eficaz em processo inflamatório crônico. Além disso, PHEX apresentou segurança para administração por via tópica, uma vez que não apresentou toxidade no modelo de irritação/corrosão dérmica, em ratos. A PHEX também não apresentou os efeitos adversos, quando comparada à dexametasona, nos modelos de atrofia cutânea e cicatrização de feridas. A atividade anti-inflamatória pode ser atribuída, ao menos em parte, às frações FH7 e FH9F3, as quais foram testadas no modelo do edema de orelha induzido pelo óleo de cróton e fenol. A fração FH7 é rica em fitol e FH9F3 é rica em sitosterol, as quais foram identificadas por métodos cromatográficos e espectrométricos. / Lacistema pubescens Mart. (Lacistemataceae), an arborea native species, is distributed in several Brazilian states and is popularly known as “espeto-vermelho, canela – vermelha, sabonete e cafezinho”. This work describes the phytochemical and cytotoxic studies against tumor cell lines, and anti-leishmanial and anti-inflammatory activities of the leaves of L. pubescens. The hexane partition (PHEX) was subjected to a chromatographic fractionation and originated 17 fractions (FH1 to FH17). Regarding to the cytotoxic activity, FH7, FH9 and FH16 fractions were more selective for tumor cells. The IC50 value was determined for FH7 and FH9F3 fractions (obtained after FH9 fractionation), and FH9F3 presented the highest response against HL60 and Jurkat cell lines. PHEX fractionation was also biomonitored for anti-leishmanial activity. The fractions FH7 and FH9 were the most selective for promastigotes. The antiamastigota activity against L. amazonensis was determined for PHEX and FH7. Regarding to the anti-inflammatory activity, it was observed that topical application of PHEX showed significant results in ear edema model induced by croton oil, phenol, histamine, arachidonic acid and ethyl phenylpropionate (EPP) in mice. These results were confirmed by histopathological analysis and enzymatic assay. A pharmaceutical formulation contained PHEX (proPHEX) was effective for chronic inflammatory process. Moreover, PHEX presented safety for topical administration, since it showed no toxicity in the irritation/corrosion model in rats. PHEX did not show the adverse effects observed for dexamethasone in cutaneous atrophy and wound healing models. The anti-inflammatory activity may be attributed, at least in part, to FH7 and FH9F3 fractions, which were tested on the ear edema induced by croton oil and phenol models. Sitosterol-rich fraction (FH9F3) and Phytol-rich fraction (FH7) were obtained from PHEX fractionation and were identified by spectroscopic methods.

Abstract α-Methylacyl-CoA racemase (Amacr) is an auxiliary enzyme of β-oxidation and participates in the elimination of methyl-branched fatty acids in peroxisomes and in mitochondria and in the synthesis of bile acids in peroxisomes. Amacr catalyzes in reversible manner the isomerization of fatty acyl-CoA esters with a methyl group in the R-configuration to the corresponding S-configuration, which allows them to serve as substrates for the next reaction in their metabolism. The substrates of Amacr include the acyl-CoA esters of 2R-pristanic acid, a metabolite derived from phytol, and 25R-THCA and 25R-DHCA (tri- and dihydroxycholestanoic acid), the bile acid intermediates derived from cholesterol. AMACR-deficiency in humans results in the accumulation of R-isoforms of its substrates. Patients with adult onset AMACR-deficiency suffer from neurological disorders. The more severe infantile form of the deficiency is characterized by liver disease. Amacr-deficient mice show a bile acid pattern similar to that of human patients with accumulation of bile acid intermediates in their body. In contrast to humans, Amacr-deficient mice are clinically symptomless on a regular laboratory chow diet. Supplementation of phytol in their diet triggers the disease state with liver abnormalities. In this study it was shown that in spite of the disruption of a major metabolic pathway, Amacr-deficient mice are able to readjust their cholesterol and bile acid metabolism to a new balanced level allowing them to live a normal life span. A double knockout mouse model deficient in Amacr and MFE-1 (peroxisomal multifunctional enzyme type 1) was generated in this work. Characterization of this mouse line showed that MFE-1 can contribute to peroxisomal side-chain shortening of C27 bile acid intermediates in both Amacr-dependent and Amacr-independent pathways. In addition, this work confirmed that Amacr-deficient mice are unable to thrive when phytol is supplemented in their chow. The main cause of death was liver failure accompanied by kidney and brain abnormalities. The detoxification of phytol metabolites in liver is accompanied by activation of multiple pathways and Amacr-deficient mice are not able to respond adequately. The results of this study emphasize the indispensable role of Amacr in detoxification of α-methyl branched fatty acids. / Tiivistelmä α-Metyyliasyyli-koentsyymi-A-rasemaasi (Amacr) osallistuu metyyli-haarautuvien rasvahappojen eliminointiin peroksisomeissa ja mitokondrioissa ja sappihappojen synteesiin kolesterolista peroksisomeissa. Amacr katalysoi käänteisesti rasvahappojen asyyli-koentsyymi-A-estereiden isomerisaatio-reaktiota, jossa stereokemiallisesti R-asemassa oleva metyyliryhmä siirtyy S-asemaan. Tämä on edellytys eliminointiketjun seuraavan reaktion tapahtumiselle. Amacr-entsyymin substraatteja ovat fytolin aineenvaihdunnassa syntyvän 2R-pristaanihapon ja kolesterolista sappihapposynteesireitin välituotteina syntyvien 25R-trihydroksikolestaanihapon ja 25R-dihydroksikolestaanihapon (25R-THCA ja 25R-DHCA) asyyli-koentsyymi-A-esterit. Ihmisellä Amacr-entsyymin puutos johtaa R-muodossa olevien substraattien kertymiseen, joka aiheuttaa neurologisia oireita aikuisiässä alkavassa sairauden muodossa. Lapsuusiässä alkavassa tautimuodossa potilaille kehittyy vakava maksasairaus. Tutkimuksen tulokset osoittivat, että Amacr-poistogeenisten hiirten elinikä ei lyhene huolimatta yhden tärkeän aineenvaihduntareitin estymisestä. Tämä on hyvä esimerkki siitä, kuinka nisäkäs pystyy mukauttamaan kolesteroli- ja sappihappoaineenvaihduntaansa vastaamaan muuttunutta tilannetta aineenvaihdunnassa. Tässä työssä tuotettiin myös kaksoispoistogeeninen hiirimalli, jonka Amacr- ja peroksisomaalinen monitoiminnallinen entsyymi tyyppi 1- (MFE-1) entsyymit ovat toimimattomat. Tämä hiirimalli paljasti, että MFE-1 pystyy osallistumaan 27:ää hiiltä sisältävien sappihappovälituotteiden sivuketjun lyhentämiseen sekä Amacr entsyymin kanssa että ilman sitä. Työn tulokset myös osoittivat, että Amacr-poistogeeniset hiiret eivät ole elinkykyisiä, jos niiden ravinto sisältää fytolia. Maksan toiminnanvajaus oli näiden hiirten tärkein kuolinsyy, mutta hiirten munuaisten ja aivojen kudosrakenteissa oli myös muutoksia. Maksassa fytolin metaboliittien vaarattomaksi tekeminen aiheuttaa villityypin hiirillä useamman aineenvaihduntareitin aktivoitumisen, mutta Amacr-poistogeeniset hiiret eivät pysty reagoimaan tähän samalla tavalla. Tämä työ osoittaa, että Amacr-entsyymin elintärkeä tehtävä on osallistua ravinnon mukana elimistöön joutuvien α-metyylihaarautuvien rasvahappojen eliminaatioon.