RECHERCHE ET ETUDE DE MOLECULES À ACTIVITE ANTITYROSINASE ET LEUR UTILISATION COMME AGENTS DEPIGMENTANTS EN DERMOCOSMETIQUE Mots clé : Tyrosinase, mélanogenèse, hyperpigmentation. La pigmentation cutanée relève en grande partie d'un phénomène biochimique bien décrit, ayant lieu dans la couche basale de l'épiderme: la mélanogenèse. Elle consiste à produire des pigments, les mélanines qui constituent pour la peau une protection naturelle contre les effets néfastes des UV. Divers dysfonctionnements de la mélanogenèse dus à des agressions extérieures (UV), aux perturbations hormonales ou au vieillissement se traduisent par l'apparition de taches d'hyperpigmentation qui peuvent être particulièrement inesthétiques. Dans ces cas, l'utilisation d'actifs capables de réduire la production des mélanines pourrait permettre de remédier à ces troubles de la pigmentation. La tyrosinase glycoprotéine membranaire et enzyme clé impliquée dans les deux premières étapes de la mélanogenèse, constitue une cible de choix pour atteindre cet objectif. Depuis plusieurs années, la population concernée a eu recours à des inhibiteurs de la tyrosinase mais leur utilisation s'avère peu efficace ou délicate du fait de l'existence de certains effets secondaires. Nous avons pour notre part mis au point et synthétisé des polyphénols de type aurone, chromone ou dérivés des acides caféique et férulique en vue d'une utilisation en tant qu'inhibiteur de la tyrosinase. Leur évaluation in vitro sur trois modèles et plus particulièrement sur la tyrosinase de mélanocytes humains en culture, a permis d'identifier quelques aurones et dérivés des acides caféique et férulique ayant un fort potentiel en tant qu'inhibiteur de la tyrosinase humaine. Après une étude de relation structure-activité, il s'avère que l'activité de ces molécules est globalement liée à la présence de groupements phénol, qui leur permettent vraisemblablement de mimer la structure des substrats naturels de l'enzyme (L-tyrosine ou L-DOPA) ou de chélater le cuivre indispensable à son activité. De plus, les molécules sélectionnées sont pour la plupart naturelles et ont montré une faible toxicité in vitro aux doses efficaces. Ceci laisse donc présager un possible développement pour une utilisation en dermocosmétique.

In this study, the aim was to find an economical plant source for polyphenol oxidase (PPO) production as an alternative to mushroom and possible application areas by characterization and immobilization of the PPOs. For this purpose, tissues of various plants of no commercial value were screened for their PPO activities. Mulberry leaf tissues showed the highest PPO activity against 4-methyl catechol which was comparable to that of mushroom. Average Km and Vmax values of free mulberry leaf PPOs were found as 7 mM and 218 U/ml, respectively. Mulberry leaf PPOs were immobilized in a polypyrole matrix and the Km and Vmax values of immobilized PPOs were calculated as 35 mM and 3 U/ml, respectively. Mulberry leaf PPO was the most active at 45&deg / C and pH 7. By using electrophoretic analysis, laccase and catechol oxidase type activities of PPOs and in addition, peroxidase activity were detected. Molecular weights of laccase, peroxidase and catechol oxidase were found to be about 62, 64 and 62-64 kDa, with pI values of 8.0-8.5, 4.5 and 10, sequentially.

Immobilization of tyrosinase in conducting copolymer matrices of pyrrole functionalized polydimethylsiloxane/polypyrrole (PDMS/PPy) were achieved by electrochemical polymerization. The polysiloxane/polypyrrole/tyrosinase electrode was constructed by the entrapment of enzyme in conducting matrices during electrochemical copolymerization. Maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were investigated for immobilized enzyme. Enzyme electrodes were prepared in two different electrolyte/solvent systems. The effect of supporting electrolytes, p-toluene sulfonic acid and sodium dodecyl sulfate on the enzyme activity and film morphology were determined. Temperature and pH optimization, operational stability and shelf-life of enzyme electrodes were also examined. Phenolic contents of green and black tea were determined by using enzyme electrodes.

ACTIVITY ANALYSIS OF IMMOBILIZED TYROSINASE ENZYME IN THE PRESENCE OF DIFFERENT INHIBITORS Narli, ISil M.Sc., Department of Chemistry Supervisor: Prof. Dr. Levent Toppare May 2006, 97 pages Immobilization of tyrosinase enzyme was performed in the matrices obtained via copolymerization of terephthalic acid bis-(2-thiophen-3-yl ethyl) ester (TATE) with pyrrole. During electrochemical polymerization of pyrrole, enzyme molecules were entrapped in the copolymer matrice. Activity measurements were performed by using Besthorn&amp / #8217 / s Hydrazone method which includes spectrophotometric analysis of quinones produced by the enzyme. Enzyme electrodes were characterized in terms of maximum reaction rate (Vmax) and Michaelis-Menten constant (Km). In addition to kinetic parameters, stability of enzyme electrodes towards environmental conditions such as pH and temperature was investigated. Usage stability and shelf-life analysis were also examined. Wines, especially red wines, contain numerous biologically active compounds, the most important of which are polyphenols, whose nutritional importance is attributed to their antioxidant power. The amounts of phenolic compounds in different red wines were analyzed by using obtained enzyme electrodes. The phenolic compound determination using free enzyme cannot reflect the actual values since there are also naturally found inhibitors in red wines. Benzoic acid, cinnamic acid and sorbic acid were utilized to understand the behavior of immobilized tyrosinase in the conducting polymer matrices toward inhibition.

Neste trabalho relata-se pela primeira vez a determinação de adenosina por um biossensor baseado em tirosinase. O biossensor foi desenvolvido mediante a modificação de um eletrodo de carbono impresso (SPE) com nanopartículas de ouro (AuNPs), tirosinase (Tyr) e Nafion, denominado biossensor Nafion/Tyr/AuNPs/SPE. As AuNPs sintetizadas possuem diâmetro médio de 15,0 ± 1,1 nm e sua função é melhorar a via de condução de elétrons entre a enzima e o eletrodo. Utilizou-se o aprisionamento com filme Nafion® para evitar a lixiviação enzimática da superfície do eletrodo. A tirosinase imobilizada apresentou boa atividade frente ao substrato catecol. Verificou-se que a adenosina atua como um inibidor do tipo não-competitivo. O biossensor é estável durante pelo menos 45 dias. Além disso, foi realizada a eletro-oxidação da adenosina para sua determinação. O biossensor apresenta sensibilidade superior em comparação com SPE, AuNPs/SPE e Nafion/AuNPs/SPE. As curvas de calibração revelaram duas faixas lineares para as concentrações de adenosina, de 1,0 × 10-5 mol L-1 até 5,0 × 10-5 mol L-1 e entre 6,0 × 10-5 mol L-1 e 1,2 × 10-4 mol L -1. O limite de detecção (3 × (desvio padrão + média dos brancos)/coeficiente angular da curva) foi de 7,0 × 10-7 mol L-1. / In this work we report for the first time the determination of adenosine by a biosensor based on tyrosinase. The biosensor was developed by modifying a screen-printed carbon electrode (SPE) with gold nanoparticles (AuNPs), tyrosinase (Tyr) and Nafion, denoted as Nafion/Tyr/AuNPs/SPE biosensor. The synthesized AuNPs have a mean diameter of 15.0 ± 1.1 nm and their function is to improve the electron conduction pathway between the enzyme and the electrode. The entrapment with Nafion® film was selected to prevent the enzyme lixiviation from the electrode surface. Immobilized tyrosinase showed good activity with the catechol substrate. It was found that adenosine acts as a non-competitive type inhibitor. The biosensor is stable for at least 45 days. In addition, the electro-oxidation of adenosine was performed for its determination. The biosensor has superior sensitivity compared to SPE, AuNPs/SPE and Nafion/AuNPs/SPE. Calibration curves revealed two linear ranges for adenosine concentrations of 1,010-5 mol L-1 up to 5,010-5 mol L-1 and from 6,010-5 mol L-1 to 1,210-4 mol L-1. The detection limit (3 × (standard deviation + mean of blanks)/slope of the curve) was 7,010-7 mol L-1.

Orientador: Marcos Antonio Corrêa / Resumo: O aumento da preocupação com a estética e saúde corporal vem elevando a demanda por produtos cosméticos contendo ativos que previnam o envelhecimento e outras alterações cutâneas. O ácido ursólico (AU), ativo natural extraído de plantas como o alecrim e o manjericão, tem se demonstrado interessante para incorporação em cosméticos por possuir potencial antioxidante, despigmentante, propriedade antimicrobiana, entre outras. Este trabalho teve como objetivo avaliar a possibilidade da utilização do AU como um ativo cosmético multifuncional através da avaliação da eficácia e citotoxicidade in vitro do AU, bem como o preparo e avaliação de uma emulsão contendo o ativo. O potencial antioxidante do AU foi avaliado por meio de métodos de inibição de radicais como o 1,1-difenil-2-picrilhidrazila (DPPH•) e o 2,2 -azino bis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS•+), tendo demonstrado atividade antioxidante, com IC50 de 235,61 µg/mL para técnica com DPPH• e 107,17 µg/mL para técnica com ABTS•+. A atividade despigmentante foi verificada através da inibição da enzima tirosinase, utilizando 3,4-dihydroxy-L-phenylalanine (L-DOPA) como substrato, e apresentando como resultado um IC50 de 338,00 µg/mL. Para a avaliação da atividade antimicrobiana diversas cepas foram testadas a partir da determinação da concentração inibitória mínima (CIM) pela técnica de diluição em microplacas, e da concentração bactericida mínima (CBM) pela replicação em placa de Petri, seguindo suplemento M100-S16 do... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Magister Scientiae - MSc / Various dermatological disorders, such as formation of black pigmented patches on the surface of the skin arise from the over-activity of tyrosinase enzyme's degenerative action. This enzyme is further implicated in the involvement of melanin in malignant melanoma, the most lifethreatening skin tumors. Although, synthetic products were found effective to combat this menace, nevertheless, overtime detrimental effect on human skin is a challenge. Investigation of natural tyrosinase inhibitors from methanol extracts of medicinal plants of Lamiaceae family using L-tyrosine as substrate on three different complementary assays (TLC bio-autography, spectrophotometry and cyclic voltammetry) was carried out accordingly. The result indicated Salvia chamelaeagnea, Salvia dolomitica, Plectranthus ecklonii, Plectranthus namaensis, and Plectranthus zuluensis, with significant zone of inhibition against tyrosinase on TLC bio-autography, spectrophotometry result showed that extracts of Plectranthus ecklonii (IC50 = 21.58 μg/mL), Plectranthus zuluensis (IC50 = 23.99 μg/mL), Plectranthus madagascariensis (IC50 = 23.99 μg/mL) and Salvia lanceolata (IC50 = 28.83) demonstrated good anti-tyrosinase activity when compared with kojic acid (IC50 = 3.607 μg/mL). On the other hand, cyclic voltammetry are in consonant with above results thereby supported the nomination of some of the extracts as strong anti-tyrosinase agents. Salvia chamelaeagnea showed strong activity in cyclic voltammetry and clear zone of inhibition on TLC bioautography, these reasons gave us justification for further chemical study to isolate the bioactive constituents. Phytochemical investigation of the bioactive extract of Salvia chamelaeagnea using different chromatographic methods including column chromatographic and semi preparative HPLC afforded six (6) known compounds viz carsonol (C1), carnosic acid (C2), 7- ethoxylrosmanol (C3), ursolic acid (C4), rosmanol (C5) and ladanein (C6). Their chemical structures were elucidated by analyses of spectroscopic (1H and 13C NMR) data as well as correlations with existing literature. The methanolic extract of S. chamelaeagnea (SC) showed moderate antityrosinase (IC50 = 267.4 μg/mL) activity, total antioxidant capacities measured as: Oxygen radicals absorbance capacity (ORAC; 14970 ± 5.16 μM TE/g), ferric-ion reducing antioxidant power (FRAP; 9869.43 ± 7.87 μM AAE/g) and trolox equivalent absorbance capacity (TEAC; 13706.5 ± 0.95 μM TE/g). Excellent total antioxidant capacities were demonstrated by C1 and C5 respectively as FRAP (9338.92 ± 1.72; 8622.73 ± 1.92) μM AAE/g; TEAC (16505 ± 0.86; 10641.5 ± 0.52) μM TE/g; ORAC (14550.5 ± 3.65; 14633.90 ± 3.84) μM TE/g and including the inhibition of Fe2+ -induced lipid peroxidation (IC50 = 32.5; 30.25) μg/mL. All the compounds except C4 are electro-active with well-defined oxidation-reduction peaks while C1 demonstrated the highest tyrosinase inhibitory activity by strongly decreased the inhibition current with time using cyclic voltammetry method. The isolated compounds especially C1, C2 and C5 are well known to combat with ageing problems and documented for their powerful activity against oxidative stress and alzheimer's diseases, which are ageing related symptoms. The isolation of such bioactive compounds indicated the synergetic effect of the results of the three methods used in this thesis. This is the first report on the evaluation of both anti-tyrosinase and total antioxidant capacities of the isolated compounds from S. chamelaeagnea. The findings therefore can be used as background information for exploitation of skin depigmentation and antioxidant agents from natural source.

Neste trabalho relata-se pela primeira vez a determinação de adenosina por um biossensor baseado em tirosinase. O biossensor foi desenvolvido mediante a modificação de um eletrodo de carbono impresso (SPE) com nanopartículas de ouro (AuNPs), tirosinase (Tyr) e Nafion, denominado biossensor Nafion/Tyr/AuNPs/SPE. As AuNPs sintetizadas possuem diâmetro médio de 15,0 ± 1,1 nm e sua função é melhorar a via de condução de elétrons entre a enzima e o eletrodo. Utilizou-se o aprisionamento com filme Nafion® para evitar a lixiviação enzimática da superfície do eletrodo. A tirosinase imobilizada apresentou boa atividade frente ao substrato catecol. Verificou-se que a adenosina atua como um inibidor do tipo não-competitivo. O biossensor é estável durante pelo menos 45 dias. Além disso, foi realizada a eletro-oxidação da adenosina para sua determinação. O biossensor apresenta sensibilidade superior em comparação com SPE, AuNPs/SPE e Nafion/AuNPs/SPE. As curvas de calibração revelaram duas faixas lineares para as concentrações de adenosina, de 1,0 × 10-5 mol L-1 até 5,0 × 10-5 mol L-1 e entre 6,0 × 10-5 mol L-1 e 1,2 × 10-4 mol L -1. O limite de detecção (3 × (desvio padrão + média dos brancos)/coeficiente angular da curva) foi de 7,0 × 10-7 mol L-1. / In this work we report for the first time the determination of adenosine by a biosensor based on tyrosinase. The biosensor was developed by modifying a screen-printed carbon electrode (SPE) with gold nanoparticles (AuNPs), tyrosinase (Tyr) and Nafion, denoted as Nafion/Tyr/AuNPs/SPE biosensor. The synthesized AuNPs have a mean diameter of 15.0 ± 1.1 nm and their function is to improve the electron conduction pathway between the enzyme and the electrode. The entrapment with Nafion® film was selected to prevent the enzyme lixiviation from the electrode surface. Immobilized tyrosinase showed good activity with the catechol substrate. It was found that adenosine acts as a non-competitive type inhibitor. The biosensor is stable for at least 45 days. In addition, the electro-oxidation of adenosine was performed for its determination. The biosensor has superior sensitivity compared to SPE, AuNPs/SPE and Nafion/AuNPs/SPE. Calibration curves revealed two linear ranges for adenosine concentrations of 1,010-5 mol L-1 up to 5,010-5 mol L-1 and from 6,010-5 mol L-1 to 1,210-4 mol L-1. The detection limit (3 × (standard deviation + mean of blanks)/slope of the curve) was 7,010-7 mol L-1.

Uma série de três complexos de cobre(II) dinucleares, contendo ligantes nitrogenados e grupos aromáticos (compostos 2, 4 e 6), foi sintetizada e caracterizada por diversas técnicas espectroscópicas (UV/Vis, IV e EPR). Esses compostos tiveram sua atividade tirosinase avaliada à temperatura ambiente, através da oxidação de L-di-hidroxifenilalanina (L-dopa), e sua citotoxicidade investigada frente a células melanomas, comparada às de complexos análogos de cobre(II) mononucleares (compostos 1, 3 e 5). A influência da luz UVB, que estimula a melanogênese, também foi verificada. A exposição das células à radiação de intensidade (13 ± 2) mJ/cm2 aumentou os danos causados, principalmente em presença das espécies dinucleares. A citotoxicidade dos diferentes complexos foi determinada frente a duas linhagens de melanomas humanos (SKMEL-05 e SKMEL-147), após 24 e 48h de incubação. Células com maior teor de melanina foram mais sensíveis aos efeitos dos complexos. Verificou-se um aumento na porcentagem de células na fase sub-G1 do ciclo celular após tratamento por 24 ou 48h com estes complexos, ao contrário do verificado frente a queratinócitos não tumorais. Testes clonogênicos também indicaram maior atividade do composto (2) contendo dois centros de cobre em sua estrutura, com diminuição significativa no número de células sobreviventes após tratamento. Ensaios complementares mostraram a redução dos íons de cobre(II) nos compostos (1) e (2) em presença de melanina e a formação de espécies reativas de oxigênio (radicais hidroxil e ânions superóxido), através de espectroscopia EPR ou do uso de sondas fluorescentes. Adicionalmente, foi constatado um aumento no nível de vacúolos citoplasmáticos após tratamento com o complexo (2), indicando indução à autofagia, corroborada pelo monitoramento das proteínas LC3 e tubulina, implicadas neste processo de morte celular. Os resultados apontam para a ocorrência de pelo menos dois mecanismos de ação dos complexos frente aos melanomas, por processo apoptótico e autofágico. Indicam ainda que esta reatividade frente a melanomas é fortemente dependente da estrutura dos complexos de cobre, sendo mais significativa para os dinucleares / A series of dinuclear copper(II) complexes containing nitrogen ligands and aromatic groups (compounds 2, 4 and 6), were synthesized and characterized by various spectroscopic methods (UV/Vis, IR and EPR). These complexes had their tyrosinase activity evaluated at room temperature, through the oxidation of L-di-hidroxyphenylalanine (L-dopa), and its cytotoxicity toward melanoma cells investigated, in comparison with the toxicity of the corresponding mononuclear complexes (compounds 1, 3 and 5). The influence of UVB light, that stimulates melanogenesis, was also verified. The exposition of the cells to radiation of intensity (13 ± 2) mJ/cm2, increased the caused damage, especially in the presence of dinuclear species. The cytotoxicity of the different complexes was determined toward two cell lines of human melanomas (SKMEL-05 e SKMEL-147), after 24 and 48h incubation. Cells containing higher leveis of melanin were more sensitive to the effects of the complexes. An increasing in the percentage of cells in the sub-G1 phase of cellular cycle was verified after treatment for 24 or 48h with these complexes. On the contrary, this effect was not observed with non-tumor keratinocytes. Clonogenic tests also indicated higher activity of compound 2 containing two copper centers in its structure, with a significant decrease in the number of survival cells after the treatment. Complementary assays show the reduction of copper(II) ions in complexes (1) and (2), in the presence of melanin, as well as the formation of reactive oxygen species (hydroxyl radicais and superoxide anions) via EPR spectroscopy or the use of fluorescent labels. Further, an increase in the levei of cytoplasmatic vacuoles was verified after treatment with complex (2), indicating induction to autophagy, which was corroborated by monitoring the proteins LC3 and tubulin, implicated in this process of cell death. The results pointed to the occurrence of at least two mechanisms of action of these complexes toward melanomas, apoptotic and autophagic processes. Also, they indicated that the reactivity of the studied compounds is strongly dependent on its structural features, being more remarkable to the dinuclear ones.

Nesta dissertação, foram estudados os métodos de síntese de filmes poliméricos como matrizes hospedeiras da enzima tirosinase vislumbrando o desenvolvimento de biossensores mais seletivos e duradouros na detecção de compostos fenólicos. Os polímeros utilizados e preparados por cronoamperometria em acetonitrila foram: polipirrol (PPI), polimetiltiofeno (PMET) e copolímero PPI-PMET. Os filmes PPI-PMET apresentaram características intermediárias às dos filmes PPI e PMET, conforme se verificaram em espectros na região do infravermelho (FTIR), voltametria cíclica e microscopia de força atômica (AFM), evidenciando, assim, a formação de uma nova matriz polimérica. O processo de superoxidação do filme PPI-PMET foi estudado visando a uma imobilização mais eficiente da enzima, quando se obteve uma condição na qual a superoxidação ocorre somente nas cadeias de PPI. Biossensores amperométricos foram preparados a partir do uso da tirosinase extraída do abacate como fonte enzimática por imobilização física e realizada de maneiras diferentes em filmes não superoxidados e superoxidados. Foi feita a otimização das respostas dos filmes variando-se o pH, potencial de trabalho, concentração da enzima e tempo de imobilização da enzima para a detecção de catecol. Os resultados indicaram que a superoxidação do PPI nos filmes PPI-PMET favoreceu a imobilização da enzima, embora não fosse tão eficiente quando se comparou aos filmes com o PPI não superoxidado. Contudo, os biossensores, em geral, apresentaram grande sensibilidade ao catecol, com um limite de detecção baixo, da ordem de 0,12 µmol/L. / The synthesis methods of polymer films as host matrices of the enzyme tyrosinase were studied in this dissertation. It was expected the development of selective and everlasting biosensors for the detection of phenolic compounds. The used polymers were prepared by chronoamperometry in acetonitrile as followed: polypyrrole (PPI), polymethylthiophene (PMET) and copolymer PPI-PMET. The PPI-PMET films showed intermediate characteristics as the PPI and PMET films, as observed in the infrared spectra (FTIR), cyclic voltammetry and atomic force microscopy (AFM), thus indicating the formation of a new polymeric matrix . The process of overoxidized of PPI-PMET film was studied in order to achieve a more efficient immobilization of the enzyme, whilst it was achieved a condition in which overoxidized occurs only in the PPI chain. Amperometric biosensors were prepared from the use of tyrosinase extracted from avocado as an enzyme source by physical immobilization and performed in different ways in both overoxidized and non-overoxidized films. It was performed the optimization of the responses of the films by varying pH, operacuonial potential, enzyme concentration and time in the enzyme immobilization for the detection of catechol. The results showed that the overoxidized PPI in the films PPI-PMET favored the immobilization of the enzyme, although not as efficient when compared to the films with nom overoxidized PPI. Neverthless, the catechol biosensors showed a high sensitivity with a low detection limit in the order of 0.12 µmol/L.