Sibutramine hydrochloride monohydrate is a weight loss agent banned from most global markets due to reports of serious adverse events including death. It is classified as a serotonin-norepinephrine reuptake inhibitor and acts within the central nervous system by inhibiting the reuptake of these neurotransmitters essentially influencing satiety and also energy expenditure. Despite its ban, sibutramine is still available for use in some countries and is often an unlisted ingredient in herbal or natural weight loss products. This leads to the oblivious and unintentional consumption of this compound which is of great concern especially if used by pregnant women. The aim of this study was to investigate the possible toxic and teratogenic effects of sibutramine by using a Spraque Dawley rat and an in ovo chick embryo model. The Sprague Dawley rat was used as in vivo model and animals were divided into three experimental groups, each consisting of 12 animals (6 males, 6 females). Rats were administered sibutramine over a 28 day period according to their assigned experimental groups namely control (C; sterile H2O), Low dose (LD; 1.32 mg/kg) and High Dose (HD; 13.2 mg/kg). On the day of termination blood was collected for various analyses which included biochemical tests for liver and kidney function, hormonal changes as well as the investigation of coagulation profiles. Brain, heart, lung, kidney and liver tissue of each animal was harvested for investigation of tissue and cellular structure. Rats were weighed daily and this data suggested that sibutramine was well tolerated by all animals, with only the female rats in the HD group showing a significant 8.2% decrease in their rate of weight gain. Biochemical data of liver and kidney function in all groups were normal. Thyroid hormone levels were comparable to control values though cortisol levels were lowered in the HD female group, a finding which correlates with the observed weight loss. Investigations of the ultrastructural morphology of the platelets and fibrin networks revealed differences between the experimental groups that are consistent with changes associated with a procoagulable state. Brain, kidney and liver tissue morphology appeared normal upon investigation, the latter confirming the biochemical findings. Examination of cardiac tissue revealed a slight increase in collagen deposition between the muscle fibers and ultrastructural analysis of lung tissue showed thickening of the alveolar walls, decreased intra-alveolar space and drastic increases in collagen deposition in the sibutramine-exposed groups. These findings were dosage dependant. The in ovo model was implemented with ephedrine, a known teratogen, as positive control. Three control groups were included to ensure the efficacy of the method. Eight experimental groups, each containing sixteen eggs, were exposed to four different concentrations of each drug (i.e. 0.5, 2.5, 5.0, 10.0 μmol). The brain, heart, liver and kidneys were harvested on embryonic day thirteen for morphological analysis. Macroscopic evaluation revealed that both drugs caused congenital abnormalities which included ventral wall and limb defects as well as growth retardation and increased mortality. Sibutramine was found to have a greater teratogenic potential than ephedrine. Histological investigation of kidney and brain samples of embryos revealed no morphological differences between the various experimental groups. Livers and hearts of embryos exposed were severely affected by both compounds in a dose dependant manner. Replacement of myocardium with adipose and connective tissue was observed in cardiac tissue, which is characteristic of muscular dystrophy. Severe liver steatosis was also evident. In conclusion, results from this animal-based study show that, at concentrations which are not toxic to the liver or kidneys, sibutramine administration led to increased coagulation, moderate cardiac and excessive lung fibrosis within the Sprague Dawley rat model. This indicates significant toxicity with the cardiac and respiratory system being more susceptible targets. In addition sibutramine was shown to possess greater teratogenic effects than ephedrine. Both drugs caused cardiac dystrophy and liver steatosis resulting in extensive liver and heart damage. Understanding the underlying mechanisms involved in the pathogenesis of these findings strongly emphasize an important area for future research. / Dissertation (MSc)--University of Pretoria, 2014. / tm2015 / Anatomy / MSc / Unrestricted

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第23092号 / 医博第4719号 / 新制||医||1050(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 黒田 知宏, 教授 上杉 志成, 教授 藤渕 航 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

O gênero Drimys é o de maior área de distribuição geográfica da família (Winteraceae), que compreende sete gêneros e cerca de 120 espécies. No Brasil, encontra-se desde a Bahia até o Rio Grande do Sul e ocorre em duas espécies, Drimys angustifolia Miers. e Drimys brasiliensis Miers, conhecidas popularmente como “casca-de-anta”. As folhas e cascas são usadas na medicina popular como antiescorbútico, estimulante, antiespasmódica, antidiarreica, antifebril, contra hemorragia uterina, antibacteriana, no tratamento de asma, bronquite, certas afecções do trato digestivo e, algumas vezes no tratamento do câncer e inseticidas. Ambas são caracterizadas pela presença de flavonóides e sesquiterpenoides. O objetivo deste trabalho foi o de avaliar a composição química, o potencial antioxidante, antiviral, inseticida, antitumoral, antifúngico, avaliação tóxica aguda e, também, formular e caracterizar duas nanoemulsões dos óleos voláteis das duas espécies do estudo. Por meio da hidrodestilação, foi possível realizar a extração dos óleos essenciais e determinar seus rendimentos que variaram entre 0,3 e 0,5% de folhas. A identificação dos componentes foi realizada utilizando a Cromatrografia Gasosa acoplada ao espectrofotômetro de massas (CG-EM), as quais apresentaram como componentes majoritários em todas as análises, o biciclogermacreno para a espécie Drimys angustifolia e a ciclocolorenona para a espécie Drimys brasiliensis. A formulação e caracterização da nanoemulsão foi realizada dentro dos parâmetros estabelecidos (tamanho de partícula, índice de polidispersão, pH) e através da microscopia eletrônica de transmissão foi confirmado o tamanho na escala nano. No que tange a atividade inseticida, ambas as espécies mostraram-se promissoras repelindo o cupim da espécie Cryptotermes brevis com índices de repelência negativos. Para avaliação inicial do potencial larvicida de Aedes aegypti, os óleos das duas espécies não foram eficientes. No entanto, a nanoemulsão de Drimys brasiliensis levou a 69% e 89% de mortalidade, correspondendo às concentrações de 0,5 μg/mL e 0,83 μg/mL, respectivamente. Com relação a atividade antioxidante, os óleos das duas espécies não foram capazes de reduzir o radical (2,2-difenil-1-picril-hidrazil) DPPH . Já na atividade antiviral frente ao VHS-1, as duas espécies mostraram-se promissoras inibindo a replicação viral. Na atividade antitumoral nas linhagens de glioma (U-138 MG) e de bexiga (T24), somente o óleo de Drimys brasiliensis reduziu a viabilidade celular das duas linhagens pelo ensaio do MTT, com resultados similares na contagem celular. Na análise da citometria pela incorporação da Anexina-V/Iodeto de propídio caracterizou-se indução de apoptose tardia. Na atividade antifúngica, as duas espécies foram eficazes contra o fungo oportunista Acremonium, presente em diversas infecções em transplantados e imunocomprometidos. Quanto à avaliação da toxicidade aguda, as duas espécies mostraram importantes sinais de toxicidade em ratos Wistar como ptose, tremor, redução da atividade motora, exolftamia, aumento da freqüência respiratória. Os animais tratados com espécie D. brasiliensis ainda apresentaram aumento na micção e diarréia. Desta maneira, é importante ressaltar que todos os ensaios biológicos, o estudo toxicológico, e a elaboração e caracterização das nanoemulsões dos óleos essenciais foram realizados pela primeira vez para as duas espécies vegetais, destacando a importância e ineditismo do presente trabalho. / The genus Drimys presents the largest geographical distribution of the family (Winteraceae), which comprises seven genera and about 120 species. In Brazil, is found from Bahia to Rio Grande do Sul and occurs in two species, Drimys angustifolia L. and Drimys brasiliensis Miers, popularly known as "Casca-de-anta". The leaves and barks are used in folk medicine as antiscorbutic, stimulant, antispasmodic, anti-diarrheal, antipyretic, against uterine bleeding, antibacterial, to treat asthma, bronchitis, certain disorders of the digestive tract and sometimes in the treatment of cancer and insecticides. Both are characterized by the presence of flavonoids and sesquiterpenoids. The objective of this study was to evaluate the chemical composition, the antioxidant, antiviral, insecticidal, antitumor, antifungal, acute toxicological evaluation and also to formulate and characterize two nanoemulsions of the volatile oils of two species of the study. By steam distillation was possible to perform the extraction of essential oils and to determine their yields ranging between 0.3 and 0.5% of leaves. The identification of components was performed using the Gas Cromatography coupled to mass spectrometer (GC-MS), which presented as major components in all analyzis bicyclogermacrene for the Drimys angustifolia species and ciclocolorenona for the Drimys brasiliensis. The formulation and characterization of nanoemulsion was performed within the established parameters (particle size, polydispersity, pH) and by transmission electron microscopy confirming the size at the nanoscale. Regarding the insecticidal activity, both species were promising repelling termites Cryptotermes brevis with negative index of repellency. To evaluate initial potential larvicidal Aedes aegypti, the oils of the two species were not efficient, however, of the Drimys brasiliensis nanoemulsion the larvae mortality of the 69% and 89%, corresponding to concentrations of 0.5 μg/mL and 0.83 μg/mL, respectively. With respect to antioxidant activity, the oils of the two species have not been able to reduce the (2,2-diphenyl-1-picrylhydrazyl) DPPH radical. In the antiviral activity against the HSV-1, the two species were promising inhibiting viral replication. In antitumor activity in glioma cell lines (U-138 MG) and bladder (T24), only the oil Drimys brasiliensis reduced cell viability of two strains by the MTT assay, with similar results in cell counts and flow cytometry analysis of the incorporation of Anexinn-V/Propidium Iodide characterized as late apoptosis. In the antifungal activity, the two species were effective against the opportunistic fungus Acremonium, present in various infections in transplant and immunocompromised. Regarding the evaluation of acute toxicity, the two species showed significant signs of toxicity in rats as ptosis, tremor, decreased motor activity, exolftamia, shortness of breath. Animals treated with species Drimys brasiliensis also showed an increase in urination and diarrhea. Thus, it is important that all biological assays, the toxicity study, and preparation and characterization of nanoemulsions of essential oils were first performed for the two species, highlighting the importance and originality of this work.

O gênero Drimys é o de maior área de distribuição geográfica da família (Winteraceae), que compreende sete gêneros e cerca de 120 espécies. No Brasil, encontra-se desde a Bahia até o Rio Grande do Sul e ocorre em duas espécies, Drimys angustifolia Miers. e Drimys brasiliensis Miers, conhecidas popularmente como “casca-de-anta”. As folhas e cascas são usadas na medicina popular como antiescorbútico, estimulante, antiespasmódica, antidiarreica, antifebril, contra hemorragia uterina, antibacteriana, no tratamento de asma, bronquite, certas afecções do trato digestivo e, algumas vezes no tratamento do câncer e inseticidas. Ambas são caracterizadas pela presença de flavonóides e sesquiterpenoides. O objetivo deste trabalho foi o de avaliar a composição química, o potencial antioxidante, antiviral, inseticida, antitumoral, antifúngico, avaliação tóxica aguda e, também, formular e caracterizar duas nanoemulsões dos óleos voláteis das duas espécies do estudo. Por meio da hidrodestilação, foi possível realizar a extração dos óleos essenciais e determinar seus rendimentos que variaram entre 0,3 e 0,5% de folhas. A identificação dos componentes foi realizada utilizando a Cromatrografia Gasosa acoplada ao espectrofotômetro de massas (CG-EM), as quais apresentaram como componentes majoritários em todas as análises, o biciclogermacreno para a espécie Drimys angustifolia e a ciclocolorenona para a espécie Drimys brasiliensis. A formulação e caracterização da nanoemulsão foi realizada dentro dos parâmetros estabelecidos (tamanho de partícula, índice de polidispersão, pH) e através da microscopia eletrônica de transmissão foi confirmado o tamanho na escala nano. No que tange a atividade inseticida, ambas as espécies mostraram-se promissoras repelindo o cupim da espécie Cryptotermes brevis com índices de repelência negativos. Para avaliação inicial do potencial larvicida de Aedes aegypti, os óleos das duas espécies não foram eficientes. No entanto, a nanoemulsão de Drimys brasiliensis levou a 69% e 89% de mortalidade, correspondendo às concentrações de 0,5 μg/mL e 0,83 μg/mL, respectivamente. Com relação a atividade antioxidante, os óleos das duas espécies não foram capazes de reduzir o radical (2,2-difenil-1-picril-hidrazil) DPPH . Já na atividade antiviral frente ao VHS-1, as duas espécies mostraram-se promissoras inibindo a replicação viral. Na atividade antitumoral nas linhagens de glioma (U-138 MG) e de bexiga (T24), somente o óleo de Drimys brasiliensis reduziu a viabilidade celular das duas linhagens pelo ensaio do MTT, com resultados similares na contagem celular. Na análise da citometria pela incorporação da Anexina-V/Iodeto de propídio caracterizou-se indução de apoptose tardia. Na atividade antifúngica, as duas espécies foram eficazes contra o fungo oportunista Acremonium, presente em diversas infecções em transplantados e imunocomprometidos. Quanto à avaliação da toxicidade aguda, as duas espécies mostraram importantes sinais de toxicidade em ratos Wistar como ptose, tremor, redução da atividade motora, exolftamia, aumento da freqüência respiratória. Os animais tratados com espécie D. brasiliensis ainda apresentaram aumento na micção e diarréia. Desta maneira, é importante ressaltar que todos os ensaios biológicos, o estudo toxicológico, e a elaboração e caracterização das nanoemulsões dos óleos essenciais foram realizados pela primeira vez para as duas espécies vegetais, destacando a importância e ineditismo do presente trabalho. / The genus Drimys presents the largest geographical distribution of the family (Winteraceae), which comprises seven genera and about 120 species. In Brazil, is found from Bahia to Rio Grande do Sul and occurs in two species, Drimys angustifolia L. and Drimys brasiliensis Miers, popularly known as "Casca-de-anta". The leaves and barks are used in folk medicine as antiscorbutic, stimulant, antispasmodic, anti-diarrheal, antipyretic, against uterine bleeding, antibacterial, to treat asthma, bronchitis, certain disorders of the digestive tract and sometimes in the treatment of cancer and insecticides. Both are characterized by the presence of flavonoids and sesquiterpenoids. The objective of this study was to evaluate the chemical composition, the antioxidant, antiviral, insecticidal, antitumor, antifungal, acute toxicological evaluation and also to formulate and characterize two nanoemulsions of the volatile oils of two species of the study. By steam distillation was possible to perform the extraction of essential oils and to determine their yields ranging between 0.3 and 0.5% of leaves. The identification of components was performed using the Gas Cromatography coupled to mass spectrometer (GC-MS), which presented as major components in all analyzis bicyclogermacrene for the Drimys angustifolia species and ciclocolorenona for the Drimys brasiliensis. The formulation and characterization of nanoemulsion was performed within the established parameters (particle size, polydispersity, pH) and by transmission electron microscopy confirming the size at the nanoscale. Regarding the insecticidal activity, both species were promising repelling termites Cryptotermes brevis with negative index of repellency. To evaluate initial potential larvicidal Aedes aegypti, the oils of the two species were not efficient, however, of the Drimys brasiliensis nanoemulsion the larvae mortality of the 69% and 89%, corresponding to concentrations of 0.5 μg/mL and 0.83 μg/mL, respectively. With respect to antioxidant activity, the oils of the two species have not been able to reduce the (2,2-diphenyl-1-picrylhydrazyl) DPPH radical. In the antiviral activity against the HSV-1, the two species were promising inhibiting viral replication. In antitumor activity in glioma cell lines (U-138 MG) and bladder (T24), only the oil Drimys brasiliensis reduced cell viability of two strains by the MTT assay, with similar results in cell counts and flow cytometry analysis of the incorporation of Anexinn-V/Propidium Iodide characterized as late apoptosis. In the antifungal activity, the two species were effective against the opportunistic fungus Acremonium, present in various infections in transplant and immunocompromised. Regarding the evaluation of acute toxicity, the two species showed significant signs of toxicity in rats as ptosis, tremor, decreased motor activity, exolftamia, shortness of breath. Animals treated with species Drimys brasiliensis also showed an increase in urination and diarrhea. Thus, it is important that all biological assays, the toxicity study, and preparation and characterization of nanoemulsions of essential oils were first performed for the two species, highlighting the importance and originality of this work.

O gênero Drimys é o de maior área de distribuição geográfica da família (Winteraceae), que compreende sete gêneros e cerca de 120 espécies. No Brasil, encontra-se desde a Bahia até o Rio Grande do Sul e ocorre em duas espécies, Drimys angustifolia Miers. e Drimys brasiliensis Miers, conhecidas popularmente como “casca-de-anta”. As folhas e cascas são usadas na medicina popular como antiescorbútico, estimulante, antiespasmódica, antidiarreica, antifebril, contra hemorragia uterina, antibacteriana, no tratamento de asma, bronquite, certas afecções do trato digestivo e, algumas vezes no tratamento do câncer e inseticidas. Ambas são caracterizadas pela presença de flavonóides e sesquiterpenoides. O objetivo deste trabalho foi o de avaliar a composição química, o potencial antioxidante, antiviral, inseticida, antitumoral, antifúngico, avaliação tóxica aguda e, também, formular e caracterizar duas nanoemulsões dos óleos voláteis das duas espécies do estudo. Por meio da hidrodestilação, foi possível realizar a extração dos óleos essenciais e determinar seus rendimentos que variaram entre 0,3 e 0,5% de folhas. A identificação dos componentes foi realizada utilizando a Cromatrografia Gasosa acoplada ao espectrofotômetro de massas (CG-EM), as quais apresentaram como componentes majoritários em todas as análises, o biciclogermacreno para a espécie Drimys angustifolia e a ciclocolorenona para a espécie Drimys brasiliensis. A formulação e caracterização da nanoemulsão foi realizada dentro dos parâmetros estabelecidos (tamanho de partícula, índice de polidispersão, pH) e através da microscopia eletrônica de transmissão foi confirmado o tamanho na escala nano. No que tange a atividade inseticida, ambas as espécies mostraram-se promissoras repelindo o cupim da espécie Cryptotermes brevis com índices de repelência negativos. Para avaliação inicial do potencial larvicida de Aedes aegypti, os óleos das duas espécies não foram eficientes. No entanto, a nanoemulsão de Drimys brasiliensis levou a 69% e 89% de mortalidade, correspondendo às concentrações de 0,5 μg/mL e 0,83 μg/mL, respectivamente. Com relação a atividade antioxidante, os óleos das duas espécies não foram capazes de reduzir o radical (2,2-difenil-1-picril-hidrazil) DPPH . Já na atividade antiviral frente ao VHS-1, as duas espécies mostraram-se promissoras inibindo a replicação viral. Na atividade antitumoral nas linhagens de glioma (U-138 MG) e de bexiga (T24), somente o óleo de Drimys brasiliensis reduziu a viabilidade celular das duas linhagens pelo ensaio do MTT, com resultados similares na contagem celular. Na análise da citometria pela incorporação da Anexina-V/Iodeto de propídio caracterizou-se indução de apoptose tardia. Na atividade antifúngica, as duas espécies foram eficazes contra o fungo oportunista Acremonium, presente em diversas infecções em transplantados e imunocomprometidos. Quanto à avaliação da toxicidade aguda, as duas espécies mostraram importantes sinais de toxicidade em ratos Wistar como ptose, tremor, redução da atividade motora, exolftamia, aumento da freqüência respiratória. Os animais tratados com espécie D. brasiliensis ainda apresentaram aumento na micção e diarréia. Desta maneira, é importante ressaltar que todos os ensaios biológicos, o estudo toxicológico, e a elaboração e caracterização das nanoemulsões dos óleos essenciais foram realizados pela primeira vez para as duas espécies vegetais, destacando a importância e ineditismo do presente trabalho. / The genus Drimys presents the largest geographical distribution of the family (Winteraceae), which comprises seven genera and about 120 species. In Brazil, is found from Bahia to Rio Grande do Sul and occurs in two species, Drimys angustifolia L. and Drimys brasiliensis Miers, popularly known as "Casca-de-anta". The leaves and barks are used in folk medicine as antiscorbutic, stimulant, antispasmodic, anti-diarrheal, antipyretic, against uterine bleeding, antibacterial, to treat asthma, bronchitis, certain disorders of the digestive tract and sometimes in the treatment of cancer and insecticides. Both are characterized by the presence of flavonoids and sesquiterpenoids. The objective of this study was to evaluate the chemical composition, the antioxidant, antiviral, insecticidal, antitumor, antifungal, acute toxicological evaluation and also to formulate and characterize two nanoemulsions of the volatile oils of two species of the study. By steam distillation was possible to perform the extraction of essential oils and to determine their yields ranging between 0.3 and 0.5% of leaves. The identification of components was performed using the Gas Cromatography coupled to mass spectrometer (GC-MS), which presented as major components in all analyzis bicyclogermacrene for the Drimys angustifolia species and ciclocolorenona for the Drimys brasiliensis. The formulation and characterization of nanoemulsion was performed within the established parameters (particle size, polydispersity, pH) and by transmission electron microscopy confirming the size at the nanoscale. Regarding the insecticidal activity, both species were promising repelling termites Cryptotermes brevis with negative index of repellency. To evaluate initial potential larvicidal Aedes aegypti, the oils of the two species were not efficient, however, of the Drimys brasiliensis nanoemulsion the larvae mortality of the 69% and 89%, corresponding to concentrations of 0.5 μg/mL and 0.83 μg/mL, respectively. With respect to antioxidant activity, the oils of the two species have not been able to reduce the (2,2-diphenyl-1-picrylhydrazyl) DPPH radical. In the antiviral activity against the HSV-1, the two species were promising inhibiting viral replication. In antitumor activity in glioma cell lines (U-138 MG) and bladder (T24), only the oil Drimys brasiliensis reduced cell viability of two strains by the MTT assay, with similar results in cell counts and flow cytometry analysis of the incorporation of Anexinn-V/Propidium Iodide characterized as late apoptosis. In the antifungal activity, the two species were effective against the opportunistic fungus Acremonium, present in various infections in transplant and immunocompromised. Regarding the evaluation of acute toxicity, the two species showed significant signs of toxicity in rats as ptosis, tremor, decreased motor activity, exolftamia, shortness of breath. Animals treated with species Drimys brasiliensis also showed an increase in urination and diarrhea. Thus, it is important that all biological assays, the toxicity study, and preparation and characterization of nanoemulsions of essential oils were first performed for the two species, highlighting the importance and originality of this work.