Lamiaceae extracts and compounds for topical application through nano delivery systems
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2017
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La resistencia a los antibióticos es uno de los principales problemas de salud que afectan a todos los continentes, ya sean países desarrollados o en desarrollo. Este tipo de infección es responsable de un elevado número de víctimas, siendo una de las más importantes causas de muerte en todo el mundo, sin una forma efectiva de terapia. Los desarrollos producidos en el campo de la nanotecnología han revelado formas más eficaces de terapéutica, mientras que se desvelan nuevas estructuras procedentes de nuevos recursos naturales nunca antes estudiados. Estas serán las nuevas terapias para este enorme problema.
Con este trabajo, se analiza el potencial y la capacidad de la 7α-acetoxi-6β-hidroxiarilanona como un nuevo compuesto para el tratamiento de infecciones asociadas con bacterias resistentes. Del mismo modo, se propone la combinación de una forma terapéutica de origen natural (extracto o compuesto aislado) con una nueva técnica de administración utilizando diferentes tipos de nanopartículas para el tratamiento de infecciones tópicas. La obtención de 7α-acetoxi-6β-hidroxiroiletanona a partir de Plectranthus grandidentatus Gürke se optimizaró, a través de siete métodos diferentes de extracción. La mayor cantidad de 7α-acetoxi-6β-hidroxiroiletanona se obtuvo mediante extracción con fluido supercrítico, suministrando una cantidad de AHR aproximadamente seis veces mayor que la del segundo método mejor evaluado. Se utilizó la 7α-acetoxi-6β-hidroxiarilanona aislada para los estudios de caracterización de sus propiedades fisicoquímicas. Se obtuvieron datos importantes, tales como la estructura molecular y cristalina de la 7α-acetoxi-6β-hidroxiarilanona a partir de la difracción de rayos X de cristal único y se estudió el comportamiento térmico del compuesto mediante calorimetría de barrido diferencial.
Se estudió el mecanismo de acción que justifica la alta actividad antibacteriana y la baja citotoxicidad de la 7α-acetoxi-6β-hidroxiroileanona sobre un tipo de cepa de Staphylococcus aureus resistente a la meticilina. Con este objetivo, se realizaron diferentes estudios sobre la interación de este posible nuevo antibiótico en la membrana y en la pared celular de la bacteria. La 7α-acetoxi-6β-hidroxiarilanona ha revelado un efecto no significativo en la permeabilidad pasiva, al mismo tiempo, las bacterias tratadas con esta royleanona muestran disrupción de la pared celular, sin revelar lisis celular.
La combinación de nanopartículas con productos naturales se ensayó utilizando dos plantas de la familia Lamiaceae. Para ello se prepararon diferentes extractos de Lavandula stoechas ssp. Luisieri (Rozeira) Rozeira y Lavandula pedunculata (Mill.) Cav. y se estableció su perfil antioxidante por inhibición de la peroxidación lipídica que confirmó dicha actividad. Debido a su alta actividad antioxidante, contenido fenólico y cantidad de flavonoides, los extractos metanólicos de Lavandula stoechas ssp. Luisieri y Lavandula pedunculata fueron seleccionados y encapsulados en nanopartículas de Poli (ácido láctico-co-glicólico). La encapsulación dio como resultado la formación de nanopartículas poligénicas de PLGA con una forma esférica bien definida y un alto rendimiento de encapsulación (˃96%). La permeación epidérmica preliminar de ambos extractos a través de la epidermis humana y su citotoxicidad in vitro en estudios de queratinocitos humanos sugirieron riesgos bajos de toxicidad.
También se estudió la combinación de nanopartículas con 7α-acetoxi-6β-hidroxiroileanona. A través de un enfoque diferente, distintas nanopartículas de plata fueron sintetizadas con éxito usando sales de citrato y borohidruro como agentes reductores. La ampicilina, como modelo, y la 7α-acetoxi-6β-hidroxi-roiletanona como nueva forma terapéutica se asociaron con ambos tipos de nanopartículas de plata sintetizadas. La caracterización mostró que se formaron nanopartículas pequeñas y monodispersas con forma esférica. La capacidad antibacteriana del antibiótico cuando se conjugó con nanopartículas de plata fue también elevada. La prueba de irritación cutánea in vivo realizada en modelos animales mostró ausencia de eritema o cambios de color de la piel.
En definitiva, el estudio desarrollado sobre esta nueva molécula muestra resultados prometedores para la futura aplicación como antibiótico. El conocimiento de sus propiedades químicas y mecánicas puede dar lugar a estructuras con mejor actividad terapéutica y menores efectos secundarios. Su combinación con la nanotecnología puede conducir a propiedades farmacológicas ventajosas para las personas infectadas con este tipo de bacterias.
Antibiotic resistance is one of the major health problem affecting every continent, whether developed or developing countries. This problem is responsible of a high number of patients and a leading cause of deaths worldwide, without an effective form of therapy. The developments in the nanotechnology field has led to more efficient delivery systems, while new structures unveiled through natural resources showed high biological activity never studied before. The combination of a new drug delivery strategy with new bioactive molecules can lead to new therapeutic forms against this type of infections. With this work, it is analysed the potencial and ability of 7?-acetoxy-6?- hydroxyroyleanone as a novel compound for the treatment of infections associated with resistant bacterias. In the same way, it is proposing that the combination of a therapeutic form of natural source (extract ou isolated compound) with an new technique of delivery using different types of nanoparticles for the treatment of topical infections. 7?-acetoxy-6?-hydroxyroyleanone extract from Plectranthus grandidentatus was optimized, through seven different methods of extraction. The highest quantity of the 7?- acetoxy-6?-hydroxyroyleanone was obtained through supercritical fluid extraction, delivering an amount of AHR about six times higher than that of the second-best method tested. 7?- acetoxy-6?-hydroxyroyleanone isolated was used for the characterization studies of their physicochemical properties. Important data, such as, molecular and crystal structure of 7?- acetoxy-6?-hydroxyroyleanone was obtained from single crystal X-ray diffraction and the thermal behavior of the compound was studied by differential scanning calorimetry. The mechanism of action of high antibacterial activity and low cytotoxicity of 7?-acetoxy-6?-hydroxyroyleanone was studied on a type of Methicillin-resistant Staphylococcus aureus strain. In this approach, different studies were carried out. 7?-acetoxy-6?- hydroxyroyleanone had a non-significant effect in passive permeability, at same time, bacteria treated with this royleanone displayed cell wall disruption, without cell lysis. The combination of nanoparticles with natural products was preliminary tested using two plants from Lamiaceae family. Different extracts from Lavandula stoechas ssp. luisieri and Lavandula pedunculata were prepared and established their antioxidant profile by lipid peroxidation inhibition and the antioxidant activity confirmed. Due to their high antioxidant activity, phenolic content and flavonoid amount, methanol extracts of Lavandula stoechas ssp.luisieri and Lavandula pedunculata were selected and encapsulated into Poly(lactic-co-glycolic acid) nanoparticles. The encapsulation of the extracts resulted in well-defined spherical shape PLGA nanoparticles and with a high encapsulation efficiency (?96%). Preliminar epidermal permeation of both extracts through ex-vivo human epidermis and their in vitro cytotoxicity in human keratinocytes studies were suggestive of their low risk of toxicity. The combination of nanoparticles with 7?-acetoxy-6?-hydroxyroyleanone was also studied in this thesis. Through a different approach, distinct silver nanoparticles were successful synthesized using citrate and borohydride salts as reducing agents. Ampicillin, as model drug, and 7?-acetoxy-6?-hidroxyroyleanone as new therapeutic compound were associated with both type of silver nanoparticles. Particle characterization showed small, spherical and monodisperse nanoparticles. The antibacterial capacity of each compound was higher after conjugation with silver nanoparticles. In vivo skin irritation test conducted in animal models showed absence of erythema or any skin color changes. The study developed on this new molecule has resulted in promising results for future application as topical formulation. The knowledge of its chemical and mechanistic properties may result in structures with better therapeutic activity and minor adverse side effects. Ultimately, their combination with nanotechnology may lead to advantageous pharmacological properties for people infected with this type of bacteria.
Antibiotic resistance is one of the major health problem affecting every continent, whether developed or developing countries. This problem is responsible of a high number of patients and a leading cause of deaths worldwide, without an effective form of therapy. The developments in the nanotechnology field has led to more efficient delivery systems, while new structures unveiled through natural resources showed high biological activity never studied before. The combination of a new drug delivery strategy with new bioactive molecules can lead to new therapeutic forms against this type of infections. With this work, it is analysed the potencial and ability of 7?-acetoxy-6?- hydroxyroyleanone as a novel compound for the treatment of infections associated with resistant bacterias. In the same way, it is proposing that the combination of a therapeutic form of natural source (extract ou isolated compound) with an new technique of delivery using different types of nanoparticles for the treatment of topical infections. 7?-acetoxy-6?-hydroxyroyleanone extract from Plectranthus grandidentatus was optimized, through seven different methods of extraction. The highest quantity of the 7?- acetoxy-6?-hydroxyroyleanone was obtained through supercritical fluid extraction, delivering an amount of AHR about six times higher than that of the second-best method tested. 7?- acetoxy-6?-hydroxyroyleanone isolated was used for the characterization studies of their physicochemical properties. Important data, such as, molecular and crystal structure of 7?- acetoxy-6?-hydroxyroyleanone was obtained from single crystal X-ray diffraction and the thermal behavior of the compound was studied by differential scanning calorimetry. The mechanism of action of high antibacterial activity and low cytotoxicity of 7?-acetoxy-6?-hydroxyroyleanone was studied on a type of Methicillin-resistant Staphylococcus aureus strain. In this approach, different studies were carried out. 7?-acetoxy-6?- hydroxyroyleanone had a non-significant effect in passive permeability, at same time, bacteria treated with this royleanone displayed cell wall disruption, without cell lysis. The combination of nanoparticles with natural products was preliminary tested using two plants from Lamiaceae family. Different extracts from Lavandula stoechas ssp. luisieri and Lavandula pedunculata were prepared and established their antioxidant profile by lipid peroxidation inhibition and the antioxidant activity confirmed. Due to their high antioxidant activity, phenolic content and flavonoid amount, methanol extracts of Lavandula stoechas ssp.luisieri and Lavandula pedunculata were selected and encapsulated into Poly(lactic-co-glycolic acid) nanoparticles. The encapsulation of the extracts resulted in well-defined spherical shape PLGA nanoparticles and with a high encapsulation efficiency (?96%). Preliminar epidermal permeation of both extracts through ex-vivo human epidermis and their in vitro cytotoxicity in human keratinocytes studies were suggestive of their low risk of toxicity. The combination of nanoparticles with 7?-acetoxy-6?-hydroxyroyleanone was also studied in this thesis. Through a different approach, distinct silver nanoparticles were successful synthesized using citrate and borohydride salts as reducing agents. Ampicillin, as model drug, and 7?-acetoxy-6?-hidroxyroyleanone as new therapeutic compound were associated with both type of silver nanoparticles. Particle characterization showed small, spherical and monodisperse nanoparticles. The antibacterial capacity of each compound was higher after conjugation with silver nanoparticles. In vivo skin irritation test conducted in animal models showed absence of erythema or any skin color changes. The study developed on this new molecule has resulted in promising results for future application as topical formulation. The knowledge of its chemical and mechanistic properties may result in structures with better therapeutic activity and minor adverse side effects. Ultimately, their combination with nanotechnology may lead to advantageous pharmacological properties for people infected with this type of bacteria.
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Diretores de Tese: Patrícia Rijo, Catarina Reis, Ana M. Díaz Lanza
Palavras-chave
HEALTH SCIENCES, ANTIMICROBIAL RESISTANCE, PLANTS, PLGA NANOPARTICLES, PHARMACOLOGY, SILVER NANOPARTICLES, DOUTORAMENTO EM CIÊNCIAS DA SAÚDE, CIÊNCIAS DA SAÚDE, RESISTÊNCIA AOS ANTIBIÓTICOS, PLANTAS, NANOPARTÍCULAS PLGA, FARMACOLOGIA, NANOPARTÍCULAS DE PRATA