Desenvolvimento de nanoemulsões contendo constituintes de Punica granatum para aplicação cutânea visando sua utilização na fotoproteção

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2015.O presente trabalho descreve o desenvolvimento de nanoemulsões do tipo óleo em água contendo constituintes de Punica granatum (romã) e hidrogéis termosensíveis incluindo nanoemulsões para aplicação cutânea visando sua utilização na fotoproteção. A fração acetato de etila foi obtida por partição líquido-líquido, a partir do extrato seco das cascas de P. granatum, e caracterizada pela quantificação de polifenóis totais, ácido gálico, ácido elágico e punicalagina - 638,1 mg/g, 238,6 mg/g, 84,7 mg/g e 296,7 mg/g - respectivamente. As nanoemulsões contendo óleo de semente de romã (EAF-PSO-NE) ou triglicerídeo de cadeia média (EAF-MCT-NE) como fase oleosa, encapsulando a fração acetato de etila, foram obtidas através de dois métodos: emulsificação-evaporação do solvente e emulsificação espontânea, respectivamente. As nanoemulsões apresentaram forma esférica e distribuição monodispersa das gotículas. O tamanho médio das gotículas obtido pela técnica de espalhamento de luz dinâmico (DLS) foi de aproximadamente 180 nm e 220 nm, para EAF-PSO-NE e EAF-MCT-NE, respectivamente. Estes dados foram confirmados por microscopia eletrônica de transmissão (MET). O teor médio de fenóis totais, ácido elágico, ácido gálico e punicalagina para EAF-PSO-NE foi de 2,3 mg/mL, 35,1 µg/mL, 1248,9 µg/mL, 425,6 µg/mL, respectivamente; para EAF-MCT-NE foi de 2,3 mg/mL, 89,9 µg/mL, 1320,7 µg/mL, 383,6 µg/mL, respectivamente. As nanoemulsões apresentaram valores de eficiência de encapsulação de acordo com a lipofilicidade de cada composto polifenólico e considerável ação antioxidade frente aos ensaios de DPPH e FRAP. As nanoemulsões foram caracterizadas como não irritantes/fotoirritantes em ensaio preliminar utilizando eritrócitos humanos. Ainda empregando eritrócitos humanos como modelo de biomembrana, verificou-se que as nanoemulsões foram capazes de interagir com a bicamada lipídica e proteger a mesma de danos oxidativos induzidos pelo iniciador de radical livre AAPH. O efeito fotoprotetor das nanoemulsões contra radiação UVB foi evidenciado pela proteção a danos no DNA celular. O fator de proteção solar (FPS) determinado in vitro foi de até 25 para EAF-PSO-NE. Estudos in vitro demonstraram que nas concentrações testadas as nanoemulsões e fração livre não foram citotóxicas ou fototóxicas. O comportamento das nanoemulsões na presença do meio de cultura foi verificado pela técnica de DLS e crio-microscopia eletrônica de transmissão (Crio-MET). Nos estudos de internalização celular as nanoemulsões demonstraram10habilidade de se acumularem ao redor da célula e também internalizar. A estabilidade físico-química (exceto teor de punicalagina) das nanoemulsões foi evidenciada por 60 dias. A fotoestabilidade das formulações foi verificada pela exposição a doses mínimas preconizadas de radiação UVA e UV-visível; as nanoemulsões foram consideradas fotoestáveis. A baixa viscosidade das nanoemulsões inviabiliza sua aplicação na pele, deste modo um copolímero termosensível foi incorporado nas mesmas. A presença das nanoemulsões nos hidrogéis foi confirmada pela técnica de DLS e por microscopia eletrônica de varredura de alta resolução (SEM-FEG). Os hidrogéis foram caracterizados físicamente e quimicamente e foi verificado que na concentração mais baixa de copolímero um hidrogel de textura agradável e de fácil aplicação em spray foi obtido. Estudos de permeação e retenção dos compostos fenólicos através de pele de orelha suína também foram conduzidos. A fração nanoemulsionada aumentou substancialmente a liberação tópica dos compostos polifenólicos quando comparada com a fração livre. A permeação e retenção dos compostos polifenólicos dependeram de suas características físico-químicas, do veículo e de possíveis interações com a pele. As concentrações dos compostos polifenólicos retidos no stratum corneum, e permeados até a epiderme viável e derme evidenciaram a possibilidade de aplicação das formulações como fotoprotetor atuando na absorção de fótons e complementando a ação antioxidante na pele. Com o intuito de visualizar a permeação e retenção a partir da aplicação tópica de nanoemulsões ou de hidrogéis contendo as nanoemulsões, um agente fluoróforo (vermelho de nilo) foi juntamente nanoemulsionado com a fração acetato de etila. As imagens obtidas por microscopia confocal de varredura laser (MCVL) corroboraram os resultados obtidos nos estudos de permeação e retenção cutânea. Assim, os resultados obtidos indicam que as nanoemulsões e hidrogéis contendo constituintes de P. granatum (óleo da semente e fração), oferecem uma estratégia promissora para liberação tópica de compostos polifenólicos (ácido gálico, ácido elágico e punicalagina) com potencial aplicação na fotoproteção.Abstract : This work describes the development of oil in water nanoemulsions containing Punica granatum (pomegranate) constituents and thermosensitive hydrogels thickened nanoemulsions for topical application aiming its use in photoprotection. The polyphenol-rich ethyl acetate fraction (EAF) was obtained by liquid-liquid partition from the dry peel extract of P. granatum and characterized by quantification of total polyphenols, gallic acid, ellagic acid, and punicalagin - 638.1 mg/g, 238.6 mg/g, 84.7 mg/g and 296.7 mg/g - respectively. The nanoemulsions containing pomegranate seed oil (EAF-PSO-NE), or medium chain triglyceride (EAF-MCT-NE) as oil phase entrapping the EAF was obtained by two methods: emulsification-evaporation of the solvent and spontaneous emulsification, respectively. Nanoemulsions presented spherical shape and monodisperse distribution of droplets. The average droplet size obtained through dynamic light scattering (DLS) was around 180 nm and 220 nm for EAF-PSO-NE and EAF-MCT-NE, respectively. These data were confirmed by transmission electron microscopy (TEM). The average of total phenolic content, ellagic acid, gallic acid and punicalagin for EAF-PSO-NE were 2.3 mg/mL, 35.1 µg/mL, 1248.9 µg/mL and 425.6 µg/mL, respectively; for EAF-MCT-NE were 2.3 mg/mL, 89.9 µg/mL, 1320.7 µg/mL and 383.6 µg/mL, respectively. The encapsulation efficiency values were according to the lipophilicity of each polyphenol compound and considerable antioxidant activity was observed for nanoemulsions through DPPH and FRAP assays. In preliminary testing using human erythrocytes the nanoemulsions were characterized as non-irritant/photoirritant. Also employing human erythrocytes as a biomembrane model, it was found that the nanoemulsions were able to interact with the lipid bilayer and protect against oxidative damage induced by the free-radical initiator AAPH. The photoprotective effect of nanoemulsions was demonstrated by cellular protection against UVB radiation induced DNA damage. The in vitro sun protection factor (SPF) was up to 25 for EAF-PSO-NE. In vitro studies showed that the concentrations tested for free EAF and nanoemulsions were not cytotoxic or phototoxic. The droplet size and zeta potential of the nanoemulsions in contact with culture medium were verified by DLS technique and cryo-transmission electron microscopy (Cryo-TEM). In the cellular internalization studies nanoemulsions demonstrated an ability to accumulate around the cell and also to internalize. The12nanoemulsions were physically and chemically stable for 60 days (except for punicalagin content). The photostability of the formulations was verified by UVA and UV-visible exposure at recommended minimum doses; nanoemulsions were considered photostable. Since the nanoemulsions exhibit low viscosities, which restrain their application to the skin surface, a thermoreversible copolymer was incorporated into the formulations. The presence of nanoemulsions in the hydrogels was confirmed by DLS technique and by high resolution scanning electron microscopy (FEG-SEM). The hydrogels were physically and chemically characterized. It was noticed that when the lowest concentration of the copolymer was employed, a hydrogel easy to spray and with pleasant texture was obtained. Permeation and retention studies of phenolic compounds across the porcine ear skin were also conducted. The nanoemulsified EAF increased substantially the topical delivery of polyphenolic compounds compared with the free EAF. The skin permeation and retention of polyphenolic compounds depended on its physico-chemical characteristics, the vehicle and possible interactions with the skin. The amounts of polyphenolic compounds retained in the stratum corneum and that permeated into the viable epidermis and dermis, highlight the possible application of the formulations as photoprotectors acting in the absorption of photons and complementing the antioxidant protection in the skin. In order to visualize the permeation and retention after topical application of nanoemulsions or hydrogels containing nanoemulsions, a fluorophore agent (nile red) was nanoemulsified together with the ethyl acetate fraction. The images obtained by confocal laser scanning microscopy (CLSM) corroborated the results obtained in the skin permeation and retention studies. Thus, the results indicated that nanoemulsions and hydrogels containing constituents of P. granatum (seed oil and EAF), offer a promising strategy for topical delivery of polyphenolic compounds (gallic acid, ellagic acid, and punicalagin) with potential application in photoprotection

Protection against oxidative damage in human erythrocytes and preliminary photosafety assessment of Punica granatum seed oil nanoemulsions entrapping polyphenol-rich ethyl acetate fraction

AbstractThe main purpose of the present study is to evaluate the ability of nanoemulsion entrapping pomegranate peel polyphenol-rich ethyl acetate fraction (EAF) prepared from pomegranate seed oil and medium chain triglyceride to protect human erythrocyte membrane from oxidative damage and to assess preliminary in vitro photosafety. In order to evaluate the phototoxic effect of nanoemulsions, human red blood cells (RBCs) are used as a biological model and the rate of haemolysis and photohaemolysis (5Jcm−2 UVA) is assessed in vitro. The level of protection against oxidative damage caused by the peroxyl radical generator AAPH in human RBCs as well as its effects on bilayer membrane characteristics such as fluidity, protein profile and RBCs morphology are determined. EAF-loaded nanoemulsions do not promote haemolysis or photohaemolysis. Anisotropy measurements show that nanoemulsions significantly retrain the increase in membrane fluidity caused by AAPH. SDS-PAGE analysis reveals that AAPH induced degradation of membrane proteins, but that nanoemulsions reduce the extension of degradation. Scanning electron microscopy examinations corroborate the interaction between AAPH, nanoemulsions and the RBC membrane bilayer. Our work demonstrates that Punica granatum nanoemulsions are photosafe and protect RBCs against oxidative damage and possible disturbance of the lipid bilayer of biomembranes. Moreover it suggests that these nanoemulsions could be promising new topical products to reduce the effects of sunlight on skin

Comparative effects of macro-sized aluminum oxide and aluminum oxide nanoparticles on erythrocyte hemolysis: influence of cell source, temperature and size

Al2O3 is the most abundantly produced nanomaterial and has been used in diverse fields, including the medical, military and industrial sectors. As there are concerns about the health effects of nanoparticles, it is important to understand how they interact with cells, and specifically with red blood cells. The hemolysis induced by three commercial nano-sized aluminum oxide particles (nanopowder 13 nm, nanopowder <50 nm and nanowire 2-6 nm × 200-400 nm) was compared to aluminum oxide and has been studied on erythrocytes from humans, rats and rabbits, in order to elucidate the mechanism of action and the influence of size and shape on hemolytic behavior. The concentrations inducing 50% hemolysis (HC50) were calculated for each compound studied. The most hemolytic aluminum oxide particles were of nanopowder 13, followed by nanowire and nanopowder 50. The addition of albumin to PBS induced a protective effect on hemolysis in all the nano-forms of Al2O3, but not on Al2O3. The drop in HC50 correlated to a decrease in nanomaterial size, which was induced by a reduction of aggregation Aluminum oxide nanoparticles are less hemolytic than other oxide nanoparticles, and behave differently depending on the size and shape of the nanoparticles. The hemolytic behavior of aluminum oxide nanoparticles differs from that of aluminum oxide

Photoprotection by Punica granatum seed oil nanoemulsion entrapping polyphenol-rich ethyl acetate fraction against UVB-induced DNA damage in human keratinocyte (HaCaT) cell line

There has been an increase in the use of botanicals as skin photoprotective agents. Pomegranate (Punica granatum L.) is well known for its high concentration of polyphenolic compounds and for its antioxidant and anti-inflammatory properties. The aim of this study was to analyse the photoprotection provided by Punica granatum seed oil nanoemulsion entrapping the polyphenol-rich ethyl acetate fraction against UVB-induced DNA damage in the keratinocyte HaCaT cell line. For this purpose, HaCaT cells were pretreated for 1 h with nanoemulsions in a serum-free medium and then irradiated with UVB (90200 mJ/cm2) rays. Fluorescence microscopy analysis provided information about the cellular internalization of the nanodroplets. We also determined the in vitro SPF of the nanoemulsions and evaluated their phototoxicity using the 3T3 Neutral Red Uptake Phototoxicity Test. The nanoemulsions were able to protect the cells' DNA against UVB-induced damage in a concentration dependent manner. Nanodroplets were internalized by the cells but a higher proportion was detected along the cell membrane. The SPF obtained (~25) depended on the concentration of the ethyl acetate fraction and pomegranate seed oil in the nanoemulsion. The photoprotective formulations were classified as non-phototoxic. In conclusion, nanoemulsions entrapping the polyphenol-rich ethyl acetate fraction show potential for use as a sunscreen product

Protection against Oxidative Damage in Human Erythrocytes and Preliminary Photosafety Assessment of Punica granatum Seed Oil Nanoemulsions Entrapping Polyphenol-rich Ethyl Acetate Fraction

The main purpose of the present study is to evaluate the ability of nanoemulsion entrapping pomegranate peel polyphenol-rich ethyl acetate fraction (EAF) prepared from pomegranate seed oil and medium chain triglyceride to protect human erythrocyte membrane from oxidative damage and to assess preliminary in vitro photosafety. In order to evaluate the phototoxic effect of nanoemulsions, human red blood cells (RBCs) are used as a biological model and the rate of haemolysis and photohaemolysis (5 J cm-2 UVA) is assessed in vitro. The level of protection against oxidative damage caused by the peroxyl radical generator AAPH in human RBCs as well as its effects on bilayer membrane characteristics such as fluidity, protein profile and RBCs morphology are determined. EAF-loaded nanoemulsions do not promote haemolysis or photohaemolysis. Anisotropy measurements show that nanoemulsions significantly retrain the increase in membrane fluidity caused by AAPH. SDS-PAGE analysis reveal that AAPH induced degradation of membrane proteins, but that nanoemulsions reduce the extend of degradation. Scanning electron microscopy examinations corroborate the interaction between AAPH, nanoemulsions and the RBCs membrane bilayer. Our work demonstrates that P. granatum nanoemulsions are photosafe and protect RBCs against oxidative damage and possible disturbance of the lipid bilayer of biomembranes. Moreover it suggests that these nanoemulsions could be promising new topical products to reduce the effects of sunlight on ski

Análise morfoanatômica das partes aéreas de Wedelia paludosa DC. (Acmela brasiliensis, Sphagneticola trilobata), Asteraceae

O presente trabalho teve por objetivo o estabelecimento de parâmetros para análise morfo-anatômica das partes aéreas (caule e folhas) da planta Wedelia paludosa DC., Asteraceae. A droga vegetal e o respectivo pó foram submetidos às análises macro e microscópica após preparação de lâminas semi-permanentes de cortes transversais e paradérmicos. O pó foi analisado após diafanização com cloral hidratado. É uma planta herbácea com caule castanho-avermelhado, folhas opostas, curto-pecioladas e membranáceas, apresentando pêlos nas duas faces, estreitada em direção à base acima do meio provida de dois pequenos lobos laterais e um terminal, maior e denteado. A planta apresenta secção circular de caule, que em crescimento primário, possui epiderme, colênquima angular, parênquima cortical e cilindro vascular, assim como oxalato de cálcio (várias drusas e alguns cristais prismáticos). A folha é simples, contendo tricomas tectores e estômato anomocítico. O mesofilo possui organização dorso-ventral e a nervura central, em corte transversal, apresenta-se côncavo-convexo com três feixes colaterais e presença de drusas de oxalato de cálcio nas células parenquimáticas. No pó obtido da droga vegetal foram verificadas as mesmas estruturas microscópicas observadas na droga vegetal rasurada. Os parâmetros morfo-anatômicos estabelecidos neste trabalho permitem o controle de qualidade macro e microscópico da planta tanto rasurada como na forma de pó

Protection against Oxidative Damage in Human Erythrocytes and Preliminary Photosafety Assessment of Punica granatum Seed Oil Nanoemulsions Entrapping Polyphenol-rich Ethyl Acetate Fraction

The main purpose of the present study is to evaluate the ability of nanoemulsion entrapping pomegranate peel polyphenol-rich ethyl acetate fraction (EAF) prepared from pomegranate seed oil and medium chain triglyceride to protect human erythrocyte membrane from oxidative damage and to assess preliminary in vitro photosafety. In order to evaluate the phototoxic effect of nanoemulsions, human red blood cells (RBCs) are used as a biological model and the rate of haemolysis and photohaemolysis (5 J cm-2 UVA) is assessed in vitro. The level of protection against oxidative damage caused by the peroxyl radical generator AAPH in human RBCs as well as its effects on bilayer membrane characteristics such as fluidity, protein profile and RBCs morphology are determined. EAF-loaded nanoemulsions do not promote haemolysis or photohaemolysis. Anisotropy measurements show that nanoemulsions significantly retrain the increase in membrane fluidity caused by AAPH. SDS-PAGE analysis reveal that AAPH induced degradation of membrane proteins, but that nanoemulsions reduce the extend of degradation. Scanning electron microscopy examinations corroborate the interaction between AAPH, nanoemulsions and the RBCs membrane bilayer. Our work demonstrates that P. granatum nanoemulsions are photosafe and protect RBCs against oxidative damage and possible disturbance of the lipid bilayer of biomembranes. Moreover it suggests that these nanoemulsions could be promising new topical products to reduce the effects of sunlight on ski

Comparative effects of macro-sized aluminum oxide and aluminum oxide nanoparticles on erythrocyte hemolysis: influence of cell source, temperature and size

Al2O3 is the most abundantly produced nanomaterial and has been used in diverse fields, including the medical, military and industrial sectors. As there are concerns about the health effects of nanoparticles, it is important to understand how they interact with cells, and specifically with red blood cells. The hemolysis induced by three commercial nano-sized aluminum oxide particles (nanopowder 13 nm, nanopowder <50 nm and nanowire 2-6 nm × 200-400 nm) was compared to aluminum oxide and has been studied on erythrocytes from humans, rats and rabbits, in order to elucidate the mechanism of action and the influence of size and shape on hemolytic behavior. The concentrations inducing 50% hemolysis (HC50) were calculated for each compound studied. The most hemolytic aluminum oxide particles were of nanopowder 13, followed by nanowire and nanopowder 50. The addition of albumin to PBS induced a protective effect on hemolysis in all the nano-forms of Al2O3, but not on Al2O3. The drop in HC50 correlated to a decrease in nanomaterial size, which was induced by a reduction of aggregation Aluminum oxide nanoparticles are less hemolytic than other oxide nanoparticles, and behave differently depending on the size and shape of the nanoparticles. The hemolytic behavior of aluminum oxide nanoparticles differs from that of aluminum oxide

Comparative effects of macro-sized aluminum oxide and aluminum oxide nanoparticles on erythrocyte hemolysis: influence of cell source, temperature and size

Al2O3 is the most abundantly produced nanomaterial and has been used in diverse fields, including the medical, military and industrial sectors. As there are concerns about the health effects of nanoparticles, it is important to understand how they interact with cells, and specifically with red blood cells. The hemolysis induced by three commercial nano-sized aluminum oxide particles (nanopowder 13 nm, nanopowder <50 nm and nanowire 2-6 nm × 200-400 nm) was compared to aluminum oxide and has been studied on erythrocytes from humans, rats and rabbits, in order to elucidate the mechanism of action and the influence of size and shape on hemolytic behavior. The concentrations inducing 50% hemolysis (HC50) were calculated for each compound studied. The most hemolytic aluminum oxide particles were of nanopowder 13, followed by nanowire and nanopowder 50. The addition of albumin to PBS induced a protective effect on hemolysis in all the nano-forms of Al2O3, but not on Al2O3. The drop in HC50 correlated to a decrease in nanomaterial size, which was induced by a reduction of aggregation Aluminum oxide nanoparticles are less hemolytic than other oxide nanoparticles, and behave differently depending on the size and shape of the nanoparticles. The hemolytic behavior of aluminum oxide nanoparticles differs from that of aluminum oxide

Photoprotection by Punica granatum seed oil nanoemulsion entrapping polyphenol-rich ethyl acetate fraction against UVB-induced DNA damage in human keratinocyte (HaCaT) cell line

There has been an increase in the use of botanicals as skin photoprotective agents. Pomegranate (Punica granatum L.) is well known for its high concentration of polyphenolic compounds and for its antioxidant and anti-inflammatory properties. The aim of this study was to analyse the photoprotection provided by Punica granatum seed oil nanoemulsion entrapping the polyphenol-rich ethyl acetate fraction against UVB-induced DNA damage in the keratinocyte HaCaT cell line. For this purpose, HaCaT cells were pretreated for 1 h with nanoemulsions in a serum-free medium and then irradiated with UVB (90200 mJ/cm2) rays. Fluorescence microscopy analysis provided information about the cellular internalization of the nanodroplets. We also determined the in vitro SPF of the nanoemulsions and evaluated their phototoxicity using the 3T3 Neutral Red Uptake Phototoxicity Test. The nanoemulsions were able to protect the cells' DNA against UVB-induced damage in a concentration dependent manner. Nanodroplets were internalized by the cells but a higher proportion was detected along the cell membrane. The SPF obtained (~25) depended on the concentration of the ethyl acetate fraction and pomegranate seed oil in the nanoemulsion. The photoprotective formulations were classified as non-phototoxic. In conclusion, nanoemulsions entrapping the polyphenol-rich ethyl acetate fraction show potential for use as a sunscreen product