Desenvolvimento tecnológico de nanoemulsões catiônicas contendo isoflavonóides de Glycine max (soja) visando ao tratamento do herpes

A atividade antiviral de compostos flavonoídicos tem sido amplamente investigada nos últimos anos. Nesse contexto, a primeira etapa da presente tese teve por objetivo avaliar a atividade anti-herpética in vitro dos principais isoflavonóides de Glycine max (soja): genisteína, daidzeína, gliciteína e cumestrol. Dentre os isoflavonóides investigados, a genisteína e o cumestrol mostraram uma interessante atividade frente aos vírus HSV-1 (cepa KOS e 29R, sensível e resistente ao aciclovir, respectivamente) e HSV-2 (cepa 333), interferindo em diferentes etapas do ciclo de replicação dos vírus. Devido à reduzida hidrossolubilidade desses isoflavonóides, foi proposta a sua incorporação em nanoemulsões de uso tópico. As formulações foram otimizadas através de um experimento fatorial completo do tipo 23 . O efeito dos fatores tipo de óleo (óleo de rícino ou miristato de isopropila), co-tensoativo iônico (oleilamina ou ácido olêico) e fosfolipídeo (DSPC ou DOPC) sobre as propriedades das nanoemulsões e a retenção da genisteína na pele de orelha suína foi investigado. Nanoemulsões compostas de miristato de isopropila/ oleilamina/DOPC apresentaram um menor diâmetro de gotícula e uma maior retenção de genisteína na pele de orelha suína enquanto que a combinação miristato de isopropila/oleilamina/DSPC mostrou o menor índice de polidispersão. A viscosidade das formulações selecionadas foi ajustada através do uso de hidroxietilcelulose visando à obtenção de produtos de uso tópico, obtendo-se hidrogéis de comportamento pseudoplástico. Na sequência, um conjunto de resultados obtidos demonstrou que a composição das formulações (especialmente o fosfolipídeo DOPC e o agente espessante hidroxietil celulose) pode influenciar a liberação e a retenção dos isoflavonóides em mucosa esofágica suína. A integridade da mucosa também desempenha um papel no aumento da permeação/retenção do cumestrol, conforme ilustrado nas imagens de microscopia confocal, utilizando vermelho do Nilo como marcador fluorescente. De maneira geral, a incorporação dos isoflavonóides nas nanoemulsões aumenta a atividade anti-herpética desses compostos in vitro. O conjunto dos resultados demonstra que as formulações desenvolvidas são potenciais carreadores de uso tópico para genisteína e cumestrol no tratamento das infecções herpéticas.The antiviral activity of flavonoid compounds has been extensively investigated in recent years. In this context, the first step of this study was to evaluate the antiherpes activity in vitro of the main isoflavonoids of Glycine max (soybean): genistein, daidzein, glycitein and coumestrol. Among the investigated isoflavonoids, genistein and coumestrol showed interesting activity against HSV-1 (KOS and 29R strains, which are acyclovir-sensitive and acyclovir–resistant strains, respectively) and HSV-2 (333 strain), interfering at different stages of the virus replication cycle. Due to the low hydrosolubility of these isoflavonoids, their incorporation into topical nanoemulsions was proposed in this study. The formulations were optimized through a 23 full factorial design. The factors effect of oil type (castor oil or isopropyl myristate), ionic co-surfactant (oleylamine or oleic acid) and phospholipid type (DSPC or DOPC) on physicochemical properties and genistein retention into porcine ear skin was investigated. Nanoemulsions composed of isopropyl myristate/ DOPC/oleylamine showed the smaller particle size and higher genistein retention into skin, whereas the formulation composed of isopropyl myristate/DSPC/oleylamine exhibited the lower polydispersity index. The viscosity of selected formulations was adjusted with hydroxyethyl cellulose to obtain topical products, which showed nonNewtonian behavior. In sequence, a set of results showed that formulations composition (especially the phospholipid DOPC and the thickening agent hydroxyethyl cellulose) could influence the release and retention of isoflavonoids in porcine esophagus mucosa. The integrity of mucosa plays a role on the increase of coumestrol permeation/retention, according to confocal microscopy images, using Red Nile as fluorescent label. In general terms, the incorporation of isoflavonoids into nanoemulsions increases the antiherpes activity of these compounds in vitro. The overall results show that the formulations developed in this study are potential topical carriers for genistein and coumestrol in the treatment of herpes infections

Nanoemulsões de uso tópico contendo cumestrol : estudos de formulação e permeação cutânea

O cumestrol é um isoflavonóide presente em diferentes leguminosas como a alfafa, soja e trevo vermelho. Estudos demonstraram uma relevante atividade antioxidante e estrogênica deste composto. Neste contexto, o objetivo deste estudo foi desenvolver nanoemulsões contendo cumestrol visando a sua aplicação tópica. Na primeira fase, nanoemulsões foram obtidas por emulsificação espontânea. Tal procedimento conduziu à obtenção de nanoemulsões monodispersas, confirmado por microscopia eletrônica de transmissão. A viscosidade das formulações permaneceu similar, enquanto que o potencial zeta foi influenciado pela presença dos tensoativos. Após espessamento, as formulações demonstraram um comportamento não-Newtoniano pseudoplástico. Na segunda etapa, o teor de cumestrol nas formulações foi determinado por CLAE. O método foi específico, linear, preciso e exato para quantificação de cumestrol nas formulações desenvolvidas. Os perfis de liberação do cumestrol, através de membrana artificial, demonstraram uma liberação imediata para todas as formulações. Por fim, estudos de permeação/retenção do cumestrol através de pele de orelha suína foram realizados utilizando-se células de difusão de Franz. Os resultados obtidos demonstram um fluxo lento do cumestrol através da pele. O conjunto de resultados obtidos demonstra o efeito da composição sobre as propriedades das formulações e distribuição do cumestrol através das camadas da pele.Coumestrol is an isoflavonoid present in various legumes such as alfalfa, soybeans, and red clovers. Studies have shown significant estrogenic and antioxidant activities for this compound. With this in mind, the main purpose of this study was to develop nanoemulsion containing coumestrol for topic application. In the first phase, nanoemulsions were obtained by spontaneous emulsification. This procedure yielded monodisperse nanoemulsions, confirmed by transmission electron microscopy. The viscosity of the formulations remained similar, while the zeta potential was influenced by the surfactants. After thickening, the formulations showed a pseudplastic non-Newtonian behavior. In the second step, the coumestrol content in the formulations was determined by HPLC. The method was specific, linear, precise, and accurate for coumestrol quantification in the developed formulations. The coumestrol release profile through artificial membrane showed immediate release for all formulations. Finally, studies of coumestrol permeation/retention through porcine ear skin were performed using Franz diffusion cells. The results showed a slow flow of coumestrol through the skin. The overall results show the effect of the composition on the properties of the formulations and coumestrol distribution through the layers of the skin

Carreadores lipídicos de tamanho nanométrico compreendendo fração enriquecida de isoflavonas agliconas da soja, processo de obtenção dos mesmos e formulações compreendendo os mesmos

Universidade Federal do Rio Grande do SulFarmáciaDepositad

Antiherpes Activity and Skin/Mucosa Distribution of Flavonoids from Achyrocline satureioides Extract Incorporated into Topical Nanoemulsions

This study investigated the inhibitory effects of Achyrocline satureioides extract (ASE) incorporated into a topical nanoemulsion on Herpes Simplex Virus type 1 (HSV-1/KOS strain) replication, as well as the distribution of the main ASE flavonoids (quercetin, luteolin, and 3-O-methylquercetin) in porcine skin and mucosa. The ASE-loaded nanoemulsion showed more pronounced effects against HSV-1 replication when compared to the ASE or pure quercetin, as determined by the viral plaque number reduction assay. All flavonoids were detected in the skin epidermis (2.2 µg/cm2) and the mucosa upper layers (3.0 µg/cm2) from ASE-loaded nanoemulsion until 8 h after topical application. A higher amount of flavonoids was detected when these tissues were impaired, especially in deeper mucosa layers (up to 7-fold). Flavonoids were detected in the receptor fluid only when the mucosa was injured. Such results were supported by confocal microscopy images. Overall, these findings suggest that the tested ASE-loaded nanoemulsion has potential to be used topically for herpes infections

Potential use of secondary products of the agri-food industry for topical formulations and comparative analysis of antioxidant activity of grape leaf polyphenols

<p>The aim of the present study was to develop a phytocosmetic using <i>Vitis</i> waste by-products, for use as a topical formulation for skin protection against ultraviolet radiation damage. The study also evaluates the free radical scavenger activity of the crude extracts of dried leaves of <i>Vitis vinifera</i> and <i>Vitis labrusca</i>, as well as the anthocyanins, flavonoid fraction and isolated compounds. Next, release and permeation studies of hydrogels were performed using Franz-type diffusion cells. Flavonoid acted more intensively in TRAP and conjugated dienes antioxidant assays, whereas anthocyanins had higher antioxidant activity in hydroxyl and nitric oxide assay. Only quercetin-3-<i>O</i>-glucuronide (5) was released from hydrogels, and the flavonoid retention in porcine ear skin after eight hours of permeation was below of limit of quantification for this compound. The polyphenols present in <i>Vitis</i> are capable of absorbing UV and visible light, justifying their potential as sunscreens for the development of a phytocosmetic.</p

Nanoemulsions containing a synthetic chalcone as an alternative for treating cutaneous leshmaniasis : optimization using a full factorial design

Nanoemulsions are drug delivery systems that may increase the penetration of lipophilic compounds through the skin, enhancing their topical effect. Chalcones are compounds of low water solubility that have been described as promising molecules for the treatment of cutaneous leishmaniasis (CL). In this context, the aim of this work was to optimize the development of a nanoemulsion containing a synthetic chalcone for CL treatment using a 22 full factorial design. The formulations were prepared by spontaneous emulsification and the experimental design studied the influence of two independent variables (type of surfactant – soybean lecithin or sorbitan monooleate and type of co-surfactants – polysorbate 20 or polysorbate 80) on the physicochemical characteristics of the nanoemulsions, as well as on the skin permeation/retention of the synthetic chalcone in porcine skin. In order to evaluate the stability of the systems, the antileishmanial assay was performed against Leishmania amazonensis 24 hours and 60 days after the preparation of the nanoemulsions. The formulation composed of soybean lecithin and polysorbate 20 presented suitable physicochemical characteristics (droplet size 171.9 nm; polydispersity index 0.14; zeta potential -39.43 mV; pH 5.16; and viscosity 2.00 cP), drug content (91.09%) and the highest retention in dermis (3.03 μg⋅g-1) – the main response of interest – confirmed by confocal microscopy. This formulation also presented better stability of leishmanicidal activity in vitro against L. amazonensis amastigote forms (half maximal inhibitory concentration value 0.32±0.05 μM), which confirmed the potential of the nanoemulsion soybean lecithin and polysorbate 20 for CL treatment

Nanoemulsions containing a synthetic chalcone as an alternative for treating cutaneous leshmaniasis : optimization using a full factorial design

Nanoemulsions are drug delivery systems that may increase the penetration of lipophilic compounds through the skin, enhancing their topical effect. Chalcones are compounds of low water solubility that have been described as promising molecules for the treatment of cutaneous leishmaniasis (CL). In this context, the aim of this work was to optimize the development of a nanoemulsion containing a synthetic chalcone for CL treatment using a 22 full factorial design. The formulations were prepared by spontaneous emulsification and the experimental design studied the influence of two independent variables (type of surfactant – soybean lecithin or sorbitan monooleate and type of co-surfactants – polysorbate 20 or polysorbate 80) on the physicochemical characteristics of the nanoemulsions, as well as on the skin permeation/retention of the synthetic chalcone in porcine skin. In order to evaluate the stability of the systems, the antileishmanial assay was performed against Leishmania amazonensis 24 hours and 60 days after the preparation of the nanoemulsions. The formulation composed of soybean lecithin and polysorbate 20 presented suitable physicochemical characteristics (droplet size 171.9 nm; polydispersity index 0.14; zeta potential -39.43 mV; pH 5.16; and viscosity 2.00 cP), drug content (91.09%) and the highest retention in dermis (3.03 μg⋅g-1) – the main response of interest – confirmed by confocal microscopy. This formulation also presented better stability of leishmanicidal activity in vitro against L. amazonensis amastigote forms (half maximal inhibitory concentration value 0.32±0.05 μM), which confirmed the potential of the nanoemulsion soybean lecithin and polysorbate 20 for CL treatment