Avaliação da eficácia e da citotoxicidade in vitro do ácido ursólico e sua incorporação em emulsão cosmética
Carregando...
Data
2018-07-04
Autores
Colombo, Fernanda Cardoso [UNESP]
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
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 CLSI. O AU demonstrou atividade antimicrobiana frente a cepa S.aureus com CIM de 25 μg/mL e CMB de 50 μg/mL; para S. epidermides com CIM de 25 μg/mL e CBM de 50 μg/mL; para E.coli, com CIM de 50 μg/mL e CBM de 50 μg/mL; a cepa P. aeruginosa revelou uma CIM de 25 μg/mL e CBM= 0. O potencial citotóxico in vitro foi avaliado através de técnica utilizando o corante 3-(4,5-dimethyl-2-thiazolyl)- (MTT) e queratinócitos humanos metabolicamente incompetentes, não tendo demonstrado citotoxicidade às células em uma concentração máxima de 50 µg/mL. Para incorporação em cosmético foi escolhida uma emulsão de alta estabilidade e como técnica de controle de qualidade do AU nessa emulsão foi desenvolvida e validada uma metodologia analítica por CLAE, utilizando os preceitos de química verde. Um estudo de estabilidade preliminar da emulsão preparada foi conduzido a fim de se avaliar a estabilidade do AU em uma emulsão estável frente a condições extremas, como temperaturas de 25, 40 e 5 ºC, luz indireta e ciclos de congelamento/ descongelamento. A emulsão preparada com AU demonstrou estabilidade para as características de pH e viscosidade, e com relação ao teor, foi mais estável a temperatura de 25 e 5 ºC. O AU, através dos ensaios in vitro realizados demonstrou caráter antioxidante, despigmentante e antimicrobiano, não sendo observada citotoxicidade às células utilizadas na faixa de concentração testada, além de ter demonstrado certa estabilidade na emulsão de escolha. Como pôde ser observado, este composto multifuncional demonstrou características in vitro de grande interesse para ser utilizado como ativo cosmético de uso tópico e possivelmente como um agente antimicrobiano, sendo assim necessário estudos mais aprofundados que possam garantir sua completa segurança e eficácia de uso.
Increased concern with body esthetics and health has raised the demand for skin cosmetics containing actives that prevent aging and other skin changes. Ursolic acid (UA), a natural active extracted from plants as rosemary and basil, has shown an interesting compound for cosmetic incorporation, to possess antioxidant activity, depigmenting, antimicrobial property, among others. This study aimed to evaluate the possibility of using UA as a multifunctional cosmetic compound through the UA efficacy and cytotoxicity in vitro, and to prepare and evaluate an emulsion containing the substance. Antioxidant potential was tested using inhibition methods of radical 1,1-diphenyl-2-picrilhidrazila (DPPH•) and radical 2,2 -azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS•+) showing antioxidant activity, with IC50= 235,61 µg/mL for DPPH• technique and 107,17 µg/mL to ABTS•+ method. The depigmenting activity was given by inhibition of the enzyme tyrosinase with3,4- dihydroxy-L-phenylalanine (L-DOPA) substrate and show IC50 de 338,00 µg/mL. For evaluation of antimicrobial activity, many strains were tested from the minimum inhibitory concentration (MIC) determined by dilution method on microplates, and minimum bactericidal concentration (MBC) by Petri dish replication, following M100-S16 supplement CLSI. The UA demonstrated antimicrobial activity, with a MIC to S. aureus strain of 25 μg/mL and MBC of 50 μg/mL; to S. epidermides a MIC of 25 μg/mL and MBC of 50 μg/mL; to E. coli a MIC of 50 μg/mL and MBC of 50 μg/mL; the P. aeruginosa strain revealed a MIC of 25 μg/mL and MBC= 0. The cytotoxicity in vitro was determined for metabolically incompetent human keratinocytes ability reduce 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide (MTT) and as result the UA didn’t present cellular cytotoxicity in a maximum concentration of 50 µg/mL. To cosmetic incorporation, a high stability emulsion was choice and as quality control technique of UA in this emulsion, an analytical methodology for HPLC was developed and validated, using green chemistry precepts. To evaluate the emulsion, a preliminary stability study was conducted, for the purpose evaluate the stability of UA in a stable emulsion, front of extreme conditions as 25, 40 e 5 ºC temperatures, indirect light and freezing/ thawing cycles. The emulsion containing UA demonstrated stability for the pH and viscosity characteristics, and about content, was more stable with 25 and 5 ºC. The UA, through in vitro assays performed demonstrated antioxidant, depigmenting and antimicrobial character, no cytotoxicity was observed in the used cells in the concentration range tested, besides showing some stability in the emulsion of choice. As noted, this multifunctional compound demonstrated in vitro characteristics of great interest for use as topical cosmetic active and possibly as an antimicrobial agent, thus further studies are needed to ensure its complete safety and efficacy of use.
Increased concern with body esthetics and health has raised the demand for skin cosmetics containing actives that prevent aging and other skin changes. Ursolic acid (UA), a natural active extracted from plants as rosemary and basil, has shown an interesting compound for cosmetic incorporation, to possess antioxidant activity, depigmenting, antimicrobial property, among others. This study aimed to evaluate the possibility of using UA as a multifunctional cosmetic compound through the UA efficacy and cytotoxicity in vitro, and to prepare and evaluate an emulsion containing the substance. Antioxidant potential was tested using inhibition methods of radical 1,1-diphenyl-2-picrilhidrazila (DPPH•) and radical 2,2 -azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS•+) showing antioxidant activity, with IC50= 235,61 µg/mL for DPPH• technique and 107,17 µg/mL to ABTS•+ method. The depigmenting activity was given by inhibition of the enzyme tyrosinase with3,4- dihydroxy-L-phenylalanine (L-DOPA) substrate and show IC50 de 338,00 µg/mL. For evaluation of antimicrobial activity, many strains were tested from the minimum inhibitory concentration (MIC) determined by dilution method on microplates, and minimum bactericidal concentration (MBC) by Petri dish replication, following M100-S16 supplement CLSI. The UA demonstrated antimicrobial activity, with a MIC to S. aureus strain of 25 μg/mL and MBC of 50 μg/mL; to S. epidermides a MIC of 25 μg/mL and MBC of 50 μg/mL; to E. coli a MIC of 50 μg/mL and MBC of 50 μg/mL; the P. aeruginosa strain revealed a MIC of 25 μg/mL and MBC= 0. The cytotoxicity in vitro was determined for metabolically incompetent human keratinocytes ability reduce 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide (MTT) and as result the UA didn’t present cellular cytotoxicity in a maximum concentration of 50 µg/mL. To cosmetic incorporation, a high stability emulsion was choice and as quality control technique of UA in this emulsion, an analytical methodology for HPLC was developed and validated, using green chemistry precepts. To evaluate the emulsion, a preliminary stability study was conducted, for the purpose evaluate the stability of UA in a stable emulsion, front of extreme conditions as 25, 40 e 5 ºC temperatures, indirect light and freezing/ thawing cycles. The emulsion containing UA demonstrated stability for the pH and viscosity characteristics, and about content, was more stable with 25 and 5 ºC. The UA, through in vitro assays performed demonstrated antioxidant, depigmenting and antimicrobial character, no cytotoxicity was observed in the used cells in the concentration range tested, besides showing some stability in the emulsion of choice. As noted, this multifunctional compound demonstrated in vitro characteristics of great interest for use as topical cosmetic active and possibly as an antimicrobial agent, thus further studies are needed to ensure its complete safety and efficacy of use.
Descrição
Palavras-chave
ácido ursólico (AU), antioxidante, despigmentante, antimicrobiana, citotóxico, CLAE, Ursolic acid (UA), Tyrosinase