In vitro evaluation of the cytotoxicity and eye irritation potential of preservatives widely used in cosmetics

Abstract The consumption of cosmetics has been increasing every year and is expected to reach $675 billion by 2020 at an estimated growth rate of 6.4% per year. Exposure to skin irritants is the major cause of non-immunological inflammation of the skin. Therefore, the safety evaluation of cosmetic preservatives should be increased. Thus, the present work aimed to evaluate the cytotoxicity as the viability endpoint and the eye irritation potential of preservatives used in cosmetics. Cytotoxicity assays were performed using MTT and NRU in human keratinocytes (HaCaT), human dermal fibroblasts, adult (HDFa), and human hepatoma cells (HepG2). The eye irritation potential was evaluated using the Hen's Egg Test-chorioallantoic membrane (HET-CAM). The evaluated preservatives were methylparaben (MP), propylparaben (PP), phenoxyethanol (PE), and a mixture of methylchloroisothiazolinone and methylisothiazolinone (CMI/MI). All preservatives showed cytotoxic potential within the permitted concentrations for use in cosmetic products. In the HET-CAM test, PE and CMI/MI, MP, and PP were classified as severe, moderate, and poor irritants, respectively. Our results indicate that proper safety evaluations are required to ensure the beneficial properties of preservatives on cosmetic products without exceeding exposure levels that would result in adverse health effects for consumers.

New and potential properties, characteristics, and analytical methods of ferulic acid: A review

Abstract Phenolic compounds are widely distributed in the plant kingdom and in the microorganisms. Cinnamic acid and its hydroxylated derivative-ferulic acid, are phenolic compounds. Ferulic acid possesses antioxidant potential, as well as anti-cancer, anti-inflammatory, and antimicrobial properties. It prevents the harmful effects of radiation both as an ultraviolet absorber and as a free radical scavenger; it is not cytotoxic. Although ferulic acid has beneficial properties, it is hardly used in cosmetic preparations and has been rarely studied in the literature. Herein, we review the literature on ferulic acid, to provide information which can contribute to further research on the compound.

Integrated approaches to testing and assessment as a tool for the hazard assessment and risk characterization of cosmetic preservatives.

The safety assessment of cosmetic products is based on the safety of the ingredients, which requires information on chemical structures, toxicological profiles, and exposure data. Approximately 6% of the population is sensitized to cosmetic ingredients, especially preservatives and fragrances. In this context, the aim of this study was to perform a hazard assessment and risk characterization of benzalkonium chloride (BAC), benzyl alcohol (BA), caprylyl glycol (CG), ethylhexylglycerin (EG), chlorphenesin (CP), dehydroacetic acid (DHA), sodium dehydroacetate (SDH), iodopropynyl butylcarbamate (IPBC), methylchloroisothiazolinone and methylisothiazolinone (MCI/MIT), methylisothiazolinone (MIT), phenoxyethanol (PE), potassium sorbate (PS), and sodium benzoate (SB). Considering the integrated approaches to testing and assessment (IATA) and weight of evidence (WoE) as a decision tree, based on published safety reports. The hazard assessment was composed of a toxicological matrix correlating the toxicity level, defined as low (L), moderate (M), or high (H) and local or systemic exposure, considering the endpoints of skin sensitization, skin irritation, eye irritation, phototoxicity, acute oral toxicity, carcinogenicity, mutagenicity/genotoxicity, and endocrine activity. In a risk assessment approach, most preservatives had a margin of safety (MoS) above 100, except for DHA, SDH, and EG, considering the worst-case scenario (100% dermal absorption). However, isolated data do not set up a safety assessment. It is necessary to carry out a rational risk characterization considering hazard and exposure assessment to estimate the level of risk of an adverse health outcome, based on the concentration in a product, frequency of use, type of product, route of exposure, body surface location, and target population.

In vitro methods to determine the antioxidant activity of caffeic acid.

Caffeic acid (CA) is a plant metabolite acting as a carcinogenic inhibitor, and exhibits a high antioxidant effect and some antimicrobial activity. Besides, this compound can be useful in the prevention of heart diseases and atherosclerosis, among others. The present study aims to determine the in vitro antioxidant activity of CA in order to increase the frequency of its use and reliability in the prevention of damage caused by free radicals and other reactive species. The tests performed were as follows: Radical anion superoxide capture; crocin bleaching assay; capturing ability of hypochlorous acid; H2O2 capture; capturing capacity of the ABTS•+/DPPH•; and SOD-like activity. The values of the CA antioxidant activity were very close to the values of standards in all tests. Besides, CA presented an antioxidant activity greater than that of ascorbic acid and trolox, and its advantages include higher stability than ascorbic acid and extraction from natural sources, as opposed to trolox.

Release and permeation profiles of spray-dried chitosan microparticles containing caffeic acid.

Caffeic acid (CA), a phenolic compound found in plants with antioxidant and antimicrobial activity, induces collagen production and prevents premature aging of the skin. The objective of this study was to develop two types of chitosan microparticles (MP) containing CA and to relate the morphology with the release and permeation profiles. One type of MP was prepared from a hydroalcoholic solution (MPI) and the other from an aqueous solution (MPII). Their morphology and size was evaluated by high-resolution scanning electron microscopy. The release profile of CA was evaluated using the cellulose membrane from the two MPs in Franz diffusion cells and the permeation profile was evaluated using human abdominal skin samples; the epidermal membranes were prepared by the heat-separation technique. MPII was spherical with a smooth surface, suitable for the controlled release of substances, whereas MPI was porous with non-internalized residual material. This result was consistent with their release and permeation profiles because MPII exhibited a slower and more controlled release than MPI. Thus, the method of preparation of MP and their composition influence the release profile of CA. Therefore, the production conditions must be closely controlled.

Bacterial cellulose skin masks-Properties and sensory tests.

BACKGROUND: Bacterial cellulose (BC) is a versatile material produced by microorganisms in the form of a membranous hydrogel, totally biocompatible, and endowed with high mechanical strength. Its high water-holding capacity based on its highly porous nanofibrillar structure allows BC to incorporate and to release substances very fast, thus being suitable for the preparation of skincare masks. AIMS: The preparation and characterization of cosmetic masks based on BC membranes and active cosmetics. METHODS: The masks were prepared by the simple incorporation of the cosmetic actives into BC membranes, used as a swelling matrix. The masks were characterized by Fourier transform infrared (FTIR), scanning electron microscopy (SEM), sensory tests, and skin moisture tests on volunteers. RESULTS: The results of sensory tests revealed the good performance of BC, being considered effective by the panel of volunteers, specially for adhesion to the skin (7.7 at the score scale), and improvement of the skin moisture (the hydration effect increased 76% in 75% of the volunteers that used vegetable extract mask formulation [VEM]), or a decrease in skin hydration (80% of the volunteers showed 32.6% decrease on skin hydration using propolis extract formulation [PEM] treatment), indicating the BC nanofiber membranes can be used to skincare applications. CONCLUSION: The results demonstrate the BC can be used as an alternative support for cosmetic actives for skin treatment.

Guava: phytochemical composition of a potential source of antioxidants for cosmetic and/or dermatological applications

ABSTRACT Guava (Psidium guajava L.) is a native fruit of the American tropics with commercial applications for its taste, flavor and aroma. Numerous pharmacological uses have been described for it, such as the antiseptic effect of its leaves, the use of the fresh fruit and tea from its leaves for the treatment of diarrhea, dysentery, diabetes mellitus, and others. However, considering its rich composition, the guava also is a potential source of antioxidants to be used in the development of new formulations for cosmetic and/or dermatological applications, the main focus of this research. Herein, we describe the study of the phytochemical composition and the antioxidant activity of a guava extract prepared with non-toxic solvents aiming its use at biological applications. High performance liquid chromatography and mass spectrometry were employed to identify the major components, while thermoanalytical measurements and hot stage microscopy were used to assess the chemical stability of guava fruit extract. The antioxidant activity was also evaluated assessing the SOD-like activity and ABTS free radical scavenger. The results show that the extract is a rich source of phenolic compounds, such as quercetin, kaempferol, schottenol, among many others. All of the components found in guava extract exhibit biological effects according to the literature data, mainly antioxidant properties.

Validation of HPLC-UV Assay of Caffeic Acid in Emulsions.

An accurate, sensitive, precise and rapid reversed-phase high-performance liquid chromatographic method was successfully developed and validated for the determination of caffeic acid (CA) in emulsions. The best separation was achieved on a 250 × 4.6 mm, 5.0 µm particle size RP18 XDB Waters column using ethanol and purified water (40:60 v/v) adjusted to pH 2.5 with acetic acid as the mobile phase at a flow rate of 0.7 mL/min. Ultraviolet detection was performed at 325 nm at ambient column temperature (25°C). The method was linear over the concentration range of 10-60 µg/mL (r(2) = 0.9999) with limits of detection and quantification of 1.44 and 4.38 µg/mL, respectively. CA was subjected to oxidation, acid, base and neutral degradation, as well as photolysis and heat as stress conditions. There were no interfering peaks at or near the retention time of CA. The method was applied to the determination of CA in standard and pharmaceutical products with excellent recoveries. The method is applicable in the quality control of CA.

Antioxidant Activity and Validation of Quantification Method for Lycopene Extracted from Tomato.

Lycopene is a carotenoid found in tomatoes with potent antioxidant activity. The aim of the study was to obtain an extract containing lycopene from four types of tomatoes, validate a quantification method for the extracts by HPLC, and assess its antioxidant activity. Results revealed that the tomatoes analyzed contained lycopene and antioxidant activity. Salad tomato presented the highest concentration of this carotenoid and antioxidant activity. The quantification method exhibited linearity with a correlation coefficient of 0.9992. Tests for the assessment of precision, accuracy, and robustness achieved coefficients with variation of less than 5%. The LOD and LOQ were 0.0012 and 0.0039 µg/mL, respectively. Salad tomato can be used as a source of lycopene for the development of topical formulations, and based on performed tests, the chosen method for the identification and quantification of lycopene was considered to be linear, precise, exact, selective, and robust.

Rheology as a Tool to Predict the Release of Alpha-Lipoic Acid from Emulsions Used for the Prevention of Skin Aging.

The availability of an active substance through the skin depends basically on two consecutive steps: the release of this substance from the vehicle and its subsequent permeation through the skin. Hence, studies on the specific properties of vehicles, such as their rheological behavior, are of great interest in the field of dermatological products. Recent studies have shown the influence of the rheological features of a vehicle on the release of drugs and active compounds from the formulation. In this context, the aim of this study was to evaluate the influence of the rheological features of two different emulsion formulations on the release of alpha-lipoic acid. Alpha-lipoic acid (ALA) was chosen for this study because of its antioxidant characteristics, which could be useful for the prevention of skin diseases and aging. The rheological and mechanical behavior and the in vitro release profile were assayed. The results showed that rheological features, such as viscosity, thixotropy, and compliance, strongly influenced the release of ALA from the emulsion and that the presence of a hydrophilic polymer in one of the emulsions was an important factor affecting the rheology and, therefore, the release of ALA.

Development and evaluation of an emulsion containing lycopene for combating acceleration of skin aging

Lycopene, a carotenoid and potent antioxidant is found in large quantities in tomatoes. Lycopene combats diseases, such as cardiovascular disease and different types of cancer, including prostate cancer. However, its topical use in emulsion form for the combat of skin aging is under-explored. The aim of the present study was to develop an emulsion containing lycopene extracted from salad tomatoes and evaluate its cytotoxicity, stability, rheological behavior, antioxidant activity and phytocosmetic permeation. The developed cosmetic comprised an oil phase made up of shea derivatives and was evaluated in terms of its physiochemical stability, spreadability, thermal analysis, rheological behavior, microbiological quality, cytotoxicity, antioxidant activity, cutaneous permeation and retention. The results demonstrate that this phytocosmetic is stable, exhibits satisfactory rheological behavior for a topical formula and is a promising product for combating skin aging.

Licopeno é um carotenóide com potente atividade antioxidante encontrado em grande quantidade no tomate e usado no combate a diversas doenças como doenças cardiovasculares e diferentes tipos de cânceres, incluindo o câncer de próstata. O objetivo desse trabalho foi desenvolver uma emulsão contendo extrato de licopeno obtido do tomate salada e avaliar a citotoxicidade do extrato, a estabilidade, o comportamento reológico, atividade antioxidante e permeação do fitocosmético. O cosmético foi desenvolvido utilizando fase oleosa contendo derivados de Karité e submetido à avaliação da estabilidade físico-química, espalhabilidade, análise térmica, comportamento reológico, qualidade microbiológica, citotoxicidade, atividade antioxidante e testes de permeação e retenção cutânea. Os resultados demonstraram que o fitocosmético é estável, apresenta comportamento reológico desejável para uma formulação tópica e é um produto promissor para ser utilizado no combate à aceleração do envelhecimento cutâneo.

Synthesis and tripanocidal activity of ferrocenyl and benzyl diamines against Trypanosoma brucei and Trypanosoma cruzi.

Trypanosoma brucei and Trypanosoma cruzi are the etiologic agents of sleeping sickness and Chagas disease, respectively, two of the 17 preventable tropical infectious diseases (NTD) which have been neglected by governments and organizations working in the health sector, as well as pharmaceutical industries. High toxicity and resistance are problems of the conventional drugs employed against trypanosomiasis, hence the need for the development of new drugs with trypanocidal activity. In this work we have evaluated the trypanocidal activity of a series of N1,N2-dibenzylethane-1,2-diamine hydrochlorides (benzyl diamines) and N1-benzyl,N2-methyferrocenylethane-1,2-diamine hydrochlorides (ferrocenyl diamines) against T. brucei and T. cruzi parasite strains. We show that incorporation of the ferrocenyl group into the benzyl diamines increases the trypanocidal activity. The molecules exhibit potential trypanocidal activity in vitro against all parasite strains. Cytotoxicity assay was also carried out to evaluate the toxicity in HepG2 cells.

Estudo da segurança de cosméticos: presente e futuro / Study of the safety of cosmetics: present and future

Cosméticos são usados pelo homem desde a antiguidade e, atualmente, o consumo tem aumentado muito, inclusive no Brasil, o terceiro maior mercado no mundo. Assim sendo, a preocupação com a segurança e eficácia destes produtos deve ser intensificada, mesmo sendo produtos raramente relacionados a reações adversas que causem danos à saúde. Para a avaliação das possíveis reações que podem ser apresentadas pelos produtos (irritação, sensibilização, efeitos sistêmicos), a Legislação Brasileira exige que os fabricantes avaliem a segurança e eficácia de seus produtos. Para este fim, em geral, são utilizados animais como modelo experimental o que está sendo cada vez mais evitado, acarretando a pesquisa por métodos alternativos para estas avaliações, que não necessitem de modelos experimentais in vivo. Desta forma, a presente revisão tem por objetivo apresentar os principais ensaios biológicos utilizados para avaliação da segurança de cosméticos, bem como ensaios in vitro que os substituam.

Cosmetics have been used since ancient times and, recently, their consumption has increased greatly in many countries, including brazil, which is the third largest consumer market in the world. Thus, concern for the safety and efficacy of these products should be heightened, even though these products are rarely related to adverse reactions that damage the health. Brazilian law requires manufacturers to subject their products to safety testing, to assess the possible reactions that could be caused by them (irritation, sensitization, systemic effects). To this end, in general, animals have been used as the experimental model, but this practice is being increasingly controlled, so that the scientific community is looking for alternative tests that do not require experimental in vivo models. Thus, this review aims to describe the main biological assays used to assess the safety of cosmetics, as well as in vitro assays that can replace them.

Design of antiseptic formulations containing extract of Plinia cauliflora / Concepção de formulações anti-sépticas que contenham extrato de Plinia cauliflora

The leaves of the Brazilian species Plinia cauliflora were used to obtain active hydroalcoholic extract and fractions enabling the development of efficient antiseptic pharmaceutical formulations. A chemical composition of 70 percent ethanol extract, aqueous and ethyl acetate fractions was analyzed by thin-layer chromatography and for phenol content. Antimicrobial activity was tested against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Lactobacillus acidophilus and Candida albicans by the agar diffusion method and the minimum inhibitory concentration was assayed by broth microdilution. Extract microbiological quality was tested to avoid contamination in the formulations. A mouthwash and a topical cream containing the extract were developed and antiseptic activity was assessed by agar diffusion. Sensory and physicochemical stability of the formulations were assayed. Chromatography indicated the presence of terpenes, flavonoids and tannins in the extract and fractions and total phenol content were found to be high. The plant samples were active against all the microorganisms tested, except for Lactobacillus acidophilus. Both topical formulations showed antiseptic activity and stability. Thus, these may be used as antimicrobials in skin infections, but would be more useful in the treatment of candidiasis.

As folhas da espécie brasileira Plinia cauliflora foram utilizadas a fim de se obter um extrato hidroalcoólico e frações ativas proporcionando o desenvolvimento de eficazes formulações farmacêuticas antissépticas. A composição química do extrato etanólico 70 por cento, fração aquosa e acetato de etila foi analisada por cromatografia em camada delgada e teor de fenóis. A atividade antimicrobiana foi testada frente a Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Lactobacillus acidophilus e Candida albicans por difusão em ágar e a concentração inibitória mínima foi determinada por microdiluição. A qualidade microbiológica do extrato foi avaliada para evitar a contaminação das formulações. Foram desenvolvidos um enxaguatório bucal e um creme tópico contendo o extrato sendo que a atividade antisséptica foi ensaiada por difusão em ágar. A estabilidade sensorial e físico-química foram testadas. A cromatografia indicou a presença de terpenos, flavonóides e taninos no extrato e frações, observando-se alto teor de fenóis totais. As amostras vegetais foram ativas contra todos os micro-organismos testados, exceto Lactobacillus acidophilus. Ambas as formulações apresentaram atividade antisséptica e estabilidade. Desta forma, infere-se que as formulações desenvolvidas podem ser utilizadas como antissépticas em infecções de pele, podendo ser mais eficazes no tratamento de candidíase.