MDGC-MS analysis of essential oils from Protium heptaphyllum (Aubl. ) and their antifungal activity against Candida specie / Análise MDGC-MS de óleos essenciais de Protium heptaphyllum (Aubl. ) e sua atividade antifúngica contra espécies de Candida

ABSTRACT Protium heptaphyllum is found in the Amazon region, and in various Brazilian states and South American countries. Also Known as almecega, it produces an oil resin used in traditional medicine as analgesic, anti-inflammatory, cicatrizant and expectorant, it is rich in pentacyclic triterpenes and essential oil. The main objective of this study was to analyze the chemical composition of P. heptaphyllumresin (OEPh) over different extraction times and to evaluate their antifungal activity against Candida species, obtained from gardeners with onychomycosis, using the disk diffusion method. The OEPh was obtained by hydrodistillation and analyzed by Multidimensional Gas Chromatography coupled with Mass Spectrometry (MDGC / MS). Candida species were obtained from lesions on the nails of horticulturist from a community garden in the city of Teresina, Piauí, Brazil. The antifungal activity in concentrations of 1000 µg/L, 500 µg/L and 250 µg/L, PROTOCOL M44-A2 (CLSI 2009) OEPh was tested. The main constituents identified were: l-limonene, α-terpineol, p-cineol, o-cymene and α-phellandrene, however, its composition varies significantly with extraction time. All species, except C. rugosa, were inhibited with halo (≥ 14 mm) at 1000 μg / L. C. krusei is naturally resistant to the drug fluconazole, but when tested with OEPh the clinical species (case 9) demonstrated sensitivity in three dilutions (halo ≤ 10 ≥ 14) and the standard strain was inhibited at concentration of 1000 μg/Lg / L (halo 14mm). A similar situation also occurred with the standard strain of C. parapsilosis (halo ≥ 11mm). OEPh has considerable antifungal activity, which merits further investigation for alternative clinical applications, since this species is widely distributed in our community, and it presents good yields, and also has important therapeutic applications.

RESUMO Protium heptaphyllum é encontrada na região amazônica, em vários estados do Brasil e países da América do Sul. Conhecida como almecega produz uma resina oleosa usada na medicina popular como analgésica, antiinflamatória, cicatrizante e expectorante, é rica em triterpenos pentaciclicos e óleo essencial. O objetivo principal do presente trabalho foi analisar a composição química do óleo essencial da resina P. heptaphyllum (OEPh) em diferentes tempo de extração e avaliarsuaatividade antifúngica contra espécies de Candida, isoladas de horticultores com onicomicoses, por método de disco-difusão. O OEPh foi obtido por hidrodestilação, analisado por Cromatografia Gasosa Multidimensinal Acoplada a Espectrometria de Massas (MDGC/MS). As espécies de Candida foram obtidas de lesões nas unhas de horticultores de uma horta comunitária na cidade de Teresina, Piauí, Brasil. Testou-se a atividade antifúngica do OEPhnas concentrações de 1000 μg/L, 500 μg/L e 250 μg/L, protocolo M44-A2 (CLSI 2009). Os principais constituintes identificados foram l- limoneno, α-terpineol, p-cineol, o-cimeno e α-felandreno, entretanto, sua composição varia significativamente em decorrência do tempo de extração. Todas as espécies, exceto a C. rugosa, foram inibidas com halo ( Χ ≥ 14 mm) na concentração de 1000 μg/L. C. krusei é naturalmente resistente ao fármaco fluconazol, mas quando testado com OEPh,a espécie clínico (caso 9) demonstrou sensibilidade nas três diluições (halo Χ ≤ 10 ≥ 14) e a cepa padrão foi inibida na concentração de 1000 μg/L (halo Χ 14mm). Fato semelhante também ocorreu com a cepa padrão de C. parapsilosis (halo Χ ≥ 11mm). O OEPh possui atividade antifúngica considerável, merecendo uma investigação mais aprofundada para aplicações clínicas alternativas, uma vez que esta espécie é amplamente distribuída em nossa comunidade, apresenta bom rendimento e, ainda, aplicações terapêuticas importantes.

Objective determination of Fitzpatrick skin type.

The overall aim of this Ph.D. project was to clarify what the subjective Fitzpatrick skin type represents with regard to the skin's reaction to UVR. Fitzpatrick skin type is used as an expression of the constitutive UV-sensitivity. It has been used for guiding dose-levels in phototherapy and is an important risk factor for skin cancer. The subjective Fitzpatrick skin type and the measured skin type PPF (pigment protection factor, calculated based on a skin reflectance measurement, predicts the UV-dose (SED) to give 1 MED) were investigated parallelly in relation to the clinically determined dose to erythema (MED) and/or pigmentation (MMD) to determine which one related best. PPF is an established method for assessing UV-sensitivity by predicting SED to MED. UV-dose to MED and/or MMD was determined after single UV-exposure to Solar Simulator on nates (n= 84) and after single and multiple (5, 6 or 12) UV-exposures (n = 24-62) on the back to four UV-sources (nUVB, Solar, bUVA and UVA1). SED to MMD was also related to wavelength. MED was only determined after a single and four UV-exposures to narrowband UVB (nUVB) and Solar Simulator (Solar). Volunteers with a broad range of constitutive pigmentation (skin types I-V) were included. Equal MMD doses (predetermined after a single UV-exposure) were used at the multiple exposures. The absolute increase in pigmentation after 6 and 12 UV-exposures, where steady-state pigmentation was reached, was independent of skin type and therefore could not enter into the calculations. But it proved that the MMD determinations after single exposure were correct and could be used at multiple UV-exposures. In contrary to what we expected, our results indicate that people may refer to the constitutive pigmentation, when they reply to the question of Fitzpatrick skin type. This applied to both erythema and pigmentation response as both dose to MED and MMD showed a better correlation to nates than to the back. As expected, our results from the back indicate that people seem to refer to sun sensitivity after multiple exposures to the sun rather than a single sun exposure, when they reply to the question of Fitzpatrick skin type. Hence, both SED to MED and SED to MMD are better correlated to skin type after respectively 4 and 5 exposures to Solar Simulator and nUVB compared to 1 exposure. Only when tanning is preceded by erythema there is a relation between SED to MMD and skin type/PPF. Thus only after nUVB and Solar. For nUVB and Solar there was a linear relation between erythema and tanning ability with the intercept different from zero. In spite of what we expected based on the literature, the correlation was better between SED to MED and skin type than between SED to MMD and skin type. This applied to single and multiple exposures and to single calculations and multiple regression analyses. The long-waved UVA1 and broadband UVA should definitely not be used for skin type determination, as there was no relation between MMD and skin type/PPF. Both nUVB and Solar can be considered. Finally, based on the objective parameters: pre-exposure pigmentation measured by skin reflectance, MED and MMD we tried to predict the Fitzpatrick skin type by multinominal logistic regression analyses to evaluate the significance of the different parameters for the subjective skin type classification and thereby hopefully enlighten what Fitzpatrick skin type represents. For single UV-exposure only the pre-exposure pigmentation worked as a predictor of Fitzpatrick skin type, and that is what PPF is based on. When this parameter was removed, only SED to MED was significant. Our model succeeds better to classify people correct after multiple UV-exposure compared to a single UV-exposure. PPF was predicted likewise and was highly correlated to SED to MED, as expected, and even higher correlated to Fitzpatrick skin type. SED to MMD was not significant. This study confirms that Fitzpatrick skin type is an unreliable predictor of UV-sensitivity with regard to MED- and MMD test. Fitzpatrick skin type in epidemiological context (risk for skin cancer) stands for burns and ability to tan may represent "cumulative" dose. SED to MED is equivalent to burns. PPF may also indirectly represent cumulative dose--the less pigmented the skin the more UVR penetrates the epidermis and will be able to accumulate and induce skin cancer. Our results indicate that Fitzpatrick skin type predominantly is determined by the skin pigmentation and that the second most important objective parameter is SED to MED (and not SED to MMD). This explains why Fitzpatrick skin type, eventhough being an unreliable predictor of UV-sensitivity, still plays an important role in epidemiology with regard to estimation of risk of skin cancer. This study showed that PPF can predict the UV-sensitivity also with regard to the tanning ability (MMD), can be applied to multiple UV-exposures and to a broader pigmentation spectrum. PPF is preferred to predict the individual UV-sensitivity rather than the subjective Fitzpatrick skin type, confirmed for both nates and back, single as well as repetitive UV-exposures. It should therefore be considered to concentrate on skin reflectance measurements.