A simple method for the quantitative analysis of tyrosol by hplc in liquid Czapek Cultures from endophytic fungi

Short Report J. Braz. Chem. Soc., Vol. 20, No. 1, 188-194, 2009. Printed in Brazil - ©2009 Sociedade Brasileira de Química 0103 - 5053 $6.00+0.00 A Simple Method for the Quantitative Analysis of Tyrosol by HPLC in Liquid Czapek Cultures from Endophytic Fungi Denise O. Guimarães,a Keyller B. Borges,b Pierina S. Bonatob and Mônica T. Pupo*,a Departamento de Ciências Farmacêuticas and bDepartamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903 Ribeirão Preto-SP, Brazil a O tirosol é provavelmente uma molécula sinalizadora em fungos endofíticos. A análise do tirosol em cultura líquida Czapek de fungo endofítico foi realizada através de cromatografia líquida de alta eficiência acoplada a detector por arranjo de diodos. As análises foram obtidas em sistema de fase móvel utilizando gradiente, modo linear, iniciando em acetonitrila/água (1:9 v/v) e terminando em acetonitrila 100% em 30 minutos com vazão de 1 mL min-1. Coluna analítica ZORBAX® ODS (250 × 4,6 mm, 5 µm) à 25 °C foi utilizada. Extração líquido-líquido de 0,5 mL do meio (pH 7,0) com acetato de etila e injeção de 20 µL após concentração do solvente sob ar comprimido originou bons resultados. Os parâmetros validados foram: linearidade 0,0125-5,0 µg mL-1 (r = 0,9967), limite de quantificação 0,0125 µg mL-1 obtidos pela média das análises; %CV (precisão) e %E (exatidão) com valores abaixo de 15% e recuperação de cerca de 80%. Além disso, o método desenvolvido apresentou valores de validação satisfatórios demonstrando eficiência na análise do tirosol em meio líquido Czapek. Tyrosol is a possible quorum sensing molecule in endophytic fungi. High-performance liquid chromatography (HPLC) coupled with diode array detector (DAD) was used for the analysis of tyrosol in liquid Czapek fungal cultures. The optimized conditions were gradient mobile phase, in linear mode, consisting initially of acetonitrile/water (1:9 v/v) and increasing up to acetonitrile (100%) in 30 minutes at a flow rate of 1 mL min-1. The column used was a ZORBAX ODS (250 × 4.6 mm, 5 µm) at 25 oC. Liquid-liquid extraction of 0.5 mL medium (pH 7.0) with ethyl acetate and injection of 20 μL after solvent evaporation under air flow gave good results. Some validation parameters obtained were: linearity 0.0125-5.0 μg mL-1 medium (r = 0.9967), quantification limit of 0.0125 μg mL-1 medium, %CV (precision) and %E (accuracy) bellow 15% and recovery around 80%. Therefore, the developed method presented satisfactory validation parameters and it was efficient for the analysis of tyrosol in Czapek medium. Keywords: tyrosol, endophytic fungi, HPLC-DAD, validation method Introduction Tyrosol (2-(4-Hydroxyphenyl)ethanol) is a wellknown phenolic compound with antioxidant properties that is present in wine and olive oil,1 and it is reported to have scavenging effects on reactive oxygen and nitrogen species that are implicated in human pathologies such as cardiovascular and thrombotic diseases.2,3 Tyrosol is produced by terrestrial fungi and showed antifungal activity against Lagenidium callinectes4 and Gibberella pulicaris.5 Recently, tyrosol has been reported as a candidate to be used in stroke therapy due to its neuroprotective effect in rats.1 Moreover, tyrosol was identified as an autoregulatory *e-mail: molecule with important implication on the dynamics of growth and morphogenesis in Candida albicans6 in a process known as quorum-sensing, which is characterized by a cellular density-dependent phenomenon. Quorumsensing effect is accomplished by the extracellular accumulation of small, self-generated chemical signaling molecules that induce bacterial population to produce the desired phenotypic effect.7 The first described quorumsensing system involved the bioluminescent marine bacterium Vibrio fischeri.8 Several chemical classes of microbial derived signaling molecules have been identified, and they might be classified in two main categories: (i) amino acids and short peptides, commonly utilized by Gram-positive bacteria, 9,10 and (ii) fatty acid derivatives, frequently utilized by Gram- Vol. 20, No. 1, 2009 Guimarães et al. negative bacteria. 11,12 Particular emphasis has been placed on the wide range of quorum-sensing systems that employ N-acyl homoserine lactones (acyl HSLs) as the signaling molecules that control the expression of diverse physiological functions.12 Several examples of signaling molecules illustrate that quorum-sensing molecules have been used by microorganisms, especially bacteria, in order to control a great variety of functional systems.13 Studies of bacterial quorum-sensing phenomenon have shown information on how bacterial chemical communication works; how chemical information is integrated, processed and transduced to control gene expression; how intra- and inter species cell-cell communication is accomplished and the intriguing possibility of prokaryote-eukaryote crosscommunication. In fungi this phenomenon has been mostly studied in Candida albicans. Due to its scavenging effects, tyrosol has been previously quantified in different matrices such as beverages14 and biological fluids (low-density-lipoprotein)15 using different analytical quantitative methods such as HPLC-DAD and HPLC-ESI-MS-MS. In our prospection study of endophytic fungi from Asteraceae species we have isolated tyrosol from several bioactive endophytic cultures. So, we hypothesized it might have some quorum-sensing role in those microorganisms. In order to check this possibility, we initially set about developing a high-performance liquid chromatography (HPLC) coupled with diode array detector (DAD) method for the analysis of tyrosol in endophytic fungi cultures. Experimental 189 times (150 mL each). The EtOAc crude extract (88.0 mg) was fractionated in a silica gel column (0.063-0.200 mm) with hexane/EtOAc (9:1 v/v); hexane/EtOAc (1:1 v/v); EtOAc and methanol. The sub fraction 25 (6.4 mg), obtained with hexane/EtOAc (1:1 v/v), was submitted to preparative thin layer chromatography in silica gel PF254 eluted with dichloromethane/methanol (9:1 v/v) yielding tyrosol (rf: 0.40, 5.0 mg). Tyrosol was extracted with acetone/methanol (4:1 v/v).18 NMR spectra were acquired in Bruker spectrometers (DRX-400 and DRX500), working at 400 and 500 MHz for 1H and at 100 and 125 MHz for 13C. The spectra were recorded in CDCl3, and the solvent signals at d 7.26 for proton, and d 77.0 for carbon, were used as reference. Mass spectra analysis was conducted in a mass spectrometer ESI-MS (Micromass Quattro LC-electrospray ionization). Chromatograms area analyses were compared in order to check the purity of isolated tyrosol and available commercial tyrosol 98% (Sigma-Aldrich Chemie, Steinheim, Germany). The purity index was 95.4% for isolated tyrosol used as standard for the quantitative validation procedures. Tyrosol stock standard solutions were prepared in methanol at concentrations of 0.250, 0.500, 1.0, 5.0, 20.0 (...truncated)