Universidde de São Pulo Bibliotec Digitl d Produção Intelectul - BDPI Deprtmento de Físic e Químic - FCFRP/DFQ Artigos e Mteriis de Revists Científics - FCFRP/DFQ 2009 A simple method for the quntittive nlysis of tyrosol by hplc in liquid Czpek Cultures from endophytic fungi Journl of the Brzilin Chemicl Society, v.20, n.1, p.188-194, 2009 http://produco.usp.br/hndle/bdpi/5846 Downloded from: Bibliotec Digitl d Produção Intelectul - BDPI, Universidde de São Pulo
J. Brz. Chem. Soc., Vol. 20, No. 1, 188-194, 2009. Printed in Brzil - 2009 Sociedde Brsileir de Químic 0103-5053 $6.00+0.00 Short Report A Simple Method for the Quntittive Anlysis of Tyrosol by HPLC in Liquid Czpek Cultures from Endophytic Fungi Denise O. Guimrães, Keyller B. Borges, b Pierin S. Bonto b nd Mônic T. Pupo*, Deprtmento de Ciêncis Frmcêutics nd b Deprtmento de Físic e Químic, Fculdde de Ciêncis Frmcêutics de Ribeirão Preto, Universidde de São Pulo, 14040-903 Ribeirão Preto-SP, Brzil O tirosol é provvelmente um molécul sinlizdor em fungos endofíticos. A nálise do tirosol em cultur líquid Czpek de fungo endofítico foi relizd trvés de cromtogrfi líquid de lt eficiênci copld detector por rrnjo de diodos. As nálises form obtids em sistem de fse móvel utilizndo grdiente, modo liner, inicindo em cetonitril/águ (1:9 v/v) e terminndo em cetonitril 100% em 30 minutos com vzão de 1 ml min -1. Colun nlític ZORBAX ODS (250 4,6 mm, 5 µm) à 25 C foi utilizd. Extrção líquido-líquido de 0,5 ml do meio (ph 7,0) com cetto de etil e injeção de 20 µl pós concentrção do solvente sob r comprimido originou bons resultdos. Os prâmetros vliddos form: lineridde 0,0125-5,0 µg ml -1 (r = 0,9967), limite de quntificção 0,0125 µg ml -1 obtidos pel médi ds nálises; %CV (precisão) e %E (extidão) com vlores bixo de 15% e recuperção de cerc de 80%. Além disso, o método desenvolvido presentou vlores de vlidção stisftórios demonstrndo eficiênci n nálise do tirosol em meio líquido Czpek. Tyrosol is possible quorum sensing molecule in endophytic fungi. High-performnce liquid chromtogrphy (HPLC) coupled with diode rry detector (DAD) ws used for the nlysis of tyrosol in liquid Czpek fungl cultures. The optimized conditions were grdient mobile phse, in liner mode, consisting initilly of cetonitrile/wter (1:9 v/v) nd incresing up to cetonitrile (100%) in 30 minutes t flow rte of 1 ml min -1. The column used ws ZORBAX ODS (250 4.6 mm, 5 µm) t 25 o C. Liquid-liquid extrction of 0.5 ml medium (ph 7.0) with ethyl cette nd injection of 20 μl fter solvent evportion under ir flow gve good results. Some vlidtion prmeters obtined were: linerity 0.0125-5.0 μg ml -1 medium (r = 0.9967), quntifiction limit of 0.0125 μg ml -1 medium, %CV (precision) nd %E (ccurcy) bellow 15% nd recovery round 80%. Therefore, the developed method presented stisfctory vlidtion prmeters nd it ws efficient for the nlysis of tyrosol in Czpek medium. Keywords: tyrosol, endophytic fungi, HPLC-DAD, vlidtion method Introduction Tyrosol (2-(4-Hydroxyphenyl)ethnol) is wellknown phenolic compound with ntioxidnt properties tht is present in wine nd olive oil, 1 nd it is reported to hve scvenging effects on rective oxygen nd nitrogen species tht re implicted in humn pthologies such s crdiovsculr nd thrombotic diseses. 2,3 Tyrosol is produced by terrestril fungi nd showed ntifungl ctivity ginst Lgenidium cllinectes 4 nd Gibberell pulicris. 5 Recently, tyrosol hs been reported s cndidte to be used in stroke therpy due to its neuroprotective effect in rts. 1 Moreover, tyrosol ws identified s n utoregultory *e-mil: mtpupo@fcfrp.usp.br molecule with importnt impliction on the dynmics of growth nd morphogenesis in Cndid lbicns 6 in process known s quorum-sensing, which is chrcterized by cellulr density-dependent phenomenon. Quorumsensing effect is ccomplished by the extrcellulr ccumultion of smll, self-generted chemicl signling molecules tht induce bcteril popultion to produce the desired phenotypic effect. 7 The first described quorumsensing system involved the bioluminescent mrine bcterium Vibrio fischeri. 8 Severl chemicl clsses of microbil derived signling molecules hve been identified, nd they might be clssified in two min ctegories: (i) mino cids nd short peptides, commonly utilized by Grm-positive bcteri, 9,10 nd (ii) ftty cid derivtives, frequently utilized by Grm-
Vol. 20, No. 1, 2009 Guimrães et l. 189 negtive bcteri. 11,12 Prticulr emphsis hs been plced on the wide rnge of quorum-sensing systems tht employ N-cyl homoserine lctones (cyl HSLs) s the signling molecules tht control the expression of diverse physiologicl functions. 12 Severl exmples of signling molecules illustrte tht quorum-sensing molecules hve been used by microorgnisms, especilly bcteri, in order to control gret vriety of functionl systems. 13 Studies of bcteril quorum-sensing phenomenon hve shown informtion on how bcteril chemicl communiction works; how chemicl informtion is integrted, processed nd trnsduced to control gene expression; how intr- nd inter species cell-cell communiction is ccomplished nd the intriguing possibility of prokryote-eukryote crosscommuniction. In fungi this phenomenon hs been mostly studied in Cndid lbicns. Due to its scvenging effects, tyrosol hs been previously quntified in different mtrices such s beverges 14 nd biologicl fluids (low-density-lipoprotein) 15 using different nlyticl quntittive methods such s HPLC-DAD nd HPLC-ESI-MS-MS. In our prospection study of endophytic fungi from Astercee species we hve isolted tyrosol from severl bioctive endophytic cultures. So, we hypothesized it might hve some quorum-sensing role in those microorgnisms. In order to check this possibility, we initilly set bout developing high-performnce liquid chromtogrphy (HPLC) coupled with diode rry detector (DAD) method for the nlysis of tyrosol in endophytic fungi cultures. Experimentl Stndrd solutions nd chemicls Tyrosol utilized for the preprtion of stndrd solutions ws obtined from the cultivtion of the endophytic fungus Glomerell cingult (Stonemn) Spuld. & H. Schrenk (code VA1) found in ssocition with the host plnt Viguier renri (Astercee). The mycelium of G. cingult grown on PDA (potto dextrose gr) Petri dishes ws inoculted on 12 Erlenmeyer flsks contining 200 ml of pre-fermenttive medium 16 nd incubted t 30 ºC with shking (120 rpm min -1 ) for 24 h. Afterwrd, the obtined myceli were trnsferred to 12 Erlenmeyer flsks contining 400 ml of fermenttive medium Czpek, 17 nd the fungus ws llowed to grow t 30 ºC nd 120 rpm min -1 for n dditionl 144 hour period. The culture broth ws seprted from the mycelium through vcuum filtrtion (400 ml) nd submitted to extrction with ethyl cette (EtOAc) three consecutive times (150 ml ech). The EtOAc crude extrct (88.0 mg) ws frctionted in silic gel column (0.063-0.200 mm) with hexne/etoac (9:1 v/v); hexne/etoac (1:1 v/v); EtOAc nd methnol. The sub frction 25 (6.4 mg), obtined with hexne/etoac (1:1 v/v), ws submitted to preprtive thin lyer chromtogrphy in silic gel PF 254 eluted with dichloromethne/methnol (9:1 v/v) yielding tyrosol (rf: 0.40, 5.0 mg). Tyrosol ws extrcted with cetone/methnol (4:1 v/v). 18 NMR spectr were cquired in Bruker spectrometers (DRX-400 nd DRX- 500), working t 400 nd 500 MHz for 1 H nd t 100 nd 125 MHz for 13 C. The spectr were recorded in CDCl 3, nd the solvent signls t d 7.26 for proton, nd d 77.0 for crbon, were used s reference. Mss spectr nlysis ws conducted in mss spectrometer ESI-MS (Micromss Quttro LC-electrospry ioniztion). Chromtogrms re nlyses were compred in order to check the purity of isolted tyrosol nd vilble commercil tyrosol 98% (Sigm-Aldrich Chemie, Steinheim, Germny). The purity index ws 95.4% for isolted tyrosol used s stndrd for the quntittive vlidtion procedures. Tyrosol stock stndrd solutions were prepred in methnol t concentrtions of 0.250, 0.500, 1.0, 5.0, 20.0, 60.0 nd 100.0 µg ml -1 (resulting in concentrtions of 0.0125, 0.0250, 0.050, 0.250, 1.0, 3.0 nd 5.0 µg ml -1 when 25 µl is used to spike 0.5 ml culture medium). The solutions were stored t 20 o C, in the bsence of light. HPLC-grde cetonitrile, methnol, ethyl cette were purchsed from Mllinckrodt Bker Inc. (Pris, USA) nd ethyl cette from Merck (Drmstdt, Germny). All other chemicls were of nlyticl-grde in the highest purity vilble. Wter ws distilled nd purified using Millipore Milli Q Plus system (Bedford, USA). Instrumenttion nd nlyticl conditions The nlyses were crried out using Shimdzu (Kyoto, Jpn) HPLC system, consisting of LC-6AD model solvent pump, system controller SCL 10A VP, column oven CTO-10AS VP, Rheodyne model 7725 injector with 20 µl loop, SPD-M10A VP diode rry detector operting t 225 nm nd softwre Clss VP for dt cquisition. The determintion of tyrosol ws performed on ZORBAX ODS column (250 4.6 mm, I.D., 5 µm prticle size, Agilent Technologies, Exton, USA), protected with 4.6 mm I.D. 12.5 mm 4.6 mm, 4-Pck endcpped gurd column (Agilent Technologies), using grdient mobile phse, in liner mode, consisting initilly of cetonitrile/ wter (1:9 v/v) nd incresing up to cetonitrile (100%) in 30 minutes t flow rte of 1 ml min -1. The column ws thermostted t 25 C.
190 A Simple Method for the Quntittive Anlysis of Tyrosol by HPLC J. Brz. Chem. Soc. Extrction procedure Preliminry recovery studies were crried out in order to verify stisfctory ph condition nd solvent to be used in the liquid-liquid extrction procedure. Tyrosol stndrd solution ws prepred t the concentrtion of 40.0 μg ml -1 (resulting in concentrtions of 1.0 nd 2.0 μg ml -1 when 25 μl of this solution ws used to spike 1 nd 0.5 ml culture medium, respectively). Aliquots of 1 ml of Czpek medium t three different phs: 5.0; 7.0 nd 9.0 were spiked with 25 µl of stndrd tyrosol solution t the concentrtion of 40.0 μg ml -1 nd extrcted with 3 ml ethyl cette (100%) or toluene/isopropnol (4:1 v/v). The tubes were cpped nd submitted to vortex mixing for 1 min nd then centrifuged t 1800 g for 4 min. The upper orgnic phses (2 ml) were trnsferred to conicl tubes nd evported under compressed ir. The dried residues were dissolved in 50 µl of the mobile phse (10% cetonitrile queous solution) nd 20 µl were chromtogrphed under the previously described conditions. In order to verify whether the phosphte buffer 1 mol L -1, ph 7.0 solution ws stisfctory to mintin neutrl ph t 7.0 for liquid-liquid extrction nother procedure ws tested: liquots of 0.5 ml of the Czpek mtrix spiked with 25 µl of the stndrd tyrosol solution (40.0 µg ml -1 ) were neutrlized with 1 ml 1 mol L -1 phosphte buffer solution ph 7.0 nd extrcted with 4 ml ethyl cette. The upper orgnic phses (3 ml) were trnsferred to conicl tubes nd evported under compressed ir. The dried residues were dissolved in 100 µl of the mobile phse (10% cetonitrile queous solution) nd 20 µl were chromtogrphed under the previously described conditions. Good results were obtined with this lst described extrction procedure, which ws used for the method vlidtion nd method ppliction by smples nlyses. In ddition, the room temperture ws set t 22 ± 2 o C in order to prevent solvent evportion during smple preprtion. Method vlidtion Clibrtions curves were obtined by spiking liquots of 0.5 ml medium with stndrd solutions of tyrosol, prepred in methnol, in the rnge of 0.250-100.0 µg ml -1, resulting in concentrtions of 0.0125-5.0 µg ml -1 in the culture medium. No internl stndrd ws used in this method. To determine the extrction recovery, medium smples (0.5 ml) were spiked with tyrosol in the concentrtions of 0.05, 1.0 nd 5.0 µg ml -1 (n = 3) nd submitted to the extrction procedure. Another set of smples were prepred extrcting 0.5 ml liquots of medium nd then spiking the extrct with the sme mount of tyrosol. The recovery ws determined by compring the res obtined before nd fter extrction nd ws expressed s percentge of the mount extrcted. The detectbility of the method ws evluted by determining the quntifiction limit (LOQ). The LOQ ws defined s the lowest concentrtion tht could be determined with ccurcy nd precision below 20% 19 over five nlyticl run nd it ws obtined using medium smples (0.5 ml, n = 5) spiked with concentrtions of 0.0125 µg ml -1 of tyrosol. The precision nd ccurcy of the method were evluted by nlyzing spiked mtrix with known stndrd tyrosol solution. The experiments were crried out by intr-dy (n = 5) nd inter-dy (n = 3) ssys using medium smples spiked with tyrosol t the concentrtions of 0.0250, 0.250 nd 3.0 µg ml -1. The results obtined were expressed s coefficient of vrition (CV, %) nd reltive error (E, %). The selectivity of the method ws evluted by nlyzing sterile medium (blnk) nd endophytic fungl culture broth under the conditions previously estblished. The stbility of tyrosol ws evluted by testing the influence of freeze ( 20 ºC) nd thw (22 ± 2 ºC) cycles, short-term room temperture nd 48- h freeze temperture. Spiked Czpek medi were prepred in sextuplicte (n = 6) t the low (0.05 µg ml -1 ) nd high concentrtion (3 µg ml - 1 ). To perform the freeze-thw cycle stbility test, these smples were stored t 20 ºC for 24 h nd thwed t room temperture. When completely thwed, the smples were refrozen for 12 h under the sme conditions. The freeze-thw cycle ws repeted once more, nd then the smples were nlyzed on the second cycle. The 48 h freeze temperture stbility test ws performed nlyzing frozen ( 20 ºC) Czpek medium smples over 48 h. This period corresponds to the mximum time the smples were kept frozen. The short-term room temperture stbility test ws ssessed nlyzing smples tht were kept on the bench-top for 4 h t room temperture (22 ± 2 ºC). After this period, the smples were nlyzed. The pek re obtined from the stbility tests ws compred to the pek re obtined with freshly prepred smples. Student s t test ws pplied, with the level of significnce set t p 0.05. Endophytic fungus isoltion nd tyrosol formtion The fungus G. cingult used in these experiments ws previously isolted s endophyte from V. renri (Astercee) nd identified by its rdna sequence. 18 The strin hs been mintined on potto dextrose gr slnts nd silic-gel (1-4 mm Synth, Brzil) nd stored t 4 o C. The
Vol. 20, No. 1, 2009 Guimrães et l. 191 Tble 1. Tyrosol optimiztion of extrction procedure Concentrtion / (µg ml -1 ; n = 4) Tyrosol extrcted with toluene/ isopropnol (4:1) Tyrosol extrcted with EtOAc 100% Tyrosol extrcted with EtOAc 100% fter ddition of phosphte buffer ph 7.0* ph 5.0 ph 7.0 ph 9.0 ph 5.0 ph 7.0 ph 9.0 ph 5.0 ph 7.0 ph 9.0 Recovery/ CV/ Recovery/ CV/ Recovery/ CV/ Recovery/ CV/ Recovery/ CV/ Recovery/ CV/ Recovery/ CV/ Recovery/ CV/ Recovery/ CV/ (%) b (%) c (%) b (%) c (%) b (%) c (%) b (%) c (%) b (%) c (%) b (%) c (%) b (%) c (%) b (%) c (%) b (%) c 1.0 31.6 0.4 33.7 6.2 27.6 8.6 66.8 11.3 74.8 3.6 49.6 9.6 90.7 8.6 92.6 13.4 92.7 12.8 Number of determintions; b men recovery; c CV, coefficient of vrition. *Concentrtion used: 2.0 μg ml -1. strin ws deposited in the Lbortório de Enzimologi Industril-FCFRP/USP. G. cingult ws cultured using two-step fermenttive process. First, suspension of the mycelium ws septiclly inoculted into 1000 ml Erlenmeyer flsks contining 200 ml of seed medium. 16 The flsks were incubted for 48 h t 30 o C on rotry shker (New Brunswick Scientific Co., Inc., model INNOVA TM 4300, New Jersey, USA) operting t 120 rpm. The resulting myceli obtined fter seed medium filtrtion were trnsferred into 2000 ml Erlenmeyer flsks contining 400 ml of Czpek medium. 17 Three different phs were dopted for the cultivtion on Czpek medium: ph 5.0, 7.0 nd 9.0. Experiments were crried out t 30 o C, with shking t 120 rpm for dditionl 20 dys. Aliquots of the Czpek filtrtes (0.5 ml) fter mycelium inocultion were submitted to the previously described extrction procedure nd nlyzed by HPLC. Results nd Discussion Method development nd liquid-liquid extrction Anlyses were crried out using ZORBAX-ODS column (250 mm 4.6 mm I.D.) nd liner grdient elution consisting initilly of cetonitrile/wter (1:9 v/v) nd incresing up to cetonitrile (100%) in 30 minutes. Anlyses of the blnk of the Czpek medium (Figure 1A) showed no interference t the retention time for tyrosol (t R = 10.05 min) (Figure 1B). In ddition, the UV profile of tyrosol is represented t Figure 1C. The utiliztion of the grdient liner mode nd the bsence of n internl stndrd were relted to the complexity of the endophytic fungi extrcts tht might be obtined from Czpek culture medium. In this cse, n internl stndrd might interfere in the extrcts nlyses. In ddition, the grdient mode contributes for better seprtion of the compounds in complex extrct. Preliminry recovery studies (Tble 1) show tht ethyl cette t ph 7.0 ws the best condition for liquid-liquid extrction of tyrosol. In order to estblish the ph of the culture broth fter fungl cultivtion, we performed the ddition of the phosphte buffer 1 mol L -1, ph 7.0 to the Czpek medium (ph 5.0, 7.0 nd 9.0) before the extrction with ethyl cette. Recovery vlues for extrction of tyrosol fter ddition of the phosphte buffer using three different phs were similr (Tble 1) illustrting tht ddition of this buffer solution ws efficient to mintin the ph of the medium t 7.0 in order to gurntee stisfctory ph for the tyrosol extrction. Method vlidtion The clibrtion curves were liner over the concentrtion rnge of 0.0125-5.0 µg ml -1 for tyrosol with liner eqution: y = 122534x + 3240.4; correltion coefficient (r) of 0.9967 nd coefficient of vrition of 13.9%. The lowest concentrtion quntified by the vlidted method ws 0.0125 µg ml -1 with coefficient of vrition of 13.7% nd error 13.9%. Recoveries were round 80% for tyrosol with coefficients of vrition lower thn 15% (Tble 2). Intr-dy (n = 5) nd inter-dy (n = 3) precision nd ccurcy presented coefficients of vrition nd reltive errors lower thn 15% (Tble 3). In spite of not using n internl stndrd, the precision results were cceptble. Stbility of tyrosol in Czpek medium ws evluted by Student s t test with the level of significnce set t p 0.05. The p-vlues obtined were bove 0.05 illustrting the stbility of tyrosol. Coefficients of vrition obtined for low nd high concentrtion used in stbility test were lower thn 15% (Tble 4). Tble 2. Recovery of tyrosol Concentrtion / (µg ml -1 ; n = 3) Recovery / (%) CV b / (%) 0.05 73.5 11.0 1.0 89.6 2.6 5.0 79.0 3.1 Men 80.7 11.8 number of determintions; b CV, coefficient of vrition.
192 A Simple Method for the Quntittive Anlysis of Tyrosol by HPLC J. Brz. Chem. Soc. Figure 1. (A) Chromtogrm referring to the nlysis of medium culture blnk. (B) Chromtogrm referring to the nlysis of tyrosol t R = 10.05 min; purity: 0.99990 t 1 mg ml -1. (C) UV-Profile of stndrd solution of tyrosol in 190-370 nm. (D) Chromtogrm referring to the nlysis of Glomerell cingult culture medi fter 144 h of incubtion.
Vol. 20, No. 1, 2009 Guimrães et l. 193 Tble 3. Precision nd ccurcy of the method for nlysis of tyrosol in Czpek medium Intr-dy (n = 5) Inter-dy (n = 3) Nominl concentrtion / (µg ml -1 ) 0.0250 0.250 3.00 0.0250 0.250 3.00 Anlyzed concentrtion / (µg ml -1 ) 0.0256 0.250 2.85 0.0250 0.231 2.66 Precision / (CV, %) b 9.8 8.5 5.9 14.5 11.3 8.9 Accurcy / (E, %) c 2.3 0.1-4.8-0.0-7.6-11.2 Number of determintions: 5 for intr-dy ssy nd 3 for inter-dy ssy; b expressed s coefficient of vrition, CV; c expressed s reltive error, E. Tble 4. Stbility test of tyrosol Nominl concentrtion / (µg ml -1, n = 6) Fresh tyrosol Tyrosol concentrtion (men) / (µg ml -1 ) CV b / (%) E c / (%) p-vlue 0.05 0.0509 2.78 1.92-3.00 3.1454 7.88 4.84 - Freeze-thw cycles 0.05 0.0497 1.64-0.46 0.10 3.00 2.9625 1.71 1.25 0.11 48 h freeze 0.05 0.0497 1.80-0.48 0.11 3.00 3.0004 10.46 0.01 0.39 Short-term room temperture 0.05 0.0504 0.74 0.86 0.39 3.00 2.9253 0.80 2.49 0.06 number of determintions; b CV, coefficient of vrition. c Expressed s reltive error, E. Method ppliction The developed method showed to be efficient for the quntittive nlyses of tyrosol in Czpek fermenttive medium. The method ws pplied for Czpek smple derived from the cultivtion of the endophytic fungus Glomerell cingult nd no interference for tyrosol pek ws observed, since the pek purity indexes were bove 0.99000 for ll nlyses. These nlyses were crried out fter inocultion of the myceli obtined from seed medium into Czpek medium t three different phs (5.0, 7.0 nd 9.0) without leving ny dditionl time of incubtion (incubtion time = 0 h) nd with 144 h of incubtion (chromtogrm of 144 h of incubtion time is shown in Figure 1D). Concentrtion of tyrosol t 0 h of incubtion ws 0.04 (CV 9.9%); 0.03 (CV 7.4%) nd 0.04 (CV 11.1%) μg ml -1 for cultures t phs t 5.0, 7.0 nd 9.0, respectively. For the 144 h incubtion time the concentrtion of tyrosol found ws 2.27 (CV 4.1%); 1.18 (CV 3.4%) nd 1.12 (CV 7.3%) μg ml -1 for cultures t ph t 5.0, 7.0 nd 9.0, respectively. The presence of tyrosol t similr concentrtions for the three different culturing conditions t 0 hour of incubtion mens tht tyrosol production probbly strts during the seed medium culture. The chromtogrm in Figure 1D refers to the nlysis of tyrosol t 144 h of incubtion of Glomerell cingult in Czpek medium. Anlysis of the pek purity index (1.00000) by DAD t t R =10.05 min shows tht no influence of other secondry metbolites were observed. Moreover, the presence of the pek t t R = 22.53 min illustrtes tht the vlidted method cn be used to verify the correltion between tyrosol nd other secondry metbolites produced by fungi in Czpek medium. Is is noteworthy tht s the production of tyrosol increses in the culture medium, the production of compound t t R = 22.53 min lso increses, s illustrted by the chromtogrm in Figure 1D. It suggests tht tyrosol might be cting s signling compound for the production of this dditionl metbolite. Tyrosol nd other ntioxidnt compounds tht re believed to ply vitl role in the prevention of coronry disese nd therosclerosis hve received specil ttention in the development of quntittive methods. Different
194 A Simple Method for the Quntittive Anlysis of Tyrosol by HPLC J. Brz. Chem. Soc. mtrices such s humn plsm, humn low density lipoproteins 15 nd ciders 14 using bsiclly HPLC or HPLC- ESI-MS-MS s min techniques hve been employed for the quntifiction of these ntioxidnt compounds. Both isocrtic nd grdient liner methods cn be found for quntittive nlyses of tyrosol nd the time of nlysis vries from short 20 through long periods 14 depending on the min objective of the study. To our concern there re no published dt for the nlysis of tyrosol in Czpek medium. Conclusions A suitble high-performnce liquid chromtogrphy method ws developed nd vlidted for the determintion of tyrosol in fermenttive Czpek medium. This method ws successfully used to quntify the formtion of tyrosol by the endophytic fungus G. cingult cultured t different phs. Therefore, this nlyticl quntittive method might be used for the detection nd quntifiction of tyrosol in Czpek medium of endophytic fungi under different culturing conditions. In ddition, the development of smple preprtion nd pproprite concentrtion procedure might extend the pplicbility of this method to other mtrices. Finlly, this method will llow further studies in order to verify the role of tyrosol s quorum-sensing signling molecule for the secondry metbolites production by endophytic fungi from Astercee species. Acknowledgments The uthors re grteful to Fundção de Ampro à Pesquis do Estdo de São Pulo (FAPESP), sub-progrm BIOTA/FAPESP (Rede BIOprospecTA), Conselho Ncionl de Desenvolvimento Científico e Tecnológico (CNPq) nd to Coordenção de Aperfeiçomento de Pessol de Nível Superior (CAPES) for finncil support nd for grnting reserch fellowships (FAPESP grnts 03/07535-5, 04/07935-6, 05/03791-2). References 1. Bu, Y.; Rho, S.; Kim, J.; Kim, M. K.; Lee, D. H.; Kim, S. Y.; Choi, H.; Kim, H.; Neurosci. Lett. 2007, 414, 218. 2. de l Puert, R.; Mrtinez Domínguez, M. E.; Ruiz-Gutierrez, V.; Flvill, J. A.; Hoult, J. R.; Life Sci. 2001, 69, 1213. 3. Bertelli, A. A.; Migliori, M.; Pnichi, V.; Longoni, B.; Origli, N.; Ferretti, A.; Cuttno, M. G.; Giovnnini, L.; Ann. N. Y. Acd. Sci. 2002, 957, 295. 4. Sofi, M.; Turnes, G.; Fenicl, W.; Biol. Bull. 1992, 182, 105. 5. Slininger, P. J.; Burkhed, K. D.; Schisler, D. A.; J. Ind. Microbiol. Biotechnol. 2004, 31, 517. 6. Chen, H.; Fujit, M.; Feng, Q.; Clrdy, J.; Fink, G. R.; Proc. Ntl. Acd. Sci. U. S. A. 2004, 101, 5048. 7. De Kievit, T. R.; Iglweski, B. H.; Infect. Immun. 2000, 68, 4839. 8. Nelson, K. H.; Hstings, J. W.; Microbiol. Rev. 1979, 43, 496. 9. Lzzzer, B. A.; Grossmn, A. D.; Trends Microbiol. 1998, 6, 288. 10. Shpiro, J. A.; Annu. Rev. Microbiol. 1998, 52, 81. 11. Dunny, G. M.; Winns, S. C.; Cell-cell Signling in Bcteri; ASM Press: Wshington, D. C., 1999. 12. Whitehed, N. A.; Brnrd, M. L.; Slter, H.; Simpson, N. J. L.; Slmond, G. P. C.; FEMS Microbiol. Rev. 2001, 25, 365. 13. Ger, C.; Srisvstv, S.; Current Sci. 2006, 90, 666. 14. Suréz, B.; Plcios, N.; Frg, N.; Rodríguez, R.; J. Chromtogr. A 2005, 1066, 105. 15. de l Torre-Crbot, K.; Juregui, O.; Cstellote, A. I.; Lmuel- Rventós, R. M.; Covs, M. -I.; Csls, I.; Lópes-Sbter, M. C.; J. Chromtogr. A 2006, 1116, 69. 16. Jckson, M.; Krwoswski, J. P.; Humphrey, P. E.; Kohl, W. L.; Brlow, G. J.; Tnk, S. K.; J. Antibiot. 1993, 46, 34. 17. Alvino, C. S.; Frbirz, S. R.; Trvssos, L. R.; Angluster, J.; Souz, W.; Mycopthologi 1992, 119, 17. 18. Guimrães, D. O.; Borges, W. S.; Kwno, C. Y.; Ribeiro, P. H.; Goldmn, G. H.; Nomizo, A.; Thiemnn, O. H.; Oliv, G.; Lopes, N. P.; Pupo, M. T.; FEMS Immunol. Med. Microbiol. 2008, 52, 134. 19. http://www.fd.gov/cder/guidnce/4252fnl.pdf, ccessed in My 2008. 20. Grizis, C.; Att-Politou, J.; Kouppris, M. A.; J. Liq. Chromtogr. Relt. Technol. 2003, 26, 599. Received: My 12, 2008 Web Relese Dte: November 18, 2008 FAPESP helped in meeting the publiction costs of this rticle.