A TLC method with densitometric detection for identification and quantitation of indole compounds in methanolic extracts of fruiting bodies of Armillaria mellea (Vahl.) P. Kumm. (Basidiomycota) ñ Honey mushroom has been developed. The TLC method with densitometric detection was validated for determination of tryptamine and serotonin contents. The use of this chromatographic-densitometric method for analysis of extracts allowed for a precise, easy and quick determination of the compounds under study. We identified three physiologically active indole compounds: tryptamine, L- tryptophan and serotonin. This analysis demonstrated the highest contents of L-tryptophan (4.467 mg/100 g d.w.). The contents of tryptamine were comparable with the contents of serotonin and amounted to: 2.740 and 2.207 mg/100 g d. w

Ekiert, Halina

Maślanka, Anna

Muszyńska, Bożena

Sułkowska-Ziaja, Katarzyna

Acta Poloniae Pharmaceutica - Drug Research

English

Jagiellonian Univeristy Repository

Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 68 No. 1 pp. 93ñ97, 2011 ISSN 0001-6837Polish Pharmaceutical SocietyOur earlier mycochemical studies of fruitingbodies from the selected representatives of higherfungi belonging to the largest taxon Basidiomycotahave demonstrated that they contain many nonhallu-cinogenic indole compounds, like L-tryptophan,tryptamine, 5-hydroxytryptophan and serotonin (1,2). The indole compounds were identified in fruitingbodies of the conditionally edible species Lactariusdeterrimus (1) as well as in fruiting bodies and invitro cultures of two edible species Tricholomaequestre and Xerocomus badius (2).In the present study, we analyzed fruiting bod-ies of another species edible after appropriate prepa-ration ñ Armillaria mellea (Vahl.) P. Kumm. (Honeymushroom). This species is common in deciduous,coniferous and mixed forests on all continents. Inmoderate climatic zone, the fruiting bodies are usu-ally formed from July to November, the most abun-dantly in October. The species Armillaria mellea canoccur in a saprophytic form developing on stumps, aparasitic form infecting roots of deciduous andconiferous trees or in a symbiotic form living in amycorrhizal association with the orchid speciesGastrodia elata occurring in Asia, Australia andNew Zealand.Chemical composition of the Armillaria melleafruiting bodies is largely known (3). The fruitingbodies contain pharmaceutically and consumption-ally valuable groups of metabolites, including car-bohydrates (4), sterols (5), sphingolipids (6), fattyacids (7), sesquiterpenoids (8), enzymes (9, 10),peptides (11) and metals (12, 13). Certain com-pounds possess potential antibacterial, fibrinolyticand anticancer properties and capability to protectbrain (14) and bone marrow cells (15). Indole com-pounds in fruiting bodies of this species have notbeen investigated so far. Knowledge of this metabo-lite group seems to be important not only from the-oretical but also pharmaceutical and toxicologicalperspective.Analysis of indole compounds in extracts offruiting bodies of the previously studied speciesTricholoma equestre and Xerocomus badius and inmycelia from in vitro cultures was performed usingthe HPLC method described by Kysilka (16) andmodified by MuszyÒska et al. (2). MuszyÒska et al.ANALYSIS OF INDOLE COMPOUNDS IN ARMILLARIA MELLEAFRUITING BODIESBOØENA MUSZY—SKA1*, ANNA MAåLANKA2, HALINA EKIERT1 and KATARZYNASU£KOWSKA-ZIAJA11Chair and Department of Pharmaceutical Botany, Jagiellonian University, Collegium Medicum, Medyczna 9, 30-688 KrakÛw, Poland.2 Chair of Inorganic and Analytical Chemistry, Jagiellonian University, Collegium Medicum, Medyczna 9, 30-688 KrakÛw, Poland.Abstract: A TLC method with densitometric detection for identification and quantitation of indole compoundsin methanolic extracts of fruiting bodies of Armillaria mellea (Vahl.) P. Kumm. (Basidiomycota) ñ Honeymushroom has been developed. The TLC method with densitometric detection was validated for determinationof tryptamine and serotonin contents. The use of this chromatographic-densitometric method for analysis ofextracts allowed for a precise, easy and quick determination of the compounds under study. We identified threephysiologically active indole compounds: tryptamine, L- tryptophan and serotonin. This analysis demonstratedthe highest contents of L-tryptophan (4.467 mg/100 g d.w.). The contents of tryptamine were comparable withthe contents of serotonin and amounted to: 2.740 and 2.207 mg/100 g d. w. Keywords: tryptamine, tryptophan, serotonin, methanolic extract, Armillaria mellea fruiting bodies, TLC withdensitometric detection93* Corresponding author: e-mail: muchon@poczta.fm94 BOØENA MUSZY—SKA et al.(1) developed also an original method for determi-nation of this group of compounds, namely TLCanalysis with densitometric detection and used it foranalysis of Lactarius deterrimus extracts. The aim ofthe present studies was to validate the method ofdetermination of tryptamine and serotonin by TLCanalysis with densitometric detection and to use itfor analysis of extracts from Armillaria mellea fruit-ing bodies. Other indole compounds, whose methodof determination had been validated previously (1),were currently examined by the TLC analysis withdensitometric detection. EXPERIMENTALApparatusLinomat IV sample applicator and TLCScanner 3 densitometer with wincats software(Camag, (Muttenz, Switzerland)Standard substances and other chemicalsThe following standard substances were used:tryptamine, L-tryptophan, 5-OH-tryptophan, sero-tonin, indole, melatonin, indolilacetic acid,indoleacetonitrile, indoloacetamide, kynureninesulfate, kynurenic acid (Sigma-Aldrich). Otherchemicals: ammonia ñ 25% solution, ethanol,petroleum ether and isopropanol all analyticalgrade, were purchased from Polish ReagentsCompany (POCh Gliwice), Origin of fruiting bodiesThe studies were conducted on Armillaria mel-lea (Vahl.) P. Kumm. fruiting bodies collected inmixed forests in southern Poland (Tunel nearKrakÛw) in September 2008 (deposited in theDepartment of Pharmaceutical Botany, JagiellonianUniversity, Collegium Medicum, KrakÛw, Poland) .Extraction and sample preparationLyophilized fruiting bodies (approximately71 g) were extracted in a percolator with petroleumether in order to remove oil fraction according tothe procedure developed in our laboratory (2).Then, the material was dried and extracted withmethanol (10 portions/300 mL) in a percolator for24 h. The extract was concentrated to 42.5 mL bydistillation in a vacuum evaporator under reducedpressure at 40OC. To remove the rest of lipids, theconcentrated extract was frozen. For the next stageof purification of the extract, we used preparativeTLC method on aluminum-backed silica gel 60plates (Merck, Art. No 1.05554.0001), on which 3mL of the extract was applied and chromatogramswere developed in mobile phase: isopropanol :25% NH3 : water (8:1:1, v/v/v). Spots were identi-fied at λ = 254 nm. Five fractions were obtainedand analyzed by the TLC method with densitomet-ric detection.TLC analysis with densitometric detectionSolutions of standard substances, i.e., threecompounds discovered in the extracts under study inpreliminary experiments, were prepared. The solu-tions were prepared by dissolving the substances inmethanol at the given concentrations: tryptamine(0.8800, 0.4400, 0.2200, 0.1100, 0.0550 and 0.0275mg/mL), serotonin (0.5300, 0.2650, 0.1325, 0.0663,0.0331 and 0.0166 mg/mL) and tryptophan, whichwas validated earlier (0.4400 mg/mL)Separation conditions for tryptamine, L-trypto-phan and serotonin were established. The chosenmobile phase was composed of: isopropanol : 25%NH3 : water (8:1:1, v/v/v) and allowed to obtain agood separation of the substances under analysis: RF~ 0.50 for L-tryptophan, RF ~ 0.58 for serotonin andRF ~ 0.73 for tryptamine (Fig. 1). Spots on chro-matograms were detected densitometrically at λ =280 nm.The method that has already been validated forL-tryptophan was chosen for the study. Conditionsof determination of tryptamine and serotonin werevalidated by evaluation of accuracy, precision, lin-earity, limit of detection and limit of quantitation.AccuracyAccuracy of the method was characterized byassessment of percent recovery for serotonin andtryptamine. The test was performed by addition ofan exactly known dose of the standard substanceamounting to 80ñ120% of the declared content. Thedetermination was performed before the addition ofthe standard substance and thereafter (Tab. 1).Figure 1. A densitogram of chromtogram after development; 1 ñL-tryptophan, 2 ñ serotonin and 3 ñ tryptamineAnalysis of indole compounds in Armillaria mellea fruiting bodies 95PrecisionPrecision was determined using model solu-tions prepared by dissolution of the substancesunder analysis in methanol. Five repetitions of eachsolution were analyzed (Tab. 1).LinearityLinearity was tested for the relationshipbetween peak area (in mm2) vs. concentration (inmg/mL). Linearity was evaluated by the calculationof linear regression equations and correlation coeffi-cients (r).Limit of detection (LOD) and limit of quantification(LOQ) Limit of detection and limit of quantificationwere calculated based on standard deviation andslope using equations: LOD = 3.3 Sy/a, LOQ = 10Sy/a, where: Sy = standard error of estimation, a =slope (Tab. 1)Analysis of extracts from fruiting bodiesStandard solutions (3 µL) and fractions of testextracts (5 µL) were loaded on 10 cm ◊ 10 cm TLCplates (coated with silica gel with fluorizing agentF254, (Merck, Art. No 1.05554.0001) as bands 1 cmlong by means of sample applicator. Chromato-grams were developed to a height of 9.5 cm in achromatographic chamber saturated with the mobilephase and were dried at room temperature.Densitometric analysis was performed at a wave-length 280 nm using Camag TLC- Scanner 3 densit-ometer with wincats software. The componentswere identified based on Rf value for spots obtainedTable 1. Validation parameters of the tryptamine and serotonin determination method.Substance RF Recovery Precision LOD LOQLinearity [%] [mm2] [µg/spot] [µg/spot]96.1 5045.795.2 4936.796.4 5092.195.3 5012.3 p = 16056.8 ◊ c + 483.6Serotonin ~ 0.58 97.5 4982.7 0.126 0.381 r = 0.99479xñ = 96.10 xñ = 5013.9 Sy = 204.09Sx = 0.930 Sx = 59.29t95% ± 1.064 t95% = ± 67.43RSD = 0.97% RSD=1.18%95.3 4293.197.3 4295.797.8 4186.295.9 4210.1 p = 5503.6 ◊ c + 532.2Tryptamine ~ 0.73 98.2 4192.1 0.171 0.518 r = 0.99749xñ = 96.90 xñ = 4235.4 Sy = 142.57Sx = 1.247 Sx = 54.55t95% = ± 1.418 t95% = ± 62.03RSD = 1.29% RSD = 1.29%xñ ñ mean value for n = 5, Sx ñ standard deviation, t95% ñ confidence interval at 95%, RSD ñ variation coefficient. p ñ area [mm2], c ñ con-centration [mg/mL]Table 2. Contents of the compounds under study in the extracts of Armillaria mellea fruiting bodies (in mg/100 g d.w.).Tryptamine L-tryptophan Serotonin2.737 4.453 2.2062.735 4.459 2.2012.748 4.477 2.2032.744 4.476 2.2172.735 4.470 2.208xñ = 2.740 xñ = 4.467 xñ = 2.207Sx = 0.0059 Sx = 0.0106 Sx = 0.0062t95% = ± 0.0073 t95% = ± 0.0132 t95% = ± 0.0077RSD = 0.22 RSD = 0.24 RSD = 0.28xñ ñ mean value for n = 5, Sx ñ standard deviation, t95% ñ confidence interval at 95%, RSD ñ variation coefficient96 BOØENA MUSZY—SKA et al.with standard substances and test extracts. The com-ponents were quantified based on peak areas. The method developed in the present study wasused for analysis of fractions obtained by prepara-tive chromatography, which were preliminarilyidentified under UV lamp. The studies aimed todetect L-tryptophan, tryptamine and serotonin, con-firmed their presence in some fractions from prepar-ative chromatography. Detailed data on the contentsof the components under study are presented inTable 2.RESULTS AND DISCUSSIONValidation of the method for determination ofserotonin and tryptamineThe conditions of thin layer chromatographywith densitometric detection in UV range allow foridentification and concomitant quantitation of trypt-mine, tryptophan and serotonin in methanolicextracts of Armillaria mellea fruiting bodies.Qualitative and quantitative analysis of these com-ponents require their prior preliminary isolationaccording to the above-described procedure of ana-lyzed preparation by extraction and subsequentextract purification by preparative TLC. The com-ponents of the extracts under study were analyzedusing the mobile phase composed of isopropanol :25% NH3 : water (8:1:1, v/v/v), which enabled sep-aration of the substances under study as demonstrat-ed with standard solutions (Fig. 1). The differencesin retention coefficient were significant, which facil-itated identification of individual components.Densitometric measurements at λ = 280 nm allowfor recording of peaks directly from chromatograms.The peaks are well resolved and easy for quantita-tive evaluation (Fig. 1). Validation parameters for the method of sero-tonin and tryptamine determination (Tab. 1) arecomparable with validation parameters for trypto-phan (1). Validation results presented in Table 1indicate that the proposed method is characterizedby a high sensitivity; LOD for serotonin is 0.126mg, and 0.180 mg for tryptamine (0.075 mg for L-tryptophan); LOQ was estimated at 0.381 mg, 0.518mg and 0.226 mg, respectively. Recovery of thecomponents under study amounts to: 96.10%,96.90% and 99.09%, respectively. The method isalso characterized by high precision, RSD for thesubstances under analysis equals to: 1.18%, 1.29%and 1.40%, respectively.Linearity of the concentration vs. peak areacurve for L-tryptophan, tryptamine and serotoninwas preserved in a wide concentration range: from0.0165 mg/mL to 0.5300 mg/mL for serotonin, from0.0275 mg/mL to 0.8800 mg/mL for tryptamine andfrom 0.0369 mg/ml to 0.5900 mg/ml for L-trypto-phan.Application of the chromatographic-densito-metric method for analysis of the extracts allowedfor a precise, relatively easy and quick determina-tion of the compounds under study.Quantitative analysis of the contents of indolecompoundsAnalysis of methanolic extracts fromArmillaria mellea fruiting bodies by TLC methodwith densitometric detection demonstrated the pres-ence of three main metabolites: L-tryptophan, trypt-amine and serotonin.We demonstrated previously that the extractsof fruiting bodies of other Basidiomycota species,contained a richer qualitative composition of thisgroup of compounds because five different com-pounds were identified in each Lactarius deterrimusand Xerocomus badius (1, 2) and four compoundswere detected in Tricholoma equestre fruiting bodies(2). Only L- tryptophan and tryptamine were com-mon to fruiting bodies of Armillaria mellea and threepreviously studied species of mushrooms. Serotoninwas found in the currently investigated Armillariamellea and in two species Tricholoma equestre andXerocomus badius examined earlier.Armillaria mellea fruiting bodies containedsignificant amounts of the metabolites: 4.467mg/100 g d.w. for L-tryptophan, 2.740 mg/100 gd.w. for tryptamine and 2.207 mg/100 g d.w. forserotonin. These contents were less diverse thanthose determined in Lactarius deterrimus fruitingbodies which ranged from 0.190 (melatonin) to2.728 mg/100 g d.w. (tryptamine) (1).Tryptophan contents in the extracts ofArmillaria mellea fruiting bodies under study weremany times higher than in previously investigatedLactarius deterrimus (0.235 mg/100 g d.w.). Highconcentration of this compound in food products,including fruiting bodies of mushrooms is not safebecause tryptophan ingested as an exogenous aminoacid at too high doses can damage the nervous sys-tem (17) and can contribute to the induction of uri-nary bladder cancer (18). Moreover, this amino acidis a precursor of all other indole compounds show-ing hormonal activity as tissue hormones. Tryptamine contents in the currently studiedextracts of Armillaria mellea fruiting bodies weremarked and almost identical as in Lactarius deter-rimus (2.728 mg/100 g d.w.). From pharmacologicaland toxicological point of view, tryptamine is not aAnalysis of indole compounds in Armillaria mellea fruiting bodies 97safe compound. It can interact with MAO inhibitorsand can even cause deadly poisoning in patients tak-ing these drugs. Tryptamine and its derivatives arepsychoactive substances showing agonistic activityat 5-HT2 receptors (18).Serotonin contents in Armillaria mellea fruitingbodies were high. The contents of this compounddetermined with another method in fruiting bodies ofTricholoma equestre and Xerocomus badius were sig-nificantly lower, amounting to 0.524 and 0.182mg/100 g d.w., respectively. Although serotonin hasrecently been proven to possess beneficial biologicalproperties, like antioxidant action and therapeuticpotential in Alzheimerís desease (19, 20), the highcontent of this compound, fulfilling the role of tissuehormone in human organism, in the fruiting bodiesunder study (2.207 mg/100 g d.w.) calls for cautionwhen this mushroom is eaten in larger amounts. Thiscaution is required also due to the proven concomi-tant presence of two other indole compounds, trypt-mine and tryptophan, in these fruiting bodies.CONCLUSIONThe present studies have demonstrated for thefirst time that extracts of Armillaria mellea fruitingbodies contain L-tryptophan, tryptamine and sero-tonin. In this work, a method of determination oftryptamine and serotonin (TLC analysis with densit-ometric detection) was developed and validated andwas successfully used for mycochemical analysis ofextracts from fruiting bodies. Tryptophan was deter-mined using a previously developed and validatedoriginal method (also TLC analysis with densito-metric detection), which was evaluated in earliermycochemical studies of Lactarius deterrimus fruit-ing bodies.High contents of L-tryptophan, tryptamine andserotonin, shown in the present study, justify furtherdetailed studies on the accumulation of indole com-pounds in this species.REFERENCES1. 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Toksykol. 25, 171 (1992).13. KalaË, P., Svoboda L., HavlÌËkov· B.: EnergyEdu. Sci. Technol. 13, 313 (2004).14. Lee S., Kim J., Kim J., Sapkota, K.: ProteinExpr. Purif. 43, 10 (2005). 15. YP., Wu K.F., Liu Y.: Zhongguo Zhong Yao ZaZhi 30, 283 (2005). 16. Kysilka R., Wurst M. J. Chromatogr. 320, 414(1985).17. SvrËek M., VanËura B.: Houby. p. 162, Artia,Praha 1983. 18. Stone T. W., Mackay G. M., Forrest C. M.,Clark C. J., Darlington L. G.: Clin. Chem. Lab.Med. 41, 852 (2003).19. Turek M., £odyga-ChruúciÒska E.: Food Chem.Biotechnol. 72, 73 (2008). 20. Ouchi Y., Yoshikawa E., Futatsubachi M., YagiS., Ueki T., Nakamura K.: J. Nucl. Med. 50,1260 (2009).Received: 16. 03. 2010

Analysis of indole compounds in "Armillaria mellea" fruiting bodies

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Analysis of indole compounds in "Armillaria mellea" fruiting bodies

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