Development, Optimization and Validation of an HPLC-PDA Method for Quantification of Taxifolin in the Bark Extract of Pinus pinaster

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1 J. Brz. Chem. Soc., Vol. 27, No. 9, , Printed in Brzil Sociedde Brsileir de Químic $ Article Development, Optimiztion nd Vlidtion of n HPLC-PDA Method for Quntifiction of Txifolin in the Brk Extrct of Pinus pinster Priscil A. de Almeid, Crl A. P. Bhering, Michele C. Alves, Mrcone A. L. de Oliveir, b Nádi R. B. Rposo, Anderson O. Ferreir c nd Mrcos A. F. Brndão*, Núcleo de Pesquis e Inovção em Ciêncis d Súde (NUPICS) nd b Grupo de Químic Anlític e Quimiometri (GQAQ), Universidde Federl de Juiz de For, Juiz de For-MG, Brzil c Lbortório de Controle de Qulidde, Ortofrm, Mtis Brbos-MG, Brzil The queous extrct of the brk of Pinus pinster hs high concentrtion of polyphenols represented by mixture of procynidins, besides txifolin, phenolic cids, cinnmic cids nd their glycosides. Its qulity control is specified in the United Sttes Phrmcopei, nd the ssy test is performed by determintion of the totl procynidins content. However, determining the individul polyphenol content my represent n dditionl qulity prmeter for this extrct. In this sense, the present study imed to develop nd optimize method of high performnce liquid chromtogrphy with photodiode rry detection (HPLC-PDA) for quntifiction of txifolin in the brk extrct of P. pinster, using 3 3 Box-Behnken fctoril design. The proposed method ws vlidted for specificity, linerity, limits of detection nd quntifiction, precision, ccurcy nd robustness nd it hs shown tht txifolin my be used s chemicl mrker for qulity control of the brk extrct of P. pinster. Keywords: nlyticl vlidtion, high performnce liquid chromtogrphy, Pinus pinster, qulity control, txifolin Introduction Over the lst decdes, polyphenols hve hd n incresing interest on understnding vitl functions of biologicl systems becuse they re importnt ntioxidnts of humn diet. 1 Due to the helth significnce of these compounds, some nlyticl methods hve been developed for their seprtion, identifiction nd quntifiction in nturl products. 2-4 Polyphenols hve wide vribility of chemicl structures, which differ in polrity nd size, from simple phenolic compounds to oligomers. Additionlly, they re found t low concentrtion levels in nturl products. 5 Therefore, smple pre-tretment is normlly required before instrumentl nlysis. 6 Liquid-liquid extrction (LLE) nd solid phse extrction (SPE) re the smple pre-tretment techniques most frequently used. Among nlyticl techniques, high performnce liquid chromtogrphy (HPLC) nd cpillry electrophoresis (CE) re the min methods. 7,8 *e-mil: mrcosbrnd2012@gmil.com P. pinster Aiton, pine commonly known s french mritime pine nd originlly occurring in the Mediterrnen region, 9 hs been investigted with regrd to its chemicl constituents nd biologicl effects. 10 The queous extrct of the brk of P. pinster hs high concentrtion of polyphenols represented by mixture of procynidins (comprising ctechin nd epictechin subunits with vrying chin lengths), besides phenolic cids (gllic, ferulic, cffeic nd p-hydroxybenzoic cids), cinnmic cids nd their glycosides forms, nd txifolin (Figure 1). 11,12 It is widely used s nutritionl supplement nd provides numerous helth benefits due to its ntioxidnt, nti inflmmtory, nd immuno-modultory effects The qulity control for the brk extrct of P. pinster is specified in the United Sttes Phrmcopei (USP) monogrph in the chpter of dietry supplements. 16 A method of HPLC with ultrviolet (UV) detection is described for identifiction test. It nlyzes four components of the extrct: cffeic cid, ctechin, ferulic cid, nd txifolin. The method cn lso be used to generte chromtogrphic fingerprints tht provide dditionl qulittive informtion. However, for quntittive purposes,

2 Vol. 27, No. 9, 2016 Almeid et l nd txifolin (purity: 87.40%) were purchsed from Sigm (USA), nd ferulic cid (purity: %) ws from Henrifrm (Brzil). The pine brk extrct (PBE) ws obtined from USP (USA). The mobile phse ws filtered through 0.45 μm filter membrne (Srtorius, Germny) nd degssed by n ultrsonic pprtus (Cristófoli, Brzil) for 30 min before use. Smple preprtion Figure 1. Chemicl composition of the brk extrct of P. pinster. method of spectrophotometry is described which provides n estimtion of the totl procynidins content (65-75%) nd it does not give quntittive mesurement of individul polyphenol content. In this regrd, txifolin, flvnonol lso known s dihydroquercetin, 17 cn be chemicl mrker for qulity control of the brk extrct of P. pinster, becuse it is present in quntittively detectble mount in the the extrct when compred with other individul polyphenol contents. 18 Furthermore, it is widely found in brks of the species within the genus Pinus, so it is n importnt flvnonol for this group. 19,20 Within this context, the im of this work ws to develop, optimize nd vlidte method of HPLC with photodiode rry detection (PDA) for quntifiction of txifolin in the brk extrct of P. pinster. The method optimiztion ws investigted by multivrite pproch, tking into ccount 3 3 Box-Behnken fctoril design. Experimentl Regents, stndrds nd smple Acetonitrile (Pnrec, Spin) nd ethyl cette (Merck, USA) were HPLC grde, nd formic cid ws nlyticl grde (Neon, Brzil). Ultrpure wter obtined in n AquMx-Ultr 370 Series (Young Lin, Kore) (18.2 MΩ cm resistivity t 25 C nd < 10 ppb totl orgnic crbon) ws used throughout nlysis. The stndrds of gllic cid (purity: %), ctechin (purity: 99.00%) The smple ws prepred by LLE with ethyl cette. The PBE (20 mg) ws ccurtely weighed nd trnsferred to beker in which 10 ml wter/methnol (2:1, v/v) were dded. The mixture ws left in ultrsonic bth for 5 min. Then, the content of the beker ws poured into seprtory funnel of 100 ml nd extrcted with ethyl cette (15 ml) by mnul shking for 1 min. After 4 min rest period, the orgnic phse ws seprted from the queous phse. To the remining queous phse, nother portion of ethyl cette ws dded (10 ml) nd the procedure ws repeted twice, mking up three extrctions. The combined orgnic phses were treted with 4 Å moleculr sieves (3 g), nd filtered to round bottom flsk through quntittive filter pper. The filtrte ws concentrted to 1/15 of the initil volume by rotry evportion under vcuum, trnsferred quntittively to 5 ml volumetric flsk nd mde up to 5 ml with ethyl cette. An liquot of 3 ml ws trnsferred to 10 ml test tube nd evported to dryness in wter bth t 50 C. The residue ws reconstituted in 2.6 ml cetonitrile, filtered in 0.45 µm filter membrne nd trnsferred to HPLC vils. Then, 20 μl were injected into the HPLC-PDA system. Instrumenttion The HPLC nlyses were performed using qulified nd clibrted Young Lin (Kore) chromtogrphy system composed of: quternry pump (YL 9110), photodiode rry detector (YL 9160), utomtic injector (YL 9150), column comprtment (YL 9130) nd softwre controller (Clrity). The HPLC-PDA method used ws modeled in conformity with the method described in the USP monogrph, ccording to the conditions vilble in our lbortory. Chromtogrphic seprtion ws chieved using octdecylsilne (C18, mm, 5 μm prticle size) column (Agilent, Brzil) mintined t 40 C nd connected with C18 pre-column ( mm, 5 µm prticle size) (Phenomenex, USA). The wvelength for UV detection ws 288 nm. The initil HPLC grdient profile (mobile phse composition nd flow rte) is shown in Tble 1. To optimize the chromtogrphic conditions, 3 3 Box Behnken fctoril design triplicted t the centrl

3 1650 Development, Optimiztion nd Vlidtion of n HPLC-PDA Method J. Brz. Chem. Soc. Tble 1. Initil HPLC grdient profile (mobile phse composition nd flow rte) time / min Solution A / % Solution B b / % Mobile phse flow rte / (ml min 1 ) % formic cid in ultrpure wter; b 0.1% formic cid in cetonitrile. point (15 runs) ws performed for the screening of significnt conditions tht ffected the seprtion of txifolin. 21 The fctors chosen for evlution nd their respective levels in the experimentl design re summrized in Tble 2. Identifiction of the compounds Solutions of gllic nd ferulic cids, ctechin nd txifolin were prepred in cetonitrile t 50 µg ml 1. PBE smples (n = 3) were chromtogrphed nd those peks corresponding to commercilly vilble stndrds were identified by UV spectr nd spiking procedure. Anlyticl vlidtion After method development nd optimiztion, the vlidtion tests were performed ccording to the Interntionl Conference on Hrmonistion 22 (ICH) nd the Brzilin Ntionl Institute of Metrology, Stndrdiztion nd Industril Qulity 23 (Inmetro) guidelines, comprising the following prmeters: Specificity The specificity of the method ws determined through the comprison of stndrd, smple nd blnk chromtogrms. Linerity The test ws conducted from the plotting of three stndrd curves, ech one constructed from five concentrtions corresponding to 40, 45, 50, 55 nd 60 µg ml 1 of txifolin. The liner regression eqution nd the determintion coefficient (R 2 ) were determined by lest squres method. After regression implementtion, the dt for ech concentrtion level were sttisticlly evluted tking into ccount homoscedsticity (Cochrn s test), residues normlity (Shpiro-Wilk s test) nd lck-of-fit into the model through n priori test hypothesis (nlysis of vrince (ANOVA)). ANOVA test consists of compring the devitions of the mens between the clibrtion line (the residul stndrd devition, s yx ) nd the y vlues from their mens (s y ) by using eqution 1, where m i is the number of mesurements, p is the clibrtion point nd m is the product between p nd m i. 24 The test is crried out by comprison between F clculted nd F criticl ; f 1 = p 2; f 2 = m p (F criticl ). If F clculted F criticl, the liner model cnnot be pplied. The txifolin quntifiction ws performed using the clibrtion curve from linerity of the stndrd. Limits of detection nd quntifiction The limit of detection (LOD) nd the limit of quntifiction (LOQ) were determined from three stndrd clibrtion curves nd were clculted s shown in equtions 2 nd 3, respectively: (1) (2) (3) Tble 2. Fctors nd levels utilized in the experimentl design for chromtogrphic condition optimiztion Experiment X X 2 b X 3 c Temperture of the column oven [( ): 35 C, (0): 40 C, (+): 45 C]; b mobile phse flow rte [( ): mintined t 1.0 ml min 1, (0): incresed until 1.2 ml min 1, (+): incresed until 1.4 ml min 1 ]; c increse in the percentge of cetonitrile in mobile phse [( ): until 15%, (0): until 20%, (+): until 25%].

4 Vol. 27, No. 9, 2016 Almeid et l where is the regression slope nd S is the stndrd devition of the y intercept. Precision The repetbility (intr-dy precision) ws determined by nlyzing six replictes consecutively by single nlyst in single dy. The intermedite precision (inter dy precision) ws evluted in the sme wy, but with two nlysts nd two dys. An injection precision of less thn 5% reltive stndrd devition (RSD) ws considered pproprite. Accurcy Accurcy represents the level of complince between individul results obtined nd reference vlue. 22 In this current work, the ccurcy of the method ws estblished by recovery tests t three concentrtion levels (40, 50 nd 60 µg ml 1 ) performed in triplicte. For this, liquots of 174, 217 nd 260 µl of the txifolin stndrd solution (concentrtion = 1000 µg ml 1 ) were dded before extrction. The results were expressed s percentge of recovery (R%), nd eqution 4, where A is the nlyte re, A S is the stndrd re nd A +S is the nlyte re with stndrd ddition, ws used. 25 R% = A +S A / A S (4) Robustness The robustness prmeter ws evluted by intentionl minor modifictions in the chromtogrphic conditions in the proposed method. 26 Thus, complete experimentl design with eight experiments (2 3 ) nd triplicte in the centrl point ws conducted, in totl of 11 experiments performed in rndom order. 27 The fctors nd their levels were: (X 1 ) temperture of the column oven [( ): 38 C, (0): 40 C nd (+): 42 C]; (X 2 ) mobile phse flow rte [( ): mintined t 1.0 ml min 1, (0) incresed until 1.2 ml min 1 nd (+) incresed until 1.4 ml min 1 ]; nd (X 3 ) increse in the percentge of cetonitrile in mobile phse [( ): until 18%, (0): until 20% nd (+) until 22%]. To evlute the significnce of deliberte vritions in ech chosen fctor, their effects were clculted. From the results of ech experiment, the coefficients for determining the sttisticl model of prediction were clculted ccording to eqution 5: b = (X t X) 1 X t y (5) where b is the mtrix of model coefficients nd X nd y re the X mtrix nd y vector, respectively. To write the eqution of the fitted model, the stndrd errors of the coefficients were clculted using eqution 6: where ε(b) is the mtrix whose min digonl represents the stndrd errors of the model estimtors (b i ) nd σ 2 is the popultion vrince of the experiments, which cn be estimted s s 2 using the center point replictes, from eqution 7: Effects were clculted in mtrix by the product X t y, where y is column vector contining the verge results of the ssy. To estimte the stndrd error of n effect, the squre root of the vlue obtined in eqution 8 ws used, nd the stndrd error of the men ws estimted using the squre root of the vlue obtined in eqution 9: With the estimted stndrd errors, it ws possible to chieve confidence intervls for the vlues of effects, using Student s t-distribution with 95% confidence, ccording to eqution 10: (6) (7) (8) (9) (10) where η is the true vlue of n effect (popultion vlue), represents the vlue obtined from the tests performed on the experiment, t v is the vlue from the Student s distribution, nd S effect is the stndrd error of n effect. Results nd Discussion Optimiztion of the HPLC-PDA method The selection of chemicl mrkers is determinnt for the qulity control of herbl medicines, 28 thus, in order to develop method suitble for qulity control of PBE, it is necessry to estblish one or more chemicl mrkers. Initilly, chromtogrphic fingerprint ws performed using USP HPLC method, nd four compounds were identified: gllic cid, ctechin, txifolin, nd ferulic cid,

5 1652 Development, Optimiztion nd Vlidtion of n HPLC-PDA Method J. Brz. Chem. Soc. in this elution order. However, low signl intensity ws obtined for these substnces in our lbortory by UV detection, nd the chromtogrphic profile showed low signl-to-noise rtio for these peks. Therefore, it ws evidenced which smple pre-tretment would be required. As txifolin showed the most intensive signl, it ws defined s chemicl mrker for method development, thus, the smple preprtion ws performed in order to ensure the best conditions for its extrction. After liquid liquid extrction, the concentrtion found for txifolin ws 1.10 ± 0.11% t 95% confidence intervl. Although txifolin cn be determined in nturl products by severl nlyticl techniques, HPLC-UV ws estblished s the most convenient method for qulittive nd quntittive nlysis of phenolic compounds. 29 Despite this, the complexity of PBE hs sustined the development of HPLC methods with mss spectrometry (MS) detection such s the method proposed by Chen et l. 18 for HPLC fingerprints nlysis using the most prominent pek (txifolin) s the reference pek. The MS detection, however, is expensive nd requires more trined nlysts, so method for txifolin determintion ws developed using UV detection, the most widespred detection in qulity control lbortories. PBE is nturl source of phenolic compounds. Thus, reverse chromtogrphy with C18-sttionry phses combined with binry elution systems contining n queous cidified solvent nd n orgnic modifier solvent (methnol nd cetonitrile, minly) re used lmost exclusively. 7,29 In this study, cetonitrile ws chosen becuse txifolin nd ferulic cid were not seprted using methnol in the preliminry investigtions, besides the ltter provided higher pressure vlues thn cetonitrile. Additionlly, it ws necessry to cidify both solvents (queous nd orgnic) with formic cid to minimize pek tiling. Within this context, the mobile phse ws initilly estblished s two solvent systems: (i) 0.1% formic cid in ultrpure wter (A) nd (ii) 0.1% formic cid in cetonitrile (B). The initil HPLC grdient profile (mobile phse composition nd flow rte) ws estblished fter screening tests. A grdient of mobile phse flow rte in the first 20 min ws lso relized in order to reduce nlysis time. In this regrd, 3 3 Box-Behnken fctoril design ws performed to optimize chromtogrphic conditions. Figure 2 shows the representtive chromtogrms obtined from fctoril design. The txifolin response (Y) took into ccount the resolution of the criticl pir nd the bseline disturbnces, common to the grdient elutions. Therefore, Y ws defined s the rtio between the resolution (R) of the criticl pir (vlue obtined between the txifolin pek nd the pek indicted by n sterisk in Figure 2) nd constnt (K; defined s 2 for the presence of bseline disturbnces nd 1 for the bsence of bseline disturbnces), ccording to eqution 11: Y = R / K (11) where R ws clculted using retention time (t R ) nd pek width t bseline (t wb ) s following (eqution 12): R = [2(t R2 t R1 )] / (t wb1 + t wb2 ) (12) Additionlly, sttisticl nlysis tools were used in order to identify significnt effects. An independent fctor hs significnt effect on given response when it hs p-vlue smller thn 0.05, considering 95% confidence level. The results obtined for ech run re listed in Tble 3. It ws found tht the temperture of the column oven (X 1 ), nd the increse of cetonitrile percentge in mobile phse (X 3 ) directly influence the txifolin response since they hve p-vlues smller thn 0.05, nd lso ll the two fctor interctions with the exception of the interction X 2 X 2. The increse of the mobile phse flow rte in the first 20 min did not ffect txifolin response (p-vlue higher thn 0.05), but it llowed the best nlysis time obtined in this study (50 min). The conditions of the centrl point (experiments 13, 14 nd 15) showed the highest response for txifolin (Tble 3) nd very stble bseline. Therefore, these conditions were selected s optiml nd they were used in the vlidtion study. Anlyticl vlidtion Using the optimized conditions, vlidtion studies were performed. The specificity results re shown in Figure 3. As one cn observe, the mtrix did not interfere with the nlysis of the compound of interest. Thus, it is possible to quntify txifolin without interference, which ws confirmed by UV spectr obtined for the txifolin pek between 200 nd 400 nm (Figure 4). For linerity, clibrtion plots (x = µg ml 1, y = mv) of txifolin mesured by the proposed method were constructed (n = 3 for ech concentrtion). The lest squres method obtined determintion coefficient greter thn 0.99 (R 2 = ), which indictes the existence of significnt liner reltionship between two vribles. The regression model dignosis ws stisfctory with no lck-of-fit becuse the vlue of F clculted is lower thn F criticl in the 95% confidence intervl (F clculted = 0.65 < F criticl(0.05) = 3.71). Moreover, the regression significnce ws higher, since the F clculted found ws t the 95% confidence

6 Vol. 27, No. 9, 2016 Almeid et l Figure 2. Representtive chromtogrms obtined from 3 3 Box-Behnken fctoril design. () Trils 1, 6, 7, nd 11 with similr chromtogrphic profiles; (b) trils 2 nd 9 with similr chromtogrphic profiles; (c) trils 3, 4, 10, nd 12 with similr chromtogrphic profiles; (d) trils 5 nd 8 with similr chromtogrphic profiles; (e) centrl point: trils 13, 14 nd 15. Chromtogrphic conditions: C18 column ( mm, 5 µm prticle size), volume of injection of 20 µl, UV detection t 288 nm, nd temperture of the column oven, mobile phse composition nd flow rte ccording to conditions described by Box-Behnken fctoril design. TAX: txifolin; *: interference pek. Tble Box-Behnken design results for the method optimiztion Experiment Txifolin response (Y) Fctor Coefficient Error p-vlue men b X b X X b X 1 X b X 2 X X 3 X b X 1 X b X 1 X b X 2 X b The best results for txifolin response; b sttisticlly significnt effects (p < 0.05). intervl. The normlity in the residues (Shpiro-Wilk s test) nd homoscedsticity (Cochrn s test) were pplied in order to verify if this model fits. Indeed, Shpiro-Wilk s W vlue (W clculted = 0.91) ws higher thn the criticl vlue (W criticl(0.05) = 0.88) nd Cochrn s C vlue (C clculted = 0.58) ws lower thn the criticl vlue (C criticl(0.05) = 0.68), which indictes no violtion of the ssumptions, thus the ANOVA is considered vlid. Therefore, the method is indeed liner, over its respective rnge. The vlues obtined for LOD nd LOQ were 6.52 nd µg ml 1, respectively. These vlues showed dequte sensitivity for the nlyticl ssy. For precision, the RSD found were 3.46, 3.62 nd 3.48% from intr-dy, first dy (n = 12); intr-dy, second dy (n = 12); nd inter-dys (n = 24), respectively. All RSD were within specifiction limits recommended ( 5%). For ccurcy, the verge recovery percentge of the stndrd dded ws clculted, nd the vlue found ws 98.23% (RSD = 1.36%), which indictes tht this method hs n cceptble ccurcy nd suitbility to quntify txifolin without interferences.

7 1654 Development, Optimiztion nd Vlidtion of n HPLC-PDA Method J. Brz. Chem. Soc. Figure 3. Chromtogrphic profiles obtined in the specificity test. () Txifolin stndrd; (b) smple; nd (c) blnk. Chromtogrphic conditions: C18 column ( mm, 5 µm prticle size) t 40 ºC, volume of injection of 20 µl, UV detection t 288 nm, nd mobile phse composition nd HPLC grdient profile ccording to Tble 1. TAX: txifolin. Figure 4. UV spectr between 200 nd 400 nm for txifolin pek. () Txifolin stndrd; (b) txifolin in the smple. For robustness nlysis, 2 3 complete experimentl design with triplicte in the centrl point ws relized. The clculted vlues for min effects nd interctions in 95% level of confidence re shown in Tble 4. From the nlysis of the results, it is possible to see tht smll vritions in these chromtogrphic prmeters did not show significnt chnges in the overll ssy vlues. Therefore, it is possible to conclude tht the method is robust for the prmeters nlyzed within the intervls investigted.

8 Vol. 27, No. 9, 2016 Almeid et l Tble 4. Fctors, levels, nd contrst coefficients mtrix for the experimentl design conducted to study robustness nd the respective clculted effects y Vectors Experiment X 1 X 2 X 3 X 12 X 13 X 23 X 123 Assy / % t R / min Effect on ssy Men 0.90 ± 0.01 Principl effect X 1 : temperture of the column oven 0.02 ± 0.03 X 2 : mobile phse flow rte 0.01 ± 0.03 X 3 : incresing of cetonitrile percentge in mobile phse 0.01 ± 0.03 Two-fctor interction X ± 0.03 X ± 0.03 X ± 0.03 Three-fctor interction X ± 0.03 t v S effect / % 0.13 Retention time. All experiments provided good fctor cpcity of the chromtogrphic column ( 1.0), symmetry of the nlyticl pek ( 2.0) nd column efficiency (number of theoreticl pltes / m 2000). Conclusions Under the conditions described, the method for quntittive determintion of txifolin in the brk extrct of P. pinster is in ccordnce with the vlidtion prmeters required by the ICH nd INMETRO guidelines. The method of HPLC-UV is vilble in qulity control lbortories, nd it is dequte for txifolin quntifiction without mtrix interferences. Therefore, the quntifiction of txifolin my be n dditionl qulity prmeter for this extrct in ssocition with the spectrophotometric determintion of the totl procynidins content. However, for this purpose, future investigtions re necessry in order to estblish limits for txifolin content. Acknowledgments This work ws supported by CAPES, CNPq, FAPEMIG nd PROPESQ/UFJF. The uthors would lso like to thnk Shrlene Loures for her help throughout the work. References 1. Vroni, E. M.; Lodi, G.; Srdell, A.; Crrssi, A.; Iriti, M.; Curr. Med. Chem. 2012, 19, Guillrme, D.; Csett, C.; Bicchi, C.; Veuthey, J.-L.; J. Chromtogr. A 2010, 1217, Hshim, S. N. N. S.; Schwrz, L. J.; Boysen, R. I.; Yng, Y.; Dnylec, B.; Hern, M. T. W.; J. Chromtogr. A 2013, 1313, Moreno, M.; Arribs, A. S.; Bermejo, E.; Zprdiel, A.; Chichrro, M.; Electrophoresis 2011, 32, Motilv, M.-J.; Serr, A.; Mcià, A.; J. Chromtogr. A 2013, 1292, Rodríguez-Medin, I. C.; Segur-Crretero, A.; Fernández- Gutiérrez, A.; J. Chromtogr. A 2009, 1216, Liu, P.; Curr. Anl. Chem. 2013, 9, Nicolou, I. N.; Kpnissi-Christodoulou, C. P.; Electrophoresis 2010, 31, Puss, J. G.; Blde, C.; Vldecntos, A.; Sev, J. P.; Fuentes, D.; Alloz, J. A.; Vilgros, A.; Butist, S.; Cortin, J.; Vllejo, R.; Plnt Ecol. 2004, 171, 209.

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