Artigo. Quim. Nova, Vol. 37, No. 6, , 2014

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1 Quim. Nov, Vol. 37, No. 6, , VOLATILE CONSTITUENTS OF Aristolochi trilobt L. (Aristolochicee): A RICH SOURCE OF SULCATYL ACETATE Drlisson de Alexndri Sntos, Péricles Brreto Alves, *, Emmnoel Vilç Cost, Clovis Roberto Pereir Frnco b, Angelit Nepel c nd Andersson Brison c Deprtmento de Químic, Universidde Federl do Sergipe, Av. Mrechl Rondon, S/N, Cmpus Universitário, São Cristóvão SE, Brsil b Deprtmento de Biologi, Universidde Federl do Sergipe, Av. Mrechl Rondon, S/N, Cmpus Universitário, São Cristóvão SE, Brsil c Deprtmento de Químic, Universidde Federl do Prná, Curitib PR, Brsil Artigo Recebido em 12/09/2013; ceito em 25/03/2014; publicdo n web em 17/06/2014 Anlysis of the voltile frction of Aristolochi trilobt stem led to the identifiction of 6-methyl-5-hepten-2-yl cette (23.31 ± 0.28%), limonene (15.43 ± 0.030%), linlool (8.70 ± 0.29%), p-cymene (7.81 ± 0.12%), bicyclogermcrene (4.21 ± 0.11%), nd spthulenol (4.17 ± 0.14%) s the mjor constituents of the essentil oil. Linlool (29.51 ± 0.49%), 6-methyl-5-hepten-2-ol (19.54 ± 0.82%), 6-methyl-5-hepten-2-yl cette (8.92 ± 0.16%), nd -terpineol (4.62 ± 0.05%) were identified s mjor constituents of the hydrolte. The compound 6-methyl-5-hepten-2-yl cette ws isolted for the first time from this plnt nd ws identified s the mjor component of the voltile frction. Keywords: Aristolochi trilobt; essentil oil; 6-methyl-5-hepten-2-yl cette. INTRODUCTION The genus Aristolochi consists of ~500 species distributed minly in Asi, Afric, South Americ, nd North Americ. 1 In recent studies, mny of these plnts hve shown diuretic, nlgesic, ntiinflmmtory, nd nti-cncer ctivity. 2 Severl species of Aristolochi, which present similrities both in terms of their botnicl chrcteristics nd their properties, cn be found in Brzil. The species most commonly used in folk medicine re A. tringulris, A. espernze, A. ridicul, A. brsiliensis, A. rcute, nd A. gignte. Considering the bove-mentioned similrities, these species shre the sme common nmes in Brzil, which include jrrinh, cipó-mil-homens, mil-homens, milone, ppo-de-peru, ervde-urubu, nd jiboinh. 3 Extensive reserch hs been crried out on the plnts of this genus, minly on extrcts of the leves, stems, nd roots. A vriety of ctivities hve been ttributed to them, these include bctericidl, nti-inflmmtory, nti-trypnosoml, nd nti-tumorl. 4-7 Chemicl compounds tht hve been identified in these plnts, both in the essentil oils nd in orgnic solvent extrcts, include porphines, mides, quinolines, lignnes, diphenyl ethers, flvonoids, benzenoids, steroids, nd terpenoids. 1 The essentil oils of the species belonging to the genus Aristolochi re comprised minly of monoterpenes nd sesquiterpenes, the commonly occurring terpenes being germcrene nd cryophyllene, which re the mjor compounds in most cses. Aristolochi trilobt L. is species of Aristolochi found in Centrl nd South Americ, nd hs severl pplictions in trditionl medicine in these regions. 8 One such medicinl use in treting injured dogs hs been reported by Lns et l., 9 who crried out study on hunters in Trinidd nd Tobgo. According to this study, species of the genus Aristolochi, in prticulr A. trilobt nd A. rugos, re widely used in tretment *e-mil: periclesblves@gmil.com for dogs tht hve been bitten by snkes or scorpions. 9 In the sme country, A. trilobt is used for treting stomch che, colic, poisoning, nd dibetes in humn ptients, s well s to fcilitte the removl of the plcent nd bortion The use of this plnt for treting snke bites is not restricted to Trinidd nd Tobgo. Studies hve reveled tht A. trilobt hs lso been used for this purpose in Brzil nd Nicrgu. 13,14 In Brzil, it hs been reported tht A. trilobt is lso used s fungicide. 15 Studies revel tht A. trilobt is used s n ntimlril gent in French Guin. 16 In Dominic, this plnt is used for treting intestinl problems. 17 An infusion (te) or plnt extrct of A. trilobt is used in folk medicine. One study determined the chemicl composition of the methnolic extrcts of the root nd stem; four ristolochic cids nd one ristolctm were identified. 18 The stem is found in mrkets nd firs in Arcju, Sergipe Stte, Brzil, nd is commonly used by the popultion in cchç (sugr cne spirit) infusions nd ingested in this form. In this pper, the first study on the chemicl composition of the essentil oil of the stem of A. trilobt is reported. The nlysis of the hydrolte is lso reported. RESULTS AND DISCUSSION The verge yield of the oil (oil mss/plnt mss) ws 0.22%, with stndrd devition of 0.05%. The chemicl constituents of the essentil oil re shown in Tble 1. The min constituents of the essentil oil were 6-methyl-5-hepten-2-yl cette (sulctyl cette) (23.31 ± 0.28%), limonene (15.43 ± 0.03%), linlool (8.70 ± 0.29%), p-cymene (7.81 ± 0.12%), bicyclogermcrene (4.21 ± 0.11%), nd spthulenol (4.17 ± 0.14%). Notble compounds in the hydrolte, due to their high content, were linlool (29.51 ± 0.49%), 6-methyl-5-hepten-2-ol (19.54 ± 0.82%), 6-methyl-5-hepten-2-yl cette (sulctyl cette; 8.92 ± 0.16%), nd -terpineol (4.62 ± 0.05%). These compounds were lso identified in the essentil oil, but in different percentges. It should

2 978 Sntos et l. Quim. Nov be noted tht oxygented compounds constituted 88.11% of the totl hydrolte composition (Tble 1). In this study, sulctyl cette (Figure 1) ws identified by nucler mgnetic resonnce ( 1 H nd 13 C NMR; both 1D nd 2D), gs chromtogrphy coupled with mss spectrometry (GC-MS), nd infrred (IR) nlyses (spectr re included in supplementry mteril). In ddition, there re only few reports of this compound in the literture. The bsolute configurtion of the chirl center ws determined by polrimetry nd by comprison with the dt in literture. The compound ws determined to be (R)-( )-sulctyl cette. 19 There is only one report of sulctyl cette from plnt source, by Mkholel nd Mnning, 20 who studied the voltile compounds ssocited with the rom of the flowers of the species Struthiol cilite. The percentge of sulctyl cette ws only 0.04%. 20 Two other ppers hve reported on the presence of sulctyl cette in nture. In the first of these studies, the uthors nlyzed the voltile compounds present in the venom of five species of wsp of the genus Polistes, genus found in the Mediterrnen region of Europe. The percentges of sulctyl cette present in the nlyzed wsp venom smples were 4.14% (P. dominulus), 0.34% (P. gllicus), 4.30% (P. nimphus), 0.86% (P. sulcifer), nd 1.78% (P. olivceus). 21 In the second study, the Figure 1. (R)-6-methyl-5-hepten-2-yl (sulctyl cette), essentil oil isolted from Aristolochi trilobt voltile compounds present in the venom of only one wsp species, P. dominulus, ws investigted, nd the percentge of sulctyl cette ws 1.78%. 22 In other publictions, sulctyl cette is cited s prticipnt in the enzymtic resolution of 6-methyl-5-hepten-2-ol (sulctol), n enntiomericlly pure pheromone of mbrosi beetles. 23,24 Thus, the identifiction of this compound s mjor component of the voltile compounds originting from plnt source, in this cse A. trilobt, is of gret relevnce. Other compounds with significnt presence in essentil oils of other species of the sme genus hve been reported. Tble 1. Constituents of essentil oil nd hydrolte of A. trilobt Compound RRI b % hydrolte ± Std. cl % oil ± Std. Dev. Dev. -pinene ± cmphene ± b-pinene ± octen-3-ol ± ± methyl-5-hepten-2-one ± 0.09 myrcene ± methyl-5-hepten-2-ol ± ± phellndrene ± d-3-crene ± p-cymene ± ± 0.01 limonene ± ± ,8-cineole ± ± 0.02 benzene cetldehyde ± 0.05 (E)-b-ocimene ± ± 0.01 cis-linlool oxide (furnoid) ± 0.04 trns-linlool oxide (furnoid) ± 0.02 linlool ± ± octen-3-yl cette ± phenylethyl lcohol ± methyl-5-hepten-2-yl cette ± ± 0.16 cmphor ± ± 0.06 cis-linlool oxide (pyrnoid) ± 0.06 borneol ± ± 0.12 terpinen-4-ol ± ± 0.14 p-cymen-8-ol ± terpineol ± ± 0.05 cis-piperitol ± 0.07 verbenone ± 0.12 trns-crveol ± 0.04 citronellol ± ± 0.08 thymol methyl ether ± crvone ± 0.01 gerniol ± 0.14 Compound RRI b % hydrolte ± Std. cl % oil ± Std. Dev. Dev. isobornyl cette ± ± 0.08 crvcrol ± 0.01 p-vinyl-guicol ± 0.04 citronellyl cette ± ± copene ± b-elemene ± (E)-cryophyllene ± ± 0.01 b-copene ± romdendrene ± (E)-b-frnesene ± ± humulene ± llo-romdendrene ± g-muurolene ± ± 0.03 germcrene D ± viridiflorene ± bicyclogermcrene ± ± 0.08 g-morphene ± g-cdinene ± selin-3,7(11)-diene ± spthulenol ± ± 0.18 cryophyllene oxide ± 0.01 globulol ± 0.13 viridiflorol ± epi-cubenol ± cdinol ± ± 0.03 eudesm-7(11)-en-4-ol ± ± 0.04 Monoterpene hydrocrbons Oxygented monoterpenes Sesquiterpene hydrocrbons Oxygented sesquiterpenes Totl compounds identified RRI cl Reltive retention index clculted using homologous series of n- lknes (C9-C18) in n polr cpillry column DB-5MS. b Anlysis crried out in triplicte.

3 Vol. 37, No. 6 Voltile constituents of Aristolochi trilobt L. (Aristolochicee) 979 For exmples, limonene, monoterpene frequently found in severl plnts of wide vriety of gener, is present in high concentrtions in the following species of this genus: A. gibertii (38.5%), A. rcut (8.7%), A. glet (10.5%), A. mlmen (10.3%), A. melstom (34.5%), A. debilis (7.3%), nd A. indic (6.9%) Another compound previously identified in Aristolochi is linlool. This compound is lso found in the voltile frction of plnts of other gener, but hs significnt presence (16.6%) in A. gignte in this genus. 29 Bicyclogermcrene nd spthulenol re lso frequently found in species of Aristolochi. In prticulr, bicyclogermcrene is one of the mjor compounds in severl species, including A. rcut (10.0%), A. chmissonis (24.0%), A. cynnchifoli (38.8%), A. espernze (22.7%), A. pulistn (40.3%), A. gignte (18.9%), A. cymbifer (8.5%), A. elegns (15.2%), A. glet (11.9%), A. mcrour (15.3%), A. melstom (9.2%), nd A. tringulris (10.7%). 31 EXPERIMENTAL Plnt mteril The plnt mteril ws collected in October 2011 from the municiplity of Estânci, Sergipe Stte, Brzil (geogrphicl coordintes: S = 11º nd W = 37º ). The plnt ws identified by Diogo Arújo (MSc) nd voucher specimen ws deposited t the herbrium of the Federl University of Sergipe (ASE) under voucher number ASE Stem distilltion of essentil oil Smples of dry A. trilobt stem were cut into smll pieces nd triturted in four-knife mill (Mrconi, model MA680). The essentil oil ws obtined through the stem distilltion process in Clevenger device. In this method, triturted stem (200 g) nd distilled wter (1500 ml) were plced in 2 liter flsk, nd distilltion initited fter coupling with the Clevenger device. Distilltion ws continued for 180 min fter the strt of the condenstion in the Clevenger device. The yield of essentil oil ws expressed s percentge (oil mss/ plnt mss). The essentil oil obtined ws stored in refrigerted mber flsk until further nlysis. Liquid-liquid extrction of hydrolte The hydrolte ws obtined by extrction of the distillte collected during stem distilltion of voltile components of A. trilobt. Initilly collected distillte (500 ml) ws extrcted with diethyl ether. The liquid-liquid extrction ws performed by wshing 250 ml of the distillte with diethyl ether (3 50 ml) t room temperture. The hydrolte extrct obtined (100 mg from 1 L of distillte) ws evported fter drying on N 2 SO 4. Generl experimentl procedures Gs chromtogrphy-mss spectrometry The oil smple ws nlyzed on Shimdzu QP5050A (Shimdzu Corportion, Kyoto, Jpn) system equipped with n AOC-20i uto- -smpler nd the gs chromtogrph ws interfced to mss spectrometer (GC/MS) with J&W Scientific DB-5MS (Folsom, CA, USA) fused-silic cpillry column (30 cm 0.25 mm i.d., composed of 5% phenylmethylpolysiloxne). Helium (99.999%) ws used s crrier gs t constnt flow of 1.2 ml min 1 nd n injection volume of 0.5 μl ws employed (split rtio of 1:83). The injector temperture ws 250 C nd the ion-source temperture ws 280 C. The oven temperture ws progrmmed to increse from 50 C (isotherml for 2 min) to 200 C with rte of 4 C/min, nd then t 10 C/min to 300 C, where it ws mintined for 10 min. Mss spectr ( D; EI) were cquired t 70 ev with scn intervl of 0.5 s. Chirl gs chromtogrphy (GC-FID) Enntioselective GC nlysis of (±)-sulctyl cette ws performed on n Agilent fused-silic cpillry column (cyclodex-b; 30 m 0.25 mm i.d., 0.25 µm film thickness) using gs chromtogrph (Shimdzu model GC 17A), equipped with flme-ioniztion detector (FID). The temperture of the oven ws progrmmed to increse from 50 C (isotherml for initil 1.0 min) to 80 C t 3 C/min; fter being held t 80 C for 10 min, the temperture ws llowed first to increse t 0.5 C/min to 95 C nd then t 15 C/min to 170 C, where it ws mintined for the finl 5 min. Helium ws used s the crrier gs t constnt flow of 1.2 ml/min, nd the injector nd detector tempertures were 200 C nd 280 C, respectively. The injection volume ws 0.5 µl (ethyl cette) with split rtio of 1:10. Identifiction of essentil oil constituents Individul components of the essentil oil were identified by computerized mtching of the cquired mss spectr with those stored in WILEY8, NIST107, nd NIST21 mss spectrl librries of the GC- MS dt system. A mixture of hydrocrbons (C9H20 C19H40) ws injected under the sme conditions nd constituents were identified by compring the spectr obtined with those in the dtbnk nd considering the reltive retention index (RRI), clculted for ech constituent s previously described. 32 Nucler mgnetic resonnce (NMR) 1D nd 2D NMR dt were cquired t 293 K in CDCl 3 on Bruker AVANCE III 400 NMR spectrometer operting t 9.4 Tesl observing 1 H nd 13 C t nd MHz, respectively. The spectrometer ws equipped with either 5-mm multinucler direct detection probe (1D NMR experiments) or 5-mm multinucler inverse detection probe (1D NOE nd 2D NMR experiments), both with z-grdient. One-bond nd long-rnge 1 H- 13 C correltions from HSQC nd HMBC NMR experiments were optimized for verge coupling constnts of 1 J (C,H) nd LR J (C,H) of 140 nd 8 Hz, respectively. All 1 H nd 13 C NMR chemicl shifts (d) re reported in ppm reltive to the TMS signl t 0.00 ppm, s internl reference, nd the coupling constnts (J) in Hz. Infrred (IR) spectroscopy A Perkin Elmer infrred spectrometer with Fourier trnsform, model BX, ws used. The spectr were obtined in the region from 4000 cm 1 to 400 cm 1. Opticl rottion Opticl rottion ws determined using Perkin Elmer polrimeter (model P-2000) t the Deprtment of Orgnic nd Inorgnic Chemistry of the Federl University of Cerá, Brzil. The mesurements were tken using monochromtic sodium light t wvelength of 589 nm nd were expressed using the nottion [α] T D, where T (in o C) is the temperture t which the mesurement ws recorded. Chloroform (CHCl 3 ) ws used for the dissolution of the smples. Isoltion of sulctyl cette Seprtions by preprtive thin lyer chromtogrphy (TLC) were crried out on freshly prepred TLC pltes. Silic gel pltes (1.0 mm thick) were prepred by evenly spreding ~30 g of Mcherey-Ngel

4 980 Sntos et l. Quim. Nov Tble 2. NMR dt (400 MHz, CDCl 3 ) for 6-methyl-5-hepten-2-yl Position 13 C 1 H mult. (J) HMBC NOE CH d (6.3) 2 & (H-2) CH 4.88 dqd (7.7; 6.3; 5.3) & (H-1) 3 3b CH ddt (13.3; 7.7; 6.3) 1.49 dtd (13.3; 7.4; 5.3) & & CH dddqq (7.4; 7.2; 6.3; 1.0; 0.8) & CH 5.08 tqq (7.2; 1.4; 1.3) & (H-7) qc CH dtq (1.3; 1.0; 0.4) 5. 6 & CH dtq (1.4; 0.8; 0.4) 5. 6 & (H4) & 1.63 (H-3) qc (C=O) CH s 9 The experiments were crried out t 295 K nd the chemicl shifts re expressed in ppm in reltion to the TMS signl t 0.00 ppm, s the internl reference, nd the coupling constnts (J) in Hz. The exct multiplicities of the signl were determined with the id of the first-order multiplet simultor/check FOMSC3. The long rnge 1 H- 13 C correltions (HMBC) were optimized for J H,C 8 Hz. silic gel 60 in 80 ml of distilled wter on glss sheets (20 20 cm). After evportion of the wter t mbient temperture, the TLC pltes were ctivted in n oven t 110 C for 30 min. For visuliztion, solution of nisldehyde in cid nd ethnol (ethyl lcohol (90 ml) + sulfuric cid (5 ml) + nisldehyde (5 ml) + cetic cid (1 ml)) ws used, followed by heting t 110 C. For ech preprtive TLC, 300 mg of oil ws pplied nd eluted twice using the pproprite solvent system. The verge yield of sulctyl cette ws 15% nd hd purity of 85%, s determined by GC-FID. (2R)-( )-6-methyl-5-hepten-2-yl cette The isoltion gve colorless oil, [α] D (c 0.2, 4 ml, CHCl 3 ). 1 H nd 13 C NMR dt of the sme re shown in Tble CONCLUSIONS Anlysis of the voltile frction of Aristolochi trilobt llowed for the isoltion nd identifiction of (2R)-( )-6-methyl-5-hepten-2-yl cette (sulctyl cette), limonene, linlool, p-cymene, bicyclogermcrene, nd spthulenol s the mjor constituents of the essentil oil. Linlool, 6-methyl-5-hepten-2-ol (sulctol), 6-methyl-5-hepten-2-yl cette (sulctyl cette), nd -terpineol were the min constituents of the hydrolte. Sulctyl cette ws identified for the first time in the genus Aristolochi, where it is present s mjor component of the voltile frction of the plnt. SUPPLEMENTARY MATERIAL 1 H nd 13 C NMR spectr (including 1D nd 2D spectr), IR spectr, MS, nd GC-FID chirl chromtogrm of sulctyl cette, long with the totl ion chromtogrm (TIC) of the essentil oil nd the hydrolte of A. trilobt re vilble t org.br in the form of PDF file, with free ccess. ACKNOWLEDGEMENTS We re grteful to CNPq nd CAPES for the finncil support, including grnts. We thnk Prof. Dr. André L. M. Porto of USP/São Crlos for kindly supplying the smples of (2R)-(-)-sulctyl cette nd the rcemic mixture. We lso thnk Prof. Gilvndete Mri P. Sntigo (UFC) for the use of the polrimeter. REFERENCES 1. Wu, T.-S.; Dmu, A. G.; Su, C.-R.; Kuo, P. C.; Studies In Nturl Products Chemistry, Att-Ur-Rhmn, ed.; Elsevier: Amsterdm, Yu, J. Q.; Lio, Z. X.; Ci, X. Q.; Lei, J. C.; Zou, G. L.; Environ. Toxicol. Phrmcol. 2007, 32, Lorenzi, H.; Mtos, F.J.A.; Plnts Medicinis No Brsil: Ntivs e Exótics, 5ª ed., Plntrum: Nov Odess, Nvrro-Grci, V. M.; Lun-Herrer, J.; Rojs-Bibriesc, M. G.; Álvrez-Fitz, P.; Ríos, M. Y.; Molecules 2011, 16, 7360; 5. Chung, Y. M.; Chng, F. R.; Tseng, T. F.; Hwng, T. L.; Chen, L. C.; Wu, S. F.; Lee, C. L.; Lin, Z. Y.; Chung, L. Y.; Su, J. H.; Wu, Y. C.; Bioorg. Med. Chem. Lett. 2011, 21, 1792; 6. Srtorelli, P.; Crvlho, C. S.; Reimão, J. Q.; Tempone, A. G.; Plnt Med. 2010, 76, 1455; 7. Hegde, V. R.; Borges, S.; Ptel, M.; Ds, P. R.; Wu, B.; Gullo, V. P.; Chn, T.-M.; Bioorg. Med. Chem. Lett. 2010, 20, Heinrich, M.; Chn, J.; Wnke, S.; Neinhuis, C.; Simmonds, M. S. J.; J. Ethnophrmcol. 2009, 125, Lns, C.; Hrper, T.; Georges, K.; Bridgwter, E.; BMC Complementry Altern. Med. 2001, 1, Lns, C.; Journl of Ethnobiology nd Ethnomedicine 2007, 3, Lns, C.; Journl of Ethnobiology nd Ethnomedicine 2007b, 3, Mhbir, D.; Gulliford, M. C.; Revist Pnmericn de Slud Públic 1997, 1, Coe, F. G.; Anderson, G. J.; J. Ethnophrmcol. 2005, 96, 307; 14. Houghton, P. J.; Osibogun, I. M.; J. Ethnophrmcol. 1993, 39, Fenner, R. Betti, A. H.; Mentz, L. A.; Rtes, S. M. K.; Rev. Brs. Cienc. Frm. 2006, 42, Vigneron, M.; Depris, X.; Dehro, E.; Bourdy, G.; J. Ethnophrmcol. 2005, 98, Quinln, M. B.; Quinln, R. J.; Noln, J. M.; J. Ethnophrmcol. 2002, 80, Jou, J. H.; Li, C. Y.; Schelonk, E. P.; Lin, C. H.; Wu, T. S.; J. Food Drug Anl. 2004, 12, Ling, S.; Pquette, L. A.; Tetrhedron: Asymmetry 1990, 1, Mkholel, T.; Mnning, J. C.; S. Afr. J. Bot. 2006, 72, Bruschini, C.; Dni, F. R.; Pierccini, G.; Gurn, F.; Turillzzi, S.; Toxicon 2006, 47, Bruschini, C.; Cervo, R.; Protti, I.; Turillzzi, S.; The Journl of Experimentl Biology 2008, 211, 2444.

5 Vol. 37, No. 6 Voltile constituents of Aristolochi trilobt L. (Aristolochicee) Nkmur, K.; Kinoshit, M.; Ohno, A; Tetrhedron 1995, 51, Ferreir, H. V.; Roch, L. C.; Severino, R. P.; Vin, R. B.; D Silv, A. B. F.; Porto, A. L. M.; Journl of the Irninin Chemicl Society 2010, 7, Knjill, P. B.; Kotoky, R.; J. Essent. Oil Res. 2009, 21, Hyshi, N.; Sugiym, Y.; Kome, H.; J. Nt. Prod. 1987, 50, Cnel, N.; Ferro, E.; Alvreng, N.; Vil, R.; Cñiguerl, S.; J. Essent. Oil Res. 2004, 16, Priestp, H. A.; Vn Bren, C. M.; Lir, P. D. L.; Prdo, H. J.; Neugebuer, M.; Myer, R.; Bndoni, A. L.; Flvour Frgrnce J. 2002, 17, Frncisco, C. S.; Messino, G. B.; Lopes, L. M. X.; Tininis, A. G.; De Oliveir, J. E.; Cpellri Jr., L.; Phytochemistry 2008, 69, Mrchesini, A. M.; Prdo, G. G.; Messino, G. B.; Mchdo, M. B.; Lopes, L. M. X.; J. Brz. Chem. Soc. 2009, 20, Silv-Brndão, K. L.; Solferini, V. N.; Trigo, J. R.; Biochem. Syst. Ecol. 2006, 34, Adms, R.P.; Identifiction of Essentil Oil Components by Gs Chromtogrphy/Mss Spectroscopy, 4ª ed., Allured Publishing Corportion: Illinois, 2007.