), AS A FUNCTION OF PLANT PROVENANCE AND PLANT PARTS. Instituto de Química, Universidade Estadual Paulista, Araraquara SP, Brasil b

Transcription

1 Quim. Nov, Vol. 37, No. 2, , INTRASPECIFIC VARIABILITY OF Holostylis reniformis: CONCENTRATION OF LIGNANS, AS DETERMINED BY MACERATION AND SUPERCRITICAL FLUID EXTRACTION (SFE-CO 2 ), AS A FUNCTION OF PLANT PROVENANCE AND PLANT PARTS Gisline F. Mrtins, Mrcos D. P. Pereir, Luci M. X. Lopes*,, Tito d Silv b, Pulo de T. Vieir e Ros c, Fernnd P. Brbos c, Gisele B. Messino d nd Antonin U. Krettli e Instituto de Químic, Universidde Estdul Pulist, Arrqur SP, Brsil b Centro de Ciêncis Sociis, Súde e Tecnologi, Universidde Federl do Mrnhão, Impertriz MA, Brsil c Instituto de Químic, Universidde Estdul de Cmpins, Cmpins SP, Brsil d Instituto Federl de São Pulo, Mtão SP, Brsil e Instituto René Rchou, FIOCRUZ, Belo Horizonte MG, Brsil Artigo Recebido em 03/07/2013; ceito em 09/10/2013; publicdo n web em 11/12/2013 Mcertion nd supercriticl fluid extrction were used to prepre extrcts from prts of plnts (Holostylis reniformis) collected in two different regions of Brzil. 1 H NMR, HPLC-DAD-ESI/MS, HPLC-DAD, GC-MS, nd chemometric techniques were used to nlyse lignns in the extrcts nd showed tht yields of SFE-CO 2 were less thn or equl to those of hexne mcertion extrcts. These nlyses, in conjunction with the concentrtions of liphtic hydrocrbons, ftty cids nd their methyl nd ethyl derivtives in the extrcts, lso llowed the chemicl composition of prts nd provennce of the plnt to be differentited. Keywords: Holostylis reniformis; Aristolochicee; lignns. INTRODUCTION Holostylis reniformis Duch. (Aristolochicee) is widely distributed in Brzil nd hs been used in trditionl Brzilin medicine s n ntirheumtic, stomchic, nd depurtive. 1 Extrcts from this species hve been shown to exhibit ntimlril ctivity nd to contin high concentrtions of 8-8 linked lignns without 9,9 -oxygention tht showed ntiplsmodil ctivity. 2 Moreover, biosynthetic studies hve shown tht isoeugenol is biosynthetic intermedite for these 2,7 lignns (ryltetrlone lignns) nd 7,7 epoxylignns (furn lignns). 3,4 Our previous studies on H. reniformis determined tht reverse- -phse HPLC-DAD 4-6 provided the most suitble conditions for obtining chromtogrphic profiles of non-polr extrcts from the roots (hexne, chloroform, nd cetone), which contined ryltetrlone lignns nd 7,7 epoxylignns. Under these conditions, t lest 10 different lignns were well chrcterized. To chieve the gol of obtining clen extrcts rich in lignns from this species, supercriticl fluid extrction (SFE) ws used, since SFE is known to be rpid, selective nd convenient method for smple preprtion prior to the nlysis of compounds in nturl product mtrices. 7 The most used supercriticl solvent is pure or modified CO 2 becuse of its criticl prmeters, especilly the criticl temperture which llows extrction t mild tempertures, decresing the tendency to degrde therml sensitive compounds. Moreover, supercriticl fluid extrction with crbon dioxide (SFE-CO 2 ) hs been used to extrct components tht present low to medium polrity from solid nd liquid phrmceuticl mtrices due to its sfety s n lterntive to the conventionl liquid extrction of lignns, such s those of Schisndr chinensis, Schisndr sphennther, nd Forsythi koren. In ddition, the on-line coupling of SFE with supercriticl fluid chromtogrphy (SFC) ws previously used for the extrction nd seprtion of neolignns such s mgnolol nd *e-mil: lopesxl@iq.unesp.br honokiol in Mgnolie cortex. 8 To contribute to dvnces in phrmcologicl tests of these extrcts nd lignns, which re needed in substntil mounts, this report describes comprtive study on two methods for obtining extrcts rich in ryltetrlone lignns from H. reniformis, nd compres the intrspecific chemicl vribility of this species collected in two different regions of Brzil (Mins Geris Stte (MG) nd Mrnhão Stte (MA)). RESULTS AND DISCUSSION Extrctions Extrcts of dried roots, leves, nd stems from plnts collected in Impertriz (MA) nd Ituiutb (MG) were prepred by supercriticl fluid extrction (SFE-CO 2 ) nd by clssicl method using mcertion (extrction with hexne, cetone, nd ethnol, successively) t room temperture. With these two methods, extrction nd concentrtion were completed within 120 min nd 8 dys, respectively. Opletl et l. nd Lojková et l. 9 observed tht the conditions for supercriticl fluid extrction t pressures bove 20 MP nd tempertures of C hd little effect on the SFE lignn yields. However, the mtrix type, including the prt of the plnt, gretly influenced the composition of lignns in extrcts obtined by SFE. 7,10 Thus, the experimentl conditions estblished for SFE-CO 2 from roots, stems, nd leves (20.0 g ech) of H. reniformis were CO 2 t 50 C, 27 MP, nd 50 min. Since the mount of rw mteril ws limited nd different from tht used by the previous SFE studies vilble in the literture, 11,12 the extrctions were conducted t high pressure, reltively high temperture nd high CO 2 flow rte in order to chieve the lrgest extrction yield in the shortest time. The extrcts obtined were then exmined with regrd to extrction yield (Tble 1) nd composition. The yields of SFE-CO 2 (E-7 to E-12) were less thn or equl to those of the hexne mcertion extrcts (E-1 to E-6) nd their chemicl composition similr, s evidenced by the following nlyses.

2 282 Mrtins et l. Quim. Nov Tble 1. Extrction yields nd provennce of H. reniformis Plnt mteril Extrct (%) Mcertion (100 g) Provennce Dry plnt prt hexne cetone ethnol SFE-CO 2 (20 g) MG Roots E-1 (3.70) E-13 (3.72) E-19 (5.92) E-7 (3.71) MG Stems E-2 (0.36) E-14 (0.83) E-20 (2.13) E-8 (0.36) MG Leves E-3 (4.42) E-15 (2.94) E-21 (7.69) E-9 (1.32) MA Roots E-4 (2.92) E-16 (2.52) E-22 (6.20) E-10 (1.31) MA Stems+roots E-5 (1.37) E-17 (1.07) E-23 (3.60) E-11 (0.89) MA Leves E-6 (4.38) E-18 (4.57) E-24 (3.41) E-12 (1.63) Qulittive nd semi-quntittive nlyses of ryltetrlone lignns Qulittive nd semi-quntittive nlyses of ryltetrlone lignns in the root nd stem extrcts were performed by using 1 H NMR, HPLC-DAD-ESI/MS, HPLC-DAD, HPLC-DAD-CD, nd GC-MS techniques. To optimize the experimentl conditions (HPLC, ESI-MS, nd GC prmeters) nd quntifiction, (-)-8 -epi-ristoligone (1) nd (-)-ristotetrlone (2), previously isolted from H. reniformis, were used s stndrd references (Figure 1). shows the typicl spectr of extrcts contining lignn 1 t high concentrtion. None of the chrcteristic signls corresponding to lignn hydrogens were observed in the 1 H NMR spectr of extrcts from the plnt leves, or in the ethnol extrcts from the roots. HPLC nlysis Bsed on n nlysis of non-polr extrcts (SFE-CO 2, hexne, nd cetone) under the optiml conditions previously determined for extrcts from the roots of H. reniformis, 4,6 11 different lignns were well-chrcterized by compring their retention times, CD, UV bsorptions, nd mss spectr: (-)-8 -epi-ristoligone (1), (-)-ristotetrlone (2), (-)-4 -O-methylenshicine (3), (-)-ristoligone (4), (-)-8 -epi-ristotetrlone (5), cgynone A (6), 8,8 -epi-ristoligone (7), cgynone B (8), (-)-8-epi-holostylone (9), (-)-holostyligone (10), nd clopiptin (12). In ddition, bsic structures were suggested for two ryltetrlone (13 nd 14) nd two furn lignns (15 nd 16) on the bsis of their mss spectr (Tble 1S, Figures 1 nd 2S). The mss spectr of ryltetrlone lignns showed chrcteristic ions corresponding to the moleculr ions nd protonted moleculr ions seen with EI nd ESI, respectively. Key ions, which my hve risen from retro Diels Alder rerrngements involving the A nd B rings nd nphthol formtion, were observed by GC-MS nd HPLC-DAD-ESI/ MS, respectively (for further detils, see supplementry mteril, Tble 1S, Figures 2S nd 3S). Reltive nd semi-quntittive nlyses by HPLC A reltive quntittive nlysis by HPLC-DAD-ESI/MS ws lso performed to compre the compositions of the root nd stem extrcts. The pek res of compounds tht showed retention times (t R ) between 6.1 nd 33.3 min in the chromtogrms were trnsformed into percentges. This nlysis (Tble 1S) showed tht cetone nd ethnol extrcts of stems+roots from MA contin significnt mount of 3, nd most of the smples from MA contin higher concentrtions of 2 thn 1. As seen in Tble 1S, plnt provennce cn be clerly seen by this nlysis. A semi-quntittive nlysis (HPLC-DAD) ws performed by the externl stndrd method for extrcts tht showed well-resolved peks with the sme retention times s 1 nd/or 2 (Figure 4S). Higher concentrtion of 1 ws lso determined in the extrcts from MG nd of 2 in those from MA (Tble 2). Figure 1. Chemicl structures of compounds H NMR nlysis 1 H NMR spectr of ryltetrlone lignns from H. reniformis re very chrcteristic. They showed hydrogen signls t d (CH 3 ), (OCH 3 ), (CH), (CH 2 O 2 ), (Ar-H), nd ~7.5 (H-6). As n exmple, Figure 1S (supplementry mteril) GC-MS nlysis The composition of the crude extrcts ws lso estblished using GC-MS nlyses by compring the liner retention index (I) of the compounds with those of stndrd smples nd with dt in the literture, s well s by nlysing their mss spectr, where seven lignns were chrcterized (1-4, 7, 11, nd 12, Tble 3). In ddition to ethyl olete, ethyl plmitte, methyl esters of 9-octdecenoic

3 Vol. 37, No. 2 Intrspecific vribility of Holostylis reniformis 283 Tble 2. Clculted concentrtions (%) of lignns 1 nd 2 in the extrcts by liner regression Extrcts Stndrds Concentrtion Extrcts Stndrds Concentrtion E E-10 1 Nc E E-11 1 Nc b E-4 1 nc E E-5 1 nc E b c E E-16 1 Nc E Concentrtion expressed s g/100 g of extrct; b Vlues clculted below LOD; c Vlues clculted below LOQ; nc: not clculted. nd 9,12-octdecdienoic cids, seven liphtic hydrocrbons (pentcosne, hexcosne, heptcosne, octcosne, noncosne, tricontne, nd hentricontne), two ftty cids (plmitic nd oleic cids) nd their methyl esters were lso identified by co-injection of uthentic smples from Aldrich (Tble 3). As observed by 1 H NMR nd HPLC nlyses, the extrcts of leves showed significnt concentrtion of liphtic esters nd hydrocrbons, but not of lignns (dt not shown). Sttisticl nlyses An gglomertive hierrchicl cluster nlysis (HCA) nd principl component nlysis (PCA) were individully pplied to dtsets of normlized chromtogrms obtined by GC-MS of the soluble hexne solutions of extrcts. These nlyses llowed the drwing of similrity plots of the corresponding extrcts to the principl components to obtin informtion bout the chrcteristic peks, which re the most discriminting for the smples observed on the plots (Figures 2, 3, nd 5S). In ddition, HCA (Figure 2) showed three distinct groups, two of which were only from roots nd group tht contined stems. Moreover, the PCA results were consistent with these three groups nd llowed stem extrcts to be differentited from stems+roots extrcts (Figure 3, Tble 2S). Positive score signls for PC1 corresponded to those extrcts of stems nd roots from MA, which were highly influenced by octcosne (vrible 21), wheres negtive vlues were influenced by 12 (vrible 17) (Figure 5S). In ddition, the hydrocrbons noncosne, tricontne, nd hentricontne (vribles 22-24) chrcterized extrcts of stems from MG while ethyl olete (vrible 9) chrcterized SFE-CO 2 extrct of stems+roots from MA (E-11). This ester nd methyl ftty esters were previously isolted from Aristolochi grndiflor nd Aristolochi long. 13 Thus, the chemicl compositions of the different prts of the plnts re significntly dissimilr. In generl, SFE-CO 2 nd hexne extrcts from the sme plnt prt hve very similr compositions, s evidenced in Figures 2 nd 3. To compre the compositions of the root nd stem extrcts, the Tble 3. Composition of extrcts determined by GC-MS I (seg) Vrible Compounds (%) Dvnol Extrcts b E-1 E-2 E-4 E-5 E-7 E-8 E-10 E-11 E-13 E-14 E Bulnesol Plmitic cid, methyl ester Plmitic cid Plmitic cid, ethyl ester ,12-Octdecdienoic cid, methyl ester Octdecenoic cid, methyl ester Oleic cid Oleic cid, ethyl ester Pentcosne Hexcosne Hydrocrbon (hexcosne type) Heptcosne Octcosne Noncosne Tricontne Hentricontne Sitosterol I: Liner retention index; b composition expressed s percentge; for extrct codes, see Tble

4 284 Mrtins et l. Quim. Nov Figure 2. Dendrogrm constructed bsed on n gglomertive hierrchicl clustering nlysis (HCA) of hexne solutions of extrcts of H. reniformis with different provennces by GC-MS (for extrct numbering, see Tble 1) Figure 4. Dendrogrm constructed bsed on n gglomertive hierrchicl clustering nlysis (HCA) of extrcts of H. reniformis of different provennces by HPLC-DAD-ESI/MS nd HPLC-DAD (for extrct numbering, see Tble 1) Figure 3. Principl component nlysis (PCA) of chemicl constituents of hexne solutions of extrcts from stems nd roots of H. reniformis by GC- -MS. The principl components (PC1 nd PC2) ccount for c. 73.6% of the informtion (for extrct numbering, see Tble 1) pek res of the compounds, which showed retention times (t R ) between 6.1 nd 33.3 min, were trnsformed into percentges. Similr HCA nd PCA nlyses using 14 extrcts from the two provennces, nd the pek re (%) for 41 compounds (chrcteristics) in HPLC chromtogrms, were performed (Tble 1S). They lso indicted tht supercriticl nd hexne extrcts from the sme plnt prts nd plnt provennce were very similr. In ddition, s expected, these nlyses showed dissimilrities between these extrcts nd those obtined from cetone nd ethnol extrction (Figures 4 nd 5, Tble 3S). Lignn 3 contributes significntly to PC1, PC2, nd PC3 positive vlues, wheres lignns 1 nd 2 contribute to PC3 positive nd negtive vlues, respectively (Figure 6S). As seen in Tbles 1S nd 3, SFE-CO 2 cn be successfully pplied for the extrction of lignns from plnts with different provennces Figure 5. Principl component nlysis (PCA) of chemicl constituents of extrcts from stems nd roots of H. reniformis by HPLC-DAD-ESI/MS nd HPLC-DAD. The principl components (PC1 nd PC2) ccount for c. 79.0% of the informtion (for extrct numbering, see Tble 1) nd lso from different plnt prts. In ddition, the richest extrcts in ryltetrlone lignns re from the roots. Mcertion with hexne ws more efficient in the extrction of lignns 1 nd 2 from roots but supercriticl fluid extrction ws more efficient in their extrction from stems. Although the nlyses by GC nd HPLC took into ccount the compound percentges in the extrct smples, the concentrtions of the compounds in the extrcts (Tbles 1S, 2 nd 3) should not be compred without considering the different conditions of nlysis, smple preprtions, nd techniques. However, chemometric nlyses showed tht both GC-MS nd HPLC-DAD-ESI/MS were relible techniques for compring plnt prt compositions nd for discriminting the plnt provennce, since PC1 nd PC2 ccounted for c. 74% nd 79% of the informtion, respectively. Furthermore, SFE-CO 2 extrction protocol optimiztion my improve both the extrction yield nd lignn purity in the obtined extrcts.

5 Vol. 37, No. 2 Intrspecific vribility of Holostylis reniformis 285 EXPERIMENTAL Stndrd compounds Aldrich kits contining 24 stndrd hydrocrbons/c 5 C 30, stright-chin lknes (Aldrich 29,850-6), 19 ftty cids/c 6 -C 24, stright-chin (Aldrich 29,851-4), nd ftty cid methyl esters/c 6 C 24 stright-chin (Aldrich 29,851-4) were used s stndrd compounds for GC-MS nlyses. Nturl compounds isolted nd identified by spectroscopic methods (minly by MS, 1 H nd 13 C NMR) from H. reniformis nd other Aristolochi species were lso used s stndrds: bulnesol, lignns: (-)-8 -epi-ristoligone (1), (-)-ristotetrlone (2), (-)-4 -O-methylenshicine (3), (-)-ristoligone (4), (-)-8 -epi-ristotetrlone (5), cgynone A (6), 8,8 -epi-ristoligone (7), cgynone B (8), (-)-8-epi-holostylone (9), holostyligone (10), glbcin (11), 6, nd clopiptin (12) (Figure 1). Plnt mterils The plnt mterils were collected in Ituiutb, MG, Brzil, nd Impertriz, MA, Brzil, in Februry 2008 nd 2010, respectively, when the vines were in the blooming stge. The distnce between these two collecting regions is bout 1510 km. 19 The plnts were identified s Holostylis reniformis Duch. by Dr. Vinícius C. Souz nd Dr. Lindolpho Cppellri Jr. Voucher specimens (ESA 88282/2008 nd ESA /2010) were deposited t the herbrium of the Escol Superior de Agricultur, Luiz de Queiroz (ESALQ), Pircicb, SP, Brzil. The mterils were seprted ccording to the plnt prts nd dried (~ 45 C). As seprtion of roots from the stems is timeconsuming, smples from plnts of MA were lso obtined without these prt plnt seprtions to provide informtion bout extrct compositions for further plnt collections. Mcertion extrction Ground plnt mterils (roots, stems, roots+stems, nd leves, g ech) from plnts collected in MA nd MG were individully subjected to mcertion extrction with hexne, cetone, nd ethnol, successively, s previously described (4 ~200 ml, 2 dys, nd shken mnully every 12 h for 2 min for ech extrction). 4,5 The solvents were then eliminted under reduced pressure in fume hood to give 18 extrcts (E-1 to E-6 nd E-13 to E-24, Tble 1). Supercriticl fluid extrction Similrly, ground plnt mterils (roots, stems, roots+stems, nd leves, 20 g ech) were individully subjected to supercriticl fluid extrction by using the extrction system previously described, 11 with slight modifictions. Briefly, column ( cm) filled with 20.0 g of the plnt mteril ws coupled to supercriticl extrction unit. The extrction system ws operted with sttic period of 5 min nd, the procedure ws then conducted using CO 2 t 50 C nd 27 MP for 50 min nd flow rte of 40 L/min. The extrcts were collected in filtering flsk cooled in n ice bth. After extrction, the pressure of the system ws reduced, nd the tubing locted fter the extrction column ws wshed with ethnol (10 ml) to recover residul mtter deposited in this re. The solvent ws eliminted in fume hood to produce 6 extrcts (E-7 to E-12, Tble 1). Ech combintion of extrction nd concentrtion ws thus completed within 120 min. 1 H NMR nlyses 1 H NMR experiments were performed on Vrin INOVA 500 spectrometer (11.7 T) t 500 MHz ( 1 H) using deuterted solvents (CDCl 3 nd DMSO-d 6 ) (P 99.9% D) nd residul solvent s n internl stndrd for 1 H NMR. d vlues re reported reltive to TMS. Smples (30 mg ech) were filtered through cotton wool, dissolved in 1.0 ml of deuterted solvent (CDCl 3 or DMSO-d 6 ), nd then subjected to 1 H NMR experiments (field: 11.7 T, temperture: 28 C, pulse: 45, relx. dely: sec, width: Hz; cq. time: sec, repetitions: 16, FT size: 65536, totl time: 80 sec). The compounds were identified by derepliction nlyses of 1 H NMR spectr of the crude extrcts nd by compring 1 H NMR dt with those reported in the literture nd/or with the uthentic smples. 2-6,15,18 Preprtion of smples nd stndrd solutions of extrcts nd their nlyses by HPLC-DAD, HPLC-DAD-CD, nd HPLC DAD-ESI/MS Extrcts nd stndrd lignns (1 mg/ml MeOH) were filtered through PVDF 0.45 µm membrne nd subjected to qulittive (HPLC-DAD-ESI/MS, HPLC-DAD, nd HPLC-DAD-CD) nd semi-quntittive (HPLC-DAD) nlyses. HPLC nlyses were performed using Shimdzu liquid chromtogrph (SPD-10 Avp) equipped with UV vis nd 341-LC polrimeter detectors, nd chromtogrms were cquired t 336 nd 254 nm using Jsco LC-NetII/ADC equipped with photodiode rry (MD-2018 Plus) nd CD (2095 Plus) detectors (rnge of 200 to 420 nm). All HPLC conditions used in this work were identicl: the smples were injected by n utomtic injector; the injection volume ws 20 µl nd the elution time ws 60 min; the columns were RP-18 (Vrin, C18, with prticle size of 5 µm, mm); the mobile phse used ws MeOH:H 2 O (7:3; v/v). HPLC- DAD-CD were used for determintion of the Cotton effect signl chrcteristic of ryltetrlone lignns t 310 nm. 15 Mss spectr (ESI-MS) were obtined on n LCQ Fleet Thermo Scientific, in positive ionistion mode (20V) recorded over mss rnge of m/z , nd flow injection into the electrospry source ws used for HPLC-DAD-ESI/MS. GC-MS nlyses Except for E-17 to E-24, which showed very low solubility in hexne, the composition of the hexne solutions of the crude extrcts ws estblished by GC-MS nlyses. These nlyses were performed on Shimdzu GCMS-QP5050A system in EI mode (70 ev) equipped with n utomtic split/splitless injector (220 C), t split rtio of 1/10, using VF-1MS fused-silic cpillry column (30 m 0.25 mm i.d.; film thickness: 0.25 µm). The oven temperture ws progrmmed from 60 C (5 min) to 280 C t rte of 4 C/min nd held t this temperture for 10 min. Helium ws used s crrier gs t flow rte of 0.8 ml/min. The injection volume ws 2 µl. Portions (0.5 mg) of ech extrct were dissolved with the GC-grde n-hexne (1 ml), filtered through PVDF 0.45 µm membrne, nd nlysed by GC MS (Tble 3). Retention indices for ll compounds were determined ccording to the eqution proposed by vn den Dool nd Krtz, 20 using n-lknes s stndrds. Adjusted retention times (RRt) for ech pek were determined by subtrcting the retention time of helium from the retention time of ech pek. Components were identified bsed on comprison of their mss spectr with those held on the NIST/EPA/NIH Mss Spectrl Dtbse (NIST08), Mss Spectrometry Dt Centre, nd those described by Adms, s well s by compring their I vlues with those of n-lknes, ftty cids nd cid methyl esters from Aldrich, nd with dt in the literture. 21 The extrcts were co-injected with severl compounds tht hd been previously isolted from H. Reniformis. 2-6,15,18

6 286 Mrtins et l. Quim. Nov Sttisticl nlysis Reltive quntittive nlysis of dt obtined from GC-MS nd HPLC-DAD-ESI/MS Agglomertive hierrchicl cluster nlyses (HCA) nd principl component nlysis (PCA) were used s sttisticl methods to suggest the structure of the set nd to nlyse the vribles in reltion to the chrcteristics being studied. GC-MS sttisticl nlysis Overll, 25 chrcteristics (chemicl compounds identified by GC-MS) were nlysed in 11 extrcts by HCA nd PCA (Tbles 3 nd 2S, Figures 2, 3, nd 5S) by using the Pirouette version 3.11 progrm. 22 The chemicl compositions were determined from the chromtogrphic profiles of 5 (6) extrcts from prts of plnts collected in MA (MG). To reduce scttering effects nd to compre smples, the chromtogrms were normlized by reducing the res under ech chromtogrm to vlue of Plots defined by PC1 (score 1), PC2 (score 2), nd PC3 (score 3) for the 25 chrcteristics were obtined for chromtogrphic dt using Pirouette version The results were obtined using n originl dt mtrix X (25 by 11) with 25 vribles, 11 smples, 3 optiml fctors, 1st derivtive, with Euclidin distnce s mesure of similrity. The vrinces of PC1 ( ), PC2 ( ), nd PC3 ( ) ccounted for 64.48%, 26.12%, nd 6.98%, respectively, of the totl PCA vrince. HPLC-DAD-MS sttisticl nlysis Anlogously, plots defined by PC1 (score 1), PC2 (score 2), nd PC3 (score 3) for 41 chrcteristics (t R determined by HPLC-DAD- ESI/MS nd HPLC-DAD, Tbles 1S nd 3S, Figures 5 nd 6S) nd 14 smples (7 (7) extrcts from prts of plnts collected in MA (MG)) were obtined for HPLC dt. In these cses, the vrinces of PC1 ( ), PC2 ( ), nd PC3 ( ) ccounted for 52.88%, 26.14%, nd 13.24%, respectively, of the totl PCA vrince. As described bove, HCA ws lso performed for these 14 extrcts nd 41 compounds (Figure 4). Evlution of linerity nd limits of detection nd quntifiction for the HPLC-DAD method The pek res of the compounds were trnsformed into percentges to compre the compositions of the root nd stem extrcts. The contents of individul compounds were mesured semi-quntittively from liner regressions using 1 nd 2, which were expressed s g/100 g extrct (Tble 2). This mesuring ws not performed in some extrcts from MA due to the presence of co-eluted compound, or in cetone nd ethnol extrcts tht did not show peks with t R in the selected rnge. The clibrtion curves were constructed by plotting pek res of 1 nd 2 ginst corresponding concentrtions in triplicte (1: 0.005, 0.100, 0.200, 0.300, 0.450, 0.600, nd mg/ml; 2: 0.044, 0.078, 0.098, 0.132, nd mg/ml). Both clibrtion curves showed good linerity with correltion coefficients (1: r 2 = , 2: r 2 = ). The liner regression equtions were y = x for 1 nd y = x for 2, where y is the pek re nd x is the concentrtion of lignn (mg/ml), nd r < The limits of detection (LOD, 1: mg/ml, 2: mg/ml) nd of quntifiction (LOQ, 1: mg/ml, 2: mg/ml) were estimted from the clibrtion curves (Figure 4S). 24 CONCLUSIONS As result of these nlyses we cn infer tht the mount of ryltetrlone lignns in extrcts from the leves, nd in ethnol extrcts from roots nd stems is not significnt. However, the hexne nd the supercriticl extrcts from roots hve similr chemicl compositions. Although the SFE-CO 2 extrction resulted in lower or equl yields, it is fster nd clener procedure thn mcertion extrction for the recovery of lignns. In ddition, HCA nd PCA showed intrspecific vribility between plnts collected from different regions of Brzil locted considerble distnce from ech other, while both GC-MS nd HPLC-DAD-ESI/MS re relible techniques for compring plnt prt compositions. SUPPLEMENTARY MATERIAL Tbles 1S 3S nd Figures 1S 6S re vilble t in PDF formt, with free ccess. ACKNOWLEDGEMENTS The uthors thnk Dr. Vinícius C. Souz nd Dr. Lindolpho Cpellri Jr. for plnt identifiction, Alexndre Cestri for GC-MS experiments, nd the Fundção de Ampro à Pesquis do Estdo de São Pulo (FAPESP), Coordenção de Aperfeiçomento de Pessol de Nível Superior (CAPES), nd Conselho Ncionl de Desenvolvimento Científico e Tecnológico (CNPq/MCT/MS/PRONEX, Brzil) for finncil support nd fellowships. REFERENCES 1. Hoehne, F. C. Em: Flor Brsílic; Lnzr, F., ed.; Grphicrds: São Pulo, 1942, vol. 15, p de Andrde-Neto, V. F.; d Silv, T.; Lopes, L. M. X.; do Rosrio, V. E.; Vrotti. F. P.; Krettli, A. U.; Antimicrob. Agents Chemother. 2007, 51, Messino, G. B.; d Silv, T.; Nscimento, I. R.; Lopes, L. M. X.; Plnt Med. 2008, 74, Messino, G. B.; d Silv, T.; Nscimento, I. R.; Lopes, L. M. X.; Phytochemistry 2009, 70, d Silv, T.; Krettli, A. U.; de Andrde-Neto, V. F.; Lopes, L. M. X.; Revist d Propriedde Industril 2005, 1795, Messino, G. B.; Wijertne, E. M. K.; Lopes, L. M. X.; Guntilk, A. A. L.; J. Nt. Prod. 2010, 73, Slnin, J.; Gltz, Z.; J. Chromtogr. B 2004, 812, Herrero, M.; Cstro-Puyn, M.; Mendiol, J. A.; Ibñez, E.; Trends Anl. Chem. 2013, 43, 67; Choi, Y. H.; Kim, J.; Yoo, K.-P.; Chromtogrphi 2003, 57, 73; Suto, K.; Ito, Y.; Sgr, K.; Itokw, H.; J. Chromtogr. A 1997, 786, Opletl, L.; Sovová, H.; Bártlová, M.; J. Chromtogr. B 2004, 812, 357; Lojková, L.; Slnin, J.; Mikesov, M.; Táborská, E.; Vejrost, J.; Phytochem. Anl. 1997, 8, Comin, L. M.; Temelli, F.; Sldñ, M. A.; J. Am. Oil Chem. Soc. 2011, 88, 707; Wng, L.; Weller, C. L.; Trends Food Sci. Technol. 2006, 17, Egydio, J. A.; Mores, A. M.; Ros, P. T. V.; J. Supercrit. Fluids 2010, 54, Justo, O. R.; Mores, A. M.; Brreto, G. P. M.; Mercdnte, A. Z.; Ros, P. T.V.; Quim. Nov 2008, 31, Teres, J. P.; Urones, J. G.; Fernndez, A.; Alvrez, M. D. V.; Phytochemistry 1984, 23, 461; Aguilr, M. I.; Espejo, O.; Cmcho, D.; Fitoterpi 1992, 63, McAlpine, J. B.; Riggs, N. V.; Gordon, P. G.; Aust. J. Chem. 1968, 21, d Silv, T.; Lopes, L. M. X.; Phytochemistry 2004, 65, d Silv, T.; Lopes, L. M. X.; Phytochemistry 2006, 67, 929.

7 Vol. 37, No. 2 Intrspecific vribility of Holostylis reniformis Lopes, L. M. X.; Pereir, M. D. P.; d Silv, T.; Krettli, A. U.; Phrm. Biol. 2012, 50, Pereir, M. D. P.; d Silv, T.; Lopes, L. M. X.; Krettli, A. U.; Mdureir, L. S.; Zukermn-Schpector, J.; Molecules 2012, 17, DistnciDoisPontos/Tel.sp, cessd em dezembro vn den Dool, H.; Krtz, P. D.; J. Chromtogr. A 1963, 11, Houss, T. G.; Rod, M.; Eight Pek Index of Mss Spectr, 4 th ed., The Royl Society of Chemistry, The Mss Spectrometry Dt Centre: Cmbridge, 1991, vols. 1 3; Adms, R. P.; Identifiction of Essentil Oil Components by Gs Chromtogrphy/Mss Spectroscopy, 4 th ed., Allured Publishing Corportion: Crol Strem, 2009; Frncisco, C. S.; Messino, G. B.; Lopes, L. M. X.; Tininis, A. G.; de Oliveir, J. E.; Cpellri Jr., L.; Phytochemistry 2008, 69, 168; com, cessd em mrço InfoMetrix; Pirouette for Windows; Version 3.11, Inc. Bothell, Bertrnd, D.; Scotter, C. N. G.; Appl. Spectrosc. 1992, 46, Thompson, M.; Ellison, S. L. R.; Wood, R.; Pure Appl. Chem. 2002, 74, 835.

8 Quim. Nov, Vol. 37, No. 2, S1-S4, 2014 INTRASPECIFIC VARIABILITY OF Holostylis reniformis: CONCENTRATION OF LIGNANS, AS DETERMINED BY MACERATION AND SUPERCRITICAL FLUID EXTRACTION (SFE-CO 2 ), AS A FUNCTION OF PLANT PROVENANCE AND PLANT PARTS Gisline F. Mrtins, Mrcos D. P. Pereir, Luci M. X. Lopes*,, Tito d Silv b, Pulo de T. Vieir e Ros c, Fernnd P. Brbos c, Gisele B. Messino d nd Antonin U. Krettli e Instituto de Químic, Universidde Estdul Pulist, Arrqur SP, Brsil b Centro de Ciêncis Sociis, Súde e Tecnologi, Universidde Federl do Mrnhão, Impertriz MA, Brsil c Instituto de Químic, Universidde Estdul de Cmpins, Cmpins SP, Brsil d Instituto Federl de São Pulo, Mtão SP, Brsil e Instituto René Rchou, FIOCRUZ, Belo Horizonte MG, Brsil Supplementry Mteril Tble 1S. Composition of extrcts determined by HPLC-DAD nd HPLC-DAD-ESI/MS Vrible b Lignn [M+H] + m/z t R (min) Extrcts (%) E-1 E-2 E-4 E-5 E-7 E-8 E-10 E-11 E-13 E-14 E-16 E-17 E-20 E t R : Retention times; composition expressed s percentge in the rnge of 6.18 to min; for extrct codes, see Tble 1. b Selected vribles: lignns nd compounds whose concentrtions were t lest 5.0% in one of the nlysed extrcts. *e-mil: lopesxl@iq.unesp.br

9 S2 Mrtins et l. Quim. Nov Tble 2S. Scores for three principl components (PCs) for the dtset of hexne solutions of extrcts from stems nd roots of H. reniformis by GC-MS Smples Fctor 1 Fctor 2 Fctor 3 E E E E E E E E E E E For extrct codes, see Tble 1. Tble 3S. Scores for three principl components (PCs) for the dtset of hexne solutions of extrcts from stems nd roots of H. reniformis by HPLC- -DAD-ESI/MS Smples Fctor 1 Fctor 2 Fctor 3 E E E E E E E E E E E E E E For extrct codes, see Tble 1. Figure 1S. Representtive 1 H NMR spectr of root extrcts of H. reniformis (collected in MG): () hexne, (b) SFE-CO 2, nd of stndrds 1 nd 2 (CDCl 3, 500 MHz)

10 Vol. 37, No. 2 Intrspecific vribility of Holostylis reniformis S3 Figure 2S. Representtive totl ion current (TIC) of hexne () nd SFE-CO 2 (b) extrcts from the roots of H. reniformis from plnts collected in MA, nd SFE-CO 2 (c) from plnts collected in MG. Peks corresponding to the identified lignns re shown (20 V, positive mode, C18, mm, UV detection t 254 nm, nd flow rte: 0.8 ml min -1 ) Figure 3S. Key ions obtined by GC-MS nd ESI-MS of 2, 3, nd 5

11 S4 Mrtins et l. Quim. Nov Figure 6S. Loding plot of principl components (PC1, PC2, nd PC3) obtined for hexne solutions of extrcts from stems nd roots of H. reniformis by HPLC-DAD-ESI/MS nd HPLC-DAD (for vrible code, see Tble 1S) Figure 4S. Linerity of response (m AU) for 1 nd 2 stndrds with HPLC- -DAD method. Concentrtions of solutions used in the clibrtion curve: () Concentrtions of 1: 0.005, 0.1, 0.2; 0.3, 0.45, 0.6, nd 0.8 mg/ml; (b) Concentrtions of 2: 0.044, 0.078, 0.098, 0.132, nd mg/ml Figure 5S. Loding plot of principl components (PC1, PC2, nd PC3) obtined for hexne solutions of extrcts from stems nd roots of H. reniformis by GC-MS (for vrible code, see Tble 3)