A

pocaTCs©

K

1

outct

gaitficitetsb

0d

J. of Supercritical Fluids 40 (2007) 354–359

GC/MS characterization of mate tea leaves extracts obtained fromhigh-pressure CO2 extraction

R.A. Jacques a, J.G. Santos b, C. Dariva b, J. Vladimir Oliveira b, E.B. Caramao a,∗a Institute of Chemistry, Federal University of Rio Grande do Sul, Av. Bento Goncalves, 9500, Porto Alegre 91501-960, RS, Brazil

b Department of Food Engineering, URI—Campus de Erechim, Av. Sete de Setembro, 1621, Erechim 99700-000, RS, Brazil

bstract

This work presents a study concerning the chemical characteristics and analytical separation process of the essential oil obtained from high-ressure carbon dioxide extraction of Ilex paraguariensis. The experiments were performed in a laboratory-scale unit in the temperature rangef 20–40 ◦C, from 100 to 250 bar. A blend of the I. paraguariensis extracts was percolated through a preparative chromatographic column,ontaining silica gel, and successively eluted with 150 mL of each of the following solvents: hexane, toluene, dichloromethane, ethyl acetate,cetone and methanol. The raw extract and its fractions were analyzed by gas chromatography coupled to mass spectrometry detection (GC/MS).

he fractionation procedure showed to be a good clean up technique due to the isolation of different classes of compounds in each fraction.hromatographic analyses allowed the identification of caffeine, fatty acids and esters, phytol, squalene, Vitamin E, stigmasterol derivatives and

aturated hydrocarbons.2006 Elsevier B.V. All rights reserved.

lean-

icdatepbpfutisr

o

eywords: Gas chromatography; High-pressure fluid extraction; Yerba-mate; C

. Introduction

The identification of organic compounds from the extractsf plants is of great importance, mainly because they can besed as an excellent source of pharmaceutical products for phy-otherapy. Since plant extracts are complex mixtures of organicompounds, they request appropriate fractionation techniqueso allow a better analysis of their individual constituents.

The main objective of sample preparation for chromato-raphic analysis is the selective dissolution of the analytes insuitable solvent and the consequent removal of the interfer-

ng compounds from the solution. Particularly useful classicalechniques of sample preparation include selective extraction,ltration, precipitation, dialysis, centrifugation and simple open-olumn chromatography. The most used extraction proceduresn phytoanalysis are solid–liquid extraction (SLE), with addi-ional stirring or mechanical agitation, percolation, solid-phasextraction (SPE) and liquid–liquid extraction (LLE); but other

echniques like supercritical fluid extraction (SFE), acceleratedolvent extraction (ASE), soxhlet extraction and liquid mem-rane extraction, are also used [1–6].

∗ Corresponding author. Fax: +55 51 33167304.E-mail address: elina@urfgs.br (E.B. Caramao).

oaAiyoa

896-8446/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.supflu.2006.07.023

up; Ilex paraguariensis; GC/MS

Supercritical fluid extraction (SFE) is a powerful techniquen the separation processes [7,8]. The solvent under supercriticalonditions has unique physicochemical properties such as highiffusivity, low viscosity and low surface tension, which make itn adequate choice for the extraction of target components fromhe solid matrix [9–11]. Today, SFE has become an acceptablextraction technique used in many areas. SFE of active naturalroducts from herbs, or more generally, from plant materials hasecome one of the most important application areas. One of theroblems with SFE is that the resulting extracts are not alwaysree from unwanted matrix components and thus require clean-p. For this purpose, a number of clean-up methods have beenested simultaneously with or after the extraction step. Interfer-ng substances are often trapped with a sorbent material and, inome cases, the solvent composition can be altered to increaseecoveries [12].

The mate tea leaves (Ilex paraguariensis) is a native kindf South America and has remarkable importance in the econ-my and in the cultural life of the southern region of Brazil anddjacent countries, such as Argentina, Uruguay and Paraguay.n average of 300,000 t of yerba-mate is produced each year

n South America [13,14]. The processed product (“mate orerba mate”) is a typical beverage prepared as a hot infusionf the dried and cut leaves. It is also used in popular medicinend is employed in commercial herbal preparations as a natu-

rcritic

rhd

otthr

ptonpamcch

2

2

dEl1fe3so

2

SaeicMtt

2

s[tj(H

ayCbelwmtcii11som

2

hmwof5b1TteofB(

2

muasp(cctw

3

R.A. Jacques et al. / J. of Supe

al medicine for arthritis, headache, constipation, rheumatism,emorrhoids, obesity, fatigue, fluid retention, hypertension, slowigestion and hepatic disorders [15].

Many reports indicate that saponins are the major constituentsf Ilex species [16–19]. The occurrence of flavanoids [20], xan-hines [21–23], aldehydes [24], hemiterpene glycosides [25],riterpenes and alkanes [26], anthocyanins [27], pentyl esters,exyl esters, and other lipophylic compounds [28] has also beeneported.

Some works regarding the extraction of mate leaves com-ounds with sub or supercritical carbon dioxide can be found inhe literatures [21,23,29,30]. However, these works are focusedn the effect of the high-pressure extraction variables and agro-omic parameters on the chemical composition of selected com-ounds (mainly methylxanthines, Vitamin E, phytol, squalenend one steroid). In this sense, the main contribution of thisanuscript relies on the development of a technique for the

haracterization and identification of important active chemicalonstituents present in mate tea leaves extracts obtained fromigh-pressure carbon dioxide extraction.

. Experimental

.1. Plant material

Mate tea leaves samples were collected in an experiment con-ucted under agronomic control at “Industria e Comercio derva-Mate Barao LTDA”, in Rio Grande do Sul, Brazil. Mate

eaves samples from plants of about 7 years old, with leaves with8 months, at complete sun exposure and without any additionalertilization added to the plants were selected for the extractionxperiments. The samples were dried in a vacuum oven (24 h at5 ◦C) and ground (40 mesh). After drying, the samples weretored under nitrogen atmosphere until the extraction to avoidxidation.

.2. Chemicals

All the solvents were bidistilled and stored in dark flasks.ome standards such as caffeine, theobromine, hexadecanoiccid, octadecanoic acid, tetradecanoic acid, phytol, squalene,icosane, docosane, hexacosane, nonacosane, tricosane, hene-cosane, heptacosane, triacontane, linoleic acid ethyl ester, tetra-osane, and Vitamin E were purchased from Sigma (St. Louis,O, USA). The CO2 employed in the extractions (White Mar-

ins S.A., 99.5% in liquid phase) was used without furtherreatment.

.3. Apparatus and extraction procedure

High-pressure extractions were performed in a laboratory-cale unit, schematically presented in previous works8,11,29,30], which consists basically of a CO2 reservoir, two

hermostatic baths, a syringe pump (ISCO 260D), a 0.2 dm3

acketed extraction vessel, an absolute pressure transducerSmar, LD301) equipped with a portable programmer (Smar,T 201) with a precision of ±0.31 bar, a collector vessel with

opw

al Fluids 40 (2007) 354–359 355

glass tube and a cold trap. Amounts of around 25 g of driederba-mate leaves were charged into the extraction vessel. TheO2 was pumped at a constant flow rate of 1 g min−1 into theed, which was supported by two 300 mesh wire disks at bothnds, and was kept in contact with the vegetable matrix for ateast 1 h to allow the system stabilization. Afterwards, the extractas collected opening the micrometering valve, and the CO2ass flow was accounted for the pump recordings. After that,

he mass of the extracted oil was weighed, the glass tube was re-onnected to the equipment and this procedure was performedn approximately 400 min. The experiments were accomplishedn the temperature range of 20–40 ◦C, and in pressures from00 to 250 bar. A whole experimental run lasted in general0 h, including all steps involved: sample weighing, temperaturetabilization (baths, extractor), depressurization, etc. Extractsbtained were dissolved in dichloromethane prior to gas chro-atography/mass spectrometry analysis (GC/MS).

.4. Clean up of the extracts

Clean up was done using silica gel as solid phase andexane, toluene, dichloromethane, ethyl acetate, acetone andethanol as elution solvents. A glass column (40 cm × 11 mm)as packed with 10.0 g of silica previously activated. On the topf the silica, it was added a thin layer of anhydrous sodium sul-ate (approximately 1.0 g). The column was conditioned with0 mL of hexane. The sample (approximately 500 mg of alend of all the high-pressure extracts) was firstly mixed with.0 g of silica and 1.0 mL of hexane to form a thin slurry.he solvent was evaporated and this dry mixture was put in

he top of the column. The elution was done with 150 mL ofach solvent. Each fraction was evaporated under a gentle fluxf nitrogen (ultra pure grade, White Martins S.A.). All theractions obtained were derivatized using the combination ofSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide) and TMCS

trimethylchlorosilane) before the chromatographic analysis.

.5. Gas chromatography analysis

The extracts were analyzed in a gas-chromatograph with aass selective detector (GC/MS Shimadzu, Model QP 5050A),

sing a capillary column OV-5 (30 m × 0.25 mm × 0.25 �m); atflow rate of 1 mL min−1; in electronic impact mode (70 eV);

plit mode injection (1:20), GC/MS interface at 280 ◦C tem-erature, column temperature program was as follows: 1001 min)–300 ◦C to 4 ◦C min−1 (30 min). Identification of theompounds was made by comparing mass spectra of standardompounds with those of the samples. Other compounds wereentatively identified comparing only the mass spectra obtainedith the Wiley library of the equipment.

. Results and discussion

The first step of this work was the investigation of the effectf temperature and pressure on the extraction yield of the high-ressure carbon dioxide extracts of mate leaves. The liquid yieldas defined here as the weight percentage of the oil extracted

356 R.A. Jacques et al. / J. of Supercritical Fluids 40 (2007) 354–359

Table 1Liquid yield of the extraction of mate tea leaves with CO2 at high pressures

Run T (◦C) P (bar) Densitya

(g/cm3)Extraction liquid yield(gextract/graw material × 100)

1 30 175 0.868 1.93 ± 0.152 20 250 0.963 1.73 ± 0.113 40 250 0.880 2.46 ± 0.194 20 100 0.854 1.19 ± 0.095 40 100 0.612 0.53 ± 0.05

a Angus et al. [31].

Fc

wtwlAilpe

oos(esOpr

fptimt

pa

Table 2Liquid yield of preparative liquid chromatographic fractionation of Ilexparaguariensis extract

Fraction Eluent % yield

1 Hexane 3.6 ± 0.232 Toluene 63.2 ± 7.223 Dichloromethane 8.8 ± 2.344 Ethyl acetate 14.8 ± 3.3656

dieicooa

ei(cfuaic

pealscp

Caffeine (peak 7), hexadecanoic acid (peaks 11, 13 and 14),squalene (peak 37), phytol (peaks 15 and 18) and Vitamin E(peak 44) stigmastenol (peaks 49 and 50), sitosterol (or stigmas-tadienol) (peaks 48 and 51) are the most abundant and important

ig. 1. Effects of temperature and pressure on the kinetics of the high-pressurearbon dioxide extraction of mate tea leaves.

ith respect to the initial charge of the raw material in the extrac-or. Table 1 presents the liquid yield of mate tea leaves extractionsith carbon dioxide at high pressures. This table also shows the

arge variation on the solvent density, which was calculated fromngus correlation [31]. The results presented in this table are

n fact mean values of three extractions. It can be observed thatiquid yields higher than 2.5% were obtained and the extractionrocess variables exert a pronounced effect on the amount ofxtracts obtained.

Fig. 1 shows the kinetic extraction curves of the extractionsf mate tea leaves, where the two well-known antagonist effectsf temperature and pressure can be observed; i.e. solvent den-ity (solvent power) at lower pressures and solute vapor pressuretemperature) at higher pressures. For example, at 100 bar thextraction yield decreases when temperature increases as a con-equence of the large reduction in the CO2 density (see Table 1).n the other hand, at 250 bar the reduction in density is lessronounced and the increase in the solute vapor pressure isesponsible for the high extraction yield.

As presented in Table 1, the extraction experiments were per-ormed covering the most common ranges of temperature andressure presented in the literature. Also, the chemical composi-ion could be strongly influenced by extraction parameters and,n this sense, to identify the chemical constituents present in

ate leaves, a blend of extracts obtained from distinct tempera-ure and pressure extraction conditions was performed.

The clean-up of extract was made taking into account theolarity of the solvents used as eluents. Different solvents weressayed, and the final order of elution was: hexane, toluene,

Fbt

Acetone 3.2 ± 0.21Methanol 6.4 ± 2.15

ichloromethane, ethyl acetate, acetone and methanol, accord-ng to the polarity increase. Table 2 shows the results of thextraction in terms of mass yield for the clean up procedure, andt can be seen that the mate tea leaves extract from high-pressurearbon dioxide extraction is constituted mainly by compoundsf low and intermediate polarity, as the main fractions werebtained with solvent elution sequence from toluene to ethylcetate.

Fig. 2 shows the total ion chromatogram (TIC) of the wholextract (blend of all extracts) and Fig. 3 presents the totalon chromatograms (TIC) and monitoring ion chromatogramsMIC) of the six fractions from the clean up procedure. MIChromatograms were obtained by monitoring the specific ionsrom some compounds, aiming at an additional “electronic cleanp” of the fractions. These ions were selected after the detailednalysis of TIC. Table 3 shows the identification of the peaksn Figs. 2 and 3 and the respective ions used in the monitoringhromatograms.

High-pressure mate tea leaves extracts showed to be a com-lex mixture of hydrocarbons, oxygen (alcohols, fatty acids,sters, etc.) and nitrogen compounds (caffeine, theobromine)greeing with what says the literatures [32,33]. In this mateeaves extract, 51 compounds were detected and identified (usingtandard compounds or the Wiley library of the GC/MS). Aompound was only considered positively identified when theercent of similarity was superior to 90%.

ig. 2. Typical total ion chromatogram of the extract of mate tea leaves obtainedy high-pressure CO2 extraction at 30 ◦C and 175 bar. Chromatographic condi-ions explained in the text.

R.A. Jacques et al. / J. of Supercritical Fluids 40 (2007) 354–359 357

F ionatit ; (f) f

caebaac

meclareTc

i

wtt(rht[fctspp

ig. 3. Total ion chromatograms (TIC) of the six fractions obtained in the fracthe text. (a) Fraction 1; (b) fraction 2; (c) fraction 3; (d) fraction 4; (e) fraction 5

onstituents of this mate leaves extract. Methyl xanthines, suchs caffeine, are known to be responsible for the stimulatingffects of the yerba-mate leaves tea [13], squalene is knowny its anti-cancer action [34,35] and stigmasterol derivativesre recognized anti oxidants [36]. Phytosterols in plant extractsre of increasing interest because of their role in the control ofholesterol levels and reduction of arterosclerosis [9].

Vitamin E, on the other hand, is a recognized substance withedicinal properties, being used against approximately 80 dis-

ases, such as cancer, cystic fibrosis cardiovascular diseases,ell membrane and DNA damage by free radicals, oxidation ofow-density lipoproteins, and disorders of the skin, eye, lungs,nd other lipid body constituents [37,38]. Fractionation of theaw extract using a preparative silica column resulted in sev-ral distinct fractions presenting different degrees of complexity.

his clean up procedure allows the selective isolation of distinctlasses of compounds according to the polarity of the eluent.

The hexanic fraction (F1) contains mainly hydrocarbons,ncluding squalene, and Vitamin E. Caffeine and theobromine

(iyu

on of the extract of mate tea leaves. Chromatographic conditions explained inraction 6.

ere found in the dichloromethane (F3) and toluene (F2) frac-ions. Alcohols and phenols such as phytol were also present inhese fractions. Fractions obtained by elution with ethyl acetateF4), acetone (F5) and methanol (F6) presented mainly unsatu-ated and saturated acids and esters. Some of these compoundsave already been reported in mate leaves extracts obtainedhrough SFE extraction [11,39] and other extraction process32,33]. It is interesting to note that in the raw extract wereound carboxylic acids and also methyl and ethyl esters. Thisan be affirmed because the free acids were derivatized forminghe TMS ester, while others appeared as alkyl esters. These sub-tances act on the central nervous system and produce varioussychic phenomena, such as increasing the perception capacity,resenting also diuretic properties [13].

It is worth noticing that the chromatogram of fraction 2

eluted with toluene) showed some unidentified compounds withntense peaks and also that this fraction had the higher massield in the fractionation procedure. This observation alloweds to conclude that some wax material was also isolated in this

358 R.A. Jacques et al. / J. of Supercritical Fluids 40 (2007) 354–359

Table 3Peak identification of compounds in the chromatograms of Figs. 2 and 3

Peak Rt (min) Name Formula/MW (g/g mol) Identification methoda

1 6.6 Octanoic acid (TMS ester) C11H24O2Si/206 std, ms2 8.8 Nonanoic acid (TMS ester) C12H26O2Si/220 std, ms3 11.5 Decanoic acid (TMS ester) C13H28O2Si/234 std, ms4 14.3 Undecanoic acid (TMS ester) C14H30O2Si/248 std, ms5 16.7 Dodecanoic acid (TMS ester) C15H32O2Si/262 std, ms6 18.6 Tridecanoic acid (TMS ester) C16H34O2Si/286 std, ms7 22.1 Caffeine C8H10N4O2/194 std, ms8 21.9 Tetradecanoic acid (TMS ester) C17H36O2/300 std, ms9 22.8 Theobromine C7H8N4O2/180 std, ms

10 23.2 Pentadecanoic acid, methyl ester C16H32O2/254 ms11 24.1 Hexadecanoic acid, methyl ester C17H34O2/270 ms12 24.5 Pentadecanoic acid (TMS ester) C18H38O2/314 std, ms13 25.5 Hexadecanoic acid, ethyl ester C18H36O2/284 std, ms14 26.9 Hexadecanoic acid (TMS ester) C19H40O2Si/328 std, ms15 28.6 Phytol C20H40O/296 std, ms16 29.1 Heptadecanoic acid (TMS ester) C20H42O2Si/342 std, ms17 29.2 Linolenic acid, methyl ester C19H32O2/292 ms18 29.9 Phytol TMS C23H48OSi/368 std, ms19 30.3 Docosane C22H46/310 std, ms20 30.7 Linoleic acid (TMS ester) C21H40O2Si/352 std, ms21 30.9 Linolenic acid (TMS ester) C21H38O2Si/350 std, ms22 31.0 Oleic acid (TMS ester) C21H42O2Si/354 std, ms23 31.4 Stearic acid (TMS ester) C21H44O2Si/356 std, ms24 32.5 Tricosane C23H48/324 std, ms25 33.7 Nonadecanoic acid (TMS ester) C22H46O2Si/370 ms26 34.6 Tetracosanol C24H50O/354 ms27 34.7 Tetracosane C24H50/338 std, ms28 35.6 Eicosanoic acid (TMS ester) C23H48O2Si/384 std, ms29 36.6 Pentacosanol C25H52O/368 ms30 36.7 Pentacosane C25H52/352 std, ms31 38.6 Hexacosanol C26H54O/382 ms32 38.7 Hexacosane C26H54/366 std, ms33 40.5 Heptacosanol C27H56O/396 ms34 40.6 Heptacosane C27H56/380 std, ms35 42.4 Octacosanol C28H58O/410 ms36 42.5 Octacosane C28H58/394 std, ms37 42.7 Squalene C30H50/410 ms38 44.2 Nonacosane C29H60/408 std, ms39 45.8 Triacontanol C30H62O/438 ms40 45.9 Triacontane C30H62/422 std, ms41 47.5 Methyl commate C or D C31H5004/486 ms42 47.7 Methyl commate C or D C31H5004/486 ms43 47.9 Untriacontane C31H64/436 std, ms44 48.8 Vitamin E C29H50O2/430 std, ms45 50.2 Dotriacontane C32H66/450 std, ms46 52.3 Stigmasterol C29H48O/412 std, ms47 53.0 Tritriacontane C33H68/464 std, ms48 53.2 Stigmasterol TMS (TMS ether) C32H56OSi/528 std, ms49 54.1 Stigmast-5-en-3-ol(3-beta) C29H50O/414 std, ms50 54.9 Stigmast-8-en-3-ol(3-beta) C29H50O/414 ms5 er)

ring t

fd

4

sc

pe4r

1 55.1 Beta sitosterol TMS (TMS eth

a Compounds were identified by injection of standards (std) and/or by compa

raction. Waxes are not appropriately separated by GC/MS, pro-ucing the hump in the base line, as can be seen in Fig. 3b.

. Conclusions

High-pressure carbon dioxide extraction of I. paraguarien-is shows to be a good technique to extract the main chemicalonstituents of this plant. It was observed that the extraction

ihPs

C32H58OSi/486 std, ms

he mass spectra with the equipment library (ms).

rocess variables (temperature and pressure) exert a pronouncedffect on the extraction yield. The higher yield was obtained at0 ◦C and 250 bar. Chromatographic analysis of high pressureaw mate extracts showed a high complexity profile, contain-

ng approximately 51 components, mainly esters, fatty acids,ydrocarbons, phytosterols, alcohols, xanthines and Vitamin E.reparative chromatographic process allowed the separation ofome important constituents in each fraction, promoting a better

rcritic

cmwa

A

fiL

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

703.

R.A. Jacques et al. / J. of Supe

haracterization of each compound. Esters and acids were elutedainly in the fractions of methanol, acetone and ethyl acetate,hile hydrocarbons and alcohols were better eluted with hexane

nd toluene in the first and second fraction.

cknowledgements

The authors thank CNPq, CAPES and FAPERGS for thenancial support and Barao Industria e Comercio de Erva Matetd. for technical assistance.

eferences

[1] E.B. Caramao, M.I. Melecchi, F.C. Abad, M.M. Miranda, P.P. Zini, Chem-ical composition of Hibiscus tiliaceus L. flowers: a study of extractionmethods, J. Sep. Sci. 25 (2002) 86.

[2] A.H. Goli, M. Barzeger, M.A. Sahari, Antioxidant activity and total phe-nolic compounds of pistachio (Pistachia vera) hull extracts, Food Chem.92 (2005) 521.

[3] A.L. Dawidowicz, D. Wianowska, PLE in the analysis of plant compounds.Part I. The application of PLE for HPLC analysis of caffeine in green tealeaves, J. Pharm. Biomed. Anal. 37 (2005) 1155.

[4] Z. Hromadkova, A. Ebringerova, Ultrasonic extraction of plantmaterials—investigation of hemicellulose release from buckwheat hulls,Ultrasonic Sonochem. 10 (2003) 127.

[5] M. Waksmundzka-Hajnos, A. Petruczynik, A. Dragan, D. Wianowska, A.L.Dawidowicz, I. Sowa, Influence of the extraction mode on the yield of somefuranocoumarins from Pastinaca sativa fruits, J. Chromatogr. B 800 (2004)181.

[6] Q. Lang, C.M. Wai, Supercritical fluid extraction in herbal and naturalproduct studies—a practical review, Talanta 53 (2001) 771.

[7] C. Dariva, M.R.A. Rodrigues, E.B. Caramao, J.G. Santos, J.V. Oliveira,Chemical composition and extraction yield of labiatae species from highpressure CO2 extraction, Chem. Eng. Trans. 1 (2002) 471.

[8] M.R.A. Rodrigues, L.C. Krause, J.G. Santos, C. Dariva, J.V. Oliveira, E.B.Caramao, Chemical composition and extraction yield of the extract of Ori-ganum vulgare obtained from sub- and supercritical CO2, J. Agric. FoodChem. 52 (2004) 3042.

[9] T.H.J. Beveridge, B. Girard, T. Kopp, J.C.G. Drover, Yield and compositionof grape seed oils extracted by supercritical carbon dioxide and petroleumether: varietal effects, J. Agric. Food Chem. 53 (2005) 1799.

10] J.W. King, E. Shle-Demessie, F. Tamale, J.A. Teel, Thermal gradient frac-tionation of glyceride mixtures under supercritical fluid conditions, J.Supercrit. Fluids 10 (1997) 127.

11] C. Dariva, M.R.A. Rodrigues, E.B. Caramao, J.G. Santos, J.V. Oliveira, Theeffects of temperature and pressure on the characteristics of the extractsfrom high-pressure CO2 extraction of Marjorana Hortensis Moench, J.Agric. Food Chem. 51 (2003) 453.

12] M. Zougagh, M. Valcarel, A. Rios, Supercritical fluid extraction: a criticalreview of its analytical usefulness, Trends Anal. Chem. 23 (5) (2004) 399.

13] F. Alikaridis, Natural constituents of Ilex species, J. Ethnopharm. 20 (1987)121.

14] M.J. Tormen, Economia Ervateira Brasileira. Erva Mate Biologia e Culturano Cone Sul, Universidade Federal do Rio Grande do Sul, Porto Alegre,Brazil, 1995, p. 27 (in Portuguese).

15] S. Gorzalczany, R. Filip, M.D.R. Alonso, J. Mino, G.E. Ferraro, C.Acevedo, Choleretic effect and intestinal propulsion of ‘mate’ (Ilex

paraguariensis) and its substitutes or adulterants, J. Ethnopharm. 75 (2001)291.

16] A.T.C. Taketa, E. Breitmaier, E.P. Schenkel, Triterpenes and triterpenoidalglycosides from the fruits of Ilex paraguariensis (Mate), J. Brazil Chem.Soc. 15 (2004) 205.

[

al Fluids 40 (2007) 354–359 359

17] A.T.C. Taketa, T. Schimittmann-Schalanger, D. Guillaume, G. Gosmann,E.P. Schenkel, Triterpenoid glycosides and a triterpene from Ilex brevicus-pis, Phytochemistry 53 (2000) 901.

18] M.A. Ouyang, Y.Q. Liu, H.Q. Wang, C.R. Yang, Triterpenoid saponinsfrom Ilex latifolia, Phytochemistry 49 (1988) 2483.

19] V.S. Pires, D. Guillaume, G. Gosmann, E.P. Schenkel, Saponins from Ilexdumosa, an Erva-mate (Ilex paraguariensis) adulterating plant, J. Agric.Food Chem. 45 (1977) 1027.

20] M.A.P. Martınez, J.P. Pelloto, N. Basualdo, Distribution of flavonoid agly-cones in Ilex species (Aquifoliaceae), Biochem. Syst. Ecol. 25 (1997)619.

21] M.D.A. Saldana, C. Zetzl, R.S. Mohamed, G. Brunner, Extraction ofmethylxanthines from guarana seeds, mate leaves, and cocoa beans usingsupercritical carbon dioxide and ethanol, J. Agric. Food Chem. 50 (2002)4820.

22] F.H. Reginatto, M.L. Ataıde, G. Gosmann, E.P. Schenkel, Methylxanthinesaccumulation in Ilex species—caffeine and theobromine in erva-mate (Ilexparaguariensis) and other Ilex species, J. Brazil Chem. Soc. 10 (1999) 443.

23] M.D.A. Saldana, R.S. Mohamed, M.G. Baer, P. Mazzafera, Extraction ofpurine alkaloids from mate (Ilex paraguariensis) using supercritical CO2,J. Agric. Food Chem. 47 (1999) 3804.

24] W. Don-Xu, C.A. Zhong-Liang, A dimeric sinapaldehyde glucoside fromIlex rotunda, Phytochemistry 41 (1996) 657.

25] H. Fuchino, H. Tachibana, N. Tanaka, Three new hemiterpene glycosidesfrom Ilex macropoda, Chem. Pharm. Bull. 45 (1997) 1533.

26] H.H. Vangenden, J. Jaarsma, Triterpenes and alkanes in developing varie-gated and albino leaves of Ilex aquifolium L. (Aquifoliaceae), Plant Sci. 72(1990) 165.

27] N. Ishikura, A further survey of anthocyanins and other phenolics in Ilexand Euonymus, Phytochemistry 14 (1975) 743.

28] H.H. Vangederen, J. Jaarsma, C. Verluis, Long chain pentyl- and hexyl-esters and other lipoids in leaf wax of Ilex aquifolium L. (Aquifoliaceae),Plant Sci. 55 (1988) 231.

29] A.A. Esmelindro, J.G. Santos, A. Mossi, R.A. Jacques, C. Dariva, Influ-ence of agronomic variables on the composition of mate tea leaves(Ilex paraguariensis) extracts obtained from CO2 extraction at 30 ◦C and175 bar, J. Agric. Food Chem. 52 (2004) 1990.

30] M.C. Esmelindro, G. Toniazzo, D. Lopes, D. Oliveira, C. Dariva, Effectsof processing conditions on the chemical distribution of mate tea leavesextracts obtained from CO2 extraction at high pressures, J. Food Eng. 70(2005) 558.

31] S. Angus, B. Armstrong, K.M. Reuck (Eds.), International ThermodynamicTables of the Fluid State, Pergamon Press, Oxford, UK, 1976.

32] E. Valduga, Caracterizacao Quımica e Anatomica da Folha de Erva-mate(Ilex paraguariensis St. Hill.) e de Especies Utilizadas na Adulteracao doMate, Master Dissertation, Curitiba, UFPR, 181p., 1994 (in portuguese).

33] M. Kawakami, A. Kobayaski, Volatile constituents of green mate androasted mate, J. Agric. Food Chem. 39 (1991) 1275.

34] H. He, Y. Cai, M. Sun, H. Corke, Extraction and purification of squalenefrom amaranthus grain, J. Agric. Food Chem. 50 (2002) 368.

35] T.J. Smith, Squalene: potential chemopreventive agent, Exp. Opin. Invest.Drugs 9 (2000) 1841.

36] I. Bousova, J. Martin, L. Jahodar, J. Dusek, V. Palicka, J. Drsata, Evaluationof in vitro effects of natural substances of plant origin using a model ofprotein glycoxidation, J. Pharm. Biomed. Anal. 37 (2005) 957.

37] L.C. Ching, S. Mohamed, Alpha-tocopherol content in 62 edible tropicalplants, J. Agric. Food Chem. 49 (2001) 3101.

38] R. Brigelius-Flohe, J.T. Salonen, The European perspective on VitaminE: current knowledge and future research, Am. J. Clin. Nutr. 76 (2002)

39] M.C. Esmelindro, G. Toniazzo, D. Lopes, D. Oliveira, C. Dariva, Effectsof processing conditions on the chemical distribution of mate tea leavesextracts obtained from CO2 extraction at high pressures, J. Food Eng. 70(2005) 588.