Influence of crude extract and bioactive fractionsof Ilex paraguariensis A. St. Hil. (yerba mate) onthe Wistar rat lipid metabolism

Pedro Ernesto de Resende a, Samuel Kaiser a, Vanessa Pittol a,Ana Lúcia Hoefel b, Raquel D’Agostini Silva b,Cláudia Vieira Marques b, Luiz Carlos Kucharski b,George González Ortega a,*a Laboratório de Desenvolvimento Galênico, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul,Av. Ipiranga, 2752, Porto Alegre, Rio Grande do Sul 90610-000, Brazilb Laboratório de Metabolismo e Endocrinologia Comparada, Departamento de Fisiologia, Instituto de CiênciasBásicas da Saúde, Universidade Federal do Rio Grande do Sul, Av. Sarmento Leite, 500, Porto Alegre, Rio Grandedo Sul 90050-170, Brazil

A R T I C L E I N F O

Article history:

Received 2 September 2014

Received in revised form 17 March

2015

Accepted 18 March 2015

Available online 17 April 2015

A B S T R A C T

The influence of yerba mate extract (YME) and two concentrated fractions of methylxanthines–

polyphenols (MXPL) and saponins (SFL) from yerba mate on the lipid metabolism of Wistar

rats fed with a high-fat diet were examined. A Box–Behnken design was employed for the

optimization of yerba mate extraction. Subsequently, one-step separation using solid phase

fractionation was applied. HPLC–PDA and UPLC/Q-TOF-MS were used for quantification and

identification of the main compounds.YME induced reductions on intra-abdominal fat weight

gain and LDL plasma level. MXPL decreased plasma levels of cholesterol, LDL and

triacylglycerols. In addition, this fraction increased lipogenesis of the muscle, hepatic gly-

cogen synthesis and lipolysis in adipose tissue. SFL enhanced lipogenesis in adipose tissue

and also fecal fat excretion comparing to both controls. Moreover, all mate preparations could

entirely revert the hepatic lipogenesis induced by the high-fat diet. Thus, the yerba mate

extract and fractions exhibited a potential anti-obesity effect on lipid metabolism of the

Wistar rat and deserve further investigation.

© 2015 Elsevier Ltd. All rights reserved.

Keywords:

Yerba mate

Ilex paraguariensis

Polyphenols

Methylxanthines

Saponins

Lipid metabolism

* Corresponding author. Av. Ipiranga, 2752, Santana, Porto Alegre, RS, Brazil, CEP: 90610-000. Tel.: +55 51 3308 5278; fax: +55 51 3308 5437.E-mail address: ortega@farmacia.ufrgs.br (G.G. Ortega).Abbreviations: YME, yerba mate extract; MXPL, methylxanthines–polyphenols concentrated fraction; SFL, saponins concentrated frac-

tion; HPLC–PDA, high performance liquid chromatography–photo diode array; UPLC/Q-TOF-MS, ultra performance liquid chromatographycoupled with electrospray time-of-flight mass spectrometry; LDL, low-density lipoprotein; HDL, high-density lipoprotein; RSM, responsesurface method; KRB, Krebs–Ringer buffer; GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic transaminasehttp://dx.doi.org/10.1016/j.jff.2015.03.0401756-4646/© 2015 Elsevier Ltd. All rights reserved.

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1. Introduction

Ilex paraguariensis A. St. Hil. (mate, Aquifoliaceae) is a SouthAmerican native evergreen tree. In several countries world-wide mate leaves and branches are industrially processed (yerbamate) and consumed as a traditional tea-like beverage, espe-cially in Argentina, Brazil, Paraguay, and Uruguay (Heck & Mejia,2007). Outstanding among the chemical constituents re-ported in yerba mate are high levels of polyphenols,methylxanthines and saponins (Borré et al., 2010).

In the past decade, several works on yerba mate biologicalproperties have focused on antioxidant, anti-mutagenic, anti-inflammatory, and anti-obesity effects. As a rule, these activitieswere related mainly to yerba mate crude extracts (Bracesco,Sanchez, Contreras, Menini, & Gugliucci, 2011). Most of thestudies were performed using aqueous extracts of yerba mate,mimicking chimarrão or tereré, the traditional way of drinkingmate tea, but without specifically considering any bioactive frac-tion. Thus, it remains uncertain whether the effects should beascribed to the polyphenols, methylxanthines or saponins(Resende et al., 2012).

Likewise many works about yerba mate were performed ac-cording with diet-induced obesity models in mice (Arçari et al.,2009; Hussein et al., 2011; Martins et al., 2009), seldom in rats(Pimentel et al., 2013) or in human beings (De Morais et al., 2009;Kim, Ko, Storni, Song, & Cho, 2012).The accumulated data haveshown fat and body weight reduction as well as an improve-ment of plasma levels of triacylglycerols, cholesterol and glucose,and regulation of gene expression involved in lipid metabolism.

Although widely studied from a chemical perspective, re-search on yerba mate technological processing revealed to berestricted regarding the preparation and biological investiga-tion of concentrated and purified fractions. Pereira et al. (2012)demonstrated that yerba mate crude extracts may behave dif-ferently from its concentrated fractions regarding plasmaglucose levels and diabetic status in Wistar rats.

Nonetheless, the separation and purification of yerba matebioactive compounds imply a rather complex optimization task,involving several chemical and technological variables simul-taneously. In this context the response surface methodology(RSM), a set of statistical techniques based on empirical models,is a useful tool to fit data gathered from an experimental design(Bezerra, Santelli, Oliveira, Villar, & Escaleira, 2008). One of thebest-accepted techniques is the Box–Behnken design, whichrequires a small number of runs, hence avoiding, time-consuming experiments (Montgomery, 2005).

Thus, this work aims to evaluate the influence of a crudehydroethanolic extract (Peixoto et al., 2010), and two bioactivefractions of yerba mate, on the lipid metabolism of male Wistarrats subjected to a high-fat diet. Moreover, polyphenol,methylxanthine and saponin contents were analytically assayedin all yerba mate preparations for better clarification of the bio-logical results obtained.

2. Material and methods

2.1. Plant materials

Leaves and branches of Ilex paraguariensis harvested in January2011 were supplied by Ervateira Barão (Barão de Cotegipe, RS,

Brazil; 27°37′S 52°23′W). A leaves:branches ratio of 70:30 (w/w)was prepared after drying and milling, to simulate the com-mercial product popularly known as yerba mate.

2.2. Yerba mate extract (YME)

The extraction of yerba mate was performed by 1-h dynamicmaceration in a magnetic stirring plate (RO 15 Power, IKA,Staufen im Breisgau, Germany) applying a three-factor at three-level Box–Behnken design with ethanol concentration (X1: 10,50 and 90% (v/v)), temperature (X2: 30, 40 and 50 °C) anddrug:solvent ratio (X3: 4, 7, 10% (w/v)). The mixtures were fil-tered at room temperature through a Whatman paper filter N°2 (Kent, UK), concentrated at 50 ± 5 °C under reduced pres-sure (Buchi R-114, Flavil, Switzerland) and freeze-dried (Modulyo4L, Edwards, Crawley, UK).

2.3. Concentrated methylxanthines–polyphenols andsaponins fractions from yerba mate extract

A 5-g sample of freeze-dried YME was placed into a 50 × 2.8-cmglass column packed with 40 g of resin Diaion HP-20 (Supelco,Bellefonte, PA, USA), and fractionated using methanol:watermixtures of 500 mL each, in a decreasing polarity gradient(Peixoto et al., 2010). The column flow rate was 5.0 mL/min.Water and 30, 40, 50, and 70% methanol fractions (v/v) werecombined, concentrated under vacuum at 50 ± 5 °C, freezedried, stored in light protected glass and coded as MXPL(methylxanthines–polyphenols concentrated fraction). Sepa-rately, the 90% methanol fraction was concentrated underreduced pressure at 50 ± 5 °C, freeze dried as usual, and codedas SFL (saponins concentrated fraction).

2.4. Quantification and characterization by HPLC–PDA

For quantification and characterization purposes, a HPLCShimadzu Prominence 20AT module (Kyoto, Japan) coupled toa photodiode array detector (PDA) SPD-M20A, controlled by LC-Solution Multi-PDA software, was used. A Gemini RP C18 column(Phenomenex, 250 × 4.6 mm i.d.; 5 µm particle size) coupled witha C18 guard column was used as the stationary phase.

2.4.1. MethylxanthinesThe methyxanthines from yerba mate were assayed by theHPLC method previously validated (Gnoatto, Bassani, Coelho,& Schenkel, 2007) employing caffeine and theobromine asexternal standards. The theobromine (Sigma-Aldrich, St. Louis,MO, USA) and caffeine (Sigma-Aldrich) standards were properlydissolved in methanol:water 40% (v/v), at concentrationsranging from 0.2 to 10.0 µg/mL. An isocratic system wasemployed, using methanol/water 40% (v/v) as mobile phase.The flow rate (1.1 mL/min) and temperature (23 ± 1 °C) werekept constant throughout the analysis. The detection wasperformed at 280 nm. All samples were properly diluted withmethanol/water 40% (v/v) seeking the linearity range ofstandard curves. The total methylxanthine content wasdetermined by the sum of caffeine and theobromine individualconcentrations.

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2.4.2. PolyphenolsThe polyphenols from yerba mate were assayed by the HPLCmethod previously validated, and the total polyphenol contentwas determined by the sum of individual concentrations ofmajor compounds previously identified as polyphenols by UVand MS/MS data (Da Silva et al., 2007). Chlorogenic acid (Sigma-Aldrich) and rutin (Sigma-Aldrich) were used as externalstandards at concentrations ranging from 2.0 to 10 µg/mL, afterdissolution in methanol/water 50% (v/v).

2.4.3. SaponinsThe saponins from yerba mate were assayed by the HPLCmethod previously reported (Borré et al., 2010) with modifica-tions employing ilexoside II as the external standard. Theilexoside II (isolated from yerba mate) standard was properlydissolved in acetontrile:water 25% (v/v), at concentrationsranging from 4.0 to 40.0 µg/mL. A gradient system was em-ployed, using phosphoric acid 0.1% (v/v) (phase A) andacetonitrile (phase B) as follows: 30–45% B (40 min), 45% B(5 min), 45–30% B (20 min). The flow rate (0.9 mL/min) and tem-perature (30 ± 1 °C) were kept constant throughout the analysis.The detection was performed at 205 nm. All samples were prop-erly diluted with acetonitrile/water 25% (v/v) seeking thelinearity range of standard curve. The total saponin contentwas determined by the sum of individual concentrations ofmajor compounds.

2.5. UPLC/Q-TOF-MS analyses of mate saponins

Saponin characterization was performed with a Waters AcquityUltra Performance LC system (Waters, Milford, MA, USA)equipped with binary solvent manager, sample manager, pho-todiode array detector (PDA), and coupled to a Q-TOF massspectrometer, in accordance with the parameters previouslydescribed (Peixoto et al., 2012; Resende et al., 2012). The massrange was over 200−1500 m/z. The data were processed byMassLynx V4.1 software (Waters). Both standards of IlexosideII and chikusetsusaponin Iva (isolated from yerba mate) wereproperly dissolved in acetonitrile/water 25% (v/v) at 5 µg/mLfor identification purpose.

2.6. Evaluation of biological activities

2.6.1. Experimental designThirty male Wistar rats (4 weeks old) were weighed, and housed(groups of 3) in steel cages (414 × 344 × 168 mm) under light/dark cycles of 12 h, at 22 ± 2 °C.The experiments were approvedby the University Ethics Committee on Research (UFRGS) (pro-tocol number 20811 – CEUA). They were performed accordingto the Guide for the Care and Use of Laboratory Animals de-veloped by the Institute of Laboratory Animal Resources of theNational Research Council, and the recommendations of theBrazilian College for Animal Experimentation.The animals weresubmitted to a standard diet (n = 6) and high-fat diet (n = 24)for 5 weeks. After this time, the animals were divided into fivegroups of six animals each and treated throughout the 30-day period of the experiment. Standard diet group (negativecontrol): animals received standard chow (composition: 8% ofmineral content, 22% of proteins, 5% of lipids, 10% of crude fiber

and 55% of carbohydrates; 2.95 kcal/g) and tap water ad libitum.High-fat diet group (positive control): animals received a high-fat diet (Pragsoluções Biociências, São Paulo, Brazil –composition: 5% of mineral content, 26% of proteins, 35% oflipids, 8% of crude fiber and 26% of carbohydrates; 5.25 kcal/g– and tap water ad libitum. Test group A: animals were treatedwith yerba mate extract (YME). Test group B: animals weretreated with methylxanthine–polyphenol concentrated frac-tion (MXPL). Test group C: animals were treated with saponinsconcentrated fraction (SFL). All the tested groups were fed withthe high-fat diet. For the test groups, freeze dried samples (YME,MXPL and SFL) were dissolved into water and administered adlibitum to animals.The amount of each preparation was definedin order that the concentrations of methylxanthine–polyphenolin the MXPL group and saponins in SFL were equivalent topresent in YME group. The preparations were renewed everyday throughout the treatment period. The intake of water con-taining the mate preparations was comparable to that measuredin both control groups.

2.6.2. Tissue and blood samplesAfter a 30-day treatment, the animals were weighed and de-capitated. Blood was collected in glass tubes, and plasma wasobtained by centrifugation for 8 min at 2150 g. Fat pads fromretroperitoneal and epididymal tissues, and samples from liverand soleus muscle were carefully excised and immediatelyweighed. All the tissues used in this study were fresh and ob-tained in the same day of decapitation of the animals.

2.6.3. Biochemical analysesTotal cholesterol, high density lipoprotein (HDL), triacylglycerols,glutamic oxaloacetic transaminase (GOT), glutamic pyruvic trans-aminase (GPT), creatinine, urea, and amylase concentrations inplasma were assayed using commercial enzymatic kits (Labtest,Lagoa Santa, MG, Brazil). The concentration of low density li-poprotein (LDL) was calculated from total cholesterol, HDL, andtriacylglycerol concentrations.

2.6.4. Glucose oxidation (14CO2 production)Glucose oxidation was performed according with Torres et al.(2001). Briefly, liver and retroperitoneal fat tissue samples wereincubated at 37 °C, for 60 min, in flasks sealed with rubber capscontaining 1.0 mL of KRB (Krebs–Ringer buffer: 118 mm NaCl,4.61 mM KCl, 2.5 mM CaCl2·2 H2O, 1.19 mM KH2PO4, MgSO4·7 H2O0.74 mM, NaHCO3, 25 mM, pH 7.4), 0.15 µCi [U-14C] glucose(55 mCi/mmol, Amersham, Little Chalfont, UK), and glucose so-lution 5 mM. The gaseous phase was saturated with a 5% CO2

and 95% O2 mixture. Small glass wells containing strips of3 mm-Whatman paper were placed above the level of the in-cubation medium. The oxidation reaction was stopped byinjecting 0.25 mL of trichloracetic acid solution 50% (v/v) throughthe rubber caps, and 0.25 mL of NaOH (2.0 M) solution di-rectly into the wells. The flasks were maintained at least 12 hat room temperature in order to capture 14CO2. The contentswere transferred to vials containing a liquid scintillation mixture(toluene – Triton X®-100 (2:1, v/v); 2,5-diphenyloxazole (0.4%,v/v) and 2-p-phenylenebis 5-phenyloxazole (0.01%,v/v)), and theradioactivity was measured using a liquid scintillation counter(LKB-Wallac, Perkin Elmer, Waltham, MA, USA) with an auto-matic curve quench correction. The 14CO2 production was

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expressed as nmol of 14C glucose incorporated into CO2 per mgof tissue, per hour.

2.6.5. Glycogen synthesis in liver and soleus muscleSamples of liver and soleus muscle (100 mg) were divided andcut into 300-mm slices. Slices were incubated with a mediumcontaining Dulbecco buffer 2.7 mM (pH 7.3), 5 mM of glucoseand 0.15 µCi [U-14C] glucose (55 mCi/mmol, Amersham). Incu-bations were saturated with carbogen (95% O2 and 5% CO2) andincubated at 37 °C for 60 min in a metabolic shaker (Dolnikoff,Martín-Hidalgo, Machado, Lima, and Herrera, 2001). Incuba-tion was stopped by placing the beaker on ice. KOH 60% (w/v)(1 mL) was added to each beaker and immediately placed intoa boiling water bath for 15 min. Ethanol was added to the tubesfor glycogen precipitation, subsequently resuspended in water,and the addition of the liquid scintillation mixture.The sampleswere assessed in a liquid scintillation counter (LKB-Wallac).

2.6.6. Determination of triacylglycerols, cholesterol andglycogen concentration in liverSlices of liver were submitted to a water bath at 100 °C. Theglycogen pellet obtained was dissolved in Milli-Q water andused to determinate the glycogen content by colorimetricmethod (Krisman, 1962). The determination of hepatictriacylglycerols and cholesterol were enzymatically assessedin a lipid extract by colorimetric kit (Labtest), according to Folch,Lees, and Stanley (1957).

2.6.7. Lipogenesis assaySamples of liver, soleus muscle and retroperitoneal adiposetissues were incubated in KRB, 0.15 µCi 14C–glucose (Amersham)and 10 mM glucose. Incubations were saturated with carbogen(95% O2 and 5% CO2), capped and incubated in a Dubnoff-type metabolic bath at 37 °C under constant shaking for 60 min.After incubation, the samples were homogenized with achloroform:methanol mixture (2:1, v/v). Lipid extraction wasperformed according to the method described by Folch et al.(1957). A saline solution was added and the tubes were agi-tated and centrifuged for 10 min at 2000 g. After evaporatingthe entire chloroform, 5 mL of liquid scintillation mixture wasadded. The samples were assessed in a scintillation liquidcounter (LKB-Wallac). The results were expressed in pmol permg of tissue per hour of incubation.

2.6.8. Lipolysis assaySamples of retroperitoneal adipose tissue (400–650 mg) weresliced and placed in incubation tubes containing 1 mL modi-fied saline solution KRB, 5 mM glucose and 1% bovine serumalbumin. Lipolysis was stimulated adding 20 µL of adrenalinesolution in ascorbic acid (2.5 10−4 M). The tubes were satu-rated with carbogen (O2/CO2, 95/5%, v/v), capped and incubatedin a Dubnoff-type metabolic bath at 37 °C and shaken for60 min. After the incubation period, the tissue reactions werestopped in an ice bath. Glycerol content was measured usinga commercial kit (R-Biopharm®, Darmstadt, Germany). Theresults were expressed as mmol of glycerol per gram of tissueper hour of incubation.

2.6.9. Fecal fat determinationFecal fat determination was accomplished by the method ofBligh and Dyer (1959). In brief, samples of feces (2.5 g) were

placed into tubes with a mixture of chloroform:methanol (1:2)and distilled water. After agitation for 30 min, chloroform andNa2SO4 1.5% (1:1) were added. Afterward, the samples were agi-tated for 2 min and centrifuged. The layer containing the fatwas removed, placed in test tubes containing 0.8 g of anhy-drous Na2SO4, mixed and filtered. The filtrate (5 mL) wascollected and placed in a previously weighed beaker.The solventand humidity were removed and it was weighed again.

2.7. Statistical analyses

Data from each experiment were expressed by the mean andrespective standard deviation (SD). Differences among groupswere tested by one-way ANOVA and Tukey test (Minitab® 14software). Values of p < 0.05 were considered significant.

3. Results and discussion

3.1. Extraction optimization and fractionation

Optimal process conditions could be straightforwardly achievedwith regard to equally satisfactory yields of polyphenols,methylxanthines and saponins from yerba mate by a three-level Box–Behnken design.The optimization process is a reliableway to guarantee the reproducibility of biological data gainedfrom vegetable extracts. In that sense, further information onthis topic is supplied in Supplementary material Appendix S1.

The procedure to separate polyphenols, methylxanthinesand saponins from each other was guided by previous workcarried out with a hydrophobic polyaromatic resin (DiaionHP-20) method (Peixoto et al., 2010). Even so, significantproduct loss was noticed after several attempts to separatemethylxanthines from polyphenols. For that reason, the integralconservation of both compound groups was preferred insteadof a plain chemical separation guided by fraction purity.Moreover, this study focused on the understanding of acomprehensive description of the chemical composition of theyerba mate concentrated fractions rather than the performanceof a research using separated fractions. In this context,the process allowed obtaining one concentrated fractioncontaining yerba mate polyphenols and methylxanthines(MXPL) (saponin-free) and one saponins concentrated fraction(SFL) (methylxanthine and polyphenol-free). The contentachieved in the concentrated fractions for polyphenols–methylxanthines (584.9 mg/g of freeze dried material) andsaponins (244.6 mg/g of freeze dried material) were about twotimes and fifteen times higher than found in yerba mate extract(YME) (398.1 mg/g and 15.0 mg/g of freeze dried material),respectively.

3.2. HPLC–PDA determination of polyphenols,methylxanthines and saponins

HPLC–PDA profiles and total contents of methylxanthines andpolyphenols in YME and MXPL preparations were compa-rable (p > 0.05), as well those of saponins in YME and SFL(p > 0.05). The polyphenols and methylxanthines in SFL, as wellas saponins in MXPL, were detected only as traces. The daily

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doses administrated of polyphenols, methylxanthines andsaponins in the different yerba mate preparations was calcu-lated considering the mean intake of 30 mL/day/animal anddetermined by the monitoring of intake/day throughout thetreatment period (Table 1).

The presence of polyphenols (caffeoylquinic derivatives andflavonoids) and methylxanthines (caffeine and theobromine)in MXPL and YME confirmed previous data from the litera-ture (Borré et al., 2010; Da Silva et al., 2007; Gnoatto et al., 2007;Resende et al., 2012; Silva et al., 2011). Also, the HPLC profileof the main saponins in YME and SFL was comparable to thatdescribed in earlier works (Borré et al., 2010; Silva et al., 2011).Since MXPL was deprived of saponins and, conversely,methylxanthines and polyphenols are missing in SFL, it allowssetting equivalent doses of the main bioactive fractions amonggroups and establishing a practical dose–effect relationship.

3.3. Saponin characterization

The structural characterization of the main saponins in YMEand SFL by UPLC/Q-TOF-MS, namely, ursolic, oleanolic, andpomolic acid derivatives, including ilexoside II andchikusetsusaponin IVa, provides a useful way to achieve a betterunderstanding of biological data from further yerba mate studies.

The analyses allowed the characterization of the major sa-ponins derived from ursolic, oleanolic, and pomolic acids,present in YME and SFL (Fig. 1). Saponins ilexoside II andchikusetsusaponin IVa were identified in YME and SFL. IlexosideII was detected at 6.11 min with m/z 951.58 [M + Na]+ to-gether with the peaks m/z 455.38 (Peixoto et al., 2012).Chikusetsusaponin IVa occurs at 10.19 minutes with m/z817.4354 [M + Na]+, together with the peak m/z 833.4175 [M + K]+

as previously noticed in Ilex paraguariensis (Dahmer et al., 2012).

3.4. Evaluation of biological activities

3.4.1. Body weight and fat/body mass indexBody weight was measured twice a week from the beginningof the experiment. The daily dose employed for each rat isshown in Table 1. The fat pad weight/body weight ratio wasexpressed in percentage.This parameter allows inference aboutthe activity in lipid metabolism induced by test groups (Fig. 2).The high-fat diet adopted in this work caused no significantincrease in the final body weight of the rats. Nonetheless, aslight increase in the fat weight/body weight ratio was noticedin retroperitoneal adipose tissue, while a significant increasein epididymal fat became evident (Fig. 2). It was observedthat in the group treated with YME, the retroperitoneal fat was

statistically equivalent to the control group (p > 0.05). Like-wise, YME demonstrated a reduction effect on epididymalweight gain, being equivalent to the standard diet group(p > 0.05), as well as MXPL, but to a lesser extent.

The treatment with YME disclosed a discrete but still sig-nificant effect on the fat pad weight/body weight ratio. It wasobserved that in the YME group, the retroperitoneal fat was sta-tistically equivalent to the control group (Fig. 2). This resultsuggests an attenuation of the weight gain tendency by themate leaves extract and agrees with previous studies using high-fat obese induced animal models (Arçari et al., 2009; Husseinet al., 2011; Martins et al., 2009; Pang, Choi, & Park, 2008). More-over, some of these works reported the reduction of theepididymal fat mass in mice (Arçari et al., 2009; Hussein et al.,2011) and rats (Silva et al., 2011), including the reduction inadipocyte size (Pang et al., 2008). On these occasions, the authorsattributed the activity, at least in part, to polyphenol andmethylxanthine contents. Nevertheless, a purified saponin frac-tion from unripe mate fruits exhibited a tendency towardepidydimal fat reduction of rats fed with standard chow, asearlier reported (Resende et al., 2012).

Also, no significant effect was detected among concen-trated fractions SFL and MXPL, despite the fat weight gaintendency when compared to the high-fat diet group.The resultswith MXPL and SFL groups clearly denote an increase of theretroperitoneal adipose tissue. This finding suggests that yerbamate bioactive fractions can act inversely in relation to theiroriginal crude extract, when administered in equivalent dosesas in YME.

3.4.2. Biochemical analyses of plasma parametersSeveral relevant biochemical parameters related to lipid me-tabolism were assayed (Table 2). The high-fat diet produced anincrease in the plasma glucose level of the rats; however, noneof the mate-treated groups exhibited any significant differ-ences in this parameter (p > 0.05). The high-fat diet groupshowed higher cholesterol levels than the control group, in-cluding those receiving yerba mate preparations. However MXPLwas able to attenuate the cholesterol level and similar behav-ior was noticed with regard to triacylglycerols. Likewise, theLDL plasma level was increased in rats fed with a high-fat diet,and remained unaffected after SFL treatment. In contrast YMEand MXPL were able to restore the LDL concentration to thelevel observed in rats of the standard diet group. ConcerningHDL, GOT, GPT, creatine, urea, and amylase levels, no signifi-cant difference could be found compared to the control groups.

No evidence of plasma glucose restoration is in accor-dance with former studies using Wistar rats and a high-fat diet

Table 1 – Daily doses of each bioactive fraction from I. paraguariensis administered orally to Wistar ratsa.

Group test Total polyphenols(mg/day/animal)(mean ± SD)*

Total methylxanthines(mg/day/animal)(mean ± SD)*

Total saponins(mg/day/animal)(mean ± SD)*

YME (yerba mate extract) 45.8 ± 2.8 5.09 ± 0.3 1.9 ± 0.1MXPL (methylxanthines–polyphenols concentrated fraction) 46.5 ± 6.6 8.3 ± 1.1 Not detectedSFL (saponins concentrated fraction) 0.05 ± 0.01 Not detected 1.75 ± 0.2

* Mean ± stand standard deviation (n = 6).a Taking into account a mean water intake of 30 mL/day/animal.

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Fig. 1 – Total ion current (TIC) of reference compounds ilexoside II (RT: 6.11 min) and chikusetsusaponin IVa (RT: 10.19 min) (A), and their fragmentation patterns (A1 andA2), respectively. TIC of saponins concentrated fraction (SFL) (B) and fragmentation patterns of ilexoside II (B1) and chikusetsusaponin Iva (B2). TIC of yerba mate extract(YME) (C) and fragmentation patterns of ilexoside II (C1) and chikusetsusaponin Iva (C2).

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(Pimentel et al., 2013). It is noteworthy that Silva et al. (2011)demonstrated for the first time that the consumption of leavesfrom gross yerba mate caused an increment of plasma glucoselevels. Similarly, a rise in plasma levels of glucose in healthyWistar rats occurred after treatment with a saponin-rich extractfrom unripe fruits of mate (Resende et al., 2012).

In contrast, a significant improvement of oral glucose tol-erance was noticed in Wistar rats treated with yerba matefractions containing methylxanthines and polyphenols, as wellas with infusions of green and roasted I. paraguariensis froma commercial source (Pereira et al., 2012). However, there aredoubts, not only as to whether the participation of differentbioactive compounds can explain this divergence about yerbamate activity in plasma glucose, but also about the animalmodel used. Thus, an antagonist effect toward a lowering ofthe plasma glucose level was noticed in high-fat diet induced

obese mice (Arçari et al., 2009; Hussein et al., 2011; Martins et al.,2009) and Sprague–Dawley rats (Pang et al., 2008) treated withyerba mate preparations. Evidently, further studies are con-sidered necessary to confirm the relationship between theconsumption of yerba mate extracts and the effect on theplasma glucose levels.

Among all yerba mate preparations assayed, no effect onthe cholesterol plasma levels was noticed, except for MXPL.Nevertheless YME and MXPL improved LDL plasma levels.Polyphenols such as catechins and quercetin may contributeto the restoration of LDL and total cholesterol plasmalevels presumably by: inhibition of intestinal absorption ofcholesterol by decreasing micellar solubility, improved excre-tion in feces, and suppression of liver cholesterol synthesis(De Morais et al., 2009). Methylxanthines, like caffeine, mayalso be involved in the inhibition of the intestinal absorption

Fig. 2 – Epydidimal and retroperitoneal adipose tissues (expressed as percentage of fat weight/body weight ratio) in ratstreated with water and standard diet (control group); water and high-fat diet; yerba mate extract (YME) and high-fat diet;methylxanthines–polyphenols concentrated fraction (MXPL) and high-fat diet; saponins concentrated fraction (SFL) andhigh-fat diet, after a 30-day treatment. a,b,c,d,e,A,B,C,D,ESame letters indicate equivalent results among treated-groups by Tukey’stest (p > 0.05) (mean ± SD, n = 6).

Table 2 – Plasma parameters in male Wistar rats treated with water and standard diet (control group); water and high-fatdiet; yerba mate extract (YME) and high-fat diet; methylxanthines–polyphenols concentrated fraction (MXPL) and high-fat diet; saponins concentrated fraction (SFL) and high-fat diet; after a 30-day treatment (mean ± SD, n = 6).

Parameter (µg/mL) Control (standard diet)(mean ± SD)*

High-fat diet(mean ± SD)*

YME (mean ± SD)* MXPL (mean ± SD)* SFL (mean ± SD)*

Glucose 102.7a ± 2.06 111.2b ± 13.51 120.2b ± 4.99 113.4b ± 5.11 114.6b ± 4.94Cholesterol 52.2a ± 5.01 64.8b ± 1.64 62.6b ± 1.34 54.3a ± 2.4 61.0b ± 4.3HDL 31.2a ± 5.1 33.7a ± 3.4 34.7a ± 4.2 38.7a ± 5.4 37.8a ± 6.3LDL 6.0a ± 1.0 10.5b ± 2.9 5.7a ± 0.6 4.3a ± 0.5 9.7b ± 3.2Triacylglycerols 92.7a ± 11.35 120.2b ± 4.6 138.0b ± 16.8 89.4a ± 18.8 146.7b ± 21.4GOT 169.6a ± 28.74 130.7a ± 15.28 191.2a ± 19.53 171.6a ± 17.27 159.7a ± 32.04GPT 60.2a ± 4.91 76.0a ± 9.96 87.25a ± 13.96 81a ± 6.85 80.8a ± 14.95Creatinine 0.3a ± 0.02 0.2a ± 0.05 0.28a ± 0.07 0.3a ± 0.03 0.3a ± 0.07Urea 36.6a ± 3.8 29.8a ± 1.9 30.6a ± 3.7 36.6a ± 2.0 31.8a ± 2.8Amylase 791.2a ± 128.4 773.5a ± 38.9 896.1a ± 80.2 854.1a ± 61.1 854.7a ± 98.5

* Mean ± standard deviation (n = 6).a,b Same letters indicate equivalent results among treated-groups by Tukey’s test (p > 0.05) for each parameter evaluated.

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of cholesterol in rats, as earlier reported (De Morais et al.,2009).

In addition, crude extracts from yerba mate leaves showeda significant activity in cholesterol and LDL parameters. More-over, a reduction or attenuation of plasma levels of totalcholesterol and LDL was found in rats, mice, and rabbits treatedwith yerba mate extracts (Arçari et al., 2009; Pang et al., 2008).A significant improvement in lipid parameters, including de-crease of cholesterol, and an additional LDL reduction inhypercholesterolemic subjects in long-term statin therapy, wasachieved using green or roasted yerba mate infusions (De Moraiset al., 2009).

Again, only MXPL showed a significant reduction in theplasma level of triacylglycerols, in agreement with former worksusing yerba mate extracts (Arçari et al., 2009; Hussein et al.,2011; Martins et al., 2009; Resende et al., 2012; Silva et al., 2011).

It seems evident that a synergistic effect occurs among thecompounds of yerba mate, via different mechanisms, contrib-uting to the improvement of plasma levels involved in lipidmetabolism. Since MXPL was the only fraction to demon-strate a reduction in cholesterol, LDL and triglyceride levels inthis study, polyphenols, and methylxanthines appear to playan important role in this activity attributed to yerba mate.

Lipid metabolism involves several partially linked, inter-dependent and cross-regulated pathways. Aiming at furtherinformation on yerba mate activity, the following assays wereperformed in different tissues: glucose oxidation, lipogen-esis, lipolysis, and fecal fat excretion.

3.4.3. Glucose oxidation (14CO2 production)The glucose oxidation assay expresses the tissue ability to me-tabolize this substrate. On the whole, hepatic and muscularglucose oxidation presented similar behavior, no matter whatyerba mate preparation was considered. However in adiposetissue, the tested groups demonstrated different effects (Fig. 3).

This parameter was decreased in hepatic, muscular, andadipose tissues of rats submitted to a high-fat diet. Neverthe-less, YME had no significant effect on glucose oxidationregardless of which tissue was assayed. Conversely,methylxanthines and polyphenols (MXPL), and saponins (SFL)increased muscle and liver metabolism.This suggests that theseparation of the bioactive compounds in yerba mate can bringforth unexpected effects compared to those observed in theyerba mate crude extract. It is noteworthy that this parameterwas markedly increased in the retroperitoneal adipose tissueexcised from rats treated with SFL.

Moreover, the glucose oxidation profiles of muscular andadipose tissue closely resemble those from lipogenesis (Fig. 4)in both tissues. Hence a possible relationship between the effectof yerba mate and both metabolic parameters is apparent,without any tangible hepatic involvement.

3.4.4. Glycogen synthesis in liver and soleus muscleIn general, glycogen synthesis in liver was lower than in soleusmuscle (Fig. 4). In both tissues the high-fat diet group exhib-ited half of the mean value compared to the standard diet group(control) (Fig. 4). On the other hand, all mate-treated groupsrevealed an increase in glycogen synthesis in the soleus muscle.In the hepatic tissue, only MXPL stimulated glycogen synthe-sis, but to a lesser extent than in the muscle.

3.4.5. Triacylglycerols, cholesterol and glycogen content inhepatic tissueThe high-fat diet induced an increase in triacylglycerols andglycogen hepatic concentration compared to the control group(p < 0.05). Although treatment with MXPL and SFL tends to in-crease both triacylglycerols and cholesterol contents, thedifferences observed were not significant (p > 0.05) comparedto the high fat diet (Table 3). Regarding the glycogen content,MXPL demonstrated a significant reduction (p < 0.05), and SFLshowed a slight decrease, but to a lesser extent.

The enhancement effect of MXPL on the hepatic glycogensynthesis, and lower contents of hepatic glycogen (Table 3),

Fig. 3 – Glucose oxidation in liver (A), soleus muscle (B) andretroperitoneal adipose tissue (C) (expressed as nmol14CO2/g/h) in rats treated with water and standard diet(control group); water and high-fat diet; yerba mate extract(YME) and high-fat diet; methylxanthine–polyphenolconcentrated fraction (MXPL) and high-fat diet; saponinconcentrated fraction (SFL) and high-fat diet, after a 30-daytreatment. a,b,c,d,eSame letters indicate equivalent resultsamong treated-groups by Tukey’s test (p > 0.05) (mean ± SD,n = 6).

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corroborated the protective effect of yerba mate polyphenolsand methylxanthines against glucose imbalance, as earlier re-ported (Pereira et al., 2012).

3.4.6. Lipogenesis assay from glucose in hepatic, muscle andadipose tissuesOnly the hepatic lipogenesis in rats fed a high-fat diet was dras-tically increased, although this condition was revertedcompletely after the administration of any yerba mate prepa-ration (Fig. 5). Basically, muscle lipogenesis remained unaffectedafter the high-fat diet (Fig. 5B). However, in adipose tissue, thisactivity was slightly decreased (Fig. 5C). The treatment withSFL induced lipogenesis but only in muscle and adipose tissues,not in hepatic tissue. In its turn, MXPL induced lipogenesis butonly in muscle tissue (Fig. 5A).

Steatosis is an accumulation of triacylglycerols in theliver. Along with an unbalanced availability of plasma fattyacids, lipid synthesis (lipogenesis) from glucose is currentlyconsidered the uppermost contributing factor for this liverdisease. As observed in Fig. 4A, the implemented high-fatdiet increased the hepatic lipogenesis from glucose signifi-cantly (p < 0.05). Conversely, all mate-preparations counteractedit irrespectively, returning the lipogenesis level to its basalvalue, thus suggesting a potential hepatic protective effect.The inhibition or lessening of hepatic lipogenic activity oftenhas been ascribed to the different vegetable polyphenols (Wuet al., 2011), as noticed in yerba mate polyphenols. Noteworthy,

yerba mate saponins also showed a comparable effect, resem-bling a former study with Platycodon grandiflorum saponins(Noh et al., 2010). Moreover, Tamura et al. (2013), demon-strated that Ilex paraguariensis extract prevents ethanol-induced liver injury in vivo, reinforcing the potential liverprotection properties of this species extracts. It thereforesuggests that both mate fractions are capable of potentiallyinhibiting steatosis.

Concerning the lipogenic activity in adipose tissue, it wasenhanced only by the yerba mate saponins fraction (Fig. 5C).Moreover, saponins from other vegetable species had en-hanced the insulin secretion and both the in vitro and in vivosensitivity toward it (Keller et al., 2011). Insulin is an impor-tant hormonal factor that stimulates lipogenesis. Yerba matepreparations are capable of stimulating insulin secretion(Pereira et al., 2012), as well as improving insulin sensitivityand insulin resistance (Arçari et al., 2011). Given that yerbamate saponins enhanced lipogenesis and glycogen synthesisin soleus muscle (Pereira et al., 2012) concomitantly, the dataobtained suggest an insulin modulation effect associatedwith SFL.

3.4.7. Lipolysis assay in retroperitoneal adipose tissueLipolysis is the process whereby the triacylglycerol is dissoci-ated into non-esterified fatty acids and glycerol. This processresults in the mobilization of several fatty acids to body tissues,including the liver, adipose tissue and skeletal muscle.

Fig. 4 – Glycogen synthesis in liver (A) and soleus muscle (B) (expressed as nmol/g/h) in rats treated with water andstandard diet (control group); water and high-fat diet; yerba mate extract (YME) and high-fat diet; methylxanthines–polyphenols concentrated fraction (MXPL) and high-fat diet; saponins concentrated fraction (SFL) and high-fat diet, after a30-day treatment. a,b,c,d,eSame letters indicate equivalent results among treated-groups by Tukey’s test (p > 0.05) (mean ± SD,n = 6).

Table 3 – Liver content (mg/100 mg tissue) of triglycerides, cholesterol and glycogen.

Group test Triglycerydes(mean ± SD)*

Cholesterol(mean ± SD)*

Glycogen(mean ± SD)*

Standard diet (control) 0.99a ± 0.15 1.01a ± 0.11 0.61a ± 0.04High-fat diet 1.56b ± 0.36 1.14a,b ± 0.14 0.78b ± 0.08YME (yerba mate extract) 1.50b ± 0.19 1.28a,b ± 0.16 0.75b ± 0.05MXPL (methylxanthines–polyphenols concentrated fraction) 1.92b ± 0.28 1.35b ± 0.22 0.60a ± 0.09SFL (saponins concentrated fraction) 1.71b ± 0.5 1.26a,b ± 0.17 0.68a,b ± 0.07

* Mean ± standard deviation (n = 6).a,b Same letters indicate equivalent contents among treated-groups by Tukey’s test (p > 0.05) for each parameter evaluated.

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Compared to the standard diet group, as a rule, lipolysis wassuppressed by the high-fat diet and all yerba mate prepara-tions (p > 0.05) (Fig. 5D). Furthermore, only MXPL induced aminor increase in lipolytic activity of retroperitoneal adiposetissue. Even if not significant, it may be due to the presenceof the methylxanthines in this fraction. As often quoted in theliterature, methylxanthines, especially caffeine, induce lipolysis

by various mechanisms (Duncan, Ahmadian, Jaworski,Sarkadi-Nagy, & Sul, 2007).

3.4.8. Fecal fat determinationThe evaluation of the fat in rat feces at the beginning of theexperiment, and 30 days after treatment, is shown in Fig. 6.

Fig. 5 – Lipogenesis in liver (A), soleus muscle (B), retroperitoneal adipose tissue (C) (expressed as pmols glucose/g/h) andlipolysis of retroperitoneal adipose tissue (D) (expressed as nmols glycerol/g/h) in rats treated with water and standard diet(control group); water and high-fat diet; yerba mate extract (YME) and high-fat diet; methylxanthines–polyphenolsconcentrated fraction (MXPL) and high-fat diet; saponins concentrated fraction (SFL) and high-fat diet, after a 30-daytreatment. a,b,c,d,eSame letters indicate equivalent results among treated-groups by Tukey’s test (p > 0.05) (mean ± SD, n = 6).

Fig. 6 – Fecal fat content (expressed mg/g) in rats treated with water and standard diet (control group); water and high-fatdiet; yerba mate extract (YME) and high-fat diet; methylxanthines–polyphenols concentrated fraction (MXPL) and high-fatdiet; saponins concentrated fraction (SFL) and high-fat diet, after a 30-day groups by Tukey’s test (p > 0.05) (mean ± SD,n = 6).

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No significant difference could be assessed among groups, apartfrom SFL.

All the groups fed with a high-fat diet and treated with yerbamate preparations exhibited an increase in the fecal fat ex-cretion compared to standard diet. The group treated with thesaponins from mate leaves (SFL) induced a 40% increase in fecalfat excretion. These results may be related to the inhibition ofthe pancreatic lipase activity by saponins (Han, Zheng,Yoshikawa, Okuda, & Kimura, 2005), including yerba mate sa-ponins (Martins et al., 2009). Thus, as the intestinal absorptionof lipids is precluded, fecal fat excretion is promoted. Since bothfecal fat loss and lipogenesis were increased by SFL in the samehigh-fat diet group, it seems that lipogenesis takes place at theexpense of the glucose metabolism in the adipose tissue.

The dissimilar results found in this work when comparedwith other studies employing yerba mate preparations may bedue to different models and strains of animals, and diets pro-vided. In this case, the selection of the correct model seemsto be critical and often depends on the mechanisms by whichnatural products mediate their actions.

4. Conclusions

Yerba mate crude extract and two of its concentrated frac-tions of methylxanthines–polyphenols and saponins were ableto influence the lipid metabolism of male Wistar rats fed witha high-fat diet. Any mate preparations induced a body weightloss in rats (Supplementary material, Table S1). However, bothcrude extract and concentrated fractions exhibited differenteffects on lipid metabolism, in particular when assessed sepa-rately. Several modifications were observed in fat weight gain,glucose oxidation, lipogenesis and lipolysis, glycogen synthe-sis, biochemical parameters, and fecal fat excretion, dependingon the material assayed. Some results agreed with formerstudies with mate crude extracts. Notwithstanding, there werealso relevant divergences, for instance, regarding body and fatweight gain, lipogenesis and glucose plasma level. Therefore,yerba mate extract and fractions revealed a potential anti-obesity effect on lipid metabolism of the Wistar rat and warrantsfurther exploration.

Conflict of interest

The authors have declared that there is no conflict of interest.

Acknowledgments

The authors are grateful to Conselho Nacional deDesenvolvimento Científico e Tecnológico (CNPq-308299/2012-4)and Coordenação de Aperfeiçoamento de Pessoal de Nível Su-perior (CAPES-PPGCF-UFRGS) for financial support. Ilexoside IIand chikusetsusaponin IVa used as reference compounds werekindly provided by Prof. G. Gosmann (Faculdade de Farmácia– UFRGS).

Appendix: Supplementary material

Supplementary data to this article can be found online atdoi:10.1016/j.jff.2015.03.040.

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