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Journal of Ethnopharmacology 144 (2012) 735–740
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Journal of Ethnopharmacology
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The activity of mate saponins (Ilex paraguariensis) in intra-abdominal and epididymal fat, and glucose oxidation in male Wistar rats
Pedro Ernesto de Resende a, Simone Gasparin Verza a, Samuel Kaiser a, Luana Ferreira Gomes b, Luiz Carlos Kucharski b, George Gonza´lez Ortega a,n
a Programa de Po´s-graduac-ao~ em Cienciasˆ Farmaceuticas,ˆ Faculdade de Farma´cia, Universidade Federal do Rio Grande do Sul, Porto Alegre 90610-000, RS, Brazil b Laborato´rio de Metabolismo e Endocrinologia Comparada, Departamento de Fisiologia, Universidade Federal do Rio Grande do Sul (UFRGS), Avenida Sarmento Leite, 500, Porto Alegre 90050-170, RS, Brazil
article info abstract
Article history: Ethnopharmacological relevance: Ilex paraguariensis A. St. Hilaire (mate) has traditionally been used in Received 10 July 2012 several South American countries to prepare tea-like beverages having stimulant effects on the CNS and Received in revised form appetite. In recent years, however, mate preparations have been recommended putatively as an 13 September 2012 appetite suppressant and slimming remedy. Moreover, studies carried out on either normal or diet- Accepted 12 October 2012 induced obese rats treated with mate extracts revealed anti-obesity and satiety effects, thus refuting Available online 23 October 2012 ethnopharmacological data. In this work, the effect of mate on the intra-abdominal and epididymal fat, Keywords: and glucose oxidation levels after oral administration in male Wistar rats, was studied using crude Ilex paraguariensis extract from leaves, unripe fruits, and a chemically well-defined purified saponin fraction (MSF). Yerba mate Material and methods: Saponin, polyphenol and methylxanthine contents in MSF were analyzed by Saponins HPLC-PDA and UPLC/Q-TOF-MS. Crude extracts from mate leaves (LAE) and unripe fruits (FHE) were Obesity Lipid metabolism assayed for comparison purposes. Male Wistar rats fed with standard diet and water ad libitum were used as the control group. Results: The fat weight and both liver and adipose glucose oxidation were reduced significantly by MSF (35, 90 and 60%, respectively), while LAE and FHE were less active. Also, a significant lowering of the blood triglycerides level was observed in rats treated with MSF and LAE. All creatinine, urea, and transaminase plasma levels remained unaffected no matter what mate preparation was considered. It is also worth pointing out that the glucose blood level was increased after treatment with FHE. This finding did not correlate either with the content of methylxanthines, polyphenols or saponins. Conclusion: A reduction in both visceral fat weight and glucose oxidation of hepatic and adipose tissue in healthy rats fed with a standard diet could be ascribed to a purified mate saponin fraction from unripe fruits. These findings agree with former studies carried out with crude mate extracts and also suggest their potential use as an anti-obesity preparation. Nonetheless, further in vivo experiments are still required to corroborate its effect on human beings. & 2012 Elsevier Ireland Ltd. Open access under the Elsevier OA license.
1. Introduction distinguishable, namely, chimarrao~ using hot water, and terere´ or caaygua using cold water. Regardless of how they are prepared, Ilex paraguariensis A. St. Hilaire (mate, caay) is a native ever- mate leaves are recommended traditionally as a CNS stimulant green tree systematically cultivated in several South American and as a remedy against fatigue (Mors et al., 2000), but also as a countries, where the intake of tea-like beverages from mate choleretic, eupectic, and appetite stimulant (Cardozo et al., 2007; leaves (yerba mate) is widespread (Silva et al., 2011). According Cruz, 1964). Nonetheless, the latter traditional indications are at to how they are prepared, two different aqueous preparations are odds with more recent uses of mate preparations, which include indications as an appetite suppressant and slimming remedy (Heck and De Mejia, 2007; Mors et al., 2000). Also, satiety and Abbreviations: MSF, Purified mate saponin fraction; LAE, Leaf aqueous extract; weight reduction effects related to the glucagon-like peptide-1 FHE, Hydroethanolic extract from unripe fruits; HDL, High density lipoprotein; are in disagreement with its ethnopharmacological use as appe- LDL, Low density lipoprotein; GOT, Glutamic-oxaloacetic transaminase; GPT, tite stimulant (Hussein et al., 2011), as well as some activities Glutamic pyruvic transaminase; KRB, Krebs-Ringer-Buffer. n Corresponding author. Tel.: þ55 51 3308 5278; fax: þ55 51 3308 5437. described in rats, mice, and human beings after treatment with E-mail address: [email protected] (G.G. Ortega). mate extracts, for instance, hypocholesterolemic (Heck and De
0378-8741 & 2012 Elsevier Ireland Ltd. Open access under the Elsevier OA license. http://dx.doi.org/10.1016/j.jep.2012.10.023 736 P.E.d. Resende et al. / Journal of Ethnopharmacology 144 (2012) 735–740
Mejia, 2007), lipolytic, hypolipidemic (Kim et al., 2012; Martins 2.3. Hydroethanolic extract from unripe fruits (FHE) et al., 2009; Paganini Stein et al., 2005; Pang et al., 2008), prevention of lipoprotein oxidation (Bracesco et al., 2011), and Selected unripe mate fruits were air-dried at 35 1C for 72 h thermogenic (Martinet et al., 1999) activities. Weight reduction (Memmert, Germany), and comminuted in a cutter mill provided induced by mate had been associated with these activities, but of a 2 mm-steel sieve (Retsch SK1, Germany). A 100 g sample was other factors may be involved, such as cholesterol binding by pretreated by immersing it in 1 L of hydroethanolic solution (40% saponins in intestinal lumen (Francis et al., 2002), appetite v/v) by 40 min, and afterward extracted using lab-scale vortex suppression (Andersen and Fogh, 2001; Pang et al., 2008), and equipment (Ultra-turrax T25 Basic, IKA, Germany) at 11.000 rpm, pancreatic lipase inhibition (Kim et al., 2009; Martins et al., 2009; for 15 min. The mixture was filtered through a Whatman paper Sugimoto et al., 2009). filter N1 2, concentrated at 5573 1C under reduced pressure In this context, the incidence of obesity and related illnesses (Buchi R-114, Switzerland), freeze-dried (Modulyo 4 L, Edwards, has more than doubled since 1980, and has become a serious USA) as usually, and coded as FHE. threat to public health worldwide (WHO, 2012). It is now well- documented that some vegetable extracts rich in saponins (Han 2.4. Purified mate saponin fraction (MSF) et al., 2001, 2002, 2005), including aqueous extracts from mate leaves, are able to modify the lipid metabolism of diet-induced A sample of about 1.0 g of FHE was dissolved in 20 mL of obese rats, leading to a substantial lessening in weight gain water, placed onto a 50 2.8-cm glass column packed with 40 g - (Arcari et al., 2009; Bracesco et al., 2011; Pang et al., 2008). of Diaion HP 20 (Supelco, USA), and fractionated using methanol:- In past decades, the main bioactive compounds of mate have water mixtures of 500 mL each, in decreasing polarity gradient. been studied extensively, with emphasis on methylxanthines, The column flow rate was 5.0 mL/min. Both 70% and 90% v/v polyphenols, and saponins. The latter are well-known amphiphilic methanol fractions were combined, concentrated under vacuum substances composed of either a steroid or triterpene core, to at 40 1C, freeze dried at once as described above for FHE, and which one or more sugar side chains are attached (Hostettmann stored in light protected glass until it was used (Pavei et al., 2007; and Marston, 2005). High saponin content occurs in mate leaves Peixoto et al., 2010). but especially in its unripe fruits (Pavei et al., 2007; Peixoto et al., 2010; Taketa et al., 2004). Many biological reports on the topic of anti-obesity are based 2.5. HPLC content assay in insufficient data about the chemical composition of extracts or are derived from tests carried out with herbal mixtures. Thus, The analyses were performed with a Shimadzu Prominence despite numerous phytochemical works on mate, it still remains LC-20AT equipment (Kyoto, Japan) coupled to a photodiode array uncertain whether the anti-obesity effect should be ascribed to detector (PDA) SPD-M20A, controlled by LC-Solution Multi-PDA the polyphenols, methylxanthines or saponins. software. The stationary phase was a Gemini-NX RP C18 column In an earlier work, we reported a significant lessening of both (Phenomenex, 250 4.6 mm i.d.; 5 mm particle size). epididymal and intra-abdominal fat in normal rats fed a standard diet and separately treated with crude aqueous extracts from a 2.5.1. Methylxantines commercial yerba mate and industrially unprocessed mate leaves An isocratic system using a methanol:water 40% (v/v) mixture (Silva et al., 2011). Interestingly, neither cholesterol nor LDL was applied, with a flow rate of 1.1 mL/min and detection at plasma levels showed significant changes at that time. Moreover, 280 nm (Gnoatto et al., 2007). Caffeine and theobromine were founded on polyphenol, methylxanthine, and saponin contents dissolved in that solvent, at concentrations ranging from 0.2 to previously assayed by HPLC, the anti-obesity effect of mate have 10 mg/mL. LAE, FHE, and MSF samples were properly diluted with been ascribed to the methylxanthine and saponin content by the same solvent seeking the linearity range of methylxanthine authors. Hence, this work aimed at evaluating the effect of a concentration. The total methylxanthine content was expressed purified mate saponin fraction from unripe fruits on the lipid as the sum of caffeine and theobromine concentrations. Each metabolism. Free-polyphenol and methylxanthine fractions were result corresponds to the mean value of three consecutive properly prepared by solid-phase separation and analyzed by determinations. HPLC-PDA and UPLC/Q-TOF-MS, for comparison purposes.
2.5.2. Polyphenols 2. Material and methods The HPLC-UV content assay of mate polyphenols was per- formed as previously reported (Silva et al., 2007). Separately, 2.1. Plant materials chlorogenic acid and rutin standard curves were prepared by dissolving in water accurately weighed samples to obtain con- Commercial yerba mate (mate leaves and stems dried by the centrations ranging from 2 to 10 mg/mL. LAE, FHE, and MSF so-called sapeco processing), with a 70:30 leaves:stems ratio (w/ samples were prepared in the same way to adjust the polyphenol w) and mate unripe fruits were collected in January 2011 and concentration within the linearity range. The total polyphenol kindly gifted by Ervateira Barao~ (Barao~ Cotegipe, RS, Brazil; content was expressed as the sum of peak areas, previously 271370S521230W). identified as the main polyphenol found in mate. Each result represents the mean value of three determinations. 2.2. Leaf aqueous extract (LAE) 2.5.3. Saponins Leaf aqueous extracts were prepared daily by a 15-min The method consisted of a gradient system of phosphoric acid infusion (80 1C) of 70 g of commercial yerba mate in 1 L of water, 0.1% (v/v) (phase A) and acetonitrile (phase B). A linear gradient thereby reproducing the traditional preparation of the tea-like elution was performed as follows: 30–45% B (40 min), 45% B beverage chimarrao~ . The mixture was filtered at room tempera- (5 min), 45–30% B (20 min), flow rate of 0.9 mL/min, detection at ture through a Whatman paper filter N1 2 (Kent, UK) just 205 nm, and temperature of 30 1C(Borre´ et al., 2010). The total before use. saponin content was calculated by means of a matesaponin-3 P.E.d. Resende et al. / Journal of Ethnopharmacology 144 (2012) 735–740 737 standard curve (6–60 mg/mL) and expressed as the sum of the 50% (v/v) through the rubber caps, and 0.25 mL of NaOH (2.0 N) numbered peak areas. solution directly into the wells. The flasks were maintained at 14 least 12 h at room temperature in order to capture CO2. The 2.6. Characterization of MSF by UPLC/Q-TOF-MS contents of the center well were transferred to vials containing scintillation liquid, and the radioactivity was measured using an The saponin characterization was performed with a Waters LKB counter (PerkinElmer, Waltham, MA, USA) with an automatic 14 Acquity Ultra Performance LC system (Waters, Milford, MA, USA) curve quench correction. The CO2 production was expressed as 14 equipped with binary solvent manager, sample manager, photo- nmol of C glucose incorporated into CO2 per mg of tissue, per diode array detector (PDA), and coupled to a Q-TOF mass spectro- min. (Mersmann and Hu, 1987; Torres et al., 2001). meter, as previously described (Peixoto et al., 2011). Briefly, ESI capillary voltage was set at þ3.0 kV for positive ion mode. The 2.7.4. Biochemical analyses mass scan was over the range of 200–1500 m/z. A Hypersil Gold Total cholesterol, high density lipoprotein (HDL), triglycerides, RP-18 column (100 2.1 mm i.d., 1.9 mm) (Thermo Scientific, transaminases (GOT and GPT), creatinine, and urea concentrations USA) was used. Elution was performed following a linear gradient obtained from plasma were assayed using commercial enzymatic procedure with formic acid 0.01% (v/v; pH 3.2) (solvent A) and kits (Labtest, Sao~ Paulo, Brazil). The concentration of low density acetonitrile:formic acid (0.99:0.01, v/v) (solvent B). The injection lipoprotein (LDL) was calculated from total cholesterol, HDL, and volume was 5 mL. The flow rate was kept constant at 0.3 mL/min, triglyceride concentrations, as usual. and the analyses were conducted at 3071 1C. A 200 mg/mL sample of MSF was previously dissolved in acetonitrile:water 2.7.5. Statistical analyses (1:1, v/v) and analyzed. Analytical data were processed by Data from each experiment were expressed by the mean and MassLynx V4.1 software (Waters, Millford, MA, USA). respective standard deviation (SD). Differences among groups were tested by One-Way ANOVA, Dunnett test, and Tukey test (software 2.7. Evaluation of biological activities s Minitab 14). Values of Po0.05 were considered significant. 2.7.1. Animals and experimental procedure Twenty-four male Wistar rats (8 weeks-old) were weighed on 3. Results and discussion the first day of the treatment, and housed in steel cages 7 1 (414 344 168 mm) under light/dark cycles of 12 h, at 22 2 C. The HPLC analysis of most relevant bioactive compounds The animals were divided into four groups (A, B, C, and D) of 6 recognized in Ilex paraguariensis revealed differences among animals each, and fed with standard diet ad libitum throughout the LAE, FHE, and MSF (Table 1). experiment period of 30 days. Test Group A: animals were treated In the LAE, the total saponin content was statistically equivalent with LAE ad libitum (24.7 mg of dried residue/mL); Test Group B: to MSF (P40.05) being higher than found in FHE (Po0.05). animals were treated with 500 mg/mL of FHE ad libitum;TestGroup Likewise, the total polyphenol content was substantially higher in C: animals were treated with 500 mg/mL of MSF ad libitum.GroupD LAE than in FHE. Methylxanthines were detected only in LAE, or negative control group: animals received only water ad libitum. having caffeine as the major compound. Neither polyphenols nor The dose used in MSF (500 mg/mL) was defined considering the methylxanthines were detected in MSF. In all cases, the saponin, saponin content in LAE bearing in mind equivalent amounts of polyphenol and methylxanthine contents agreed with earlier che- saponins in both preparations. The intake of water containing the mical data (Borre´ et al., 2010; Peixoto et al., 2011; Silva et al., 2011). mate preparations was comparable to that measured in the control group. All experimental procedures followed the Guide for the Care and Use of Laboratory Animals developed by the Institute of 3.1. Saponins characterization and UPLC-analyses Laboratory Animal Resources of the National Research Council and the recommendations of the Brazilian College for Animal Experi- The purification process of mate saponins by hydrophobic mentation. The experimental protocol was approved by the UFRGS polyaromatic resin has enabled the characterization by UPLC/Q- Ethical Committee on Research (protocol CEUA 36-n1116). TOF-MS of 10 triterpenic saponins derived from ursolic, oleanolic, pomolic, and acetylursolic acids (Fig. 1)(Peixoto et al., 2011; Taketa et al., 2004). Ilexoside II , the most prevalent saponin in 2.7.2. Fat pads, liver and blood samples mate leaves and unripe fruits (Taketa et al., 2004) occurs in MSF After 30-day treatment, the animals were weighed and decapi- at 7.94 min retention time. Its identity was corroborated by tated. Blood was collected in glass tubes, and plasma was analysis of the MS fragmentation patterns (Peixoto et al., 2011). obtained by centrifugation at 2150g for 8 min. Separately, visceral fat pads from retroperitoneal and epididymal tissues, and samples Table 1 from liver were carefully excised and weighed at once. The fat HPLC content assay of saponins, polyphenols, and methylxanthines in aqueous weight/body weight ratio was calculated and expressed as per- extract from mate leaves (LAE), ethanolic extract from mate unripe fruits (FHE) cent. This parameter allows inference about the activity in lipid and the purified mate saponin fraction (MSF) administered orally, ad libitum,to metabolism induced by test groups. Wistar rats. Content assay LAE (mg/mL) FHE (mg/mL) MSF (mg/mL) 14 (meana7SD) (meana7SD) (meana7SD) 2.7.3. Glucose oxidation ( CO2 production) Liver and retroperitoneal fat tissue samples were incubated at Total saponins 409.86b75.61 214.6774.23 399.09b718.84 37 1C, for 60 min, in flasks sealed with rubber caps containing Total polyphenols 4904.0970.703 9.7970.021 NDc 14 1.0 mL of KRB (Krebs-Ringer-Buffer), 0.15 Ci [U- C] glucose Total methylxanthines 630.5271.13 NDc NDc (Amersham, USA), and glucose solution 5 mM. The gaseous phase Caffeine 483.7272.06 NDc NDc Theobromine 146.6170.92 NDc NDc was saturated with a 5% CO2 and 95% O2 mixture. Small glass wells containing strips of 3 mm-Whatman paper were placed a Mean value of three determinations. above the level of the incubation medium. The oxidation reaction b Equivalent saponins concentrations (Po0.05). was stopped by injecting 0.25 mL of trichloracetic acid solution c ND: not detected. 738 P.E.d. Resende et al. / Journal of Ethnopharmacology 144 (2012) 735–740
Fig. 1. UPLC-PDA chromatogram of the purified mate saponin fraction (MSF), at 205 nm.
Fig. 2. Epydidimal and retroperitoneal fat pad (expressed as fat weight/body weight ratio) in rats treated with water (control group), aqueous extract from mate leaves (LAE) (24.7 mg of dried residue/mL), ethanolic extract from mate Fig. 3. Glucose oxidation of hepatic and adipose tissues (expressed as nmols 14 unripe fruits (FHE) (500 mg/mL), and the purified mate saponin fraction (MSF) CO2/g/h) in rats treated with water (control group), aqueous extract from mate (500 mg/mL) after 30-day treatment (mean7SD, n¼6). Significant difference with leaves (LAE) (24.7 mg of dried residue/mL), ethanolic extract from mate unripe control group is designated as Po0.05 (Dunnett test). fruits (FHE) (500 mg/mL), and the purified mate saponin fraction (MSF) (500 mg/ mL) after a 30-day treatment (mean7SD, n¼6). Significant differences with the control group are designated as *Po0.05 (Dunnett test); those with LAE group as a b 3.2. Evaluation of biological activities Po0.05; and those with FHE group as Po0.05 (Tukey test).
3.2.1. Body weight significant reduction in retroperitoneal nor epididymal fat The individual consumption of a mate preparation was around (P40.05). Silva et al. (2011) also reported a reduction of intra- 40 mL per day, disregarding the animal group considered. No abdominal fat in rats treated with crude extract from industrially significant effect on body weight gain was observed in all groups unprocessed mate leaves. On that occasion the effect was ascribed evaluated after 30-day treatment. This finding agrees with pre- to the methylxanthine and saponin content. Although both vious studies carried out on rabbits and rats fed with a standard polyphenols and methylxanthines are missing in MSF, it is rather diet and treated with mate aqueous extracts (Mosimann et al., unlikely that only mate saponins are implicated in the visceral fat 2006; Silva et al., 2011). On the other hand, dissimilar results reduction. Thus, mate methylxanthines and polyphenols also can were noticed in diet-induced obese rats, which showed a sub- induce both satiety and body weight reduction via glucagon-like stantial decrease in body weight gain (Arc-ari et al., 2009; Bracesco peptide-1 and serum lectin, for instance (Hussein et al., 2011). et al., 2011; Hussein et al., 2011; Pang et al., 2008). Therefore, it seems possible that mate extracts can elicit differentiated effects, 14 depending on whether animals have been induced to obesity 3.2.3. Glucose oxidation ( CO2 production) previously or not, as earlier suggested (Jenkins and Atwal, 1994). The glucose oxidation measurement reveals the consumption of the glucose substrate and consequently the metabolic activity of tissues. The metabolic activity of liver was higher in the control 3.2.2. Fat weight/body weight ratio group than in adipose tissues (Fig. 3). All mate preparations were Though all groups evaluated had a comparable body weight able to decrease the hepatic glucose oxidation significantly, but gain, a significant reduction of about 35% in retroperitoneal fat MSF to a greater extent than LAE and FHE. Thus, rats treated with was noticed in rats treated only with MSF when compared to a MSF presented a reduction of nearly 60% and 90% in glucose control group (Fig. 2). FHE and LAE did not produce any oxidation activity in the adipose and hepatic tissue. On the P.E.d. Resende et al. / Journal of Ethnopharmacology 144 (2012) 735–740 739
Table 2 Plasma parameters in rats treated with water (control group), aqueous extract from mate leaves (LAE), ethanolic extract from mate unripe fruits (FHE), and the purified mate saponin fraction (MSF) after a 30-day treatment.
Parameter Control LAE FHE MSF (meana7SD) (meana7SD) (meana7SD) (meana7SD)
Glucose (mg/mL) 126.50712.42 128.0077.21 156.83b79.37 137.1779.93 Cholesterol (mg/mL) 72.6078.96 68.1777.86 64.6778.59 61.0075.87 HDL (mg/mL) 28.5073.73 29.3373.93 26.5073.39 26.8073.42 LDL(mg/mL) 13.8176.23 14.4072.70 19.1375.54 16.3373.21 Triglycerides (mg/mL) 215.25720.61 128.25b729.53 180.00b736.13 128.60b743.43 GOT (mg/mL) 175.00725.14 176.50716.62 187.83719.76 204.60733.61 GPT (mg/mL) 75.3376.74 82.8377.28 74.6778.24 73.80710.83 Creatinine (mg/mL) 0.2970.05 0.3170.04 0.2870.04 0.3070.02 Urea (mg/mL) 57.5077.23 55.5072.43 57.5077.94 60.0076.52
a Mean value of six determinations. b Significant differences with control group (Po0.05).
contrary, in rats treated with LAE this activity was almost rats (Han et al., 2002), while ursolic acid isolated from Actinidia unaltered in adipose tissue. In view of the fact that methyl- arguta roots enhanced lipolysis in rat fat cells and prevented the xanthines occur in LAE but they were not detected in MSF and elevation of plasma triglycerides in rats (Kim et al., 2009). In both FHE (Table 1), it is, therefore, possible that methylxanthines from cases, the inhibition of the pancreatic lipase was postulated as mate leaves may be related with this opposite activity. being the driving mechanism. Moreover, isolated saponins and saponin-containing foods also have consistently shown a hypo- cholesterolemic effect (Malinow et al., 1980), but it seems to be 3.2.4. Biochemical analyses subordinated to a rich-cholesterol diet (Oakenfull et al., 1983). In general, the levels of glucose, cholesterol, HDL, LDL, trigly- Regarding LAE, FHE, and MSF no evidence of either hepatic or cerides, GOT and GPT, creatinine, and urea obtained from plasma renal toxicity could be detected through analysis of creatinine, (Table 2) did not differ from those previously found in normal rats urea and transaminase (GOT and GPT) parameters. As reported treated with aqueous extracts from either commercial mate earlier, no noticeable toxicity, neither acute or chronic, could be leaves or gross yerba mate (Silva et al., 2011). Rats treated with assessed previously in mice and rats treated with mate saponins FHE showed a significant increase of plasma level of glucose, (Peixoto et al., 2010). which was comparable to that caused by gross yerba mate (Silva et al., 2011). Again, studies on the anti-obesity activity of mate seem to lead to opposite results at times, depending upon 4. Conclusions whether normal rats or diet-induced obese rats were used. Thus, rats submitted to a high-fat diet and treated with an aqueous A purified mate saponin fraction from unripe fruits induced extract from mate leaves showed a reduction of the glucose the reduction of fat weight, plasma triglyceride level, and both plasma level (Arc-ari et al., 2009; Bracesco et al., 2011; Pang liver and fat tissues glucose oxidation in healthy rats fed with a et al., 2008). standard diet. These results agreed with former works carried out By comparing the LAE, FAE, and MSF chemical composition, no with crude mate extracts as well as other vegetable extracts and relationship could be established among saponin, methyl- isolated saponins. Nonetheless, it did not exclude the involve- xanthine, and polyphenol contents, and the increasing plasma ment of mate polyphenols and methylxanthines. However, level of glucose. It is noteworthy that the analysis of mate unripe further evidence is still needed to establish to what extent diet- fruits and leaves showed low sugar contents in both cases (data induced obesity and the chronic intake of mate preparations may omitted). A plausible explanation still appears to be open to influence these findings. discussion. Regarding the plasma level of triglycerides, all mate prepara- tion LAE, FHE and MSF were able to reduce it significantly Acknowledgments (Table 2). This activity seems associated with the saponins contents, owing to the fact that both LAE and MSF had an equal The authors are grateful to Conselho Nacional de Desenvolvi- saponin content, while methylxantines and polyphenols are mento Cientı´fico e Tecnolo´ gico (CNPq) and Coordenac-ao~ de missing in MSF. In addition, FHE has reduced the triglycerides Aperfeic-oamento de Pessoal de Nı´vel Superior (CAPES) for finan- level, but to a minor extent. This finding seems to be consistent cial support. with its low saponin content, namely, only one-half of that calculated in LAE and MSF (Table 1). References The total cholesterol, HDL, and LDL level did not change in any animal group. This result is consistent with previous reports on Andersen, T., Fogh, J., 2001. Weight loss and delayed gastric emptying following a the anti-obesity effect of mate, independent of the use of either South American herbal preparation in overweight patients. Journal of Human normal or diet-induced obese rats (Han et al., 2001, 2002, 2005; Nutrition and Dietetics 14, 243–250. - Silva et al., 2011). Nonetheless, a quite different behavior has been Arcari, D.P., Bartchewsky, W., Dos Santos, T.W., Oliveira, K.A., Funck, A., Pedrazzoli, J., De Souza, M.F., Saad, M.J., Bastos, D.H., Gambero, A., Carvalho, P.O., Ribeiro, reported in rats treated with a hypercholesterolemic diet and M.L., 2009. 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