The Activity of Mate Saponins (Ilex Paraguariensis) in Intra-Abdominal and Epididymal Fat, and Glucose Oxidation in Male Wistar Rats
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Journal of Ethnopharmacology 144 (2012) 735–740 Contents lists available at SciVerse ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep 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: ortega@farmacia.ufrgs.br (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 Â 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 Â 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.