Oxidized LDL is considered to be an important factor in the atherogenic progression of coronary artery disease (CAD). Different lipid oxidation products have been identified in lesions and plasma of patients with vascular disease. For most lipid oxidation products found in vivo, the exact mechanisms for their generation are unknown and may involve multiple pathways [1], including oxygen and nitrogen free radical chain reactions, pro-oxidant action of metal ions (Fe³⁺, Cu²⁺) and cell lipoxygenase peroxidation activity [1,2,3], among others.

Simultaneous production of nitric oxide (^•NO) and superoxide anion (O₂⁻) by vascular cells may explain peroxynitrite (ONOO−) formation within the vascular wall. Peroxynitrite-modified LDL binds to scavenger receptors, leading to accumulation of cholesteryl esters involved in the production of atherosclerotic lesions [4]. Lipoxygenase is an enzyme that incorporates one molecule of oxygen into unsaturated fatty acids in a regiospecific and stereospecific manner. In mammalian tissues, there are four lipoxygenase isoenzymes, named according to the position of oxygenation in arachidonic acid. Lipoxygenases oxygenate not only free fatty acids, but also esterified fatty acids such as phospholipids, cholesteryl esters and cholesteryl ester in LDL [2]. Transition-metals are powerful initiators of lipid peroxidation, which may be available in free form in animal and human atherosclerotic lesions [5].

The oxidation process generates a huge variety of carbonyl compounds, such as malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), acrolein and glyoxal. These compounds may directly modify LDL and may also react with proteins, thereby impairing their biological activity [2,6]. Different lipid oxidation products are identified in lesions and plasma of patients with vascular disease.

Carbonyl scavenging agents, i.e., substances capable of reducing the accumulation of advanced lipoperoxidation end products, are of potential interest because they may reduce or retard oxidative damage [7]. Dietary polyphenols play a major role in maintaining the antioxidant defense of organisms that are exposed to several free radical generating factors, since they are able to scavenge such chemical species [8]. Leaves from yerba maté (Ilex paraguariensis) are a potential source of polyphenols such as phenolic acids (chlorogenic and caffeic acids), also present in hot infusions made from either the dried green leaves (“chimarrão”) or the roasted leaves (maté tea) [9]. In addition to polyphenols yerba maté contains caffeine and saponins [10]. The antioxidant activity of green yerba maté beverages has already been described in both in vitro and in vivo models [10,11]. There is a lack of studies on maté tea (the roasted product from yerba maté) in human models.

The aim of this study was to evaluate the antioxidant activity of maté tea on LDL from young female volunteers after acute and prolonged maté tea intake (one week). We used three different pathways for LDL oxidation, mimicking those that occur in the arterial wall: transition metal, peroxynitrite and lipoxygenase, in accordance with Gugliucci and Menini [12].

In order to characterize the plant material used in this study, we present the chromatographic profiles of maté tea at 272 and 323 nm (Figure 1), the absorption spectra of caffeoylquinic acids as well as chlorogenic acids and xanthines contents (Table 1).

The baseline lipid profile and body mass index (BMI) for all subjects were within normal values (Table 2). Analysis of the three-day dietary food record at baseline (first week) and after the maté tea supplementation week showed good overall compliance with the recommended diets. There were no differences regarding daily nutrient intake before and after the supplementation period (p > 0.05) (Table 3).

Maté tea intake did not significantly affect conjugate diene formation when SIN-1 or lipoxygenase were used, either at T₁ or T₂. Nonetheless, we observed a nonsignificant decrease in the maximal rate of conjugated diene formation when SIN-1 was used. In contrast, there was a significant decrease in the susceptibility of LDL to oxidation after daily maté tea intake (T₂), compared with baseline (T₀), when peroxidation was induced with copper (Table 4).

Figure 2 shows the SDS-PAGE profile for Apo B, in which lane 2 represents native LDL Apo B. The oxidation of Apo B, which results in high molecular weight aggregates that do not penetrate the separating gel, leads to modification in the protein eletrophoretic behaviour. After peroxidation, there is a marked decrease in lane 2 intensity, observed in the eletrophoresis gel and followed by the decrease in the optical density (% OD). Maté tea intake inhibited this effect at T₂ but not at T₁ (Figure 2 a,b).

In order to evaluate whether acute and prolonged intake of maté tea would protect LDL from peroxidation, and to investigate the possible mechanisms, we used three different peroxidation pathways, as described by Gugliucci and Menini [11] and recommended by Halliweel [4].

We did not observe any significant decrease in susceptibility to LDL oxidation when SIN-1 or lipoxygenase were the peroxidation agents after acute (T₁) or prolonged (T₂) intake of maté tea (Table 4). On the other hand, maté tea intake for one week significantly decreased LDL susceptibility to copper oxidation (T₂), while no significant effect was observed one hour after maté tea consumption. In a previous study, a dose-dependent increase in LDL protection was observed when Cu⁺⁺, SIN-1 and lipoxygenase were used as oxidants [13], after the ingestion of the green yerba maté beverage “chimarrão”. The fact that polyphenol content decreases and considerable degradation of caffeoylquinic esters occurs during the roasting process [10], and that the abovementioned study evaluated reactive thiobarbituric substances, rather than diene conjugate formation, might explain the discrepancies in the results.

LDL peroxidation is a slow process that occurs in the sub-intimal space, by contact between LDL and reactive species. The oxidation process generates a huge variety of lipid peroxidation products that can react with lysine residues in Apo B, with several implications relating to the atherogenesis process [6]. The effects of LDL Apo B modification can be followed by SDS-PAGE. Modifications in Apo B include cross-linking along the protein chain, with the formation of aggregates that will not enter the electrophoresis gel [13,14,15,16,17,18,19,20,21]. We observed that the consumption of maté tea for one week increased the protection of LDL Apo B against peroxidation with copper (Figure 2). There was a noticeable increase in optical density (OD) for lane T₂ after oxidation, which corresponds to the native Apo B state (Figure 2a), despite individual differences (Figure 2b). This result is in agreement with the findings of Gugliucci and Menini [11] and Gugliucci [22]. Other medicinal plants, traditionally used in China, like Scolopendra subspinipes, have shown a similar effect in vitro [23].

Ilex paraguariensis infusions contain a large amount (approximately 30%) of polyphenols. About 3% of the weight of yerba maté leaves are in the form of caffeoylquinic acids and its isomers, generically named as chlorogenic acids (CGA), esters from caffeic acid and quinic acid [9,24]. Maté tea, besides the aforementioned chlorogenic acids, monocaffeoylquinic acid (CQA) and dicaffeoylquinic acid (diCQA), dicaffeoylshikimic acid and caffeoylshikimic acid are also found [10]. However, due to the roasting process, phenolic compounds can be lost while compounds with antioxidant properties are generated by the Maillard reaction [9]. Most notably of mate’s biological activities is its high antioxidant capacity, which has been shown to be higher than green tea [9]. This high antioxidant capacity is attributed and is directly proportional to its chlorogenic acid and total phenolic acids contents [25]. Studies with coffee, an important food source of CGAs, have shown that these hydrophilic compounds are absorbed by the body and can be detected in human plasma within 0.5 to 4 hours after consumption [19].

Owing to an ortho-dihydroxy phenyl ring, CGAs are both efficient free radical scavenger and metal ion chelators, mechanisms by which these compounds protect LDL against metal-catalyzed oxidation [26]. Furthermore, CGAs may: increase the resistance of LDL to oxidative modification as a result of their incorporation into LDL [27]; regenerate alfa-tocopherol through the reduction of the α-tocopheroxyl radical, and thus protecting biological structures in non polar environments [28,29]. These different mechanisms of action may support the hypothesis that polyphenols from maté tea and/or their metabolites may diffuse into the arterial wall and enhance antioxidant capacity in the aqueous phase, possibly on the surface of the lipoprotein particles, thereby inhibiting Cu²⁺-induced LDL oxidation [20,22].

Besides substantial amounts of phenolic acids, the leaves of maté also contain caffeine and saponins [10,24]. These bioactive compounds are recognized to influence the central nervous system [30] and exert anti-inflammatory activities [31]. However, the potential biological relevance of antioxidant properties of these compounds is yet uncertain [32]. Maté tea has been shown to affect plasma oxidative stress parameters as well as the level of leukocyte antioxidant enzyme gene expression in young women [33] and to decrease fatty acid peroxidation in mice liver [34], indicating that polyphenols from maté tea promote protection against peroxidation by several mechanisms in different biological structures. There is considerable evidence indicating that polyphenols present in fruits and vegetables have antioxidant activity in vitro.

We hypothesize that prolonged maté tea intake may protect LDL, because of the ability of phenolic acids to inhibit copper-induced LDL autoxidation, rather than through scavenging nitrogen free radical species or inhibiting lipoxygenase activity, in the model used in this study [35]. In vivo studies aiming to evaluate cellular and molecular targets are helpful for elucidating whether and how these phytochemicals are effective as dietary coadjutants in health maintenance. Although the number of volunteers in this study was small, each individual was her own control and a similar trend could be observed for the results, despite individual differences. Polyphenols from maté tea were bioavailable to protect LDL against ex-vivo copper oxidation, as already observed for green maté “chimarrão”.

In conclusion, we observed that prolonged maté tea consumption prevented LDL oxidation, expressed as diene conjugate formation, and prevented LDL Apo B structural changes when copper is the oxidative agent, but not in the presence of other pro-oxidants such as peroxynitrites and lipoxygenase. Results from previous experiments showed that green maté “chimarrão” was able to prevent LDL peroxidation. This study showed that maté tea presents similar in vivo antioxidant capacity, despite the roasting process.

The authors want to express their gratitude to FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) for the financial support (#06/58019-5 and #07/51952-0), to Leão Jr (Curitiba, Paraná) for providing the instant maté tea and for the grant-in-aid to author Matsumoto.