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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. Acknowledgments
  9. References

The inhibitory effects of maté tea (MT), a beverage produced with leaves from Ilex paraguariensis, in vitro lipase activity and on obesity in obese mice models were examined. For the in vitro experiment, porcine and human pancreatic lipase (PL) activities were determined by measuring the rate of release of oleic acid from hydrolysis of olive oil emulsified with taurocholate, phospholipids, gum arabic, or polyvinyl alcohol. For the in vivo experiments, animals were fed with a standard diet (SD, n = 10) or high-fat diet (HFD, n = 30) for 16 weeks. After the first 8 weeks on the HFD, the animals were treated with 1 and 2 g/kg of body weight of MT. The time course of the body weight and obesity-related biochemical parameters were evaluated. The results showed that MT inhibited both porcine and human PL (half-maximal inhibitory concentration = 1.5 mg MT/ml) and induced a strong inhibition of the porcine lipase activity in the hydrolysis of substrate emulsified with taurocholate + phosphatidylcholine (PC) (83 ± 3.8%) or PC alone (62 ± 4.3%). MT suppressed the increases in body weight (P < 0.05) and decreased the serum triglycerides and low-density lipoprotein (LDL)-cholesterol concentrations at both doses (from 190.3 ± 5.7 to 135.0 ± 8.9 mg/dl, from 189.1 ± 7.3 to 129.3 ± 17.6 mg/dl; P < 0.05, respectively) after they had been increased by the HFD. The liver lipid content was also decreased by the diet containing MT (from 132.6 ± 3.9 to 95.6 ± 6.1 mg/g of tissue; P < 0.05). These results suggest that MT could be a potentially therapeutic alternative in the treatment of obesity caused by a HFD.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. Acknowledgments
  9. References

One of the most important strategies in the treatment of obesity includes the development of nutrient digestion and absorption inhibitors, in an attempt to reduce the energy intake through gastrointestinal mechanisms, without altering any central mechanisms (1,2). Pancreatic lipase (PL) inhibition is one of the most widely studied mechanisms used to determine the potential efficacy of natural products as antiobesity agents (3,4,5). Orlistat, one of the two clinically approved drugs for obesity treatment, has been shown to act by inhibiting PL. Although it is one of the best-selling drugs worldwide, it has certain unpleasant gastrointestinal side effects such as oily stools, oily spotting, and flatulence, among others. The success of orlistat has prompted research for the identification of new PL inhibitors that lack some of these unpleasant side effects. At present, the potential of natural products for the treatment of obesity is still largely unexplored and might be an excellent alternative strategy for the development of safe and effective antiobesity drugs (4). PL or triacylglycerol acyl hydrolase (EC 3.1.1.3), the principal lipolytic enzyme synthesized and secreted by the pancreas, plays a key role in the efficient digestion of triglycerides. PL is responsible for the hydrolysis of 50–70% of the total dietary fats. It removes fatty acids from the α and α′ positions of dietary triglycerides, yielding β-monoglycerides and long chain saturated and polyunsaturated fatty acids as the lipolytic products (6). Many polyphenolics such as flavones, flavonols, tannins, and chalcones are active against PL. Many reports have described the inhibition of PL in the presence of unfermented (green tea) (7) and semifermented (oolong tea) (8) extracts of Camellia sinensis L. The polyphenol level in yerba maté is higher than in green tea, and is the same as that in red wines (9). In addition to the polyphenols such as flavonoids (quercetin and rutin) and phenolic acids (chlorogenic and caffeic acids), yerba maté is also rich in caffeine and saponins (10,11). Yerba maté (Ilex paraguariensis) is a plant from the subtropical region of South America, widely consumed in Brazil, Argentina, Paraguay, and Uruguay. The leaves are dried or dried and roasted to prepare different beverages, such as chimarrão and tererê (both from dried green maté leaves) and maté tea (MT), prepared from roasted maté leaves, also used to produce soft drinks. Studies reporting the biological effects of MT, especially with respect to the antioxidant properties of the constituents of I. paraguariensis extracts in animal models have already been widely reported (12,13,14,15,16), while evidence that maté extracts act directly or indirectly on obesity is more limited (17,18). Some hypolipidemic effects (12,18) as well as the ability of maté components to prevent lipoprotein oxidation (19,20,21) have also been reported. Although the effect of some polyphenols in the inhibition of digestive enzymes has been investigated (22), this is the first report on the inhibitory activity of MT against PL. So, the aim of the present study was to quantify the inhibition of PL in vitro by MT, as well as to determine the kinetic parameters of the inhibition reaction. Furthermore, the preventive effects of the tea on the development of obesity in mice fed a high-fat diet (HFD) were examined.

Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. Acknowledgments
  9. References

Reagents

Human PL (from pancreatic juice, BCR-693), porcine PL (type II, 100–400 U/mg protein using olive oil), L-α-phosphatidylcholine (PC; from egg yolk), phosphatidylserine (from bovine brain), phosphatidylethanolamine (from egg yolk), taurocholic acid sodium salt hydrate, and bovine serum albumin were purchased from Sigma Chemical (St Louis, MO). The enzymatic reagents for the cholesterol and triglyceride determinations were purchased from Roche Diagnostics (Indianapolis, IN). All other chemicals were of analytical grade.

MT preparation

The beverage used in this study was prepared by dissolving 27 g of lyophilized instant MT (obtained from a local market, produced by Leão Junior, Curitiba, Brazil) in 1 l of water using a homogenizer. All the MT aliquots used in this study were from the same batch and contained 348.80 ± 16.35 mg/g of phenolic compounds, determined by the Folin–Ciocalteau method using 5-caffeoylquinic acid as the standard for the calibration curve (y = 4.843x + 0.0149); 5.82 ± 0.17 mg/g of caffeine, 32.25 ± 0.50 mg/g of 5-caffeoylquinic acid, 0.58 ± 0.01 mg/g of caffeic acid, and 3.30 ± 0.35 mg/g of theobromine, as determined by high-performance liquid chromatography.

In vitro enzyme activity assays

The lipase activity was determined by measuring the rate of release of oleic acid from emulsified olive oil (23). The substrate emulsion (5 ml in a 30-ml centrifuge tube) was prepared by ultrasonification of olive oil (56 mmol/l) in a solution containing 9 mmol/l taurocholate, 1 mmol/l PC, 0.1 mmol/l cholesterol, 15 mg of bovine serum albumin/ml, 100 mmol/l NaCl, 2.0 mmol/l Tris/HCl (pH 8.0), and 1 mmol/l CaCl2 (standard assay). After the addition of 750 µl of a MT solution (0.5–5.0 mg/ml) dissolved in 100 mmol/l Tris–HCl (pH 8.0) or not (no MT = control), the assay tube was preincubated for 5 min at 37 °C. The enzyme reaction was started by the addition of 750 µl porcine PL solution containing 0.1 mg/ml of 100 mmol/l Tris–HCl (pH 8.0). After incubation for 30 min at 37 °C, the concentration of free acids in the reaction mixture was measured using oleic acid as the standard. The enzymatic activity was expressed as µmol of oleic acid released per minute of reaction (µmol/min/l). The inhibitory activity of each sample was reported as the relative percentage compared with the control value. For comparative purposes, in one of the experiments, the inhibitory effect of MT was tested not only on porcine lipase, but also on recombinant human lipase. This assay was performed in a manner similar to that described earlier.

Influence of several olive oil emulsifying reagents

The inhibitory activity of MT against porcine lipase was determined using the standard assay described earlier but with other emulsifiers in the reaction mixture. The substrate emulsion was prepared by ultrasonification of olive oil (56 mmol/l) in a solution containing a fixed concentration of each of the emulsifying reagents listed in Table 1.

Table 1. In vitro lipase activity assays using several substrate emulsifying reagents
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Measurement of the kinetic constants

In order to measure the Michaelis–Menten constant, Km, the inhibition constant, Ki, and the Vmax, a series of substrate concentrations (0–100 mmol/l) were tested in the assay system. Each analysis was performed with and without MT. Lineweaver–Burk plots were fitted to determine the mechanism of the effect of MT on the PL activity, using the SigmaPlot software (Aspire Software International, Ashburn, VA). The inhibition constant, Ki was calculated from the following equation:

  • image

where Km, app and Km represent the Km with or without MT, [l] represents the concentration of MT.

Animals and diets

Forty male 6-week-old Swiss strain mice (Sw/Uni) (27.3 ± 0.4 g), free of specific pathogens, were obtained from Multidisciplinary Center of Biological Investigation (State University of Campinas, Campinas, Brazil). The animals were maintained on a 12:12 h artificial light–dark cycle and housed in individual cages. After a random selection, the mice were introduced to the standard diet (SD, n = 10) or HFD (n = 30) for 16 weeks. The compositions of the experimental diets are shown in Table 2. After the first 8 weeks on the HFD, the obesity status was observed and the animals were randomly divided into three subgroups according to the intervention: group 1 (n = 10) received an aqueous extract of MT (1 g/kg of body weight), group 2 (n = 10) received an aqueous extract of MT (2 g/kg), group 3 (control group) (n = 10) received pure water. The three groups were treated for 8 weeks and the solutions were administered by intragastric gavage. The total food intake by each group was recorded at least twice weekly, and the body weight of each mouse was recorded once weekly. At the end of the experiment, the mice were deeply anaesthetized (1:1 xylazine–ketamine) and killed by a transcardiac perfusion with 70 ml isotonic saline solution (4 °C) over a period of 6 min. After 8 weeks of feeding on the diet indicated, the blood and tissues were collected and stored at −80 °C until analyzed. The experiments were performed in accordance with the principles outlined by the Brazilian College for Animal Experimentation and were approved by The Ethics Committee of the São Francisco University, Bragança Paulista, SP, Brazil.

Table 2. Composition of the experimental diets
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Biochemical analysis

The serum was obtained by centrifugation of the blood at 800 g for 10 min and the total cholesterol, triglyceride, and high-density lipoprotein-cholesterol concentrations were immediately determined using an automatic analyzer (COBAS-MIRA System of Roche Diagnostics, Indianapolis, IN). Low-density lipoprotein (LDL)-cholesterol was calculated from the formula: LDL-cholesterol (mg/dl) = total cholesterol − triglyceride/5 − high-density lipoprotein-cholesterol. The lipids were extracted from the fresh liver homogenate using Folch's method (24), and the extracts evaporated in a vacuum and weighed. The total fat content was calculated as an absolute value and as a percentage of the final body weight.

Statistical analysis

The data were expressed as the mean ± s.e.m. Comparisons among the groups of data were carried out using the one-way ANOVA followed by the Dunnett multiple Comparisons test. The statistical significance for the expression of the analysis was also assessed by ANOVA and the differences identified were pinpointed by an unpaired Student's t-test. An associated probability (P value) of <5% was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. Acknowledgments
  9. References

In vitro inhibitory effect of MT on the activity of lipases

The inhibitory action of MT against porcine and human lipases was determined using different concentrations (0.5–5.0 mg/ml) of MT. As shown in Figure 1, MT inhibited the enzyme activities in a dose-dependent way and its inhibitory activity against both lipases reached a maximum at 3.0 mg/ml, corresponding to 9 mg of tea/g substrate (83 ± 2.1% inhibition against porcine lipase and 79 ± 1.3% against human lipase). The inhibitory activity of MT was the same at 3.0 and at 5.0 mg/ml for both lipases (17 ± 3.4 and 21 ± 2.1% inhibition against porcine and human lipases, respectively, at 3.0 mg/ml, and 22 ± 1.9 and 18 ± 2.2% inhibition against porcine and human lipases, respectively, at 5.0 mg/ml, P < 0.05). Under our assay conditions, the half-maximal inhibitory concentration of MT was determined to be 1.5 mg/l (or 4.5 mg of MT/g of substrate).

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Figure 1. Effect of maté tea (MT) on the porcine (circles) and human (squares) lipase activities in a reaction mixture containing olive oil emulsified with taurocholate and phosphatidylcholine. The values are means ± s.e.m., n = 3.

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In addition, the inhibitory activity of MT at 3.0 mg/ml against porcine lipase was determined in the substrate emulsified with different emulsifiers, as described in the Methods and Procedures section. As shown in Figure 2, the MT inhibited the lipase activity in substrates containing taurocholate with PC (A: 83 ± 3.8% inhibition) and PC alone (E: 62 ± 4.3% inhibition), but was partially reduced when incubated with taurocholate alone (D: 48 ± 2.4% inhibition), but not in the presence of the other phospholipids (B, C, F, G: <30% inhibition). When the olive oil was emulsified with gum arabic (H) and polyvinyl alcohol (I), the inhibition was only 10%.

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Figure 2. The effect of several substrate emulsifying reagents on the inhibition of porcine pancreatic lipase activity. The emulsifiers (B–I) were used as described in the Methods and Procedures section, and the lipolytic activity was measured in the reaction mixture containing 3.0 mg/ml of maté tea. The values are means ± s.e.m., n = 3. Different (*P < 0.05, **P < 0.01) from the standard assay (A taurocholate–phosphatidylcholine emulsified olive oil) according to Dunnett's test.

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As shown in Figure 3, the Lineweaver–Burk analysis was carried out in order to characterize the mechanism of the inhibitory effect of MT on the porcine lipase activity. As the MT concentration varied, the value for the y-intercept in the equation (1/V = Vmax = 33.3 µmol/l/min) for each curve remained at a fixed point, which indicated that the inhibition of PL by MT was of a competitive type. The value for Km without MT was 28.6 mmol/l, and with the addition of 1.0 and 3.0 mg/ml of MT, the values shifted to 49.3 and 92.3 mmol/l, respectively. The Ki value was 12.9 mmol/l.

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Figure 3. Lineweaver–Burk plots of the oleic acid released from taurocholate–phosphatidylcholine emulsified olive oil in the presence of various concentrations of maté tea: 0 mg/l (squares), 1.0 mg/ml (open circles), and 3.0 mg/ml (filled circles). The values are means ± s.e.m., n = 3.

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Effects of MT on weight gain, serum parameters, and liver lipids

Figure 4 shows the changes in body weight gain (expressed as % of initial body weight) for the mice fed the HFD with or without MT, and for the mice fed the SD. The mice fed on HFD for 16 weeks showed a significantly higher body weight gain than mice fed on the SD (HFD, 98.7% vs. SD, 53.1%; P < 0.05). In mice fed on HFD and then treated with MT in the last 8 weeks, the body weight gain (HFD + MT 1 g/kg, 78.2% and HFD + MT 2 g/kg, 76.4%) was significantly suppressed as compared to the group fed on HFD with pure water (HFD, 98.7%; P < 0.05). It is interesting that both doses of MT suppressed the weight gain without affecting food consumption. The energy intake per mice differed between the SD and HFD groups throughout the whole experimental period, but it did not differ between the group fed HFD alone and the groups fed HFD plus MT (data not shown).

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Figure 4. The effect of maté tea (MT) on body weight gain in mice fed the experimental diets. Changes in body weight after each treatment period are shown as the percentage of the initial body weight. Open squares, standard diet; filled squares, high-fat diet + pure water; filled circles, high-fat diet + 1 g MT/kg of body weight; and open circles, high-fat diet + 2 g MT/kg of body weight. The values are means ± s.e.m., n = 3.

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As shown in Table 3, the HFD-induced hyperlipidemia, with increases in total serum cholesterol, LDL-cholesterol, and triglyceride as compared to the SD group. The animals fed on HFD and treated for the last 8 weeks with MT (1.0 and 2.0 g/kg of body weight) showed significantly reduced total serum cholesterol and LDL-cholesterol levels as compared to the HFD alone. Serum triglyceride was also significantly reduced (P < 0.05) by the MT containing HFD and the levels obtained were the same as those of the SD rats. Additionally, the HFD doubled the amount of total liver lipids as compared to the SD group. Thus the addition of MT significantly reduced (P < 0.05) lipid accumulation in the liver, as compared to those fed on HFD alone. At these doses, the total liver lipid decreased by ∼30% (P < 0.05).

Table 3. Serum parameters and liver lipids in mice fed experimental diets
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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. Acknowledgments
  9. References

PL inhibition is one of the most widely studied mechanisms for the determination of the potential efficacy of natural products as antiobesity agents (4). In the first set of experiments, the composition of the substrate emulsion containing taurocholate, phospholipid, and cholesterol used in the porcine PL inhibition assay, was selected to mimic in vivo conditions. To determine possible mechanisms involved in PL inhibition by MT, a different substrate emulsifying reagent was used in vitro. In this case, it was observed that the inhibition of the lipolytic activity of PL toward olive oil emulsified with taurocholate-PC by MT was strongly dose dependent. It was thus suggested that the inhibition by MT depended on how the substrate was presented to the lipases, and that the phospholipid species, especially the choline moieties, profoundly affected the lipase inhibitory activity of MT. Because the major bile phospholipid is PC, accounting for >90% of the total amount, MT may be an effective inhibitor in vivo. Furthermore, the concentration of bile salts in the substrate used in this study was estimated to be approximately the same as that occurring in the digestive tract (25).

The kinetic results indicated that MT competitively inhibited PL activity in a concentration-dependent manner with a half-maximal inhibitory concentration value of 1.5 mg MT/ml (or 4.5 mg of MT/g of substrate), whereas a drastic decrease in lipolytic activity (>80% that of the control) was observed in the presence of 3.0 mg MT/ml. The present results were similar to those obtained with other PL inhibitors, such as oolong, green, and black teas (inhibition ratios were 100, 75, and 55% at a concentration of 2.0 g/l, respectively) (26), and superior to those reported for green tea extract (66.5% of inhibition with 60 mg/g triolein) (7).

The lipolytic reaction occurs at the substrate surface (lipid–water interface) and is dependent on surface adsorption of the enzyme. It may therefore be reasonable to consider that the MT compounds efficiently inhibit PL activity by interacting with the emulsified olive oil–TC-PC micelle, adsorbing to the substrate surface and retarding the lipolytic reaction. It is known that high-molecular weight polyphenols (for example, condensed tannins) have a high affinity for proteins, forming insoluble complexes with proteins and other macromolecules. The process is established by the interaction of at least two hydroxyl groups of the polyphenols with the protein (22). Juhel et al. (7) showed that the activities of both gastric and PLs are markedly reduced (96.8 and 66.5% inhibition, respectively) in the presence of green tea extract containing 25% catechins, mainly in the form of epigallocatechin-3-gallate. On the other hand, Han et al. (27) reported that epicatechin gallate and epigallocatechin gallate had no effect on PL activity, but that tea saponin (a mixture of theasaponins E1 and E2) isolated from oolong tea inhibited PL activity in vitro (26). Furthermore, they found that the caffeine isolated from oolong tea was a substance accelerating norepinephrine-induced lipolysis. Recently, Nakai et al. (8) demonstrated that the dimeric compounds of the flavan-3-ol gallate esters from oolong tea, such as proanthocyanidins, oolonghomobisflavans, and theasinensins, were more active in the inhibition of PL than epigallocatechin gallate, and that the presence of galloyl moieties within the structure was required to enhance PL inhibition. This does not rule out that other components present in MT might be implicated in some inhibitory effect. The main polyphenols in green yerba maté are the caffeoylquinic acids or dicaffeoylquinic acids, generically known as chlorogenic acids, esters from caffeic acid and quinic acid. In MT, besides the aforementioned chlorogenic acids, caffeoylshikimic acid and dicaffeoylshikimic acid, which contain one less water molecule than their analogous caffeoyl- and dicaffeoylquinic acids, are also found (28). In this study, no relationship between the potency of PL inhibition and the active constituents in MT could be established.

The effect of MT on obesity was tested using mice fed a HFD containing 1 and 2 g/kg of MT. The MT prevented the HFD-induced increases in body weight (Figure 4) and decreased the serum triglyceride, cholesterol, and LDL-cholesterol concentrations after they had been increased by HFD (Table 3). These effects did not depend on decreased food or energy intakes because there were no significant differences between the HFD and HFD plus MT groups. The results in animals did not show a dose–response effect probably because we were working with higher doses which reach the maximum effects. Another possibility that could be considered is a pharmacokinetic alteration. A portion of the administered polyphenol might be absorbed from the intestine, metabolized, and excreted into urine. Probably, higher dose (>1.0 g/kg) of MT polyphenol might be excreted in the feces without absorption, but the mechanism for this remains to be elucidated.

These results suggest that MT has an antiobesity function: it prevents the hydrolysis of dietary fat in the small intestine and reduces the subsequent intestinal absorption of dietary fat. The hypolipidemic effects of the I. paraguariensis extract shown in this study were in agreement with previous reports on rats fed a hypercholesterolemic diet (12,18). Paganini Stein et al. (12) showed that rats fed the hypercholesterolemic diet for 30 days and which were treated for the last 15 days with I. paraguariensis extract (500 mg/kg) showed significantly reduced serum cholesterol (30% reduction) and triglyceride (60.4% reduction) levels as compared to those fed the hypercholesterolemic diet alone. Similar results was recently reported by Pang et al. (18) suggesting that the I. paraguariensis extract might have a protective effect against HFD-induced obesity in rats through an enhanced expression of uncoupling proteins and elevated AMPK phosphorylation in the visceral adipose tissue.

In conclusion, this investigation demonstrated that MT efficiently inhibited in vitro porcine and human PL activities. Additionally, it showed that the treatment had potent antiobesity activity on HFD-induced obese mice. These results suggest that MT is able to suppress dietary fat absorption from the small intestine of mice by inhibiting pancreatic activity.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. Acknowledgments
  9. References

We are grateful to the Brazilian Research Funding Agency (FINEP) and to Leão Junior S/A for their financial support.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. Acknowledgments
  9. References