herbs and plant extracts that offer similar benefits without the side effects.

Tea from the plant Camellia sinensis is one of the most popular beverages consumed worldwide [5]. Tea is rich in antioxidant polyphenolic flavonoids (catechins, flavonols, theaflavins, and thearubigins) that possesses various phar- macologic effects such as antihypertensive, antiarteriosclero- tic, hypoglycemic, and hypocholesterolemic activities [5-8]. Green tea polyphenols are composed of numerous types of

catechins. The major catechins present in green tea are (−)–

epigallocatechin 3-O-gallate (EGCG), (−)–epicatechin 3-O-

gallate (ECG), (−)–epigallocatechin (EGC), and (−)-epica-

techin. Among them, EGCG is primarily responsible for the

beneficial effect of green tea [5-8]. There is some evidence for the possible preventive role of green tea and its active components in CVD and other diet-dependent diseases [1,2,5,8,9]. Green tea reduces adipose tissue weight in animal models of obesity [7] and has a pronounced effect on lipid metabolism in hyperlipidemia models [10]. The visceral weight loss and lipid profile that improve the effect of green tea in rats may have been due to an enhancing thermogenesis and fat oxidation [11], inhibiting gastric and pancreatic lipases [12], or suppressing appetite and reduction in food intake [13]. The health-promoting effects of green tea have been mainly attributed to the catechins content. It is well- known that those components are better extracted by hydroalcoholic solvents, and therefore, hydroalcoholic green tea extract has been used in most human and animal studies [14]. However, a possible hepatotoxicity due to the lipophilic green tea components extracted by hydroalcoholic solvents has been mentioned by Bun et al [15]. In addition, sporadic cases of liver disorders (acute hepatitis or hepatocellular necrosis) after the ingestion of green tea supplements based on hydroalcoholic extracts have been indicated in other reports (13 cases of hepatic attack have been described among patients receiving such extracts) [16]. Due to the possible hepatotoxicity effect of extracts obtained by hydroalcoholic solvent, green tea aqueous extracts (GTAEs) have been recognized as more desirable from the perspective of health safety. On the other hand, the hepatotoxicity of green tea extract can also be attributed to EGCG or its metabolites, which are polar substances soluble both in water and hydroalcoholic solvents [14]. However, an adequate dose of EGCG might also promote a decrease in body weight, body fat, energy absorption, and plasma lipid concentrations. We hypothesize that GTAE depending on dose improves cardiovascular risk indicators (body weight, visceral fat content, and atherogenic index [AI] of plasma) and does not have unfavorable effect on protein availability. Accordingly, in rats fed with a high-fat (HF) diet, we evaluated the effect of ingesting 1.1% and 2.0% GTAE on body  weight,  visceral  fat  contents,  lipids  profile,  and a

 

1. Methods and materials

 

1. Animals and treatments

Eighteen 11-week-old male Wistar rats with initial body weights ranging from 266 to 292 g were obtained from the Department of Toxicology at the Medical University of Poznan, Poland. The animals were kept in individual cages in a temperature controlled room at 22° ± 2°C, with 55% to 60% humidity and a 12-h dark/12-h light cycle. Rats were fed with chow diet (Labofeed B), purchased from Labofeed company (Andrzej Morawski Feed Production Plant, Kcynia near Bydgoszcz, Poland), and with tap water ad libitum for 1 week before the experiment. All animals were humanely treated in accordance with the World Health Organization’s guideline for animal care, and the animal study design was approved by the Local Bioethical Committee on Animal Research at the Department Animal Physiology and Biochemistry, Poznan University of Life Sciences (July 2008).

After 7 days of adaptation to laboratory conditions, the experimental animals were randomly divided into 3 groups of 6 each. There was no difference in body weight among the groups at the beginning of the experiment. One of the groups received a control HF diet (approximately 50% energy from fat) (Table 1), and the other 2 groups received the same HF diet enriched with 1.1% and 2.0% of GTAE for a total of 56 days. The sources of fat in the rat’s diet were sunflower oil (10%) and lard (20%) (Table 2). Each ingredient in the diet was weighed (within 0.5% of the amount needed) and mixed. The mixture was then formed into equally sized pellets and placed into a temperature- and humidity- controlled room to remove the moisture from the pellets. The rats received water and pellets ad libitum. Food consumption (of individual rats) was checked every day, from differences between amounts of the diet supply each day and amounts of the remaining diet. The body weight was measured every 7 days using an electronic balance. Food efficiency ratio (FER) was determined as follows: body weight gain (gram)/food intake (gram).

 

1. Preparation  of  GTAE

The GTAE was prepared according to the method presented by Gramza and Regula [18] from the Japanese Sencha Fukuju Green Tea, which was bought at a specialty store (The House of Tea). The tea leaves (100 g) were ground and then boiled in double-distilled water (1000 mL), followed by stirring for 15 minutes at 70°C (the procedure

 

Table 1

Macronutrient content of the diets

Macronutrient composition         Control        1.1% GTAE      2.0% GTAE

AIN 93 mineral mix c

GTAE d

 

35.0            35.0                    35.0

0               11                       20

 

diet, its moisture content was measured [22]. To determine the apparent protein digestibility, 1-g samples of diet and

Modified from the AIN-93G diet [17].

1. Individual ingredients of the diet are as follows: caseine (Lacpol Ltd, Gdynia, Poland); wheat starch (Hortimex Plus Ltd, Konin, Poland); L-cysteine (Sigma Chemical Co, St Louis, Mo); and sucrose, sunflower oil, lard, and potato starch were purchased from a local supermarket.
2. Mineral mix contains the following components (per gram  mineral

mix): sodium chloride, 259 mg; magnesium oxide, 41.9 mg; magnesium sulfate, 257.6 mg; chromium potassium sulfate, 1.925 mg; cupric carbonate,

1.05 mg; sodium fluoride, 0.2 mg; potassium iodate, 0.035 mg; ferric citrate,

21 mg; manganous carbonate, 12.25 mg; ammonium molybdate, 0.3 mg; sodium selenite, 0.035 mg; zinc carbonate, 5.6 mg (individual ingredients of the mineral mix were bought in Sigma Chemical Co).

1. Vitamin mix contains the following components (per gram  vitamin

mix): retinyl acetate, 0.8 mg; cholecalciferol, 1.0 mg; DL-α-tocopheryl acetate, 10.0 mg; menadione sodium bisulfite, 0.05 mg; biotin, 0.02 mg; cyanocobalamin, 1 mg; folic acid, 0.2 mg; nicotinic acid 3 mg; calcium pantothenate, 1.6 mg; pyridoxine-HCl, 0.7 mg; riboflavin, 0.6 mg; thiamin HCl, 0.6 mg (individual ingredients of the mineral mix were bought in Sigma Chemical Co).

1. Doses of 1.1% and 2.0% GTAE given to the diet correspond to an

intake in humans of 5 and 8 cups of green tea per day, respectively.

 

was repeated 3 times). Collected extracts were centrifuged after filtration (2700g−1, 15 minutes) and then lyophilized under vacuum (Multi Branch Trade & Manufacturing Company “Elena,” Zelazkow, Poland). One gram of GTAE dry matter contained 7.0% EGCG, 4.1% EGC, and 1.8%   ECG,   as   determined   by   high-performance   liquid

chromatography (HPLC). The HPLC analyses of green tea catechin contents were performed on a Waters Alliance HPLC System 2695 (Milford, Mass, USA) equipped with an X-Terra RP18 5 μm column (Milford) according to the method described by Andrade et al [19], with slight modifications by the authors. The 1.1% and 2.0% levels of GTAE in the diet were equivalent to human intakes of 5 and 8 cups of green tea beverage (2.0 g green tea [GT] in 200 mL of water) per day, respectively, as estimated based on an energy intake of 8374 kJ per day [20,21].

 

1. Collection of blood and internal organ samples

At the end of the test (day 56 of the experiment) and after 16 hours of starvation, the  animals  were  weighed and euthanized by intraperitoneal injection of thiopental (40 mg/kg body weight). They were dissected to collect blood for biochemical studies and to harvest and clean their internal organs (eg, liver, spleen, and visceral fat) for weight

 

feces were analyzed for nitrogen content using the Kjeldahl method (%N × 6.25) [23]. The Kjeltec 1026 autoanalyzer (Foss Tecator, Höganäs, Sweden) was used. Apparent protein digestion were calculated using the following equation: [(N of the ingested food − N of the feces)/(N of the ingested food)] ×100.

Fat content (in feces and the rat’s diets) was determined using Avanti Soxtec System (Model: 2055 Manual Extraction Unit; Foss Tecator, Höganäs, Sweden)  [24]. The apparent fat digestion was calculated using the following equation: [(lipid of the ingested food − lipid of the feces)/(lipid of the ingested food)] × 100. The energy value of diet and feces was assessed by bomb calorimetry

(Calorymetr   KL-11   Microprocessor   System   “Mikado”,

Precyzja Company, Bydgoszcz, Poland).

1. Plasma lipids profile analyses

 

Total cholesterol, HDL-C, and TAG concentrations were determined by colorimetric methods [25,26] using Olympus AU 560 equipment (Olympus Japan, Tokyo, Japan). The low-density lipoprotein cholesterol (LDL-C) and AI were calculated using the following formulas:

AI = ðTC − HDL−CÞ = HDL−C [27]; and LDL−C = TC − HDL−C −ðTAG =5Þ [28].

1. Statistical analyses

 

Each datum was presented as the mean ± SD. A power analysis indicated that a sample size of 6 rats per group would be suitable for detecting a difference between the 3 groups for most analyzed variables:

 

1. body weight from 0 to 8 weeks of treatment (gram) (β = 50%, α = 5%);

• FER (β = 60%, α = 5%);

1. energy value of feces (kJ/g) (β = 95%, α = 5%);
2. visceral fat contents (gram) (β = 50%, α = 5%);

• AI plasma (β = 91%, α = 5%);

1. apparent digestion of protein (%) (β = 80%, α = 5%);
2. apparent digestion of fat (%) (β = 91%, α = 5%);
3. liver weight/body weight ratio (β = 70%, α = 5%).

Difference between groups’ means was compared using 1-way analysis of variance (ANOVA). If a significant F ratio

was obtained, Tukey’s HSD (Honestly Significant Differ- ence) was used to locate differences between means. Differences in body weight over time (from 0 to 8 weeks) and between the treatments (Control, 1.1% GTAE and 2.0% GTAE) were determined using ANOVA with repeated measures model. If a significant F ratio was obtained, Tukey’s HSD was used to locate differences between means. Differences were considered significant at P b .05. All data were analyzed using Statistica Software (version 8.0; Statsoft Inc., Tulsa, Okla, USA).

 

1. Results

1. Food intake, FER, apparent digestion of protein and fat, body weight, and visceral fat content

 

The food intake, FER, apparent digestion of protein and fat, energy value of feces, visceral fat contents of the rats fed with the HF diet, and the same diet enriched with 1.1% or 2.2% of GTAE are presented in Table 3.

There were no significant differences in daily food intake between the groups, whereas FER representing body weight gain relative to food intake was significantly lower in the 2.0% GTAE group compared to the control (0.106 ± 0.008 vs

0.123 ± 0.012, respectively). A significant (P b .05) reduction in the apparent digestion of protein was observed in both HF diets enriched with GTAE (1.1% GTAE: 82.6 ± 1.8%; 2.0% GTAE: 84.3 ± 0.8%) when compared to the control group

(93.3 ± 1.5%), whereas the apparent digestion of fat was unchanged. The energy content of feces from both groups supplemented with GTAE was considerably higher (1.1% GTAE: 19.7 ± 1.3 kJ/g; 2.0% GTAE: 19.3 ± 1.2 kJ/g) in

comparison with the control group (14.7 ± 2.2 kJ/g) (Table 3).

By week 8, the administration of the HF diet induced body weight gain in all experimental groups of rats (Fig. 1), but only addition of 2.0% GTAE to the diet a significant (P b

.05) reduced the body weight gain by 5.6% of the rats in comparison to the control group. A tendency for a difference in body weight was observed on weeks 5 to 8 of the study (P b .05, respectively) (Fig. 1). The smaller body weight gain

 

observed in the rats receiving the 2.0% GTAE was also reflected by significantly (P b .05) lowered visceral fat contents compared to the control group (2.0% GTAE: 3.7 ±

0.8 g per 100 g vs Control: 4.5 ± 0.2 g per 100 g) (Table 3).

1. Plasma biochemistry

 

The lipid profiles and AI of plasma of the rats on HF diet and the same diets supplemented with 1.1% and 2.2% of GTAE are shown in Table 4. The AI of plasma was not significantly different between the 2 GTAE groups (0.6 ± 0.06), but both had significantly lower (P b .05) values than the control group (0.7 ± 0.03). The lower AI observed in both GTAE groups was achieved in 2 different ways: in the 2.0% GTAE group, by lowering the LDL-C level (27.6 ±

2.5 mg/dL); and in the 1.1% GTAE group, by elevating the HDL-C level (63.3 ± 6.1 mg/dL). The TAG concentrations were not significantly different between the groups.

1. Organ weights

 

The liver weight/body weight ratio was similar in all the experimental groups; the only significant (P b .05) elevation (12.0%) in this ratio for the 1.1% GTAE group compared to the other was observed. There were no significant differ- ences in the spleen weight/body weight ratios between all the groups (Table 5).

 

1. Discussion

According to Kris-Etherton et al [29], regular consump- tion of a diet rich in active compounds (ie, flavonoids) has been shown to protect against CVD and metabolic disease. Green tea is a functional food that may have beneficial health effects in ameliorating these diseases [30-32].

Green tea contains an abundance of naturally occurring

polyphenols called catechins, of which EGCG is the most prevalent [6]. We manufactured GTAE in which the concentrations of EGCG, EGC, and ECG were 7.0%, 4.1%, and 1.8%, respectively, of  the dry weight  of the powder. The 3 major catechin concentrations were similar to

 

Table 3

Daily food intake, FER, apparent digestion of protein and fat, energy value of feces, and visceral fat content of treatment and control groups

	Control	1.1% GTAE	2.0% GTAE
Food intake (g/d) FER aApparent digestion of protein (%)	14.9 ± 0.60.123 ± 0.012B93.3 ± 1.5B	14.5 ± 0.50.116 ± 0.013AB82.6 ± 1.8A	14.5 ± 0.70.106 ± 0.008A84.3 ± 0.8A
Apparent digestion of fat (%)Energy value of feces (kJ/g)Visceral fat content (g per 100 g body weight (BW))	99.2 ± 0.1 14.7 ± 2.2A 4.5 ± 0.3B	98.8 ± 0.119.7 ± 1.3B4.0 ± 0.8AB	99.1 ± 0.119.3 ± 1.2B3.7 ± 0.8A

Rats were fed HF diets (approximately 50% energy from fat)—control HF diet (control) and HF diets enriched in 1.1 and 2.0% GTAE daily for 8 weeks—1.1% GTAE and 2.0% GTAE groups, respectively.

Each value in the table represents means ± SD of 6 rats.

Comparisons were made using 1-way ANOVA analysis with Tukey post hoc tests. Means in the column with different superscripts are significantly different (P b .05).

a  Body weight gain (gram) per food intake (gram).

those presented by Goto et al [33], where the EGCG, EGC, and ECG contents ranged (depending on the grade of the Sencha teas) from 7.9% to 9.3%, 4.0% to 4.3%, and 1.6% to 1.7%, respectively. Based on the studies presented by Bose et al [20] and Shrestha et al [21], GTAE intakes of 1.1% and 2.0% in rats are equivalent to human intakes of 5 and 8 cups of green tea (2.0 g GTAE in 200 mL of water) per day, respectively, as estimated based on a daily energy intake of 8374 kJ.

 

In the current study, supplementation with both 1.1% and 2.0% GTAE (where EGCG content was 0.8 and 1.4 g/kg diet) reduced body weight gain in rats fed with an HF diet (approximately 50% energy from fat), but this effect was dose dependent. After 8 weeks of experiment, a significantly lower body weight gain (5.6%) was observed for the 2.0% GTAE group compared to the control group. These differences were observed as early as 5 weeks into the feeding period.

Table 4

Plasma biochemistry of the treatment and control groups

 

 

Table 5

Organ weights of rats fed the control and treatment diets

                                                                                                                                                                                                                                              

Control                  1.1% GTAE          2.0% GTAE

 

TAG (mg/dL)             32.8 ± 6.7                 36.5 ± 5.1               32.8 ± 8.6 AI a                                     0.7 ± 0.03B                                   0.6 ± 0.06A                                  0.6 ± 0.06A

Rats were fed HF diets (approximately 50% energy from fat)—control HF diet (control) and HF diets enriched in 1.1 and 2.0% GTAE daily for 8 weeks

 

Liver weight/body weight ratio

Spleen

weight/body weight ratio

 

0.025 ± 0.001A            0.028 ± 0.003B             0.025 ± 0.002A

0.0016 ± 0.0001   0.0015 ± 0.0001   0.0015 ± 0.0001

—1.1% GTAE and 2.0% GTAE groups, respectively.

Total cholesterol, HDL-C, and TAG levels were determined by colorimetric

methods after the 8-week feeding period.

Each value in the table represents means ± SD of 6 rats.

Comparisons were made using 1-way ANOVA analysis with Tukey post hoc tests.

Means in the column with different superscripts are significantly different (P b .05).

a  Atherogenic index of plasma was calculated as follows: AI = (TC −

HDL-C)/ high-density lipoprotein −control HDL-C.

 

Rats were fed HF diets (approximately 50% energy from fat)—control HF

diet (control) and HF diets enriched in 1.1 and 2.0% GTAE daily for 8 weeks

—1.1% GTAE and 2.0% GTAE groups, respectively.

Rats after the 8-week feeding period were killed, and liver and spleen were

harvested and weighed.

Each value in the table represents means ± SD of 6 rats.

Comparisons were made using 1-way ANOVA analysis with Tukey post hoc tests.

Means in the column with different superscripts are significantly different (P b .05).

We found that 8-week 2.0% GTAE treatment also significantly decreased visceral fat content (17.8%) in comparison to control rats fed with an HF diet.

According to study obtained by Bose et al [20] in which a 16-week dietary EGCG treatment (3.2 g EGCG/kg diet) significantly decreased body weight gain (33%-41%) and fat accumulation in mice fed with an HF diet (60% energy from fat), it is plausible that the reduced body weight gain may be because of the presence of EGCG. Considering that, in our study, the dose of approximately 1.4 g EGCG per kilogram diet was almost one-half smaller and the treatment time was twice shorter (8 weeks), it is clear that differences in the reduction of body weight gain in both studies are proportional to dose, regardless of the animal model.

In addition, in another animal obesity model study [34,35], the decrease in body fat accumulation was observed after an even shorter time (29 days). This decrease occurred without a change in food intake but with a significant reduction in food digestion. The EGCG dose was much higher (5- and 10 g EGCG/kg diet) than that in our study and the study conducted by Bose et al [20]. The authors reported that doses of 5 g EGCG/kg diet reduced body fat gain by 3.7 g and that the additional reduction with the 10 g EGCG/kg diet was only 1.7 g. These findings indicate that supplementation of a rat’s diet with 5-g EGCG/kg diet already achieves close to the maximum obtainable effect.

In our study, the effect of lower body weight gain (5.6%) in the supplemented groups was observed despite no changes observed in food intake in the 3 groups of rats. Although our study was underpowered to see small significant differences between groups, it was calculated that rats from both GTAE groups consumed 2.8% (500 kJ/8 wk) less food than rats from control group. It seems that it had a minor importance for observed differences in body weight gain. In the experiment by Kao et al [13], male Sprague-Dawley rats, given EGCG orally, consumed approximately 15% less food than did the control rats and similarly lost 5% of their initial body weight. Results of both studies indicated that different mechanisms are involved in reduction of body weight gain during GTAE ingestion.

However, feces energy content significantly increased

(34.3% and 31.4%) in both GTAE-enriched groups in comparison with control, indicating reduced digestion of food and decreased long-term energy absorption. The results revealed that the higher energy value of feces observed in the 1.1% and 2.0% GTAE groups resulted from a reduction in the apparent digestion of protein but not of fat. This finding agrees with the results obtained by Unno et al [35] and Onishi et al [36]. The first of these studies demonstrated that the apparent digestion of protein was 95.8% for controls and 89.3% for rats receiving a diet with 1.0% tea catechins, mostly as gallate forms. In the second study, even doses of green tea polyphenols as small as 0.2% and 0.4% (green tea extract (GTE) used by Onishi et al were 5 times lesser than those used in our study) decreased both protein digestion in

 

the small intestine and the activity of microflora in the large intestine of rats. Unno et al [35] explained that phenolic group of catechins binding to proteins through hydrophobic hydrogen bonds and creating catechin-protein complexes could limit the access of proteolytic enzymes (mainly pepsin and trypsin) to the substrate.

According to our results  and results  obtained by the aforementioned authors, it is interesting if a higher dose of EGCG than 1.4 g/kg diet could reduce, in a larger extent, the apparent digestion of protein. If this is the case, excessive consumption of green tea extract could be unfavorable, especially for undernourished individuals.

Some epidemiological studies have suggested that drinking between 5 and 10 cups of green tea per day is associated with lower plasma cholesterol concentrations [37,38]. In animal models of HF diets, dramatic increases in serum low-density lipoprotein, TAGs, and TC levels but relatively slight changes in high-density lipoprotein were observed [10]. However, Tijburg et al [39] found that GTE included in the drinking water did not significantly decrease plasma TC levels in the cholesterol-fed hypercholesterol- emic rabbits. In the present study, the administration of GTAE at the level of 2 g per 100 g of HF diet significantly reduced total and plasma LDL-C concentrations in compar- ison with the control and 1.1% GTAE diets. However, the 1.1% GTAE diet increased TC because of the increased plasma HDL-C. This finding is in conflict with the cholesterol-lowering effect of GTE (15 g/L or 130 mg powdered green tea per day) seen in normal male animals fed with an HF (200 g of lard per kilogram and 1 g of cholesterol per kilogram of diet) or high-sucrose (50% sucrose diet containing 15% butter) diet [40,41]. Consistent with our findings, however, Shrestha et al [21] reported the TC and HDL-C raising effect of a high fructose diet with 1.0% GTE. The investigators indicated that the mechanism of this effect appears unrelated to changes in hepatic expression of scavenger receptor class B type 1 (SR-B1), a protein present in hepatocytes that takes cholesterol from high-density lipoprotein, or adenosine 5c-triphosphate (ATP) -binding cassette transporter A1 (ABCA1), a protein that mediates cholesterol uptake and transport, and is, therefore, difficult to explain [21]. Despite this finding, a significantly lower AI was observed in both the 1.1% and 2.0% GTAE groups. These observations probably indicate that there are different mechanisms to achieve the beneficial effects on plasma lipid modulation for lower and higher doses of GTAE. In addition,

significantly  higher  liver  weight–to–body  weight  ratio

(12.0%) was observed in 1.1%GTAE rats in comparison

with other 2 groups. This mild liver hypertrophy could be observed not only in the abnormal condition but also in the case of reverse cholesterol transport.

It was concluded that GTAE may have preventive effects on the accumulation of visceral fat but only in higher doses. Although both doses improved cardiovascular risk indica- tors, they, in addition, inhibited protein digestion. The metabolic abnormality, expressed as increased liver weight, was associated with the lower dose of GTAE only. For that reason, results obtained in our study indicate on some difficulties in reconciling of high effectiveness in prevention of cardiovascular risk factors with low influence on dietary protein digestion.

 

Acknowledgment

The authors thank Malgorzata Tubacka for her assistance during animal study and Elsevier Language Editing Services for editorial assistance. This research was supported by the Polish State Committee for Scientific Research (NN312331635).

 

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