Single dose of green tea extract decreases lipid digestion and absorption from a test meal in humans *

1Department of Pediatric Gastroenterology & Metabolic Diseases, Poznan University of Medical Sciences, 60-572 Poznań, Poland; 2Department of Human Nutrition and Hygiene, Poznan University of Life Sciences, Poznań, Poland; 3Department of Internal Diseases, Metabolism and Dietetics, Poznan University of Medical Sciences, Poznań, Poland; 4Department of Neonatology, Poznan University of Medical Sciences, Poznań, Poland


IntRoduCtIon
Green tea is known worldwide for its high content of polyphenolic compounds and multifactorial beneficial effects on human health.Green tea contains a broad range of catechins: (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin gallate (EGCG) (Raederstorff et al., 2003;Lee et al., 2009;Lee et al., 2009;Tanaka et al., 2010;Kim et al., 2011).EGCG is the most abundant (50% of the total amount of catechins in green tea) and possessing the most potent antioxidant activity catechin (Collins et al., 2007;Lee et al., 2009).Green tea and its components show various biological and pharmacological effects like antibacterial, anti-inflammatory and anti-carcinogenic activities (Kim et al., 2009;Shrestha et al., 2009;Huang et al., 2011).Numerous studies conducted in humans have revealed that green tea extract (GTE) decreases weight and body fat gain (Mochizuki & Hasegawa, 2004).Lee et al. (2009) showed that green tea EGCG inhibits lipid accumulation in cultured adipocytes via stimulation of lipolysis.It is believed that green tea increases energy ex-penditure and for this reason it may have application in a supportive treatment of obesity (Mochizuki & Hasegawa, 2004;Moon & Lee, 2007;Bose & Lambert, 2008;Lee et al., 2009;Lee et al., 2009).The role of green tea as an inhibitor of lipid hydrolysis is widely discussed.However, there are no data evaluating impact of pure GTE on lipid digestion and absorption in humans.The aim of the present study was to assess the influence of GTE on lipid digestion and absorption.

MAteRIAl And MethodS
The study comprised 32 healthy volunteers aged 23 to 30 years (Table 1) with normal exocrine pancreatic function (fecal elastase-1 concentration > 200 μg/g) (Walkowiak et al., 2005).Exclusion criteria included: antibiotic therapy within preceding month, acute or chronic diarrhea, celiac disease, pancreatitis, severe systemic disease.In all subjects, 13 C-labelled mixed triglyceride breath test ( 13 C MTG-BT) was performed twice in a random order, with and without GTE ingestion (GTE and placebo tests). 13C MTG-BT was performed after overnight fasting.Each subject received 150 mg of 13 C mixed triglyceride with 12.5 g butter mixed on a slice of bread and GTE (4 g; EGCG content -257.6 mg, caffeine content 136 mg) obtained as described below or placebo.Breath samples were collected at baseline (fasting) and at 30,60,90,120,150,180,210,240,270,300,330 and 360 minutes after test meal ingestion.The samples were analyzed with an IRIS 13 C-Analyser System (Wagner, Bremen, Germany).Cumulative percentage dose recovery (CPDR) was considered to reflect digestion and absorption of lipids, values lower than 13% were considered to be abnormal (Lisowska et al., 2011).
The green tea extract was prepared according to the method described by Bajerska et al. (2011) 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 was repeated 3 times).Collected extracts were filtered through filter paper, centrifuged (at 2700 × g, for 15 min), frozen and lyophilized under a vacuum (Multi Branch Trade & Manufacturing Company "Elena," Zelazkow, Poland).
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. (2002) with slight modifications by the authors.
Results are expressed as medians and 1st-3rd quartile distribution.The statistical significance of differences in CPDR between GTE and placebo tests was determined with the use of Wilcoxon-rank test.The level of significance was set at p<0.05.Statistical analysis was performed using STATISTICA 8.0.(StatSoft Inc. 2008).The protocol of the investigation was approved by the Bioethical Committee of Poznan University of Medical Sciences, Poland.

ReSultS
In all subjects, CPDR values were normal in a placebo test (36.8%<30.1-43.3%>).These results were significantly higher (p=0.021)than those obtained in GTE test (28.8%<23.1-37.2%>).A tendency towards increasing differences between the two tests was observed throughout whole test, reaching a statistical significance at 300 minutes (Table 2).Interestingly, in six subjects the CPDR values in the test with GTE consumption were abnormal (range 5.8-12.0%).No correlation was found between the effect of GTE ingestion and demographic data (sex, age, BMI).

dISCuSSIon
In the present study, a single dose of GTE taken with a test meal decreased lipid digestion and absorption.Based on the information that the mean EGCG content of a single cup (200 mL) of tea prepared with 2 g of green tea leaves ranges between 4.62 mg and 406.4 mg (Bhagwat et al., 2011), dose of green tea extract (4 g) used in our study was equivalent to intakes at least several cups of green tea and, in some cases, more than 10 cups per day.To the best of our knowledge, this is the first study evaluating the impact of pure GTE on lipid digestion and absorption in humans.Currently, the role of green tea as an inhibitor of lipid hydrolysis is widely discussed.A study conducted on mice provided evidence that EGCG increases fecal excretion of lipids supporting the claim that EGCG decreases lipid digestion and absorption (Raederstorff et al., 2003;Unno et al., 2005;Moon & Lee, 2007;Bose & Lambert, 2008;Kim et al., 2011;2012;Sae-tan et al., 2011).A study on rats with inserted mesenteric lymph cannulas has revealed that green tea inhibits the intestinal absorption of dietary li-pids, including triacylglycerol, cholesterol and lipophylic compounds like α-tocopherol (Raederstorff et al., 2003;Unno et al., 2005;Koo & Noh, 2007;Shrestha et al., 2009;Rains et al., 2011).EGCG has been suggested to regulate various enzymes related to lipid metabolism, such as pancreatic and gastric lipase (Juhel et al., 2000;Raederstorff et al., 2003;Koo & Noh, 2007;Moon & Lee, 2007;Rains et al., 2011).Some authors attribute the  U n c o r r e c t e d P a p e r i n P r e s s Green tea and lipid digestion and absorption competence to inhibit pancreatic lipase activity in vitro to tea saponins and not catechins (Han et al., 1999).Gondoin et al. (2010) have shown that strictinin (a tea leaf catechin fraction) has the strongest inhibitory effect on pancreatic lipase activity in vitro.In a study conducted on rats, Ikeda et al. (2005) proved that GTE containing 59.6 g of catechins per 100 g of dry weight dosedependently inhibits pancreatic lipase activity.In that study rats were cannulated in the thoracic duct.On the other hand, Zhong et al. (2006) provided evidence that a beverage containing 0.1 g black, 0.1 g green and 0.1 g mulberry teas caused malabsorption of carbohydrates but did not affect triacylglycerol absorption as evaluated with the use of 13 C MTBT.In contrast to the latter study, we assessed the influence of pure GTE on lipid absorption and digestion.Some authors claim that the inhibition of lipid absorption stems from the EGCG ability to form complexes with lipids and lipolytic enzymes by interfering with emulsification and micellar solubilization of lipids (Lee et al., 2009;Chan et al., 2011;Kim et al., 2012).EGCG may influence the size of lipid droplets and thereby prevent efficient emulsification.It has been shown that a larger fat droplet size and reduced surface area impede fat digestion by digestive enzymes (Unno et al., 2005).The results obtained in the present study support the concept that pure GTE inhibits lipid digestion and absorption.However, the mechanism responsible for this action in humans remains to be elucidated.

ConCluSIonS
A single dose of GTE taken with a test meal decreases lipid digestion and absorption.