To assess possibility of polyphenol-enriched oolong tea to reduce dietary lipid absorption in humans.
Twelve healthy adult subjects, three males and nine females, aged (mean±s.d.) 22.0±1.8 years, respectively, were randomly divided into two groups. The participants were followed a double-blind placebo-controlled crossover design, including 7-day washout periods and 10-day treatment periods. During the treatment periods, subjects were given about 38 g of lipids from potato chips (19 g each within 30 min after lunch and dinner) and total 750 ml beverages (placebo- or polyphenol-enriched oolong tea) at three meals. Blood samples were collected for biochemical examination at days 8, 18, 25 and 35 of the study period. On the last 3 days of each treatment period, feces were collected to measure the excretion of lipids.
Lipid excretion into feces was significantly higher in the polyphenol-enriched oolong tea period (19.3±12.9 g/3day) than in the placebo period (9.4±7.3 g/3day) (P<0.01). Cholesterol excretion tended to increase in polyphenol-enriched oolong tea period (1.8±1.2 g/3day) compared with that of placebo (1.2±0.6 g/3day) (P=0.056).
The results of this study indicated that polyphenol-enriched oolong tea could increase lipid excretion into feces when subjects took high-lipid diet.
Obesity is one of the most important risk factors for various diseases in modern society. Excess lipid ingestion is the most common factor of obesity. For the affiliation between dietary lipid and obesity, and even for cardiovascular diseases, reduction of lipid intake is a logical nutritional recourse.
Dietary lipid consumption is usually high in many people. The digestion of lipids by pancreatic lipase is an essential step to absorb dietary lipids. Studies in mice suggested that inhibition of digestive lipase activity could significantly affect dietary lipid absorption and could increase lipid excretion into the feces (Carriere et al., 2001; Huggins et al., 2003). Orlistat (Xencial; Hoffmann-La Roche, Basel, Switzerland) is presently used as a curative medicine effective in the treatment of obesity by inhibiting lipase (Hartmann et al., 1993; Hussain et al., 1994; Zhi et al., 1994; Guerciolini et al., 2001), although, the use of the drug is recommended only within the serious obesity patients.
Several studies have shown that tea has many kind of health benefits in humans (Bell and Goodrick, 2002; Weisburger, 2002). Among them, oolong tea has been studied for its antihypertensive effect (Yang and Koo, 2000), antioxidant properties (Kuroda and Hara, 1999; Siddiqui et al., 2004), effect on reduction of cardiovascular disease risk (Yang and Koo, 1997) and antiobesitic properties (Han et al., 1999; Rumpler et al., 2001; Komatsu et al., 2003). We have previously shown that oolong tea has antihyperglycemic effect (Hosoda et al., 2003). So far, one of the mechanisms of antiobesitic effect of oolong tea is thought to be increased energy expenditure (Han et al., 1999; Rumpler et al., 2001; Komatsu et al., 2003).
Another important profile of oolong tea for antiobestic effect is inhibition of lipase activity, which is essential for lipid absorption (Han et al., 1999, 2001). Oolong tea polymerized-polyphenols (OTPP) are produced by semifermentation and heating process, which is thought to be one of the major moieties of oolong tea. The inhibitory activity of OTPP on lipase was stronger than that of the total extracts from oolong tea or green tea in vitro (Nakai et al., 2005b). By the further analysis on structure–activity relationship of purified compounds from OTPP fraction against the inhibitory activities of lipase, highly active compounds have galloyl-ester moieties, and the inhibitory activity of OTPP was significantly decreased by the elimination of galloyl-ester moieties using tannin acylhydrolase, which suggested that the lipase inhibition potency of OTPP could be explained by the ester bound galloyl moieties. In our previous in vivo study, OTPP significantly suppressed both the lymphatic triglyceride (TG) absorption (P<0.05) and serum TG elevation when high lipid diet was given (P<0.05) (Nakai et al., 2005a). In human study with high-lipid load had demonstrated that polyphenol-enriched oolong tea suppressed postprandial serum TG and chylomicron (Hara et al., 2004).
Although oolong tea is known to influence lipase activity, there is no direct proof on the lipid absorption in humans. Estimation of lipid excretion in fecal matter is one of the keystones to examine dietary lipid absorption. Therefore, in the present study we prospectively investigated the effect of polyphenol-enriched oolong tea on fecal lipid excretion in humans.
Subjects and methods
Healthy university students were recruited through a disclosure process from Providence University in Taiwan. Complete medical and nutritional histories were obtained by a questionnaire. Smokers, competitive athletes and persons who were engaged in intense physical activities or who had a history of weight loss were not eligible for inclusion in the study. Twelve healthy adult subjects, three males and nine females, aged 22.0±1.8 years, were included. The study was explained to subjects and the informed consents were obtained from all the participants. The study was approved by the Ethical Committee on Human Experimentation of the Providence University in Taiwan and was conducted in accordance with its rules and regulations. The study protocol was conformed to the Helsinki Declaration.
The diets were given throughout the whole periods. The different menus were prepared at each meal and each day; however, they were same between the two treatment periods and also the two washout periods. They met the recommended dietary allowances (RDA) of Taiwan (2001). During the treatment periods, each group was given 38 g lipids from potato chips.
The concentrations of OTPP in the oolong tea were analyzed by high-performance liquid chromatography (HPLC) with ultraviolet detection at 280 nm by directly injecting the oolong tea. The analysis was performed with a reversed-phase HPLC column (TSKgel ODS-80Ts QA, 4.6 × 150 mm, Toso Co., Tokyo, Japan) at 40°C at a flow rate of 1 ml/min. Gradient elution was performed with a solvent system of solvent A, 0.05% trifluoroacetic acid in water and solvent B, 0.05% trifluoroacetic acid in 100% acetonitrile, using a gradient program (solvent B content: isocratic elution of 10% for 5 min, gradient elution of 10–15.6% for 6 min, gradient elution of 15.6–17% for 10 min, gradient elution of 17–80% for 1 min, isocratic elution of 80% for 15 min). The quantitative analysis of OTPP was made using oolonghomobisflavan B as a standard. (Nakai et al., 2005b).
Placebo beverage was prepared with tea flavor and caramel for coloring. Double-blind crossover design was carried out in this study; all the participants were unable to differentiate between the two experimental beverages.
The subjects were randomly divided into two groups. They were followed by a crossover design including 7-day washout periods and 10-day treatment periods (Figure 1). During the treatment periods, each group was given 38 g lipids a day from potato chips (19 g lipids each within 30 min after lunch and dinner) and total 750 ml beverages (placebo- or polyphenol-enriched oolong tea) at three meals (250 ml at each meal). Subjects were allowed to take water freely. All the participants were instructed to maintain their habitual patterns of physical activity throughout the entire study period.
The physical characteristics were measured and 10 ml of blood was collected after 12 h fasting in the early morning. The blood samples were collected for biochemical examination at days 8, 18, 25 and 35 of the study period. Capsules containing activated charcoal powder were given to the subjects before breakfast at days 15 and 32 and after dinner at days 17 and 34 as a marker of the stool (Gades and Stern, 2003, 2005). Fecal samples were collected for the analysis of lipid excretion from the first appearance of the marker in the stool until the marker disappeared in two experimental periods.
Analyses of blood
Blood was collected into tubes containing heparin as an anticoagulant. Plasma was prepared from the heparinized blood by immediate centrifugation at 3000 r.p.m. for 15 min at 4°C. All the plasma analyses were performed by UM Clinical Laboratory (Taichung, Taiwan). The methods were as follows: TG and total-cholesterol by enzymatic–colorimetric method, high-density lipoprotein-cholesterol by precipitation method, total protein by timed end point method, iron by guanidine/ferrozine method, glucose by oxygen rate method using glucose oxygen electrode, glutamyl oxaloacetic transaminase and glutamyl pyrubic transaminase by enzymatic rate method, serum alkaline phosphatase (ALP) by kinetic rate method, uric acid time end point method and γ-glutamyl transpeptidase by dry-type chemical analysis method and hemoglobin A1c by using HPLC.
Analysis of fecal and potato chips lipids
Potato chips and feces samples were collected during the final 3 days of each treatment period from subject. They were weighed, oven-dried, homogenized and stored at −20°C for later analysis of macro-nutrients concentration at a central laboratory of Providence University, Taiwan. After fecal neutral sterols were extracted by petroleum ether, the concentration of fecal cholesterol was measured by the Lieberman-Burchard method (Folch et al., 1957; Huang et al., 1961).
All the data were expressed as means±standard deviation (s.d.) for the subjects in each experimental period. Statistical differences of the means before and after the study were tested by two-tailed paired Student's t-test, followed by the Wilcoxon's-signed rank test. Significance was accepted at the 95% confidence interval (P<0.05). All the analyses were performed with Statistical Package for the Social Science 10.0 (SPSS 10.0).
Nutrient intakes and physical characteristics
Table 2 shows the profile of dietary intakes, estimated by the standard food composition tables for Taiwanese. The lipid concentration of potato chips was about 33 g/100 g, which was lower than nutritive component table (about 35 g/100 g) shown on the potato chips bag. We gave 115 g potato chips a day/subject, which contained about 38 g lipids.
The physical characteristics of the subjects were shown in Table 3. Mean ±s.d. values of the physical characteristics of the 12 subjects were not changed. During the study, we did not see any significant differences in the lifestyles and none of the subjects reported any detrimental side effects.
Effect on blood biochemical parameters
Result of the blood biochemical examinations is shown in Table 3. Although there was no difference in plasma TG concentration between the two periods before the treatment (75.3±27.7 vs 75.1±32.8), after the treatment placebo period had higher concentration than that of the polyphenol-enriched oolong tea period (86.9±40.7 vs 72.3±31.0, P<0.05). No other differences between initial and final values in all the parameters in both placebo and polyphenol-enriched oolong tea periods were found, except for ALP which had significantly increased in both periods, almost in the same extent.
Effect on fecal lipid extraction
Table 4 shows the results of fecal excretion. Total fecal lipid excretion in the polyphenol-enriched oolong tea period (19.3±12.9 g/3 day) was significantly higher than that in placebo period (9.4±7.3 g/3 day) (P<0.01). The difference of the lipid excretion was 3.3 g/3 day, which was equivalent to 29.7 kcal/day. Total fecal cholesterol excretion was also increased in the polyphenol-enriched oolong tea period (1.8±1.2 g/3 day) compared with placebo period (1.2±0.6 g/3 day), although the difference was not statistically significant (P=0.056).
In this study, we could demonstrated that polyphenol-enriched oolong tea increased fecal lipid excretion when healthy subjects took high-lipid diet. After high-lipid diet, polyphenol-enriched oolong tea consumption significantly increased lipid excretion in feces compared with placebo beverage (19.3 g/3 day vs 9.4 g/3 day, P<0.01). Previous human clinical experiments in which control subjects were given 2400–2500 kcal diet and 75–85 g lipids a day showed total lipid excretion in fecal material to be about 3–5 g/day (Hartmann et al., 1993; Hussain et al., 1994; Guerciolini et al., 2001). These were in agreement with our results from the placebo group (3.1 g/day). Proportion of lipids in feces (total fecal lipid/total fecal dry weight × 100%) were 13 and 21% (P<0.001, data not shown) in placebo- and polyphenol-enriched oolong tea, respectively. We have also found that the fecal cholesterol contents were enhanced in polyphenol-enriched oolong tea group compared with placebo period (P=0.056). We did not measure the total fecal lipid content before the treatment because this study was done by placebo-controlled crossover design.
The medical treatments of obesity and its related diseases are the prime concern in health improvement. Dietary lipids contain TG, phospholipids (primarily phosphatidylcholine) and sterols (mainly cholesterol), which represent a highly concentrated form of energy. The excess dietary lipid uptake is widely thought to be one of the main factors of obesity. Inhibiting TG digestion has been developed as a mean to reduce lipid absorption. In recent years, Orlistat was found to interfere with the absorption of dietary lipid (Hochuli et al., 1987; Hartmann et al., 1993; Hussain et al., 1994; Zhi et al., 1994; Guerciolini, 1997, 2001; Gades and Stern, 2002, 2003, 2005). Orlistat is a powerful lipase inhibitor that reduces dietary lipid absorption, reduces body weight and increases fecal lipid excretion. However, about 20% of patients who used Orlistat therapy suffered from steatorrhea, deficiencies of lipid-soluble vitamins and essential fatty acids, which became more serious when a high-lipid diet was taken (Carriere et al., 2001). In order to suppress these side effects to the minimum, low-lipid diet is recommended. Orlistat is not recommended to be used for a long time.
Recently, the inhibitory effects on obesity by green tea were reported (Muramatsu et al., 1986; Matsumoto et al., 1998; Han et al., 1999, 2001; Kao et al., 2000). Although many studies have been explaining how tea influences dietary lipid metabolism, the detailed mechanism is not yet fully understood. It has been demonstrated that tea catechins, such as (−)-epicatechin, (−)-epigallocatechin, (−)-epicatechingallate and (−)-epigallocatechingallate(EGCG) activate hepatic lipid metabolism and result in the suppression of diet-induced obesity in rats (Chan et al., 1999; Yang et al., 2001; Murase et al., 2002; Raederstorff et al., 2003). Ikeda et al. (1992) have found that EGCG, the major component of catechins in green tea, inhibits pancreatic lipase in vitro and cholesterol absorption, and lymphatic TG absorption in rats.
Han et al. (1999) suggested that the antiobesity action of oolong tea extract might be owing to inhibition of pancreatic lipase activity, acceleration of lipolysis in adipose tissue and other mechanisms in mice. The characteristic feature of oolong tea components is that it contains numerous kinds of polymerized-polyphenols, which are derived from tea catechins by polyphenol oxidases or by heating process, which is called semifermented process; on the other hand, black tea polyphenol is a product of well-fermented processes. The polymerized polyphenols in oolong tea are different from those in black tea, although they are not found in green tea. Some studies indicated antiobesitic action of the oolong tea compounds could be mainly owing to polyphenols and then caffeine (Han et al., 1999, 2001). As shown in Table 1, the major compounds of polyphenol-enriched oolong tea except polymerized-polyphenols are total catechins and caffeine. Previously, OTPP, EGCG, gallocatechin gallate and caffeine, which are the major compounds of oolong tea extract, were examined (Nakai et al., 2005b). The results suggest that OTPP showed the strongest inhibition against pancreatic lipase activity in vitro. Moreover, oolong tea extract was shown to delay lymphatic absorption of TG more effectively than green tea extract in rats and mice fed high-lipid and high-sucrose diet (Nakai et al., 2005a), but caffeine did not show any influence on TG absorption. Recently, we have shown that oolong tea enriched with polymerized-polyphenols suppressed postprandial serum TG elevation in adult humans more effectively than oolong tea enriched with catechins (Hara et al., 2004). Caffeine is known to have no influence on lipase activity (Han et al., 1999, 2001). These information suggests that the effects of oolong tea on the decrease in lipid absorption and fecal lipid excretion are mainly owing to the OTPP but not caffeine.
In our previous study, we observed that polyphenol-enriched oolong tea suppressed postprandial serum TG elevation after high-lipid meal load in high-risk and mild hyperlipidemia adults (Hara et al., 2004). Several studies have demonstrated the effect of oolong tea on improving moderate cardiovascular disease (Yang and Koo, 1997) and obesity (Han et al., 1999; Rumpler et al., 2001; Komatsu et al., 2003) by decreasing serum cholesterol and TG concentrations. This is also supported by the animal studies, which have shown the reduced TG levels in mice administered with oolong tea (Han et al., 1999, 2001; Kurihara et al., 2002).
In this study with high-lipid diet, we have observed that serum ALP increased within the normal range both in polyphenol-enriched oolong tea and placebo groups after treatment, which could be explained as that ingestion of high-lipid diet induced a rise in intestinal-specific ALP, which resulted in elevated serum ALP (Sasaki et al., 2004). The serum ALP levels elevate in healthy humans or animals when lipids are administered in a test meal or a high-lipid diet is consumed (McDowell and Ross, 1966; Young et al., 1981; Deems et al., 1994). On the other hand, polyphenolic compounds have been shown to inhibit iron absorption (Zijp et al., 2000; Samman et al., 2001). In this study, ingestion of polyphenol-enriched oolong tea for 10 days (3 times/day) did not significantly influence serum iron levels.
In this study, we could elucidate that polyphenol-enriched oolong tea partly might exert its antiobesitic action through inhibition of lipase activity by influencing intestinal absorption of dietary lipid, which corresponded to the mechanism of Orlistat. Nevertheless, Orlistat is a potent medicine of weight loss therapy for only obesity people. The result indicated that polyphenol-enriched oolong tea increased fecal lipid excretion without any side effects; so, it could be acceptable as daily beverage and be expected to control the absorption of lipids for the healthy people.
We may be able to conclude from this study that oolong tea increases fecal lipid excretion. This finding together with the previous one that showed the increase of energy expenditure suggests that oolong tea contributes to weight loss.
I am indebted to the students at Providence University, Taichung, Taiwan. Financial support for the study was obtained from Suntory Ltd, Osaka, Japan. My special thanks are owing to Afework Kassu for valuable comments and criticisms.
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