Dietary flavonoids as antioxidants in vivo:Conjugated metabolites of (-) -epicatechin and quercetin participate in antioxidative defense in blood plasma

Dietary flavonoids as antioxidants in vivo:Conjugated metabolites of (-) -epicatechin and quercetin participate in antioxidative defense in blood plasma

Junji Terao

Department of Nutrition, The University of Tokushima School of Medicine, Tokushima, Japan

Abstract:Flavonoids are present in mainly plant foods and have attracted much atten-tion in relation to disease prevention. Their antioxidant activity at least partly accounts for their potential health effect, because oxidative stress leads to a variety of patho-physiological events. It is essential to know the bioavailability of flavonoids involving intestinal absorption, metabolic conversion and urinary excretion, in order to evaluate their in vivo antioxidant activity after intake. Here (-)-epicatechin and quercetin were selected as typical flavanol- and flavonol-flavonoids present in vegetables, fruits and tea. Our rat study suggests that their metabolic conversion begins in the intestinal mucosa where the activity of uridine-5'-diphosphoglucuronosyltransferase (UGT) is at its highest. Both flavonoids accumulated mostly as glucuronide and sulfate conjugates in blood plasma after oral administration. No intact quercetin was found in the circulation. However, on the oral administration of these flavonoids, the antioxidative ability of rat plasma was enhanced indicating that conjugated metabolites participate in the anti-oxidant defense in blood plasma. Therefore, the intake of vegetables, fruits and tea rich in flavonoids may help to prevent oxidative damages in the blood. J. Med. Invest. 46:159-168, 1999

Keywords:flavonoid, quercetin, (-)-epicatechin, antioxidant, glucuronidation

INTRODUCTION
Flavonoids are polyphenol compounds containing a unique C6-C3-C6 structure (diphenyl propane struc-ture) and more than 4,000 varieties of flavonoids are distributed in the plant kingdom. They are mostly present as glycosides in which phenolic hydrogen or hydrogens are substituted for the sugar moiety. Flavonoids can be classified as calcones, flavones, flavanones, flavanols and flavonols. Quercetin, a typical flavonol, possesses additional phenolic OH groups at the5- and 7-position of the A ring and3'- and 4'-position of the B-ring (Fig.1). Quercetin glycosides, such as rutin, quercitrin and quercimeritrin, are common flavonoids present in fruits and vegeta-bles. On the other hand, tea catechins consist of four flavanol-type compounds containing additional phenolic OH groups at the 5 and 7 position. Further-more, (-)-epicatechin and (-)-epicatechin gallate contain OH groups at the3'and4'position of the B ring, and (-)-epigallocatehin and (-)-epigallocatechin, at the 3', 4'and 5'position, respectively. Interesting-ly, epicatechin gallate and epigallocatechin gallate are the derivatives of epicatechin and epigallocatechin in which the gallate group is esterified to an OH group at the 3-position of the flavanol structure. Thus, tea catechins are composed of free flavanols and their gallate esters.
Daily intake of flavonoids by humans is estimated at 25mg (1). However, this value only covers five aglycones including quercetin and the total intake of flavonoids from plant food may reach several hundred mg/day (2). This level is not low as com-pared with that of vitamin E or vitamin C. In1936,Szent-Gyorgyi (3) claimed that two flavonoids from citrus fruits reduced capillary fragility and perme-ability in humans and he named them vitamin P. At present, flavonoids are not involved in the category of vitamins. Nevertheless, they are recog-nized as having a potential beneficial effect in disease prevention (4-9). Epidemiological studies strongly suggest that consumption of fruits, vegetables and teas lowers the risk of coronary heart disease (10). It should be noted that an inverse relationship be-tween the intake of flavonoids and coronary heart disease risk was also reported (11, 12). The so-called French paradox, the lack of a positive correlation between a high intake of saturated fat and the occurrence of coronary heart disease is related at least partly to the consumption of red wine (13),which is rich in flavonoids including epicatechin and quercetin.
The antioxidant activity of flavonoids has been frequently mentioned in connection with their physiological function in vivo, because oxidative stress is known to participate in the initial process of atherosclerosis leading to coronary heart dis-ease (6). A number of studies have revealed that flavonoids act as antioxidants by scavenging reac-tive oxygen species (ROS) and/or chelating metal ion responsible for the generation of ROS. The structure-activity relationship of flavonoids and their antioxidant activity is well documented (14). We have already carried out a kinetic study of the inhib-itory effect of several flavonoids on lipid peroxidation in solution and in liposomal membranes (15, 16). The results implied that flavonoids act as interfacial antioxidants in the lipid/water biphasic system, because the hydrophilic property of the flavonoids facilitates their localization at the interface of the lipid bilayers resulting in an effective inhibition of the initial attack by aqueous radicals (17). However, the in vivo function of dietary flavonoids cannot be estimated without a knowledge of their absorption and metabolic fate. Thus, much study has been done on the absorption and metabolism of flavonoids in recent years. We are also investigating the absorption rate and metabolic process of flavonoids, in particu-lar, (-)-epicatechin and quercetin, in rat and human. Here we review recent studies on the absorption and metabolism of these two flavonoids.

ABSORPTION AND METABOLIC PATHWAY OF DIETARY (-)-EPICATECHIN
In1971, Das et al. (18) detected (+)-catechin and its metabolites in human urine after oral adminis-tration of (+)-catechin. This was the first evidence that catechins are absorbed into the human body. It was recently confirmed that tea catechins are absorbed into human body and accumulated in the blood plasma by the intake of tea catechin concen-trate (19, 20). A rat study (21) demonstrated that a main component of tea catechin, (-)-epigallocatechin gallate, was widely distributed in several tissues including the liver and kidney. On the other hand, Hackett (22) showed that oral administration of epicatechin results in urinary excretion of glucuronide and sulfate conjugates of epicatechin and 3'-O-methyl epicatechin, indicating that absorbed catechins are mostly subject to metabolic conversion into conju-gates. It is also suggested that conjugated metabo-lites come to bile from liver and are then reabsorbed into the body after hydrolysis and ring-cleavage (enterohepatic circulation) (23). It is therefore likely that tea catechins accumulate in human plasma through enterohepatic circulation.
In general, liver is a main tissue for the metabo-lism of xenobiotic substances. However, intestinal mucosa, kidney and other tissues also possess enzy-matic activity for metabolism such as glucuronidation, O-methylation, and hydroxylation (P-450). It is un-clear how and where catechins are metabolized after intestinal absorption. Here we used (-)-epicatechin and measured the enzymatic activities for its meta-bolic conversion in several rat tissues (24). The enzymes are uridine-5'-diphosphoglucuronosyl transferase (UGT) for glucuronidation, phenol-sulfotransferase (PST) for sulfation and catechol-O -methyltransferase (COMT) for methylation. Figure 2 shows that the UGT activity was strongest for the preparation from the intestine. The only organ to present activity of PST was the liver. The liver is the main organ for COMT and the activity in the kidney was lower than that in the liver but was higher than in the other tissues. Thus, we propose a metabolic pathway of orally administered (-)-epicatechin in rats as shown in Fig. 3. Absorbed epicatechin is likely to be immediately conjugated with glucuronic acid in the intestinal mucosa. Conjugation in the intestinal mucosa is plausible because the glucuronidation of phenolic compounds in the intestinal mucosa has been reported elsewhere (25). The second step for metabolic conversion is conjugation with sulfate. The final step is methylation resulting in O-methylated catechin, which seems to be the final product of ab-sorbed EC. We therefore postulate that epicatechin is metabolized to glucuronyl conjugates in the intestinal mucosa and these enter the portal vein and are metabolized further in liver and other tis-sues. Finally, they are excreted from the body via bile or urine. The antioxidant activity of catechins has been often discussed based on the results of in vitro studies. However, the activity of their metabolites should be taken into account when estimating their in vivo effectiveness.

EX VIVO EFFECT OF (-)-EPICATECHIN ON THE ANTIOXIDATIVE DEFENSE IN RAT BLOOD PLASMA
Blood plasma is a well-organized defense system which utilizing antioxidant enzymes such as extra-cellular superoxide dismutase and glutathione per-oxidase, and low-molecular weight antioxidants such as vitamin E, vitamin C, uric acid and so on. It is of interest to know the role of dietary catechins in antioxidative defense in blood plasma, because catechins are found to be mainly present as conju-gated and methylated metabolites. It remains unclear whether or not catechins possess any antioxidant activity after intestinal absorption and metabolic conversion. Thus, we measured the changes in the oxidizability of rat plasma after oral administration of (-)-epicatechin (26). Male Wistar rats were fasted for 12-15hr and then administered (-)-epicatechin(10or50mg/200g body weight) dissolved in 2.0ml of water intragastrically by direct stomach intubation. The rats were anesthetized with diethylether at1hr and 6hr after administration. Rat plasma was ob-tained and the concentration of (-)-epicatechin and its metabolites were determined by HPLC analysis and a method of enzymatic hydrolysis which we developed. Table 1 shows the profiles of metabolites in rat plasma after (-)-epicatechin administration. One hr after an intragastric administration of 20mg (-)-epicatechin, the concentration of total (-)-epicatechin
metabolites that had accumulated was 42.3μM. 70% and 30% were nonmethylated and O-methylated epicatechins, respectively. As little as 7% of the metabolites were in a non-conjugated form. After6hr administration, approx. 84%of metabolites were
cleared from plasma. Administration of 50mg (-)-epicatechin increased the level of total metabo-lites in plasma to 103.0μM and 73.3μM at 1hr and 6hr, respectively. 13% free (-)-epicatechin was detected in the plasma indicating saturation of the conjugation reaction.
Fig.4 shows the accumulation of cholesteryl ester hydroperoxides (CE-OOH) and consumption of α-tocopherol in copper ion-induced oxidation of diluted rat plasma. This figure clearly shows that oral administration of (-)-epicatechin hindered the accumulation of CE-OOH and retarded the con-sumption of α-tocopherol. It is therefore likely that oral administration of (-)-epicatechin expands the antioxidative capacity of rat blood plasma, although (-)-epicatechin is mostly present as its metabolites. This implies that some (-)-epicatechin metabolites act as antioxidants in plasma by scavenging radicals and/or chelating metal ion.
Bors (27) suggested that the O-dihydroxyl structure in the B-ring (catechol structure) is essential to the free radical-scavenging activity for flavanol-type flavonoids. Metabolites posses-sing a O-dihydroxyl structure seem to be responsible for the antioxidant activity of orally administered (-)-epicatechin. A human study (28) demonstrated that inges-tion of tea improves the antioxidant capacity of blood plasma. It is therefore likely that conjugated metabolites with a catechol structure are responsible for the in vivo antioxidant activity of tea catechins. Re-cently Okusio et al. (29) and Harada et al. (30) detected (-)-epicatechin-5-O-glucuronide as a (-)-epicatechin metabo-lite in rat blood plasma. This conjugated metabolite contains a catechol structure and thus may be responsible for the antioxidant activity of dietary catechins.

ABSORPTION OF QUERCETIN AND QUERCETIN GLYCOSIDES
In1975, Gugler et al. (31) reported that less than 1% of quercetin was absorbed into the human body following oral adminis-tration of quercetin aglycone. Ueno (32) et al. demonstrated using C14-labeled quercetin that 20% of quercetin was absorbed from the digestive tract and present in bile and urine as glucuronide and sulfate conjugates within48hrs. We are interested in the effects of the vehicles for quercetin in oral administration on the efficiency of intestinal absorption and accumulation in blood(33). Quercetin's solubility in the vehicles used for its administration was compared with respective absorption profiles. We compared water and propylene glycol as vehicles for the administration in the rat. If quercetin's solubility in propylene glycol is taken as 1 (complete solubilzaition), the relative solubility in water is 1.6 x10-5. The results shown in Fig.5 clearly reveal that the extent of quercetin absorption depends on the solubility in the vehicle used for the administration. It should be emphasized that the vehicles substantially affect the efficiency of the absorption. Alcohol as a vehicle may elevate the absorption of quercetin from the digestive tract because of its high solubility.
Quercetin is, in general, present in the form of glycosides in plant foods and thus the absorption of quercetin glycosides need to be clarified to estimate the physiological function of dietary quercetin. Water-soluble glycosides seem to be little absorbed because of their poor solubility in bile acid micelles in the intestinal tract. However, in the large intestine, glycosides are hydrolyzed to release aglycone by the action of β-glycosidase in anaerobic enterobacteria (34). Furthermore, a part of the aglycone is subjected to ring scission (35). Thus, flavonoids improve their lipophilicity resulting in high solubility in bile acid micelles. Nevertheless, there have been contradic-tory results on the intestinal absorption of quercetin glycosides. Manach et al. (36) suggested that the absorption of rutin (quercetin-3-rutinoside) in rats is slower than that of the respective aglycone because hydrolysis in the large intestine is required. On the other hand, Holmann (37, 38) et al. claimed that quercetin glucosides are absorbed more easily than quercetin aglycone in humans. Papanga et al(39) and Aziz et al. (40) reported that quercetin glucosides are present in human plasma without metabolic conversion. However, Manach et al. (41) suspected that intact quercetin glucosides are present in blood circulation without metabolic conversion. They sug-gested that conjugated metabolites including sulfates and glucuronides exclusively accumulate in the plasma after intake of quercetin glucosides from plant food (42). It is still obscure whether or not quercetin glucosides are absorbed directly or ab-sorbed after hydrolysis in the tract. We pointed out that β-glucosidase activity is present in rat intestinal mucosa homogenate (43). Thus we suggest that quercetin glucosides are hydrolyzed in the intestinal mucosa and incorporated into the cell in which glucuronidation occurs. The participation of the glucose transporter in the cellular intake of quercetin glucosides from diet has been reported (44, 45).However, there is no direct evidence that this trans-port system is responsible for the absorption of quercetin glucosides (46). Our recent results suggest that not glucosides but glucuronide conjugates ac-cumulated in human plasma after intake of onion rich in quercetin glucosides (unpublished data).

QUERCETIN METABOLITES ACT AS ANTI-OXIDANTS ON LIPID PEROXIDAITON IN RAT PLASMA
Absorbed quercetin seems to be metabolized via the same pathway as (-)-epicatechin, in which glucuronidation occurs at first (Fig6). It was re-ported that in a rat experiment isorhamnetin (3'-O-methylated quercetin) and4'-O-methylated quercetin accumulated after oral administration of quercetin(47). However, we found that glucuronide and sul-fate conjugates without methylation also accumu-lated in blood plasma after oral administration (33)and thus supposed that some conjugates in circu-lation act as antioxidants. For example, in metabolic profiles of quercetin in the rat obtained after oral ad-ministration of 2mg and 5mg of quercetin aglycone in propylene glycol, respectively (Table 2) (48), at1hr and6hr after the administration neither free quercetin nor free isorhamnetin was found in the plasma, indicating that all of the absorbed quercetin is present as conjugated metabolite in the circula-tion. At 2.0mg, 13μM of quercetin accumulated at1hr and 9μM after 6hr. When the dose was el-evated to10mg, the concentration of quercetin was 89.4 and 35.3μM, after 1and 6hr, respectively. The antioxidant activity of quercetin-treated plasma was determined after dilution by measuring the accu-mulation of CE-OOH during the copper ion-induced lipid peroxidation of rat plasma (Fig.7). Plasma after administration of quercetin enhanced the resistance against CE-OOH accumulation indicating that quercetin metabolites participate in the anti-oxidative defense in blood plasma. It can be con-cluded that some conjugated metabolites of quercetin inhibit copper ion-induced plasma oxidation. Thus, quercetin possesses antioxidative activity for copper-ion induced oxidation of plasma even after its metabolic conversion. We are now trying to clari-fy the metabolites responsible for the antioxidant defense in blood.

CONCLUSION
(-)-Epicatechin and quercetin are obtained by the daily intake of vegetables, fruits and tea. Intestinal absorption and the subsequent metabolic conver-sion should be taken into account when elucidating their physiological functions. Although little is clear as to their absorption and metabolism in humans, studies indicate that both flavonoids are partly ab-sorbed into the body and largely accumulated as glucuronide and sulfate conjugates. Conjugated metabolites containing a catechol group are likely to be responsible for the increase of plasma anti-oxidative capacity. Although the conjugation with glucuronide and sulfate is a step in the detoxifica-tion to lose the physicochemical activity, intermedi-ate products should retain their activity. Metabolites may be at least partly responsible for the physiologi-cal function of dietary flavonoids.

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Received for publication June 1, 1999;accepted July 29, 1999.

Address correspondence and reprint requests to Junji Terao, Ph.D., Department of Nutrition, The University of Tokushima School of Medicine, Kuramoto-cho, Tokushima 770-8503, Japan and Fax:+81-88-633-7087.