Lipid-lowering efficacy of hesperetin metabolites in high-cholesterol fed rats

Clinica Chimica Acta 327 (2003) 129 – 137 www.elsevier.com/locate/clinchim

Lipid-lowering efficacy of hesperetin metabolites in high-cholesterol fed rats Hae Kyung Kim a, Tae-Sook Jeong b, Mi-Kyung Lee a, Yong Bok Park c, Myung-Sook Choi a,* a

Department of Food Science and Nutrition, Kyungpook National University, 1370 Sank-Yuk Dong, Puk-Ku, Taegu 702-701, South Korea b Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yusong, Taejon 305-600, South Korea c Department of Genetic Engineering, Kyungpook National University, Taegu 702-701, South Korea Received 18 July 2002; received in revised form 4 October 2002; accepted 4 October 2002

Abstract Background: Hesperetin is a naturally occurring flavonoid that has hypolipidemic properties. Methods: Male rats were fed a 1 g/100 g high-cholesterol diet for 5 weeks along with hesperetin (0.02%, 0.066 mmol/100 g diet) and hesperetin metabolites. The hesperetin metabolites, m-hydroxycinnamic acid (m-HC), 3,4-dihydroxyphenylpropionic acid (3,4-DHPP), and 3-methoxy4-hydroxycinnamic acid (ferulic acid), were supplemented based on an equivalent amount of hesperetin. Results: The supplementation of hesperetin and its metabolites significantly lowered the plasma total cholesterol and triglyceride concentrations compared to the control group. The hepatic HMG-CoA reductase and acyl-CoA:cholesterol acyltransferase (ACAT) activities were significantly lower in the hesperetin and its metabolite supplemented groups than in the control group. The excretion of acidic sterol was significantly higher in the hesperetin, m-HC, 3,4-DHPP, and ferulic acid supplemented groups than in the control group. Conclusions: These results demonstrated that the hesperetin metabolites played as potent a role as hesperetin in plasma lipid-lowering activities in vivo, and further suggest that cholesterol biosynthesis and esterification were concomitantly reduced by hesperetin and its metabolites, as indicated by the decreased HMG-CoA reductase and ACAT activities. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Hesperetin and its metabolites; HMG-CoA reductase; Acyl-CoA:cholesterol acyltransferase; Cholesterol metabolism; Flavonoids

1. Introduction Epidemiological studies have shown an inverse correlation between the intake of dietary flavonoids and death from coronary disease [1,2]. As such, there * Corresponding author. Tel.: +82-53-950-6232; fax: +82-53950-6229. E-mail address: [email protected] (M.-S. Choi).

is considerable interest in investigating the hypolipidemic or antiatherogenic nature of these compounds. Among naturally occurring citrus flavonoids, hesperidin, hesperetin, naringin, and naringenin have already been evaluated as potential agents for improving the metabolism of cholesterol in vivo [3,4]. In addition, hesperetin and naringenin have also been found to exhibit estrogenic, anticarcinogenic, and antioxidative properties [5].

0009-8981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 8 9 8 1 ( 0 2 ) 0 0 3 4 4 - 3

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Hesperetin belongs to the class of flavonoids called flavanones, which are abundant in citrus fruits, such as grapefruit and oranges [6]. Hesperetin is derived from the hydrolysis of its aglycone, hesperidin (hesperetin 7rhammnoglucoside) [7]. Honohan et al. [8] showed that the intestinal metabolite of 3-[14C] hesperetin in rats is 3,4-dihydroxyphenylpropionic acid (3,4DHPP), 3-methoxy-4-hydroxycinnamic acid (ferulic acid), and m-hydroxycinnamic acid (m-HC). The current authors previously reported that hesperetin inhibits HMG-CoA reductase and lowers the plasma cholesterol level in rats [9]. However, due to the substantial metabolism of hesperetin in the intestine or liver, it is unclear whether all these metabolites actually influence the metabolic pathways involved in the cholesterollowering action through the systemic circulation or tissues. Accordingly, the current study investigates to compare the lipid-lowering efficacy of hesperetin and its metabolites in rats fed a high-cholesterol diet. The effect on the key enzymes in the liver involved in the regulation of cholesterol homeostasis and the excretion of fecal sterols was also examined.

2. Materials and methods 2.1. Animals and diets Fifty male Sprague – Dawley rats weighing between 50 and 55 g were purchased from Bio Genomics (Seoul, Korea), which shares research technology on the commercial production of experimental animals with the Charles River Laboratory (Wilmington, USA). The animals were individually housed in stainless steel cages in a room with controlled temperature (24 jC) and lighting (alternating 12-h periods of light and dark). All the rats were fed a pellitized commercial chow diet for 10 days after arrival. Next, the rats were randomly divided into 5 groups (n = 10) and fed a high-cholesterol diet (1%, wt/wt) or high cholesterol diet supplemented with hesperetin (0.02% or 0.066 mmol/100 g diet), m-HC, 3,4-DHPP, or ferulic acid (Sigma and Aldrich) for 5 weeks. The structure of the experimental materials is shown in Fig. 1. The hesperetin dose in present study was only one fifth of our previous dose [9], which the 0.1%

Fig. 1. Structure of experimental materials.

hesperetin lowered plasma cholesterol concentration. We supposed that the lower dose of hesperetin could be still effective for cholesterol-lowering. The amount of each hesperetin metabolite was equivalent to a 0.066 mmol/100 g diet that is based on the mmol of hesperetin in the diet. The composition of the experimental diet, as shown in Table 1, was based on the AIN-76 semisynthetic diet [10,11]. The animals were given food and distilled water ad libitum throughout the experimental period. The food consumption and weight gain were measured daily and weekly, respectively. The feces collected during the last 3 days using metabolic cages were used for determining the fecal sterol. At the end of the experimental period, the rats were anesthetized with ketamine following a 12-h fast. Blood samples were collected from the inferior vena cava to determine the plasma lipid profile. The livers were removed and rinsed with physiological saline. All samples were stored at 70 jC until analyzed. This experimental design was approved by the Committee of Kyungpook National University for the care and use of laboratory animals.

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Table 1 Composition of control and experimental diets (g/100 g diet) Component

Control

Hesperetin

m-HC

3,4-DHPP

Ferulic acid

Casein D,L-Methionine Corn starch Sucrose Cellulose powder Corn oil Mineral mixturea Vitamin mixtureb Choline bitartrate Cholesterol Hesperetinc m-HCd 3,4-DHPPe Ferulic acidf Total

20.0 0.3 15.0 49.0 5.0 5.0 3.5 1.0 0.2 1.0 – – – – 100

20.0 0.3 15.0 48.98 5.0 5.0 3.5 1.0 0.2 1.0 0.02c – – – 100

20.0 0.3 15.0 48.989 5.0 5.0 3.5 1.0 0.2 1.0 – 0.011d – – 100

20.0 0.3 15.0 48.988 5.0 5.0 3.5 1.0 0.2 1.0 – – 0.012e – 100

20.0 0.3 15.0 48.987 5.0 5.0 3.5 1.0 0.2 1.0 – – – 0.013f 100

a

AIN-76 mineral mixture (Harlan Teklad, USA). AIN-76 vitamin mixture (Harlan Teklad, USA). c Hesperetin group: 0.02% hesperetin (Mwt. 302.3). d m-HC group: 0.011% m-hydroxycinnamic acid (Mwt. 164.16). e 3,4-DHPP group: 0.012% 3,4-dihydroxyphenylpropionic acid (Mwt. 182.18). f Ferulic acid group: 0.013% 3-methoxy-4-hydroxycinnamic acid (Mwt. 194.2). b

2.2. Plasma and hepatic lipids The plasma cholesterol and HDL-cholesterol concentrations were determined using a commercial kit (Sigma) based on a modification of the cholesterol oxidase method of Allain et al. [12]. The HDL-fractions were separated using a Sigma (St. Louis, MO) kit based on the heparin-manganese precipitation procedure [13]. The plasma triglyceride concentrations were measured enzymatically using a kit from Sigma, a modification of the lipase-glycerol phosphate oxidase method [14]. The hepatic lipids were extracted using the procedure developed by Folch et al. [15]. The dried lipid residues were dissolved in 1 ml of ethanol for the cholesterol and triglyceride assays. Triton X-100 and a sodium cholate solution (in distilled H2O) were added to 200 Al of the dissolved lipid solution to produce final concentrations of 5 g/l and 3 mmol/l, respectively. The hepatic cholesterol and triglycerides were analyzed with the same enzymatic kit as used in the plasma analysis. 2.3. HMG-CoA reductase and ACAT activities The microsomes were prepared according to the method developed by Hulcher and Oleson [16] with a

slight modification. Two grams of liver tissue were homogenized in 8 ml of an ice-cold buffer (pH 7.0) containing 0.1 mol/l of triethanolamine, 0.02 mol/l of EDTA, and 2 mmol/l of dithiothreitol, pH 7.0. The homogenates were centrifuged for 10 min at 10,000g and then at 12,000  g at 4 jC. Next, the supernatants were ultracentrifuged twice at 100,000  g for 60 min at 4 jC. The resulting microsomal pellets were then redissolved in 1 ml of a homogenation buffer for protein determination [17] and finally analyzed for HMG-CoA reductase and acyl-CoA:cholesterol acyltransferase (ACAT) activities. Using freshly prepared hepatic microsomes, the HMG-CoA reductase activities were determined by slightly modifying the method of Shapiro et al. [18]. Briefly, an incubation mixture (120 Al) containing microsomes (100 – 150 Ag) and 500 nmol of NADPH (dissolved in a reaction buffer containing 0.1 mol/l of triethanolamine and 10 mmol/l of EDTA) was preincubated at 37 jC for 5 min. Next, 50 nmol of [14C]HMG-CoA (specific activity; 2.1420 GBq/mmol; NEMk Life Science Products, Boston, MA) was added and the incubation was continued for 15 min at 37 jC. The reaction was terminated by the addition of 30 Al of 6 mol/l HCl, then the resultant reaction mixture was incubated at 37 jC for a further 15 min to

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Table 2 Effects of hesperetin metabolite supplementation on weight gains, food intakes, and organic weights in high-cholesterol fed rats* Weight gains (g/day)

Control Hesperetin m-HC 3,4-DHPP Ferulic acid

8.40 F 0.22 8.53 F 0.17 8.44 F 0.13 8.97 F 0.19 8.51 F 0.17

Food intakes (g/day)

Organ weights (g)

25.05 F 0.47 25.97 F 0.31 25.90 F 0.46 26.16 F 0.47 25.87 F 0.44

Liver

Heart

Kidney

17.75 F 0.76 19.40 F 0.74 17.48 F 0.52 18.93 F 0.50 18.93 F 0.50

1.45 F 0.04 1.32 F 0.03 1.30 F 0.03 1.36 F 0.04 1.32 F 0.02

3.30 F 0.06 3.15 F 0.06 3.14 F 0.12 3.21 F 0.10 3.08 F 0.04

* Mean F S.E.

convert the mevalonate into mevalonolactone. The incubation mixture was centrifuged at 10,000  g for 5 min, and the supernatant spotted on a Silica Gel 60 F254 TLC plate with a mevalonolactone standard. The plate was developed in benzene/acetone (1:1, v/v) and air-dried. Finally, the Rf 0.3 –0.6 region was removed by scraping using a clean razor blade, and the 14C radioactivity determined using a liquid scintillation counter (Packard Tricarb 1600TR; Packard Instrument, Meriden, CT). The results were expressed as pmol mevalonate synthesized per min per mg protein. Using freshly prepared hepatic microsomes, the ACAT activities were determined according to the method developed by Erickson et al. [19] and modified by Gillies et al. [20]. To prepare the cholesterol substrate, 6 mg of cholesterol and 600 mg of Tyloxapol (Triton WR-1339, Sigma) were each dissolved in 6 ml of acetone, mixed well, and completely dried in N2 gas. The dried substrate was then redissolved in 20 ml of distilled water to a final concentration of 300 Ag of cholesterol/ml. Next, reaction mixtures containing 20 Al of a cholesterol solution (6 Ag of cholesterol), 20 Al of a 1 mol/l potassium-phosphate buffer (pH 7.4), 5 Al of 0.6 mmol/l bovine serum albumin, 50– 100 Ag of the microsomal fraction, and distilled water (up to 180 Al)

were preincubated at 37 jC for 30 min. The reaction was then initiated by adding 5 nmol of [14C]-Oleoyl CoA (specific activity; 2.0202 GBq/mmol; NEMk Life Science Products) to a final volume of 200 Al; the reaction time was 30 min at 37 jC. The reaction was stopped by the addition of 500 Al of isopropanol/ heptane (4:1, v/v), 300 Al of heptane, and 200 Al of 0.1 mol/l potassium phosphate (pH 7.4), then the reaction mixture was allowed to stand at room temperature for 2 min. Finally, an aliquot (200 Al) of the supernatant was subjected to scintillation counting. The ACAT activity was expressed as pmol cholesteryl oleate synthesized per min per mg protein. 2.4. Fecal sterols The fecal neutral sterols were determined using a simplified micro-method developed by Czubayko et al. [21]. The gas –liquid chromatography was carried out using a Hewlett-Packard gas chromatograph (Model 5890; Palo Alto, CA) equipped with a hydrogen flame-ionization detector and Sack-5 capillary column (30 m  0.25 mm i.d., 0.25 Am film; Supelco, Bellefonate, PA, USA). Helium was used as the carrier gas. The temperatures were set at 230 jC for

Table 3 Effects of hesperetin metabolite supplementation on plasma lipids in high-cholesterol fed rats* Groups Control Hesperetin m-HC 3,4-DHPP Ferulic acid

TC1 (mmol/l) a

3.03 F 0.20 2.26 F 0.08b 2.41 F 0.14b 2.44 F 0.12b 2.36 F 0.11b

TG (mmol/l) a

0.97 F 0.06 0.69 F 0.06b 0.72 F 0.05b 0.64 F 0.04b 0.60 F 0.05b

HDL-C (mmol/l) 0.63 F 0.04 0.79 F 0.05 0.61 F 0.06 0.63 F 0.04 0.70 F 0.08

HDL-C/TC (%) a

21.09 F 2.29 35.28 F 1.94c 25.56 F 3.34ab 26.02 F 1.83ab 29.34 F 3.23bc

Means in the same column not sharing a common superscript are significantly different ( p < 0.05) between groups. * Mean F S.E. 1 TC, total cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol. 2 Atherogenic index : (total cholesterol HDL-cholesterol)/HDL-cholesterol.

Atherogenic index2 3.75 F 0.40a 1.88 F 0.19b 2.86 F 0.62ab 2.89 F 0.38ab 2.35 F 0.34b

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the column and 280 jC for the injector/detector temperature. 5-a-cholestane (Supelco) was used as the internal standard. The daily neutral sterol excretion was calculated based on the sum of the cholesterol, coprostanol, and coprostanone found in each sample. The fecal bile acid was extracted with tbutanol and quantified enzymatically with 3-ahydroxysteroid dehydrogenase [22]. 2.5. Statistical analysis All data is presented as the mean F S.E. The data was evaluated by one-way ANOVA using an SPSS program, and the differences between the means assessed using Duncan’s multiple-range test. Statistical significance was considered at p < 0.05.

3. Results 3.1. Food intakes, weight gains, and organ weights There were no differences in the weight gains, food intakes and organ weights between the groups (Table 2). 3.2. Plasma and hepatic lipids The supplementation of hesperetin and its metabolites significantly lowered the plasma total cholesterol and triglyceride concentrations compared to the control group (Table 3). Although the plasma HDLcholesterol level did not differ between the groups, the ratio of HDL-cholesterol to total cholesterol was significantly higher in the hesperetin and ferulic acid supplemented groups compared to the control group.

Fig. 2. Effects of hesperetin metabolite supplementation on hepatic HMG-CoA reductase activity in high-cholesterol fed rats (mean F S.E.). The means not sharing a common letter are significantly different between groups ( p < 0.05).

The atherogenic index, coronary heart disease risk factor, was also lowered in the hesperetin and ferulic acid supplemented groups of high cholesterol-fed rats (Table 3). However, the hepatic cholesterol and triglyceride content did not differ between the groups (Table 4). 3.3. Hepatic HMG-CoA reductase and ACAT activities The hepatic HMG-CoA reductase was significantly lower in the hesperetin, m-HC, 3,4-DHPP, and ferulic acid supplemented groups compared to the control

Table 4 Effects of hesperetin metabolite supplementation on hepatic lipids in high-cholesterol fed rats*

Control Hesperetin m-HC 3,4-DHPP Ferulic acid * Mean F S.E.

Total cholesterol (mmol/g)

Triglyceride (mmol/g)

0.25 F 0.01 0.23 F 0.02 0.26 F 0.02 0.26 F 0.01 0.27 F 0.01

0.11 F 0.01 0.10 F 0.01 0.09 F 0.01 0.08 F 0.01 0.10 F 0.01

Fig. 3. Effects of hesperetin metabolite supplementation on hepatic ACAT activity in high-cholesterol fed rats (mean F S.E.). The means not sharing a common letter are significantly different between groups ( p < 0.05).

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Table 5 Effects of hesperetin metabolite supplementation on fecal sterol contents in high-cholesterol fed rats* Neutral sterol

Acidic sterol

Total fecal sterol

18.44 F 1.67a 31.06 F 1.33b 36.45 F 1.61c 43.91 F 0.92d 43.38 F 1.65d

170.73 F 8.17a 200.28 F 4.99b 211.91 F 6.95b 213.62 F 4.40b 208.87 F 6.68b

mg/day Control Hesperetin m-HC 3,4-DHPP Ferulic acid

152.30 F 9.60 169.21 F 6.02 175.46 F 8.41 169.71 F 6.38 165.49 F 7.15

The daily neutral sterol excretion was calculated from the sum of the cholesterol, coprostanol, and coprostanone from the gas chromatographic peaks, while the bile acids were measured as total bile acids using an enzymatic method. The total fecal sterol refers to the sum of the neutral sterols and bile acids. Means in the same column not sharing a common superscript are significantly different ( p < 0.05) between groups. * Mean F S.E.

group (Fig. 2). The ACAT activity also was significantly lower in the hesperetin and hesperetin metabolite supplemented groups than in the control group (Fig. 3). Accordingly, when compared to the control group, the hesperetin and hesperetin metabolites apparently inhibited hepatic cholesterol biosynthesis and esterification. 3.4. Fecal sterols The daily excretion of fecal sterols is shown in Table 5. The fecal neutral sterol excretion did not differ between the groups, whereas the excretion of acidic sterol was higher in the hesperetin, m-HC, 3,4DHPP, and ferulic acid groups than in the control group. Overall, the effect of supplementing hesperetin or its metabolites resulted in marked changes in the total fecal sterols under cholesterol-fed conditions.

4. Discussion The role of naringenin and the structurally related citrus flavanone hesperetin in the prevention and treatment of disease has recently received considerable attention [23], with particular interest in the use of these flavanones as anticancer [24] and antiatherogenic [25] compounds. Investigation of naturally occurring compounds as regulators of cholesterol metabolism has particular therapeutic importance for the treatment of

hyperlipidemia. As such, flavonoids may represent another beneficial group of naturally occurring hypolipidemic compounds. Dietary hesperidin is deglycosylated into hesperetin by intestinal bacteria prior to absorption [26]. Booth et al. [27] showed that 330 mg/kg hesperidin given to rabbits yields hesperetin, m-hydroxyphenylpropionic acid, m-HC, 3,4-DHPP and ferulic acid in the urine. However, it is unknown whether the role of each metabolite is different in the metabolism of cholesterol. Therefore, the present study was designed to compare the effects of hesperetin and equivalent amounts of its metabolites on the lipid metabolism in high cholesterol-fed rats. It was observed that hesperetin and its structurally related metabolites lowered the plasma cholesterol and triglyceride levels in rats fed a high-cholesterol diet, accompanied by the inhibition of hepatic HMGCoA reductase and ACAT activities and an increase in the fecal acidic sterols. In the plasma lipids, the HDLC/total-C ratio was higher in the hesperetin and ferulic acid supplemented groups than in the control group due to a decrease in the total cholesterol concentration. Consequently, the atherogenic index was significantly lowered by the hesperetin and ferulic acid supplements. Hesperetin and ferulic acid seemed to be more potent than m-HC and 3,4-DHPP in decreasing the atherosclerotic risk based on the HDL-C/totalC ratio and their atherogenic index. Among the plant flavonoids previously investigated for their possible cholesterol-lowering potential, the best known are isoflavones from soybean, consisting mainly of genestein. The principal citrus flavonoids, hesperetin from oranges and naingenin from grapefruit, are structurally similar to genestein. Recently, Kurowska et al. [28] reported that consumption of 750 ml orange juice daily increased HDL-C concentrations by 21% and triglyceride concentrations by 30% in subjects with hypercholesterolemia. In our previous study, 3,4-dihydroxycinnamate was also very potent in lowering plasma cholesterol and hepatic lipids [29]. Recently, the effects of hesperetin in the human hepatoma cell line HepG2 indicated that hesperetin reduces the secretion of apolipoprotein (apo) B-containing lipoproteins in a dose-dependent manner [30]. The reduced hepatic secretion of apo B-containing lipoproteins, such as VLDL, would be expected to contribute to a lower plasma triglyceride or cholesterol

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concentration in rats supplemented with hesperetin or its metabolites, however, this was not measured in the current study. A previous report by the current authors already speculated on the cholesterol-lowering action of a 0.1% (wt/wt) hesperetin diet via the inhibition of HMG-CoA reductase and ACAT activities in highcholesterol fed rats [9]. The present results indicate that hesperetin supplementation at a low dosage (0.02%) was sufficient to exert a hypolipidemic effect in high-cholesterol fed rats. The supplementation of the hesperetin metabolites, m-HC, 3,4-DHPP, and ferulic acid induced a plasma lipid-lowering action at a dose of 0.066 mmol/100 g diet along with a simultaneous decrease in HMG-CoA reductase and ACAT activities and an increase in acidic sterol excretion. The two key enzymes involved in the regulation of cholesterol metabolism are HMG-CoA reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway, and ACAT, the cholesterol-esterifying enzyme in tissues, including the small intestine. The inhibition of HMG-CoA reductase decreases cholesterol synthesis and its inhibitors are very effective in lowering serum cholesterol in most animal species, including humans [31 – 33]. The present study indicated that the supplementation of hesperetin or its metabolites significantly inhibited hepatic cholesterol biosynthesis and esterification. The blockade of cholesterol synthesis by an inhibitor of HMG-CoA reductase results in a lower intracellular supply of cholesterol, thereby triggering an over-expression of hepatic LDL receptors and enhancing the clearance of circulating LDL particles [34]. ACAT is another key enzyme involved in the esterification and absorption of cholesterol, secretion of hepatic LDL-cholesterol, and cholesterol accumulation in the arterial wall [35,36]. An accumulation of esterified cholesterol is one of the major metabolic changes in an atherosclerotic lesion [37]. A reduction in plasma cholesterol may lower the incidence of coronary heart disease through the reduction of esterified cholesterol in atherosclerotic lesions [38,39] and possibly in the liver. Therefore, ACAT inhibitors are also expected to be cholesterol-lowering and antiatherosclerotic agents. The inhibition of ACAT reduces the hepatic production of apo-B containing lipoproteins [40], possibly by limiting the availability of newly synthesized cholesteryl ester for association with apo B during the assembly of apo B-containing

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lipoproteins, such as VLDL, thereby reducing the plasma triglyceride concentration. Wilcox et al. [41] and Borradaile et al. [30] provided evidence that the citrus flavonoids naringenin and hesperetin not only decrease cholesterol synthesis but also inhibit ACAT activity in HepG2 cells. Thus, the inhibition of hepatic ACAT could be one of the mechanisms whereby hesperetin exerts its hypocholesterolemic and hypotriglyceridemic effects. However, the hepatic lipid profile was somewhat different from the plasma lipid changes, as the hepatic triglyceride and cholesterol contents did not differ between the groups. Although the mechanism underlying this effect is still unclear, Wilcox et al. [42] found that naringenin added to HepG2 cells lead to a significant decrease in the intracellular cholesteryl ester mass, whereas the mass of free cholesterol and triglyceride remained unaffected. Whether the in vivo effects of naringenin or hesperetin will be similar to the lipid changes found in HepG2 cells still needs to be verified. The supplements of hesperetin and its metabolites did not produce an overall change in the neutral sterol excretion, although the hesperetin, m-HC, 3,4-DHPP, and ferulic acid supplements significantly increased the excretion of acidic sterol compared to the control group. These results would suggest that, in highcholesterol fed rats, the supplementation of hesperetin and its metabolites promotes sterol excretion through the enhanced excretion of acidic sterol. Previous studies using 3-[14C]-hesperetin in rats indicate that the intestinal absorption of aglycone flavonones may be greater than 90% [8]. Intestinal microflora have been shown to further metabolize aglycone flavonones [27], for instance converting hesperetin into m-HC, 3,4-DHPP, and ferulic acid in the intestine. It is interesting that a major route for flavonoid metabolism in rats has been shown to be excretion in bile [43]. This generally occurs following the conjugation of flavonoid polar hydroxyl groups with glucuronic acid, sulfate, or glycine. When flavonoids are present in the bile, they can either be excreted or reabsorbed. In support of the hepatic regulation of hesperetin metabolites, it was previously demonstrated that approximately 40% of a dose of hesperetin-3-[14C], given either orally or intraperitoneally to rats, was recovered as 14C, a byproduct of hepatic metabolism [8], suggesting that at least 40% of the hesperetin dose reached the liver. Therefore,

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further studies are required to examine the distribution of hesperetin in various tissues. Together with the inhibition of ACAT and HMGCoA reductase, the current results suggest that hesperetin and its metabolites, m-HC, 3,4-DHPP, and ferulic acid, are all very potent for lowering plasma lipids. Accordingly, hesperetin and its metabolites supplementation (0.066 mmol/100 g diet) may provide potential benefits to animal fed high-cholesterol diet through improving the lipid metabolism. However, more extensive investigation of the in vivo action of hesperetin metabolites in animal models and humans is still required to further elucidate the usefulness of these flavonoids.

Acknowledgements This work was supported by a grant from the Korean Ministry of Science and Technology (M10015-00-0012, 01-J-BP-01-B-66).

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