Download Antiatherogenic Properties of Naringenin, a Citrus Flavonoid

Cardiovascular Drug Reviews
Vol. 17, No. 2, pp. 160–178
© 1999 Neva Press, Branford, Connecticut
Antiatherogenic Properties of Naringenin, a
Citrus Flavonoid
Lisa J. Wilcox, Nica M. Borradaile, Murray W. Huff
The Departments of Medicine and Biochemistry and The John P. Robarts Research Institute,
University of Western Ontario, London, Ontario N6A 5K8, Canada.
Key Words: Naringenin—Flavonoids—Hesperetin—Atherosclerosis—Lipids—Antioxidant.
INTRODUCTION
An inverse association between flavonoid intake and coronary heart disease (CHD) has
been suggested by a number of epidemiological studies (42,44,54,78). As a consequence,
there is considerable interest in investigating the antiatherogenic nature of these compounds. Flavonoids are naturally occurring molecules abundant in fruit, vegetables, nuts,
seeds, and beverages, such as tea and wine. Over 4000 different flavonoids have been
identified in the human diet, where the daily intake ranges from levels as high as 1 g (59)
to a more conservative estimate of 23 mg (43). Flavonoids are characterized by their
polyphenolic structure (Fig. 1). Variations in this structure give rise to the major classes
of flavonoids, including the flavonols, flavones, flavanones, catechins, anthocyanidins,
isoflavones, dihydroflavonols, and chalcones.
Naringenin belongs to the class of flavonoids called the flavanones. The flavanones are
abundant in citrus fruits such as grapefruit (Citrus paradisi) and the oranges (Citrus
sinensis). The role of naringenin and the related citrus flavanone hesperetin in the prevention and treatment of disease has recently received considerable attention (71), with
particular interest in the use of these flavanones as anticancer (36) and antiatherogenic
(85) compounds. This review focuses on the potential antiatherogenic roles of citrus
flavonoids, with particular focus on naringenin.
CHEMISTRY
The chemical name of naringenin is 2,3-dihydro-5,7-dihydroxy-2-(4-hydroxyphenyl)4H-1-benzopyran-4-one (Fig. 1), and it has a molecular weight of 272.26 (C15H12O5).
Naringenin is almost insoluble in water and is soluble in organic solvents such as alcohol.
Naringenin is derived from the hydrolysis of glycone forms of this flavanone, such as
naringin or narirutin (67). Naringin (naringenin-7-rhamnoglucoside), the bitter principle
Address correspondence to Dr. M. Huff. The John Robarts Research Institute, 4-16, University of Western
Ontario, 100 Perth Drive, London, Ontario N6A 5K8, CANADA. Fax: 519-663-3789 E-mail: [email protected]
160
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FIG. 1. Chemical structures of naringenin and related flavonoids.
of grapefruit (Citrus paradisi), is found in the juice, flower, and rind of the fruit and
constitutes up to 10% of the dry weight. Unlike the aglycone naringenin, naringin is
relatively soluble in water (1 mg/mL in water at 40°C) (67). Naringin is present in
grapefruit juice at concentrations of up to 800 mg/L (81) and occurs as a mixture of chiral
isomers that vary markedly in proportion depending on the maturity of the fruit and the
method of purification. Naringin and other naringenin glycosides can be found in a variety
of other sources including propolis (73) and Prunus davidiana (14). Monotes engleri
contains a prenylated form of naringenin (6-(1,1-dimethylallyl)naringenin) (86).
A number of flavonoids including hesperetin, are structurally related to naringenin.
Hesperetin, S-2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one (Fig. 1), has a molecular weight of 302.28 (C16H14O6). Like naringenin,
hesperetin is relatively lipophilic, being soluble in organic solvents and only slightly
soluble in water (67). Hesperetin, which differs from naringenin by the substitution of a
methoxy group at position R6 and the addition of a hydroxy group at position R5, is
derived from the hydrolysis of its glycone form hesperidin (hesperetin 7-rhamnoglucoside) (67). Hesperidin is the predominant flavonoid in lemons and sweet oranges. As a
citrus flavanone, hesperetin might possess antiatherogenic actions in common with the
structurally related naringenin. Therefore, this review will also briefly describe the antiatherogenic properties of hesperetin.
PHARMACOKINETICS
As naringenin is generally present in foods bound to sugars as ␤-glycosides (i.e.,
naringin), it was originally thought that absorption from the diet would be negligible.
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However, a number a studies have detected naringenin in human urine (3,29,50,64,92) and
plasma (3,29) following oral doses of pure naringin (3,50) or grapefruit juice (3,29,64,92).
Hesperetin has been detected also in human urine (3,92) and plasma (3) following doses
of pure compound or orange juice. These results showed that naringenin and hesperetin
can be absorbed from the diet. In fact, studies using 3-[14C]-hesperetin in rats indicate that
intestinal absorption of aglycone flavanones may be greater than 90% (47).
The glycoside form of naringenin, naringin, is not detected in either human or animal
urine suggesting that naringin is deglycosylated prior to intestinal absorption. It has been
shown that the intestinal microflora of rats (9,34) and humans (29,46) can cleave the
glycosidic bonds of naringin, liberating the aglycone form naringenin. Studies by Furh et
al. (29) have shown large interindividual differences in the ability of humans to convert
naringin to naringenin as evident in feces, suggesting that the presence or absence of
certain bacterial strains in the gut may explain the interindividual variability observed in
studies examining grapefruit-juice–drug interactions (6) (discussed later). In a recent
study, Kim et al. (46) identified a number of bacteria in the human intestine that are
capable of transforming naringin to naringenin and hesperidin to hesperetin, including
Fusobacterium K-60, Eubacterium YK-4, and Bacteroides JY-6.
Intestinal microflora have been shown to further metabolize naringenin. Booth et al. (9)
originally showed that oral doses of naringin administered to rats yielded 4-hydroxyphenylpropionic acid, 4-hydroxycinnamic acid, and 4-hydroxybenzoic acid sulfate in the urine.
Griffiths and Smith (34) showed that rat intestinal microflora resulted in the generation of
4-hydroxyphenylpropionic acid and naringenin only, suggesting that 4-hydroxycinnamic
acid and 4-hydroxybenzoic acid may be metabolites that are generated by hepatic enzymes. Honohan et al. (47) showed that the major intestinal metabolite of 3-[14C] hesperetin in rats was 3-phenylpropionic acid, while hepatic enzymes mediated further breakdown to benzoic acid and CO2. More recently, Kim et al. (46) have shown that human
intestinal bacteria can metabolize naringin to naringenin and then to 4-hydroxybenzoic
acid, phloroglucinol, 2,4,6-trihydroxybenzoic acid, and 4-hydroxyphenylacetic acid, demonstrating species differences in the intestinal metabolism of naringin and naringenin.
A large percentage of naringenin absorbed in humans appears in the urine as naringenin
glucuronides (29,50,64), indicating that conjugation, presumably within the intestine or
liver (29), may play a major role in the metabolism of this compound. Naringenin has also
been reported to be a substrate for a UDP glucuronosyl transferase (33). Furh et al. (29)
showed that excretion of naringenin glucuronides in humans reaches levels more than
100-fold higher than the concentration of naringenin excreted in the urine. Hackett et al.
(38) have shown that a major route for flavonoid metabolism in rats is excretion in the
bile. This generally occurs following conjugation of flavonoid polar hydroxyl groups with
glucuronic acid, sulfate, or glycine. Naringenin present in the bile may either be excreted
or reabsorbed, therefore raising the possibility of enterohepatic recycling of naringenin.
In addition to conjugation, naringenin may be further metabolized by hepatic enzymes.
Nielsen et al (74) have recently examined the metabolism of naringenin and hesperetin in
rat liver microsomes. The major metabolite observed for both naringenin and hesperetin
was the flavonoid eriodictyol. Eriodictyol was generated by the addition of a hydroxy
group to the R6 position of naringenin and the demethylation of the R6 methoxy of
hesperetin. The hydroxylation of naringenin was shown to be mediated by hepatic cytochrome P450 1A (CYP1A) (74).
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Naringenin has been detected in the plasma following oral administration of naringin or
grapefruit juice but is generally reported to be below accurate detection limits (3,29) and
has not been reported to exceed 4 ␮M (29). However, due to the lipophilic nature of
naringenin, it is possible that it accumulates within tissues, particularly membranes, and
eventually reaches greater concentrations than those observed in the plasma. This accumulation would most likely occur in tissues such as the liver and intestine. In support of
this, it has been demonstrated that approximately 40% of a dose of hesperetin,-3-[14C]
given either orally or intraperitoneally to rats, was recovered as 14CO2, a by-product of
hepatic metabolism (47). This suggests that at least 40% of the hesperetin dose reached the
liver. Similar experiments using radiolabelled naringenin in either humans or rats have not
yet been reported. Clearly further studies are required to examine the tissue distribution of
naringenin.
TOXICITY
Toxicity studies of naringenin are scarce; however, flavonoids are generally considered
to have low toxicity. In one study (9), a single, 2-g oral dose of naringin was administered
to a human volunteer with no deleterious effects. Naringin has also been administered to
humans at oral doses of 500 mg with no adverse responses (3,50). Kim et al. (46) have
reported on the in vitro cytotoxicity for various flavonoids in a number of cells lines. The
IC50 (50% inhibition concentration) for cell growth was > 1 mM for both naringenin and
hesperetin in the human hepatoma cell line HepG2, the Macacus’ rhesus monkey kidney
cell line MA-104, and the human lung cancer cell line A549. The results of these studies
indicate that these flavonoids have relatively low toxicity in cell culture.
POTENTIAL CARDIOPROTECTIVE ACTIVITIES
An association between flavonoid consumption and reduced cardiovascular mortality
was first revealed in the Zutphen Elderly study, in which 805 men from the Netherlands,
aged 65–84 years, were studied over 5 years (42). Flavonoid consumption (with quercetin
and kaempferol representing 95% of the measured flavonoid intake) was inversely associated with mortality from CHD. Relative risks for CHD mortality and first myocardial
infarction were approximately 50% lower in the highest tertile of flavonoid intake (mean
intake 41.6 mg/day) compared to the lowest tertile (mean intake 12.0 mg/day) (p ⳱ 0.015,
95% Cl 0.20–0.88). Further results from the Zutphen Elderly study (54), showed an
inverse association between dietary flavonoid intake and the incidence of stroke. Similar
associations between flavonoid intake and reduced CHD were reported in men and women
from Finland (57) and men from the Seven Countries Study, which included cohorts from
Finland, Greece, Yugoslavia, Japan, Netherlands, Italy and the United States (44). The
relationship between intake of flavonols and flavones and the risk for fatal and nonfatal
CHD was examined in the Health Professionals Follow-up Study (78), which involved
34,789 male health professionals. The intake of flavonoids was not associated with risk for
total CHD; however, a nonsignificant trend for an inverse association with CHD mortality
and flavonoid intake was observed in men with previous CHD. In contrast to these reports,
a recent study by Hertog et al. (45) revealed increased mortality from ischemic heart
disease in Welsh men consuming high amounts of flavonols, mainly from tea. Therefore,
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although inconclusive, the epidemiological evidence suggests a protective effect that
flavonoids have against cardiovascular disease. To date, no study has specifically examined the association between the intake of flavanones, the group of flavonoids to which
naringenin belongs, or citrus flavonoids and CHD.
ANTIOXIDANT, ANTITHROMBOTIC, ANTIINFLAMMATORY, AND
VASODILATORY ROLES IN ATHEROSCLEROSIS
Much of the observed association between flavonoid intake and reduced CHD mortality
has been attributed to their antioxidant properties (25,76,84). Cells of the arterial wall
including macrophages, smooth muscle cells, and endothelial cells can oxidize or otherwise modify low density lipoproteins (LDL) (40,83). Modified LDL can be a ligand for
receptor-mediated processes leading to significant accumulation of cholesteryl esters (CE)
in macrophages and smooth muscle cells (40,83). These CE-rich cells, known as foam
cells, are the hallmark of the early atherosclerotic lesion. Central to this issue is whether
flavonoids are present within the subendothelial space of the arterial wall in concentrations sufficient to protect lipoproteins such as LDL from oxidation. There is some evidence to suggest that flavonoids can be incorporated into lipoproteins within the liver or
intestine and subsequently be transported within the lipoprotein particle (30,55). Naringenin has been shown also to associate with and penetrate lipid membranes (84). Therefore flavonoids, including naringenin, may be ideally located for protecting LDL from
oxidation.
Flavonoids have been shown to inhibit the oxidation of LDL in vitro (18,19,27,39, see
ref. 5 for review). Furthermore, the addition of the flavonoids quercetin and catechin to the
diet have been shown to reduce LDL oxidation ex vivo in rats (27) and was found to
decrease atherosclerotic lesion area in apoE-deficient mice (39). The mechanisms
whereby flavonoids inhibit LDL oxidation are unclear. They may protect ␣-tocopherol in
LDL from oxidation, possibly by being preferentially oxidized themselves, or they may
reduce the formation or release of free radicals. Flavonoids can react with superoxide
anions (1), hydroxyl radicals (49), and lipid peroxy radicals (91). These compounds may
also act by chelating iron (1,72) which is thought to catalyze processes leading to the
appearance of free radicals.
A number of studies have investigated the ability of flavonoids to act as antioxidants.
Saija et al. (84) found that naringenin was able to inhibit Fe2+-induced linoleate peroxidation with an IC50 of 565 ␮M and autooxidation of rat cerebral membranes (ARCMs)
with an IC50 of 322 ␮M. The inhibitory activity of naringenin was relatively weak
compared to the structurally-related flavanone hesperetin, which inhibited Fe2+-induced
peroxidation and ARCM, with IC50s of 17 ␮M and 148 ␮M, respectively. Interestingly,
this study demonstrated also that naringenin and hesperetin interact with liposomes of
dipalmitoylphosphatidyl-choline. This may result from an ability of naringenin to anchor
to the polar head groups of phospholipids or to penetrate and associate with the lipid
bilayer. This suggests that some physiological effects of naringenin may be caused by its
interaction with biological membranes.
Naringenin has been shown also to inhibit microsomal lipid peroxidation (IC50 of 465
␮M) (63), nonenzymatic lipid peroxidation (33% inhibition at a concentration of 1 mM)
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(76), and ascorbic acid-induced malondialdehyde (MDA) formation (by 21% at a concentration of 100 ␮M) (Fig. 2). Hesperetin, at the same concentration, showed a similar
level of inhibition. Naringenin, however, had no effect on ferrous sulfate–induced MDA
production (76). In the studies mentioned earlier, neither naringin nor hesperidin were able
to inhibit lipid peroxidation, suggesting that the effect was specific to aglycone flavonoids.
In addition to direct oxidant scavenging, flavonoids may inhibit enzymes involved in
generating pro-oxidant molecules. For example, flavonoids have been shown to inhibit the
generation or release of free radicals derived from lipoxygenase (LOX) (18). It has been
suggested that LOX is involved in the early events of atherosclerosis by inducing plasma
LDL oxidation in the subendothelial space of the arterial wall (18). Laughton et al. (63)
investigated the effects of the glycoside naringin on 5-LOX and cyclooxygenase (COX)
in rat peritoneal leukocytes, which generate products of both LOX and COX pathways
when activated. Naringin was found to be a poor inhibitor of 5-LOX (IC50 > 500 ␮M) and
COX (IC50 ⳱ 320 ␮M). In a related study, Corvazier and Maclouf (17) demonstrated that
naringenin (500 ␮M) is an irreversible inhibitor of both LOX and COX pathways. Furthermore, naringenin has been shown to inhibit myeloperoxidase (MPO) (approximate
IC50 ⳱ 150 ␮M) (22). MPO secreted by macrophages and activated neutrophils is a
potent catalyst of LDL oxidation in vitro and colocalizes with macrophages in human
atherosclerotic lesions (40). MPO generates a range of reactive species, including the
potent oxidizing agent hypochlorous acid (40). Therefore, naringenin in addition to direct
antioxidant roles, may inhibit the oxidation of LDL by mechanisms involving inhibition
of LOX, COX and MPO.
Recent studies by Hayek et al. (39) demonstrated that cholesteryl ester accumulation in
cultured macrophages incubated with LDL derived from mice consuming the flavonoids
FIG. 2. Effect of naringenin and hesperetin on malonaldehyde (MDA) formation induced by 1.0 mM ascorbic
acid in rat brain mitochondrial suspensions. The flavonoids were examined at concentrations of 0.1 mM, 1 mM,
and 4 mM and the values are expressed as percentage inhibition relative to controls. Adapted from ref. 76.
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catechin and quercetin was significantly lower than the accumulation observed in the
presence of LDL from control mice. This effect has been attributed to the reduced
oxidative modification of LDL. Whether a similar mechanism occurs with the intake of
flavonoids, such as naringenin, is unknown. Furthermore, it has not been determined if
naringenin has a direct effect on foam cell formation, independent of an effect on LDL.
As discussed later, we have recently shown that naringenin inhibits cholesterol esterification in cultured hepatocytes (94). It is possible that a similar mechanism exists in
macrophages and smooth muscle cells.
Flavonoids have been shown to have a number of antithrombotic actions (17,62). The
effect of flavonoids on the oxidative metabolism of arachidonic acid (AA) in human
platelets and neutrophils was investigated by Corvazier and Maclouf (17). Naringenin was
found to inhibit thromboxane B2 production in platelets stimulated with either thrombin
or AA with an IC50 of approximately 175 to 200 ␮M, whereas the glycoside form naringin
was inactive. Naringenin also inhibited the formation of oxygenated metabolites in platelets stimulated with thrombin and inhibited AA-induced platelet aggregation with an IC50
of 500 ␮M. Landolfi et al. (62) demonstrated that naringenin is a weak inhibitor of AAor collagen-induced platelet aggregation (IC50 of 90 ␮M and > 200 ␮M, respectively), but
it had no effect on the production of thromboxane B2, 12-hydroxyheptadecatrienoic acid
(HHT), or hydroxyeicosatetraenoic acid (HETE) from AA. Nevertheless, naringenin appears to be a relatively weak inhibitor of these processes when compared to the flavonol
quercetin, which inhibits platelet aggregation with an IC50 of 25 ␮M and thromboxane B2
production with IC50 of 44 to 55 ␮M (17).
Flavonoids have also been shown to have antiinflammatory activities (68). The antiinflammatory roles of naringenin may be of particular interest with respect to atherosclerosis, as this disease is increasingly being viewed as one with a strong inflammatory
component (for review see ref. 83). The earliest form of the atherosclerotic lesion, the fatty
streak, is an inflammatory lesion consisting of monocyte-derived macrophages and T
lymphocytes. It is tempting to speculate that flavonoids may be useful in inhibiting a wide
range of inflammatory responses, including those proposed to play roles in the progression
of CHD. Middleton and Kandaswami (68) have reviewed the effect of flavonoids on
immune and inflammatory functions. Flavonoids can effect the function of T cells, B cells,
NK cells, macrophages, mast cells, basophils, neutrophils, eosinophils and platelets, each
of which are involved in immunity and inflammation. Cytokines and growth factors
secreted by these cells may control processes involved in atherosclerosis, such as macrophage activation, scavenger receptor expression, smooth muscle proliferation, and nitric
oxide production (80). Habtemariam (37) examined the effect of flavonoids on TNF␣–
induced cytotoxicity in murine fibroblast L-929 cells. Although eriodictyol, a hepatic
metabolite of naringenin, offered protection against TNF-induced cytotoxicity with an
EC50 (50% effective concentration) of 4–6 ␮M, naringenin itself was not protective and
actually potentiated the effect when examined at concentrations up to 250 ␮M. Some
flavonoids also exert antiinflammatory effects by inhibiting cytokine-induced gene expression (31). Apigenin (25 ␮M), the flavone equivalent of naringenin, blocks the cytokine-induced upregulation in human endothelial cells of intercellular adhesion molecule
(ICAM-1) and vascular adhesion molecule (VCAM-1), both of which have been implicated in inflammation and atherosclerosis. However, at concentrations up to 50 ␮M,
naringenin did not inhibit the expression of these molecules (31). Therefore, although
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related flavonoids may affect cytokine-induced gene expression, this has not yet been
demonstrated for naringenin.
Flavonoids may have beneficial effects on cardiovascular diseases involving vasodilation. Herrera et al. (41) examined the effects of flavonoids on the noradrenaline-, KCl-,
and phorbol myristic acid (PMA)-induced contractions of rat aortic rings (10−5M, 80␮M,
and 10−7M, respectively). Naringenin (IC50 ⳱ 46 ␮M to 96 ␮M) and hesperetin (IC50 ⳱
88 ␮M to 139 ␮M) displayed a concentration-dependent inhibition of the agonist-induced
contractile responses. Sodium nitroprusside potentiated the vasodilation by naringenin,
therefore suggesting the potential involvement of cGMP-specific phosphodiesterases. The
ability of naringenin to block the effect of PMA suggests that naringenin may inhibit
contraction through inhibition of protein kinase C (PKC).
LIPID AND LIPOPROTEIN METABOLISM
While the majority of research has focused on the antioxidant roles of flavonoids, a
number of reports have suggested that these compounds may also influence atherogenesis
through an effect on lipid and lipoprotein metabolism (4,8,10,14,15,51,88,94,96). Investigation of naturally occurring compounds as regulators of triglyceride and cholesterol
metabolism has particular therapeutic importance, as evidenced by the discovery of the
first HMG-CoA reductase inhibitors derived from fungal fermentation products, which are
now widely used for the treatment of hyperlipidemia. Flavonoids might represent another
beneficial group of naturally occurring hypolipidemic compounds. Studies in rats have
shown that the flavonoids quercetin (8), hesperidin (70), marsupin (51), pterosupin (51),
liquiritigenin (51), biochanin A (88), formononetin (88), and pratensein (88) cause significant reductions in serum total cholesterol (TC) and triglyceride (TG). In nonhuman
primates, dietary genistein (Figure 1), the isoflavone analog of naringenin, significantly
reduces plasma LDL and VLDL cholesterol levels (4).
Studies in hyperlipidemic rats (14) fed high-fat diets showed that i.p. administration of
a methanolic extract from Prunus davidiana and its flavonoid components catechin,
naringenin 7-O-glucoside (prunin), and hesperetin 5-O-glucoside for 3 days resulted in a
significant reduction in blood TG and TC. Furthermore, naringenin 7-O-glucoside or
hesperetin 5-O-glucoside, when administered alone at doses of 20 mg/kg and 10 mg/kg,
respectively, showed significant hypocholesterolemic effects. In a related study by the
same investigators (15), the effects of naringenin 7-O-glucoside (prunin) in streptozotocin-diabetic rats was examined. Single i.p. administration of prunin (10 mg/kg) caused a
significant decrease in plasma glucose, TG, and TC (Fig. 3).
In a recent study Kurowska et al. (61), reported that replacing the drinking water of
rabbits with either grapefruit or orange juice resulted in significant reductions in the
elevation of serum LDL cholesterol and hepatic cholesteryl ester (CE) levels induced by
feeding a semipurified, cholesterol-free, casein-containing diet. This model is characterized by an overproduction of hepatic apoB-containing lipoproteins (56). It was hypothesized that the hypocholesterolemic effects of the juices were due to their flavonoid
components (naringenin in grapefruit juice and hesperetin in orange juice). Subsequently,
the effects of these flavonoids on the secretion of apolipoprotein B(apo B)-containing
lipoproteins by the human hepatoma cell line HepG2 was examined. Naringenin and
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FIG. 3. Effect of a single dose administration of naringenin 7-O-␤-D-glucoside on the concentrations of serum
glucose, triglyceride, and cholesterol in streptozotocin-diabetic rats. Values were determined 5 h after naringenin
administration and are reported as the mean ± SEM for six animals; p < 0.05. Adapted from ref. 15.
hesperetin dose-dependently reduced the accumulation of apoB in the culture media
(76–81% at 200 ␮M) over 24 hours (10). Reduced hepatic secretion of apoB-containing
lipoproteins would be expected to contribute to a hypocholesterolemic effect of naringenin
in vivo.
Recent studies by Wilcox et al. (94) have further investigated the effect of naringenin
on lipid and lipoprotein metabolism in HepG2 cells. Significant reductions in apoB
secretion were observed (83% reduction at 200 ␮M, p < 0.002) (Fig. 4) after a 24-hour
incubation with naringenin. Importantly, we demonstrated that naringenin (200 ␮M)
reduced the secretion of newly synthesized CE, TG, and phospholipid (PL) (Fig. 5).
Naringenin (200 ␮M) significantly reduced intracellular CE mass, which was largely due
to a dose-dependent inhibition of CE synthesis (up to 89% at 200 ␮M of naringenin) (Fig.
4). The mass of free cholesterol and TG were unaffected. This effect on hepatic CE
synthesis may be explained, in part, by an inhibition of hepatic ACAT as evidenced by a
reduction in ACAT activity in isolated porcine hepatic microsomes (IC50 ⳱ 200 ␮M).
Previously, we have shown that inhibition of ACAT reduces the hepatic production of
apoB-containing lipoproteins (11), possibly by limiting the availability of newly synthesized CE for association with apoB during assembly of the lipoprotein. Therefore, inhibition of hepatic ACAT may be the mechanism whereby naringenin exerts its hypocholesterolemic effects. Inhibition of hepatic ACAT has also been demonstrated for a flavonoid structurally different from naringenin, baicalein (IC50 of approximately 100 ␮M in
isolated rat hepatic microsomes) (96). Whether reduced apoB secretion and inhibition of
ACAT activity are general features of flavonoids remains to be determined.
Inhibition of ACAT activity and apoB secretion by naringenin might occur via a direct
inhibition of ACAT, following receptor-mediated signalling events or following direct
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FIG. 4. A. Effect of naringenin (10–200 ␮M) on apoB secretion and cholesterol esterification by HepG2 cells.
The accumulation of apoB in the media was measured using a noncompetitive ELISA assay following a 24 h
incubation with naringenin. B. [14C]Oleic acid (0.08 ␮Ci) incorporation into cholesteryl esters was determined
after a 5 h incubation in the presence of [14C]oleic acid and naringenin. Adapted from ref. 94.
interaction with cellular kinases (68). Another possible mechanism may be through interaction with the plasma membrane transporter, multidrug resistance p-glycoprotein (pgp). Recently, Conseil et al. (16) have shown that flavonoids can bind mouse p-gp and
modulate its activity. Flavonoids are thought to have bifunctional interactions at the
vicinal ATP-binding site and steroid-interacting regions of p-gp (16). Naringenin was
shown to bind purified p-gp with 50% binding at 28.5 ␮M (16). Binding at the ATPbinding site is thought to antagonize ATP binding and thus inhibit the ATPase activity of
this protein. This may contribute to the ability of naringenin to inhibit the activity of p-gp
(87). Inhibition of p-gp activity has been shown to inhibit cholesterol esterification in cell
lines, including HepG2 (20) and the intestinal cell line CaCo-2 (20). The secretion of apoB
in CaCo-2 cells was also found to be reduced following inhibition of p-gp. It is possible
that this mechanism contributes to the inhibition of cholesterol esterification and apoB
secretion by naringenin in HepG2 cells.
In further studies from our laboratory, we discovered a potentially novel mechanism for
the regulation of apoB secretion by naringenin. Naringenin (200 ␮M) caused a significant
50% decrease in the mRNA for the microsomal triglyceride transfer protein (MTP) (94),
a protein known to be essential for the assembly and hepatic secretion of apoB-containing
lipoproteins (93). Together with the inhibition of ACAT (discussed earlier), these results
suggest potential mechanisms whereby naringenin may reduce the plasma concentrations
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FIG. 5. Effect of naringenin (200 ␮M) on the incorporation of [14C]acetic acid (0.5 ␮Ci) into secreted cholesteryl ester, triglyceride, phospholipid, and free cholesterol from HepG2 cells. Values are given as the mean
± SEM from experiments with triplicate samples. From ref. 94.
of apoB-containing lipoproteins, such as VLDL and LDL. Whether naringenin produces
similar effects in vivo remains to be determined.
ESTROGENIC ROLE
Flavonoids which contain a phenolic group and 6-membered rings with different degrees of desaturation, such as naringenin, structurally resemble estradiol and other steroid
hormones, thyroid hormone, retinoic acid, nucleosides, and folic acid. This structural
similarity raises the possibility that flavonoids or their metabolites could bind steroid
receptors. Although specific flavonoid receptors have not been identified, naringenin and
other flavonoids can bind to the estrogen receptor (7,60,69). Using an estrogen receptor
reporter construct, Balaguer et al. (7) showed that naringenin elicits a dose-dependent
induction in activity with maximal activity at 50 ␮M and an EC50 of 1 uM. Furthermore,
Kuiper et al. (60) showed that naringenin can bind to both estrogen receptors, ER␣ and
ER␤. Importantly, naringenin competed more effectively with 17-␤-estradiol for binding
to ER␤ (IC50 for competition ⳱ 590 nM) than for ER␣. Interaction of naringenin with
ER␤ may be relevant for cardiovascular effects as this receptor is present in significant
amounts in arterial tissue (77). In fact, estrogen has been reported to have a number of
beneficial cardiovascular effects. Estrogen has been shown to protect against foam cell
formation (90), and estrogen replacement has also been shown to reduce CHD risk (21)
and plasma TC and LDL cholesterol in postmenopausal women (12,95). This reduction
has been attributed, in part, to increased clearance of LDL (12,95). This is consistent with
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171
the observation that estrogen induces an increase in hepatic LDL receptor expression (58).
Whether naringenin affects clearance of apoB-containing lipoproteins by mechanisms
similar to those mediated by estrogen is unknown.
Flavonoids, including naringenin, have been shown to bind to the estrogen receptor
with binding affinities of 1000- to 10000-fold less than that of 17-␤-estradiol (60). Therefore, the question arises as to whether the in vivo concentrations can reach sufficiently
high concentrations to exert a biological effect. However, flavonoids may accumulate in
fatty tissue and reach concentrations sufficient to activate steroid nuclear receptors. Alternatively, the flavonoid could be metabolized to a form with enhanced estrogen activity.
Naringenin may also influence estrogenic actions by altering the metabolism of the steroid. Kao et al. (53) have shown that naringenin competitively inhibits human aromatase
(estrogen synthetase), the enzyme that converts androgen to estrogen, with a Ki of 5.1 ␮M
(53). Huang et al. (48) report that naringenin inhibits estrone sulfatase, a key enzyme in
17-␤-estradiol synthesis, with an IC50 < 10 ␮M in human hepatic microsomes. Naringenin and hesperetin were found to inhibit rat hepatic microsomal glucuronidation of
estrone and estradiol with an IC50 of approximately 25 ␮M (97). Relative to other flavonoids, naringenin and hesperetin were potent competitive inhibitors of estrone glucuronidation at a flavonoid concentration of 10 ␮M and noncompetitive inhibitors at a
concentration of 50 ␮M.
Ruh et al. (82) showed that, in addition to its weakly in vitro estrogenic effects, (Fig.
6), naringenin exhibits antiestrogenic activity in vivo. In rats, naringenin (30 mg/rat) was
shown to inhibit the 17-␤-estradiol–induced increase in uterine weight, induction of
progesterone receptor binding, [3H]thymidine uptake, and uterine peroxidase activity.
Naringenin also attenuated the estrogen-induced increase in cell proliferation in MCF-7
human breast cancer cells. These studies showed that, in addition to estrogenic effects,
naringenin may significantly attenuate estrogenic activity. The balance between these two
actions in vivo remains to be determined.
In addition to the estrogenic actions discussed above, naringenin may also act as a weak
progestin. Rosenberg et al. (79) have shown that naringenin (10 ␮M) acts as a weak
progestin in mammary cancer cell lines. Naringenin was approximately 104-fold less
potent than the agonist norgestrel. In contrast, the related flavanone hesperetin acts as an
antiandrogen and antiprogestin at concentrations of 10 ␮M. The reason for the difference
between naringenin and hesperetin, which are structurally quite similar, is unclear.
SIGNAL TRANSDUCTION PATHWAYS
The citrus flavonoids may interact directly with intracellular signal transduction pathways. Since second messenger signalling plays an important role in the development and
progression of atherosclerosis (75), it can be predicted that the modulation of these
signalling pathways may alter the progression of the disease. For example, the interaction
of compounds such as flavonoids with the proteins involved in signal transduction could
modulate the expression of inflammatory molecules within the lesion and/or modulate the
activity and/or expression of proteins involved in cholesterol trafficking in smooth muscle
and immune cells within the lesion, as well as in hepatocytes. The growth factor- and
cytokine-mediated signalling involved in the atherogenic process was recently reviewed in
detail by Pomerantz et al. (75). Briefly, ligand activation of receptor tyrosine kinase or
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172
L. J. WILCOX ET AL.
FIG. 6. Effect of naringenin on pS2-luciferase reporter vector activity in MCF-7 human breast cancer cells.
Estradiol induction of pS2 gene expression is mediated by a palindromic estrogen responsive element-like
sequence that acts as an enhancer for the nuclear estrogen receptor. Values are the means ± SEM of four
measurements for each treatment and are reported as a percentage of induction relative to 1 nM estradiol.
Adapted from ref. 82.
membrane-associated G-protein–mediated signalling pathways can lead to the formation
of inflammatory mediators such as prostaglandins and leukotrienes via phosphospholipase
A2 (PLA2). Signalling pathways can also regulate the expression and/or activity of a
number of proteins involved in cholesterol trafficking, including the LDL-receptor, the
scavenger receptor, HMG CoA-reductase, ACAT, and HDL-receptors.
Recently, a number of flavonoids, including hesperetin, have been shown to inhibit
PLA2 activity in vitro (66). In general, hydroxyl groups at positions 5, 6, and 7 on the A
ring have been implicated in the PLA2-inhibitory activity of flavonoids (13,32,66). This
is of interest since naringenin, like hesperetin, has hydroxyl functional groups at positions
5, and 7 (which correspond to R2 and R3 in Figure 1): however, the ability of naringenin
to inhibit this enzyme has not been examined. Neither naringenin nor hesperetin have been
shown to significantly alter phosphatidylinositol 3-kinase (Pl3 kinase) activity (2) or PKC
activity (2,26,89). The role of naringenin as an inhibitor of rat brain PKC was investigated
by Ferriola et al. (26). Naringin and hesperetin showed only a small (approximately 10%)
and nonsignificant inhibition of PKC activity. Although the effect of the active metabolite
of naringin, naringenin, was not investigated, it has been reported that naringenin inhibits
PKC activity by less than 20% in human breast cancer cells (89). Similar results have been
observed with hesperetin (89). Collectively, these observations indicate that naringenin is
not an effective inhibitor of PKC activity. The effects of the citrus flavonoids on other
Cardiovascular Drug Reviews, Vol. 17, No. 2, 1999
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173
signal transduction enzymes, such as phospholipase C (PLC) and mitogen-activated protein kinase (MAPK), have not yet been examined. Further research is also required to
determine whether the observed inhibition of ACAT activity by naringenin (94) could be
partially due to modulation of signalling pathways involved in the regulation of expression
of this enzyme.
NARINGENIN DRUG INTERACTIONS
Naringenin-drug interactions and the effects of naringenin on cytochrome P450 enzymes were first suggested in studies that investigated the effects of grapefruit juice on the
metabolism of dihydropyridine calcium channel blockers (6). It was subsequently shown
that grapefruit juice increased the oral bioavailability of felodipine (6) and cyclosporin
(23). Inhibition of selected cytochrome P450 isozymes by citrus flavonoids may explain
the altered bioavailability of some coadministered drugs. More direct evidence to indicate
that naringenin effects drug metabolism was provided by studies that demonstrated a
direct inhibition of specific cytochrome P450 enzymes (24,35) by the aglycone naringenin. The glycoside naringin (the primary form of naringenin in grapefruit juice) did not
affect human cytochrome P450 activities in vitro (28,35). Fuhr et al. (28) reported that
naringenin is a potent competitive inhibitor of CYP1A2-mediated caffeine 3-demethylation in hepatic microsomes. Guengerich et al. (35) have shown that naringenin competitively inhibits CYP3A4 in vitro (approximate IC50 100 ␮M), in human liver microsomes.
Hesperetin, although less effective than naringenin, was also shown to possess CYP3A4inhibitory activity. Therefore, naringenin may increase the oral bioavailability of drugs
with marked first-pass metabolism that involve metabolism by CYP3A4 or CYP1A2.
Naringenin-drug interactions may have important consequences in relation to the potential antiatherogenic uses of flavonoid compounds in hyperlipidemic individuals. Grapefruit juice has been shown to increase the serum concentrations of simvastatin (65) and
lovastatin (52), two HMG-CoA reductase inhibitors commonly used for the treatment of
hypercholesterolemia. This is thought to occur by inhibition of CYP3A4-mediated firstpass metabolism of the HMG-CoA reductase inhibitors in the small intestine, as discussed
earlier.
SUMMARY
There is significant experimental evidence to suggest that naringenin and other citrus
flavonoids may be potentially useful as pharmacological agents in the treatment or prevention of atherosclerosis. However, due to the substantial metabolism of naringenin by
intestinal and hepatic mechanisms, it is not clear whether orally administered naringenin
will reach the systemic circulation and other tissues in sufficient concentrations to influence the many metabolic pathways involved in atherogenic processes. While orally administered naringenin might influence apoB-containing lipoprotein production from the
intestine, as we have shown in hepatocytes, the effects of orally administered naringenin
on hepatic apoB production in vivo requires further investigation. In light of the inhibitory
effects of naringenin on intestinal CYP450 enzymes, oral administration of naringenin as
an antiatherogenic drug may have important implications in individuals currently receiving drugs such as calcium channel blockers and statins.
Clearly naringenin possesses a number of antiatherogenic activities (Table 1), the
majority of which have been investigated in vitro. If these effects occur in vivo, naringenin
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L. J. WILCOX ET AL.
TABLE 1. Anti-atherogenic properties of naringenin
Effect
Antioxidant
Inhibit lipid peroxidation
Inhibit autooxidation of rat cerebral membranes
Inhibit lipoxygenases and cyclooxygenases
Inhibit myeloperoxidase
Antithrombotic and Vasodilatory
Inhibit thromboxane B2 production
Inhibit platelet aggregation
Inhibit smooth muscle cell contraction
Lipid Metabolism
Reduce apoB secretion from hepatocytes
Inhibit acyl-CoA:cholesterol acyltransferase
Steroid Hormone Activity/Metabolism
Agonist of estrogenic activity
Agonist of progesterone activity
Inhibit estrone sulfatase
Inhibit aromatase
Inhibit glucuronidation of estrone and estradiol
CYP450 Enzyme Activity
Inhibit CYP3A4
IC50/ED50
Reference
465–565 ␮M
322 ␮M
>200 ␮M
150 ␮M
(63,84)
(84)
(63,17)
(22)
175–200 ␮M
90–500 ␮M
46–96 ␮M
(17)
(62,17)
(41)
50–100 ␮M
50–100 ␮M
(10,94)
(94)
ⱕ1 ␮M
10 ␮M
ⱕ10 ␮M
5 ␮M
25 ␮M
(7,60)
(79)
(48)
(53)
(97)
100 ␮M
(35)
represents a potentially useful antiatherogenic agent by interfering with processes directly
related to the early events of atherosclerotic lesion formation including foam cell formation. However, more extensive investigation of the in vivo action of naringenin in animal
models of atherosclerosis and in humans are required to further elucidate the usefulness
of this flavanone.
Acknowledgments: This work was supported by a grant from the Heart and Stroke Foundation
of Ontario (T-3371 to M. W. Huff). L. J. Wilcox is a recipient of a Medical Research Council of
Canada Studentship. N. M. Borradaile is a recipient of a Heart and Stroke Foundation of Canada
Studentship. M. W. Huff is a Career Investigator of the Heart and Stroke Foundation of Ontario. We
thank Linda de Dreu, Cindy Sawyez, Dawn Telford for expert technical assistance.
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