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Vol. 52, Issue 4, 673-751, December 2000
,Chebeague Island Institute of Natural Product Research, Chebeague Island, Maryland (E.M., C.K.); and Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, Massachusetts (T.C.T.)
Abstract
I. General Aspects
A. Introduction
B. Synthesis
C. Metabolism and Disposition
D. Adverse Reactions
II. Effects on Mammalian Enzyme Systems
A. Kinases
B. Phospholipase A2
C. ATPases
D. Lipoxygenases and Cyclooxygenases
E. Phospholipase C
F. Cyclic Nucleotide Phosphodiesterase
G. Adenylate Cyclase
H. Reverse Transcriptase
I. HIV-1 Proteinase
J. HIV-1 Integrase
K. Ornithine Decarboxylase
L. Topoisomerase
M. Glutathione S-Transferase
N. Epoxide Hydrolase
O. Glyoxalase
P. Xanthine Oxidase
Q. Aromatase
R. 11--Hydroxysteroid Dehydrogenase
S. Catechol-O-methyltransferase
T. Aldose Reductase
U. Monoamine Oxidase (FAD-Containing)
V. Aldo-Keto-Reductase Family of Enzymes
W. Hyaluronidase
X. Histidine Decarboxylase and DOPA Decarboxylase
Y. Malate Dehydrogenase
Z. Lactic Dehydrogenase and Pyruvate Kinase
AA. Aldehyde and Alcohol Dehydrogenases
BB. Amylase
CC. RNA and DNA Polymerases
DD. Human DNA Ligase I
EE. Ribonuclease
FF. Sialidase
GG. Cytochrome P450 Systems
HH. Elastase
II. Nitric-Oxide Synthase
III. Modulation of the Functions of Inflammatory Cells
A. T Lymphocytes
B. B Lymphocytes
C. Natural Killer Cells
D. Macrophages and Monocytes
E. Mast Cells and Basophils
F. Neutrophils
G. Eosinophils
H. Platelets
I. Adhesion Molecule Expression
IV. Effects of Flavonoids on Other Cells
A. Smooth Muscle and Cardiac Muscle Cells
B. Effects on Nerve Cells
C. Calcium Homeostasis
V. Endocrine and Metabolic Effects
VI. Antiviral Effects
VII. Antitoxic, Hepatoprotective, and Cytoprotective Effects
VIII. Antioxidant Activity
A. Influence of Flavonoids on Reactive Oxygen Species Production by Phagocytic Cells
B. Effect of Flavonoids on Lipid Peroxidation and Oxyradical Production
IX. Actions in Relation to Coronary Artery Disease and Vascular Disorders
X. Flavonoid-Vitamin C Interactions
XI. Cancer-Related Properties
A. Microbial Mutagenicity Studies
B. Genetic Effects of Flavonoids in Mammalian Cells
C. Mutagenicity Studies in Vivo
D. Carcinogenicity of Flavonoids?
E. Anticarcinogenic Effects
F. Apoptosis and Cancer
G. Antiproliferative Activity
H. Differentiating Effects
I. Adhesion/Metastasis/Angiogenesis
J. Effect on Heat Shock Proteins
K. Effect on Multidrug Resistance
XII. Effects on Xenobiotic Metabolism
XIII. Concluding Remarks
Acknowledgments
References
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Abstract |
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Flavonoids are nearly ubiquitous in plants and are recognized as the pigments responsible for the colors of leaves, especially in autumn. They are rich in seeds, citrus fruits, olive oil, tea, and red wine. They are low molecular weight compounds composed of a three-ring structure with various substitutions. This basic structure is shared by tocopherols (vitamin E). Flavonoids can be subdivided according to the presence of an oxy group at position 4, a double bond between carbon atoms 2 and 3, or a hydroxyl group in position 3 of the C (middle) ring. These characteristics appear to also be required for best activity, especially antioxidant and antiproliferative, in the systems studied. The particular hydroxylation pattern of the B ring of the flavonoles increases their activities, especially in inhibition of mast cell secretion. Certain plants and spices containing flavonoids have been used for thousands of years in traditional Eastern medicine. In spite of the voluminous literature available, however, Western medicine has not yet used flavonoids therapeutically, even though their safety record is exceptional. Suggestions are made where such possibilities may be worth pursuing.
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I. General Aspects |
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A. Introduction
Over 4000 structurally unique flavonoids have been identified in
plant sources (Harborne et al., 1975
; Harborne, 1985a
,b
, 1986
).
Primarily recognized as the pigments responsible for the autumnal burst
of hues and the many shades of yellow, orange, and red in flowers and
food (Timberlake and Henry, 1986
; Brouillard and Cheminant,
1988
), the flavonoids are found in fruits, vegetables, nuts,
seeds, herbs, spices, stems, flowers, as well as tea and red wine. They
are prominent components of citrus fruits (Kefford and Chandler, 1970
)
and other food sources (Herrmann, 1976
) and are consumed regularly with
the human diet. These low molecular weight substances, found in all
vascular plants, are phenylbenzo-pyrones (phenylchromones) with an
assortment of structures based on a common three-ring nucleus. They are
usually subdivided according to their substituents into flavanols (a),
anthocyanidins (b), and flavones, flavanones, and chalcones (c) (Table
1 and Fig. 1). This basic structure is comprised of
two benzene rings (A and B) linked through a heterocyclic pyran or
pyrone (with a double bond) ring (c) in the middle (Fig. 1). This
subdivision is primarily based on the presence (or absence) of a double
bond on position 4 of the C (middle) ring, the presence (or absence) of
a double bond between carbon atoms 2 and 3 of the C ring, and the
presence of hydroxyl groups in the B ring (Fig. 1). In the flavonoid
structure, a phenyl group is usually substituted at the 2-position of
the pyrone ring. In isoflavonoids, the substitution is at the
3-position. Flavonoids and tocopherols (vitamin E) share a common
structure, i.e., the chromane ring. There have been several efforts to
quantitate the amounts of different flavonoids in assorted food plants
(Bilyk and Sapers, 1985
; Hertog et al., 1992
; Rice-Evans and Packer, 1998
). Establishing these kinds of data will help nutrition scientists, for example, with studies of flavonoid pharmacodynamic effects and may
lead to a better understanding of whether there is an optimal
consumption level for flavonoids. On average, the daily USA diet was
estimated to contain approximately 1 g of mixed flavonoids expressed as glycosides (Kühnau, 1976
). However, according to Hertog et al. (1992)
, the average intake of mixed flavonoids was only
23 mg/day based on data from the 1987-88 Dutch National Food Consumption Survey (Hertog et al., 1993b
). The flavonoid
consumed most was quercetin, and the richest sources of flavonoids
consumed in general were tea (48% of total), onions, and apples
(Hertog et al., 1993b
). The amount of 23 mg/day was mostly flavonols
and flavones measured as aglycones (Hertog et al., 1993b
). The
corresponding amount of daily aglycones consumed in the USA would be
about 650 mg/day, since Kühnau had estimated 1 g/day to be the
daily flavonoid-glycoside consumption. Although there is a 5-fold
difference between the estimates of Kühnau and Hertog, it should
be stressed that recent evidence indicates that flavonoid-glycosides
are much more readily absorbed (than the aglycones) by humans (Hollman
and Katan, 1998
). Moreover, both the amount and the source could vary
appreciably in different countries. For instance, the amount consumed
could be considerably higher in the Mediterranean diet, which is rich in olive oil, citrus fruits, and greens. These quantities could provide
pharmacologically significant concentrations in body fluids and
tissues. Nevertheless, flavonoid dietary intake far exceeds that of
vitamin E, a monophenolic antioxidant, and that of
-carotene on a
milligram per day basis (Hertog et al., 1993b
). A resurgence of
interest in traditional Eastern medicine during the past two decades,
together with an expanded effort in pharmacognosy, has rekindled
interest in the flavonoids and the need to understand their interaction
with mammalian cells and tissues.
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Flavonoids may have existed in nature for over one billion years
(Swain, 1975
) and thus have interacted with evolving organisms over the
eons. Clearly, the flavonoids possess some important purposes in
nature, having survived in vascular plants throughout evolution (Swain,
1975
). The very long association of plant flavonoids with various
animal species and other organisms throughout evolution may account for
the extraordinary range of biochemical and pharmacological activities
of these chemicals in mammalian and other biological systems. Unique
examples are the inhibition of gamete membrane fusion in sea urchins
caused by quercetin during egg fertilization (Eckberg and Perotti,
1983
) and modulation of mammalian sperm motility by quercetin
(Nass-Arden and Breitbart, 1990
). Also, prenatal exposure to genistein
does indeed influence sexual differentiation in rats (Levy et al.,
1995
) and thus raises the question of analogous effects in humans.
Flavonoids have important effects in plant biochemistry and physiology,
acting as antioxidants, enzyme inhibitors, precursors of toxic
substances, and pigments and light screens (Harborne et al., 1975
;
McClure, 1986
). In addition, these compounds are involved in
photosensitization and energy transfer, the actions of plant growth
hormones and growth regulators, the control of respiration,
photosynthesis, morphogenesis, and sex determination, as well as
defense against infection (Smith and Banks, 1986
). Reports indicate
that plant flavonoids cause the activation of bacterial
(Rhizobium) modulation genes involved in control of nitrogen
fixation, which suggests important relationships between particular
flavonoids and the activation and expression of mammalian genes (Firmin
et al., 1986
; Peters et al., 1986
; Djordjevic et al., 1987
; Zaat et
al., 1987
).
The flavonoids have long been recognized to possess anti-inflammatory,
antioxidant, antiallergic, hepatoprotective, antithrombotic, antiviral,
and anticarcinogenic activities, discussed below separately (Gabor,
1979
, 1986
; Havsteen, 1984
; Cody et al., 1986
; Farkas et al., 1986
;
Selway, 1986
; Cody et al., 1988
; Welton et al., 1988
; Das, 1989;
Middleton and Kandaswami, 1993
; Carroll et al., 1998
; Hertog and Katan,
1998
). The flavonoids are typical phenolic compounds and, therefore,
act as potent metal chelators and free radical scavengers (Hughes and
Wilson, 1977
; Torel et al., 1986
; Clemetson, 1989
; Pratt, 1992;
Kandaswami and Middleton, 1994
). They are powerful chain-breaking
antioxidants. The flavonoids display a remarkable array of biochemical
and pharmacological actions, some of which suggest that certain members
of this group of compounds may significantly affect the function of
various mammalian cellular systems. Anti-inflammatory flavonoids were discussed by Lewis (1989)
, and their potential utility as therapeutic agents was emphasized. In 1955, the New York Academy of Medicine published a series of papers discussing bioflavonoids and the capillary
(Miner, 1955
). As early as 1950, there was evidence of antiallergic
activity, including information on vitamin C-flavonoid interaction. In
1952, Schoenkerman and Justice suggested that treatment with rutin plus
an antihistamine conferred a clinical benefit to patients with allergic disease.
Of historical importance is the observation that a mixture of two
flavonoids called citrin and hesperidin were considered to
possess vitamin-like activity (Scarborough and Bacharach, 1949
; Kühnau, 1976
; Hughes and Wilson, 1977
). The term vitamin P was coined to indicate that this material had the property of decreasing capillary permeability (and fragility), prolonging the life of marginally scorbutic guinea pigs, and reducing the signs of
hypovitaminosis C in experimental animals. Although so-called vitamin P
was shown ultimately not to fulfill the definition of a vitamin and the term was appropriately abandoned, there was nonetheless a strong indication that the flavonoids had potent antioxidant-dependent and
vitamin C-sparing activity (Clemetson, 1989
). This will be discussed in
detail later. At present, flavonoids are considered to be secondary,
nonessential dietary factors without any documented relevance to human
health and/or disease. As the contents of this review will indicate,
however, this position may need to be modified in view of the
pleiotropic, potentially health-promoting, and disease-preventing
activities of the flavonoids that have come to be appreciated, at least
in experimental situations. Moreover, some flavonoids also have
anticarcinogenic properties (Hertog et al., 1992
, 1993b
, 1995
).
The flavonoids do not have carcinogenic potential in experimental
animals (Aeschbacher et al., 1982
).
Alcoholism is a prevalent human disorder, and the search for effective
remedies continues. For about 2000 years, the Chinese have recognized
the antidipsotropic effect of Radix puerariae, an herb used
in Chinese traditional medicine for the treatment of alcohol abuse.
Keung and Vallee (1993)
took advantage of the propensity for alcohol of
the Syrian golden hamster to study the effect of extracts of R. puerariae and of daidzin and daidzein, two isoflavones found in
the extracts. The isoflavone compounds effectively reduced ethanol
consumption in the Syrian golden hamsters by approximately 50%, thus
pointing the way to the development of a new class of therapeutic
agents for alcoholism.
Another briefly reported observation of potentially great significance
is the finding of quercetin in bovine retinal tissue (Pautler et al.,
1986
). Do ingested flavonoids accumulate in various tissues and
modulate their functions? An excellent review of flavonoids in health
and disease has been published recently (Rice-Evans and Packer, 1998
).
Das et al. (1994)
conducted a careful
structure-system-activity-relationship study of flavonoids with special
respect to carcinogenicity, mutagenicity, and cancer-preventing
activities. They concluded, in spite of some ongoing controversy, that
not only are the "vast majority of flavonoids and isoflavonoids
completely innocuous, but may be beneficial in a variety of human
disorders". The naturally occurring flavonoids will be the primary
focus of this review, with occasional reference to synthetic compounds.
The review is not exhaustive; it is intended to acquaint the reader
with this interesting group of natural plant compounds. There has been, in recent years, a major rekindling of interest in pharmacognosy. Flavonoids turn out to be present in many natural therapeutically utilized products. For example, a drug profile on Ginkgo
biloba shows that flavonoids are a major component (Kleinjnen and
Knipschild, 1992
).
B. Synthesis
The flavonoids are formed in plants and participate in the
light-dependent phase of photosynthesis during which they catalyze electron transport (Das, 1994
). They are synthesized from the aromatic
amino acids, phenylalamine and tyrosine, together with acetate units
(Heller and Forkmann, 1993
). Phenylalamine and tyrosine are
converted to cinnamic acid and parahydroxycinnamic acid, respectively, by the action of phenylalamine and tyrosine ammonia lyases (Wagner and
Farkas, 1975
). Cinnamic acid (or parahydroxycinnamic acid) condenses
with acetate units to form the cinnamoyl structure of the flavonoids
(Fries rearrangement). A variety of phenolic acids, such as caffeic
acid, ferulic acid, and chlorogenic acid, are cinnamic acid
derivatives. There is then alkali-catalyzed condensation of an
ortho-hydroxyacetophenone with a benzaldehyde derivative generating chalcones and flavonones (Fig. 1), as well as a similar condensation of an ortho-hydroxyacetophenone with a benzoic
acid derivative (acid chloride or anhydride), leading to
2-hydroxyflavanones and flavones (Heller and Forkman, 1993
). The
synthesis of chalcones and anthocyanidins has been described in detail
by Dhar (1994)
. Biotransformation of flavonoids in the gut can release
these cinnamic acid (phenolic acids) derivatives (Scheline, 1991
).
Flavonoids are complex and highly evolved molecules with intricate
structural variation. In plants, they generally occur as glycosylated
and sulfated derivatives.
C. Metabolism and Disposition
The fate of orally and parenterally administered flavonoids in
mammals and the significance of biliary excretion was reviewed by
Griffiths and Barrow in 1972. Since then, progress in understanding flavonoid pharmacokinetics has been slow. Published studies of flavonoid metabolism are not extensive, and were reviewed again recently (Hollman and Katan, 1998
). Such studies are essential to
enhance our understanding of the possible importance of flavonoids in
human health and disease. The subject has been reviewed by Griffiths
and Barrow (1972)
, Hackett (1986)
, and Scheline (1991)
and will not be
exhaustively reviewed here. Considerable information is available
regarding the metabolism of flavonoids in animals and to a very limited
extent in humans (Hackett, 1986
; Scheline, 1991
).
Ring scission occurs under the influence of intestinal microorganisms,
which also account for the subsequent demethylation and dehydroxylation
of the resulting phenolic acids (cinnamic acid derivatives and simple
phenols). Intestinal bacteria also possess glycosidases capable of
cleaving sugar residues from flavonoid glycosides. Such glycosidases do
not appear to exist in mammalian tissues. Flavonoids can undergo
oxidation and reduction reactions, as well as methylation,
glucuronidation, and sulfation in animal species. An early evaluation
of the absorption and metabolism of (+)-catechin in humans was
presented by Das (1971)
. Oral administration (83 mg/kg) resulted in
rapid absorption, metabolism, and excretion of the flavonoid within
24 h. Eleven metabolites were detected in urine. No quercetin
could be found in plasma after oral administration of up to 4 g in
humans (Gugler et al., 1975
; Shali et al., 1991
). Hepatic metabolism of
quercetin and catechin by isolated perfused rat liver has been
demonstrated in studies by Shah et al. (1991). The flavonoids were
converted into sulfated and/or glucuronidated metabolites, which were
excreted in the bile. Recent improvements in analytical techniques have
made possible the determination of baicalein and baicalin (the
glycoside of baicalein) in rat plasma by high pressure liquid
chromatography with electrochemical detection (Wakui et al.,
1992
). Oral administration of these flavonoids to rats led to readily
measurable concentrations of the compound in plasma (100-10,000
ng/ml). This assay would be suitable for clinical pharmacokinetic
studies. More recently, Ferry and coworkers (1996)
performed a phase I
clinical trial of quercetin; pharmacokinetic patterns were established
following i.v. bolus administration. The plasma concentrations achieved
inhibited lymphocyte protein tyrosine phosphorylation, and there was
some evidence of antitumor activity.
Silibinin (two diastereomers), the principal component in extracts of
Silybum marianum, can be measured in plasma by refined chromatographic assays (Rickling et al., 1995
), permitting
pharmacokinetic studies. Silibinin is absorbed following oral
administration of silymarin. The several plasma concentration peaks
detected could be caused by enterohepatic circulation of the compound.
The significant biliary route of excretion of baicalin and baicalein
was also noted by Abe et al. (1990)
. Chronic exposure to soya (soy
milk) in the diet did not modify the metabolic pathways of the
isoflavones daidzein and genistein but did alter the time courses of
their excretion (Lu et al., 1995
).
In long overdue studies,
Hertog et al. (1993a)
in The Netherlands measured the flavonoid content
of several foods, their consumption by elderly males, and the
relationship to the development of coronary artery disease. The
flavonoids measured were quercetin, kaempferol, myricetin, apigenin,
and luteolin. The principal sources of dietary flavonoids were tea,
onions, and apples. Flavonoid consumption was significantly inversely
related to mortality from coronary artery disease (after adjustment for
multiple variables). The authors concluded that the regular ingestion
of flavonoid-containing foods may protect against death from coronary
artery disease in elderly men. The same group measured the content of
potentially anticarcinogenic flavonoids of 28 vegetables, wine, and
fruits frequently consumed in The Netherlands (Hertog et al., 1992
). Again, the measured flavonoids were quercetin, kaempferol, myricetin, apigenin, and luteolin. The mean daily intake of these five antioxidant flavonoids was 23 mg/day, which exceeds the intake of other familiar antioxidants such as
-carotene (2-3 mg/day) and vitamin E (7-10 mg/day) and is about one-third the average intake of vitamin C (70-100
mg/day) (Hertog et al., 1993b
). If The Netherlands investigators had
measured total flavonoid intake, including all sources of these
chemicals, and had estimated the flavonoid glycoside content (Kühnau, 1976
), the daily intake could have been considerably higher. The total aglycone consumption according to Kühnau (1976)
was 650 mg/day in the USA. It would be useful to have comparable data
for other countries. On the other hand, Rimm and coworkers (1996)
did
not find a strong inverse association between intake of flavonoids and
total coronary heart disease. The authors suggested that flavonoids may
exert a protective effect in men with established coronary artery disease.
One of the few recent pharmacokinetic studies of flavonoids in humans
was conducted by Cova et al. (1992)
using diosmin, the 7-rhamnoglucoside of diosmetin, 5,7,3'-trihydroxy-4'-methoxyflavone. Five healthy volunteers received 10 mg/kg of body weight of diosmin. Diosmin and diosmetin were measured in blood and urine by high performance liquid chromatography and liquid chromatography-mass spectrometry techniques. Only diosmetin (the aglycone) could be detected in plasma. The time course of diosmetin plasma concentrations indicated rapid initial distribution and prolonged final elimination half-life of 31.5 h. Neither diosmin nor diosmetin could be
detected in urine. The metabolites in urine were
m-hydroxyphenylpropionic acid and several other phenolic
acids. The prolonged presence of diosmetin in blood suggests an
enterohepatic circulation. The apparent volume of distribution of
approximately 62.1 liters points to an extensive uptake of diosmetin by
tissues. Using more recent analytical techniques, some Netherlands
investigators (Hollman et al., 1996
) measured plasma quercetin
concentrations following ingestion of fried onions containing quercetin
glycosides equivalent to 64 mg of quercetin aglycone. Peak plasma
levels of 196 µg/ml were achieved after 2.9 h with a half-life
of absorption of 0.87 h. The distribution phase half-life was
3.8 h and the elimination phase half-life was 16.8 h. Thus,
oral dietary (cooked vegetable) quercetin can be absorbed and reach
tissues and plasma where antioxidant and other activities could be
exerted. What is true for quercetin is very likely true also for other
flavonoids in other vegetable sources. Thus, the cumulative
concentration of quercetin plus other flavonoids could be substantially
greater than that shown for quercetin alone. The possible importance of
quercetin metabolites and their antioxidant properties has been
discussed by Morand et al. (1998)
. Rats fed quercetin in the diet
(0.2%) generated measurable quantities of metabolites with antioxidant
properties. Rats adapted to this diet also had a total "antioxidant
status" much greater than the control animals. In studies of
absorption of quercetin and kaempferol from the diet of human subjects,
de Vries and coworkers (1998)
found that these flavonols (from tea and
onions) could be used as biomarkers for dietary intake.
Hollman and Katan (1998)
reviewed the bioavailability and health
effects of dietary flavonols in humans. They found that quercetin glycosides from onions were more readily absorbed than the pure aglycone; absorbed quercetin was eliminated slowly from the blood, suggesting that the enterohepatic circulation may be operative. In
related studies, Hollman et al. (1995)
concluded that quercetin-glucose conjugates were more readily absorbable; the suggestion was made that
the glycosides may be absorbed via the intestinal sugar uptake route.
Determination of the urinary metabolites of deuterated rutin was
performed by Baba et al. (1981)
following oral administration of 10 mg/kg rutin-d or 50 mg/kg unlabeled rutin. Several metabolites appeared
(consistent with scission of the C ring), but no unchanged rutin (or
quercetin) was detected in the urine.
Isoflavonoid phytoestrogens and mammalian lignans, occurring in animal
and human biological fluids and in feces, are diphenolic compounds with
molecular weights similar to those of steroid estrogens. The mammalian
compounds are produced from plant sources and isoflavonoids by
intestinal microflora (Axelson and Setchell, 1981
; Setchell et al.,
1981
; Borriello et al., 1985
). Bannwart et al. (1984)
described the
presence of the phytoestrogenic isoflavone daidzein in human urine by
GC-MS.2 The isoflavonoids
have been shown to bind with relatively high affinities to the estrogen
receptors of human mammary tumor cells (Martin et al., 1978
). They may,
therefore, be implicated in the inhibition of breast carcinoma cell
growth mediated by estrogen.
Wheat fiber is recognized to be a potentially important anticancer food
material, as is the case with soy isoflavones, such as genistein.
Interestingly, therefore, Tew et al. (1996)
found that a fiber-rich
diet produced a marked decrease in plasma genistein concentrations
after 24 h following soy dosing and reduced total urinary
genistein excretion. Urinary daidzein was not related to fiber intake.
The significance of this observation in relationship to the future
design of flavonoid-rich diets must be taken into consideration. When
human volunteers consumed soya flour, the urinary excretion of
genistein, daidzein, and glycitein increased after 24 h as
did the isoflavonoid metabolites equol and
O-desmethylangolensin. The experiments also indicated that
individual subjects exhibited preferred metabolic pathways (Kelly et
al., 1995
).
The plasma concentrations of four isoflavonoids, daidzein, genistein,
O-desmethylangolensin, and equol, were very high in Japanese
men consuming a low fat diet with a high content of soy products
(Adlercreutz et al., 1993
). The geometric mean plasma total and
individual isoflavonoid levels were 7 to 110 times higher in the
Japanese men than in the Finnish men. These phytoestrogen levels may
inhibit the growth of prostate cancer in Japanese men, which may
explain the low mortality from prostatic cancer in that country.
Genistein concentrations in urine of subjects consuming a traditional
soy-rich Japanese diet were in the micromolar range, while these
concentrations were 1/30th or less of those in urine of omnivores
(Adlercreutz et al., 1991
).
The most important information derived from recent studies is the fact
that most flavonoids, except catechins, exist in nature as glycosides.
Moreover, at least quercetin glucosides were absorbed better than the
aglycone quercetin-
-glucoside (Hollman and Katan, 1998
).
Consequently, the amount of flavonoid glycosides consumed is a better
indication than the amount of aglycones, thus raising the lower level
estimated for the flavonoid aglycones. Finally, supplementation of the
diet should more appropriately use flavonoid glycosides instead of aglycones.
D. Adverse Reactions
Adverse reactions to flavonoids in humans appear to be rare.
Studies of Salama and Mueller-Eckhardt (1987)
indicated that (+)-catechin and its metabolites can bind tightly to erythrocyte membranes and that this generates new antigenic sites with consequent development of autoantibodies presumed to be the cause of hemolytic anemia in six patients who had taken the catechin. The hemolytic anemia
disappeared after discontinuation of catechin ingestion although the
subjects continued to ingest cross-reactive dietary flavonoids.
Some flavonoids are capable of quinone formation, a familiar pathway
leading to contact sensitization. However, as reviewed by Schmalle et
al. (1986)
, the flavonoids are not potent contact allergens and are not
distinguished as contact sensitizers in the dermatologic literature,
even though essentially all human beings have daily physical contact
with flavonoid-containing foods and plants. Hausen et al. (1990)
have
described the development of contact allergy to the Australian
blackwood, which is known to be an important cause of contact
dermatitis in this region; several hydroxyflavans proved to be
allergenic. Some flavonoids and their related phenolic compounds could
have toxic effects. However, such flavonoids are not found in our food supply.
While there is a popular impression that flavonoids have
"antiaging" properties, possibly through their antioxidant
activity, note that quercetin may significantly reduce the life span of mice, (an effect was noted mainly in the "shorter-living" males (Jones and Hughes, 1982
).
On balance, the flavonoids appear to be remarkably safe nutrients with a wide range of biochemical and pharmacologic activities that strongly suggest their possible role as health-promoting, disease-preventing dietary supplements.
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II. Effects on Mammalian Enzyme Systems |
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Flavonoids have been demonstrated to affect the activity of many mammalian enzyme systems in vitro. Some evidence indicates that they can also do so in vivo; however, the question remains how flavonoids enter the cells and whether they could accumulate in certain organ cells. Notable structure-activity relationships have been detected in many cases and are mentioned. The following listing is not exhaustive and aims to familiarize the reader with the extent of enzyme modulatory activities recorded.
A. Kinases
Protein kinase C (PKC), the ubiquitous, largely
Ca2+- and phospholipid-dependent, multifunctional
serine- and threonine-phosphorylating enzyme, is involved in a wide
range of cellular activities, including tumor promotion, mitogenesis,
secretory processes, inflammatory cell function, and T lymphocyte
function, among others (Nishizuka, 1986
, 1988
, 1995
). PKC has been
shown to be inhibitable in vitro by certain flavonoids (Graziani et
al., 1981
; Gschwendt et al., 1983
; End et al., 1987
; Hagiwara et al.,
1988
; Ferriola et al., 1989
; Picq et al., 1989
). Graziani et al. (1983)
demonstrated that quercetin inhibited the phosphorylating activity of
the Rous sarcoma virus transforming gene product both in vitro and in
vivo. In addition, quercetin was competitive toward the nucleotide
substrates ATP and GTP. Mitogen activated protein (MAP) kinase
in human epidermal carcinoma cells was strongly inhibited by
quercetin (30 µM) (Bird et al., 1992
).
Ferriola et al. (1989)
used a partially purified rat brain PKC
preparation and found that fisetin, quercetin, and luteolin were the
most active flavonoid inhibitors of this enzyme. Experiments utilizing
different protein substrates (histone and protamine) and different
activators [diacylglycerol and tetradecanoylphorbol acetate (TPA)]
showed that fisetin (and luteolin) competitively blocked the ATP
binding site on the catalytic unit of PKC. Several other ATP-utilizing
enzymes inhibited by flavonoids were affected by competitive binding of
the flavonoid to the ATP binding site (vide infra). Structure activity
studies suggested that addition of one hydroxyl group at position 3 largely eliminated inhibitory activity (Alexandrakis et al., 1999
).
Myosin light chain kinase (MLCK) catalyzes the phosphorylation of MLCs
in many cell types. It is essential for the development of active
tension in smooth muscle and for movement or migration of other cells.
It is of interest, therefore, that kaempferol was an active and
relatively specific inhibitor (IC50, 0.45 µM) of purified bovine aorta MLCK (Rogers and Williams, 1989
). Kaempferol was specific for MLCK by a factor of 30 or greater as compared with
several other kinases. As in other systems with different flavonoids,
kaempferol acted competitively with ATP. Avian MLCK was also inhibited
by several flavonoids, maximally with compounds with C2-C3 unsaturation
and polyhydroxylation of two of the three ring structures (Jinsart et
al., 1991
). Methoxylation or glycosylation markedly reduced or
abolished activity.
A large number of protein tyrosine kinases (PTK) have been described.
They are found in many different types of cells and are implicated in
the regulation of cell transformation and cell growth, gene expression,
cell-cell adhesion interactions, cell motility, and shape (cf. Huang,
1989
; Taniguchi et al., 1995
; Qian and Weiss, 1997
). PTK was inhibited
by genistein (Akiyama et al., 1987
). In addition to affecting PTK and
PKC activity, quercetin was also capable of inhibiting nuclear kinase
II-catalyzed phosphorylation of isolated nuclear proteins in HeLa cells
using GTP as phosphate donor (Friedman et al., 1985
). This result is of
interest because it shows that quercetin could inhibit a GTP-dependent phosphorylation reaction and raised the question whether intact cell
nuclear protein phosphorylation could be affected by flavonoids and
thus affect many non-ATP-dependent aspects of cell function.
Another flavonoid-sensitive kinase is rabbit muscle phosphorylase
kinase. Kyriakidis et al. (1986)
found quercetin and fisetin to be
effective inhibitors of nonactivated phosphorylase kinase, while the
flavanone hesperetin stimulated the enzyme. Quercetin acted as a
competitive inhibitor of ATP binding and was more effective as an
inhibitor of the enzyme when stimulated by ethanol or alkaline pH.
Cochet et al. (1982)
examined the effect of quercetin and several other
flavonoids on inhibition of cyclic nucleotide-independent protein
kinase (G type casein kinase) and two other kinases. The G type kinase,
which utilizes GTP as well as ATP, was selectively inhibited by several
flavonoids. Kinetic evaluation showed that quercetin behaved as a
competitive antagonist. Fisetin, chrysin, and kaempferol were also
active. The importance of the pattern of A and B ring hydroxylation,
C2-C3 unsaturation, and C4 keto were again recognized as strongly
affecting inhibitory activity. Srivastava (1985)
showed quercetin to be
an effective inhibitor of phosphorylase kinase and also of protein
tyrosine kinase. ATP competitively blocked quercetin's inhibitory
activity with protein tyrosine kinase, but not with phosphorylase
kinase. The data suggested once more that quercetin competed for the
ATP binding site of the tyrosine kinase. It is currently unknown how
the flavonoids enter the cell and react in the compartment where the
kinases are localized. One possibility is that the flavonoids have no effect on kinases in quiescent cells and only interfere with the ATP
binding site when the enzyme trans-locates upon activation.
Kakeya et al. (1993)
isolated a unique substrate-competitive tyrosine
kinase inhibitor from the plant Desmos chinensis; they named
it "desmal" and determined its structure to be
8-formyl-2',5,7-trihydroxy-6-methylflavanone. Desmal showed competitive
inhibition of phosphorylation with respect to histone and
noncompetitive inhibition with respect to ATP (in contrast to some
other flavonoid inhibitors of phosphorylation noted above). Desmal also
inhibited EGF-induced inositol phosphate formation. Moreover, desmal
inhibited intracellular tyrosine phosphorylation in EGF
receptor-overexpressing NIH 3T3 (ER12) cells.
Human cytomegalovirus DNA can induce a serine-threonine protein kinase
with a molecular mass of 68 kDa in human diploid lung fibroblasts. This p68 kinase catalytic activity was inhibitable by
quercetin acting competitively with respect to the nucleotide substrate
(Michelson et al., 1985
).
In studies of NK cell-mediated cytotoxicity, Nishio et al. (1994)
found
that genistein decreased the affinity of the tyrosine kinase
p56lck to the
-chain of the interleukin (IL)-2
receptor, a crucial event in IL-2-stimulated signaling events. In
addition, genistein decreased the fast Na+
current in a concentration-dependent manner with an
IC50 of 9 µM in human uterine leiomyosarcoma
cells (Kusaka and Sperelakis, 1996
). These investigators also studied
the effect of genistein and daidzein on regulation of L-type
Ca2+ channels in freshly isolated uterine smooth
muscle cells. Genistein decreased L-type Ca2+
current concentration dependently, while daidzein had no effect (Kusaka
and Sperelakis, 1995
).
Rat liver cyclic AMP-dependent protein kinase catalytic subunit could
be inhibited by a variety of flavonoids (Jinsart et al., 1992
). Again,
C2-C3 unsaturation and polyhydroxylation of two or more flavonoid rings
favored the development of inhibitory activity. Methoxylated and
glycosylated agents were much less active. Several flavonoids inactive
against MLCK were good inhibitors of cyclic AMP-dependent protein
kinase catalytic subunit.
Recent evidence indicates that flavonoids can induce the
phosphorylation of a 78-kDa protein, which was recently shown to be
homologous to moesin (Theoharides et al., 2000
). Further work showed
that this phosphorylation was caused by a Ca2+-
and phorbol ester-independent PKC isozyme "
" (Wang et al., 1999
). The possibility that the increase in phosphate
incorporation may be due to inhibition of a phosphatase is unlikely
because there has not been any such evidence. Preliminary data
from our studies suggest that flavonoids reduce
intracellular calcium ion levels, thus reducing secretion and
activating a Ca2+-independent PKC isozyme. The
combined effect is regulation of secretion.
B. Phospholipase A2
Phospholipase A2
(PLA2), an enzyme involved in many cell
activation processes, catalyzes the hydrolysis of phospholipids
esterified at the second carbon in the glycerol backbone. Arachidonic
acid is commonly esterified in this position, and the action of
PLA2 releases arachidonic acid for subsequent
metabolism via the cyclooxygenase (CO) and lipoxygenase (LO) pathways.
PLA2 is likely an important intra-and
extracellular mediator of inflammation (Pruzanski and Vadas, 1991
).
Quercetin was found to be an effective inhibitor of
PLA2 from human (Lee et al., 1982
) and rabbit
(Lanni and Becker, 1985
) leukocytes. Quercetagetin,
kaempferol-3-O-galactoside, and scutellarein inhibited human
recombinant synovial PLA2 with
IC50 values ranging from 12.2 to 17.6 µM (Gil
et al., 1994
).
C. ATPases
Flavonoids can affect the function of plasma membrane transport
Na+- and K+-ATPases (Rodney
et al., 1950
; Carpenedo et al., 1969
; Lang and Racker, 1974
),
mitochondrial ATPase, and Ca2+-ATPase (Deters et
al., 1975
; Cantley and Hammes, 1976
). The Mg 2+-ectoATPase of human leukocytes was inhibited
by quercetin (Long et al., 1981
). Rabbit muscle sarcoplasmic reticulum
Ca2+-ATPase was effectively inhibited by several
flavonoids that were also active inhibitors of antigen-induced rat mast
cell histamine release (Fewtrell and Gomperts, 1977a
). Inhibition of
Ca2+-ATPases by flavonoids such as quercetin was
demonstrated (Shoshan et al., 1980
; Shoshan and MacLennan, 1981
), and
quercetin acted as a competitive inhibitor of ATP binding to the
enzyme. Others have described quercetin inhibition of hog gastric
H+,K+-ATPase where the
inhibition was competitive with respect to ATP (Murakami et al., 1992
).
In studies of contractile proteins of rabbit skeletal muscle, Zyma et
al. (1988)
found quercetin to cause conformational changes in the
structure of myosin with a coincident increase in ATPase activity. At
higher concentrations, quercetin inhibited actomyosin
superprecipitation as well as ATPase activity. Inhibition of
Ca2+ transport across erythrocyte membranes by
quercetin has also been described (Wuthrich and Schatzmann, 1980
).
Fischer et al. (1987)
showed that quercetin inhibited platelet and
sarcoplasmic reticulum Ca2+-ATPase activities in
a concentration-dependent manner. Quercetin proved to be a competitive
inhibitor of the calcium pump ATPase with respect to ATP. Inhibition of
Na+,K+-ATPase apparently
was not related to the cardiac glycoside-specific (ouabain) binding
site(s) of this enzyme (Hirano et al., 1989a
).
D. Lipoxygenases and Cyclooxygenases
Arachidonic acid released from membrane phospholipids or other
sources is metabolized by the LO pathway to the smooth muscle contractile and vasoactive leukotrienes (LT),
LTC4, LTD4, and LTE4, as well as to the potent chemoattractant,
LTB4 (Lewis and Austen, 1984
). These molecules
are intimately involved in inflammation, asthma, and allergy, as well
as in multiple other physiologic and pathologic processes. Yamamoto and
coworkers (1984)
studied the effect of several benzoquinone and
flavonoid compounds on various enzymes of the LT biosynthetic pathway.
For instance, cirsiliol (3',4',5-trihydroxy-6,7-dimethoxyflavone)
proved to be a potent inhibitor of 5-LO (IC50,
0.1 µM) derived from rat basophilic leukemia cells and guinea pig
peritoneal polymorphonuclear leukocytes. The partially purified 5-LO of
rat basophilic leukemia cells was also strongly inhibited by cirsiliol
(Furukawa et al., 1984
). Hoult et al. (1994)
studied the effects of
flavonoids on 5-LO and CO in rat peritoneal leukocytes and human
polymorphonuclear leukocytes stimulated with the nonphysiological
cation ionophore A23187. 5-LO was best inhibited by polyhydroxylated
compounds. The authors considered that 5-LO, but not CO, inhibition
could be caused by a combination of iron ion-reducing/iron
ion-chelating abilities and was not dependent on lipid peroxyl
scavenging. Laughton et al. (1991)
had also indicated that a
combination of iron-chelating and iron ion-reducing properties was
required for selective peritoneal leukocyte 5-LO inhibition by phenolic compounds.
Differential inhibition of LT biosynthetic enzymes was further
documented when cirsiliol was shown to have approximately 10-fold less
activity against the 12-LO enzyme and negligible effect on CO of bovine
vesicular gland. Partially purified mouse epidermal cell LO was
inhibited potently by flavone derivatives bearing appropriate patterns
of hydroxylation, but not by flavone itself (Wheeler and Berry, 1986
).
Baicalein was reported to selectively inhibit platelet 5-LO (Sekiya and
Okuda, 1982
). Artonin E (5'-hydroxymorusin) was a potent and fairly
selective inhibitor of porcine leukocyte 5-LO (Reddy et al., 1991
).
Hypolaetin (a catecholic flavonoid), but not its 8-glucoside, proved to
be a good inhibitor of stimulated rat peritoneal leukocyte 5-LO,
although it was inactive as a CO inhibitor (Moroney et al., 1988
).
Interestingly, these investigators found more CO inhibition and less LO
inhibition with flavone compounds containing few hydroxyl substituents,
including absence of the 3',4'-dihydroxy pattern in the B ring.
In contrast, Kalkbrenner et al. (1992)
found that nonplanar flavans
were more potent inhibitors of rat seminal vesicle LO than planar
flavones and flavonols. No flavanones caused inhibition except
silibinin, a flavanon-3-ol. Kinetics of inhibition varied with the
class of flavonoid. On the other hand, Swies et al. (1984)
found that
ram seminal vesicle CO was stimulated by quercetin and several other
flavonoids at high substrate arachidonic acid concentrations, whereas
at low substrate concentration quercetin was inhibitory.
Baumann et al. (1980a)
also examined the effect of several flavonoids
on arachidonic acid peroxidation. Luteolin (3',4'-dihydroxyflavone), morin, galangin, and (+)-catechin were moderately active inhibitors of
rat renal medulla CO. Landolfi et al. (1984)
found that flavone, chrysin, apigenin, and phloretin depressed CO activity and inhibited platelet aggregation. In early experiments, Fiebrich and Koch (1979)
showed that the three pharmacologically active compounds of silymarin,
namely, silybin, silydianin, and silychristin, inhibited CO.
Ferrandiz et al. (1990)
studied some unusual flavonoids for their
effect on arachidonic acid metabolism via the LO (5-HETE and
LTB4) and CO (TxB2,
PGE2, 6-keto-PGF1
)
pathways in rat peritoneal leukocytes. IC50 of
less than 10 µM was found for sideretoflavone, oroxinidin,
quercetagetin-7-glucoside, and tambuletin against both pathways. Also,
eight naturally occurring isoprenylated flavones were studied for their
effect on 5-LO activity purified from porcine leukocytes. Artonin E
(5'-hydroxymorusin) was the most potent inhibitor, with an IC
50 of 0.36 µM. Butenko et al. (1993)
also showed baicalein to be an inhibitor of LTC4
production via inhibition of 5-LO; the resultant anti-inflammatory
activity was greater in the rat adjuvant arthritis model than in the
rat carrageenan-induced paw edema model.
Rao and coworkers (1985)
found differential effects of the inhibitors
on membrane- and cytosol-associated LO activity. Quercetin was an
effective inhibitor of 12-LO activity in human platelets. Inhibitory
activity of some chalcone derivatives on mouse epidermal 12-LO and CO
was studied by Nakadate et al. (1985b)
. Effects of chalcones on 12-LO
were much greater than on CO. Inhibitory activity was related to the
chalcone's having a cinnamoyl or 4-hydroxycinnamoyl residue in the
molecule. Skin tumor formation and TPA-induced ornithine decarboxylase
activation were also strongly inhibited by some LO inhibitors (Aizu et
al., 1986
).
E. Phospholipase C
No direct measurements of the effect of flavonoids on PLC have
been reported. However, as reviewed in a later section, evidence strongly suggests that PTK-dependent phosphorylation of PLC-
is required for activation of the enzyme; consequently, inhibition of
PTK with such flavonoids as genistein blocks PLC activation and
formation of inositol trisphosphate (IP3) and
diacylglycerol (DAG). Earlier work of Cockcroft (1982)
indirectly
indicated quercetin inhibition of PLC activity in stimulated rat mast
cells, but the mechanism of action was not established.
F. Cyclic Nucleotide Phosphodiesterase
The cyclic nucleotides, cAMP and cGMP, mediate many biological
processes through their ability to stimulate cyclic
nucleotide-dependent protein kinases, which in turn phosphorylate
cellular protein substrates and evoke specific responses. cAMP and cGMP
are formed from ATP and GTP by the catalytic activity of adenylate and
guanylate cyclases stimulated by various agonists. Their activity is
terminated by the cyclic nucleotide phosphodiesterases (PDE). The
cyclic nucleotides are involved in regulation of many cellular
processes, such as cell division, smooth muscle contractility,
secretory functions, immunological processes, and platelet aggregation, to name a few. Flavonoid inhibition of PDEs from many cellular sources
has been described (Ruckstuhl and Landry, 1981
; Beretz et al., 1986
).
The minimal structural requirements for PDE inhibitor activity include
a flavone, flavonol, or flavylium skeleton (Beretz et al., 1979
).
Ferrell et al. (1979)
proposed that the flavonoid inhibitory activity
on PDE could be ascribed to the structural mimicry of the pyrimidine
ring in cAMP and the pyranone ring of active flavonoids.
G. Adenylate Cyclase
Landolfi et al. (1984)
reported that flavone, chrysin, and
apigenin decreased the platelet cyclic AMP response to prostacyclin, an
effect attributed to inhibition of adenylate cyclase. The isoflavone prunetin was also active, while the flavones 7-hydroxyflavone, apigenin, galangin, and kaempferol were less active.
H. Reverse Transcriptase
Selected naturally occurring flavonoids have been shown (Spedding
et al., 1989
) to inhibit three reverse transcriptases (RT) [avian
myeloblastosis RT, Rous-associated virus-2 RT, and Moloney murine
leukemia virus (MMLV) RT] when poly(rA)oligo(dT) 12-18 or rabbit
globin mRNA were used as template. Amentoflavone, scutellarein and
quercetin were the most active compounds, and their effect was
concentration-dependent. The enzymes exhibited differential sensitivity
to the inhibitory effects of the flavonoids. These flavonoids also
inhibited rabbit globin mRNA-directed MMLV RT-catalyzed DNA synthesis.
Amentoflavone and scutellarein inhibited ongoing new DNA synthesis
catalyzed by Rous-associated virus-2 RT. Kinetic studies were performed
in an attempt to elucidate the mechanism of action of amentoflavone and
scutellarein (Spedding et al., 1989
). Inhibition of Moloney murine
leukemia strains of RT by baicalein (5,6,7-trihydroxyflavone) was
described by Ono et al. (1989)
. Baicalein inhibition of RT was
competitive with respect to the template primer (rA) n (dT) 12-18 and
noncompetitive with respect to the substrate dTTP. In other
experiments, Ono et al. (1990)
found that baicalein, quercetin,
quercetagetin, and myricetin were potent inhibitors (there was
significant activity at 1-2 µg/ml) of RTs from Rauscher murine
leukemia virus and HIV. The inhibition noted with baicalein was very
specific, whereas quercetin and quercetagetin proved also to be potent
inhibitors of DNA polymerase
and DNA polymerase I, respectively.
Moloney murine and Rous associated virus-2 RT were also inhibited by
baicalin (Baylor et al., 1992
). This flavone caused a
concentration-dependent inhibition of human T cell leukemia virus type
1 (HTLV-1) replication in infected T and B cells and selectively
inhibited the HTLV-1 p19 gag protein without otherwise adversely
affecting the cells. Inoue and coworkers (1989)
found inhibitory
activity against avian myeloblastosis RT with fisetin, quercetin,
myricetin, and baicalein. The effect of flavonoids on MMLV RT was
studied by Chu et al. (1992)
, who found that flavononols and flavonols
were active, while flavones and flavanones were not. There was no
requirement for a double bond at C2-C3.
Nakane and Ono (1990)
found two components of green tea, namely
(
)-epigallocatechin gallate and (
)-epicatechin gallate, to
differentially inhibit the activities of RT and cellular DNA and RNA
polymerases. RT was most strongly inhibited, as were DNA polymerases
and
. The authors suggested the possibility that these compounds
might exert selective inhibition of HIV RT at appropriate concentrations.
I. HIV-1 Proteinase
This enzyme is a necessary component for the processing and
replication of HIV-1. Brinkworth et al. (1992)
suggested that certain
flavones may be potential nonpeptidic inhibitors of the enzyme.
Gardenin A, myricetin, morin, quercetin, and fisetin exhibited activity
with IC50 values in the 10 to 50 µM range.
Lineweaver-Burk analysis indicated competitive inhibition for fisetin
and quercetin.
J. HIV-1 Integrase
Yet another enzyme involved in HIV replication could be inhibited
by quercetin, namely the integrase (Fesen et al., 1993
). This
inhibition required at least one ortho pair of phenolic hydroxyl groups
and at least one or two additional hydroxyl groups (Fesen et al.,
1994
).
K. Ornithine Decarboxylase
The effects of flavonoids on ornithine decarboxylase (ODC) have
not been studied in depth. ODC catalyzes the transformation of
ornithine to the polycationic bases, putresine, spermine, and spermidine; these compounds exert regulatory effects on cell growth. Studies by Kato et al. (1983)
showed that quercetin (10-30
µmol/mouse) markedly suppressed the stimulatory effect of TPA on ODC
activity and on skin tumor formation in mice initiated with
dimethylbenzanthracene. Such inhibition may be related to the
activation of the catalytic site, which is under nonconventional
regulation by small molecules (Theoharides and Canellakis,
1975
). Also, the synthetic flavonoid, flavone acetic acid, was
shown to inhibit the activity of ODC in stimulated human peripheral
blood lymphocytes and human colonic lamina propria lymphocytes (Elitsur
et al., 1990
). Nakadate et al. (1985a)
reported that quercetin
suppressed ODC induction by teleocidin. Topical application of the
flavonoid silymarin to mice inhibited TPA-induced epidermal ODC
activity and TPA-induced ODC mRNA expression (Agarwal et al., 1994
).
Topical application of apigenin, a close chemical relative of
quercetin, also proved to be an effective, dose-dependent inhibitor of
ODC activity and papilloma formation (Wei et al., 1990
).
L. Topoisomerase
DNA topoisomerases are enzymes that introduce transient breaks in
linear DNA sequences. They participate in several genetically related
processes, including replication, transcription, recombination, integration, and transposition (Okura et al., 1988
). DNA topoisomerase II is an important cellular target for several antineoplastic DNA
intercalators and nonintercalators. Flavonoids had apparently different
effects on these enzymes. Markovits et al. (1989)
found that genistein
inhibited mammalian DNA topoisomerase II as well as protein tyrosine
kinase. Two flavones, fisetin and quercetin, also showed the same
activity (Yamashita et al., 1990
). Okura and coworkers (1988)
showed
that both topoisomerase I and II were sensitive to genistein by
increasing the DNA-enzyme complex in L1210 cells and interfering with
enzyme-induced DNA relaxation (pBR22 DNA). Genistein selectively
suppressed the growth of the ras-transformed NIH 3T3 cells, but not the
normal NIH 3T3 cells, and inhibited topoisomerase II-catalyzed ATP
hydrolysis (Robinson et al., 1993
). In contrast, baicalein, quercetin,
quercetagetin, and myricetin, known inhibitors of RT, unwound DNA and
appeared to promote mammalian DNA topoisomerase-mediated site-specific DNA cleavage (Austin et al., 1992
).
M. Glutathione S-Transferase
Glutathione S-transferase (GST) isozymes participate in
detoxification processes by catalyzing the formation of
xenobiotic-glutathione (GSH) conjugates. Anionic and cationic GST
isozymes were differentially inhibited to varying degrees by quercetin
in vitro (Das and Ratty, 1986
). Flavonoid administration in
vivo, however, induced this activity (Trela and Carlson, 1987
). Rat
liver GST was effectively inhibited in vitro by several other
flavonoids. This activity was again closely related to the pattern of
hydroxylation and presence of a C2-C3 double bond (Merlos et al.,
1991
).
N. Epoxide Hydrolase
Epoxide hydrolase catalyzes the hydration of arene oxides
(generated by cytochrome P450 enzymes) to yield dihydrodiols, which can
be converted to diol epoxides by cytochrome P450-dependent multifunction oxidases (MFOs). Diol epoxides generated from polynuclear aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BP), may function as ultimate carcinogens (Dipple et al., 1984
). Flavone and
7,8-benzoflavone both stimulated epoxide hydrase activity, and flavone
fed to rats increased the activity of the enzyme in liver microsomes
(Alworth et al., 1980
).
O. Glyoxalase
Glyoxalase substrates may be important in the regulation of cell
division. Glyoxalases detoxify
-ketoaldehydes (glyoxalase I) by
facilitating their oxidation to inert
-hydroxy acids (glyoxalase II). Quercetin, fisetin, myricetin, and several other flavonoids were
potent inhibitors of glyoxalase I (Klopman and Dimayuga, 1988
).
P. Xanthine Oxidase
Xanthine oxidase catalyzes the formation of urate and superoxide
anion from xanthine. Bindoli et al. (1985)
, in early experiments, demonstrated the inhibitory action of quercetin on both xanthine oxidase and xanthine dehydrogenase activity. Hayashi et al. (1988)
also
found several flavonoids to be effective inhibitors of cow milk
xanthine oxidase. Quercetin and several other flavonoids were weak (100 µM) inhibitors of the enzyme; inhibitory activity did not correlate
consistently with flavonoid-induced cytochrome c reduction
(Iio et al., 1986
). Chang et al. (1993)
also found that baicalein and
quercetin were potent inhibitors of xanthine oxidase. These authors
also noted that xanthine oxidase serum levels were increased in
patients with hepatitis and brain tumor; they suggested that selected
flavonoids might be useful in treating these disorders.
Q. Aromatase
The conversion of androstenedione to estrone is catalyzed by
aromatase. Inhibition of aromatase (human estrogen synthetase) by
several naturally occurring flavonoids (including quercetin, chrysin,
apigenin, and others) was described by Kellis and Vickery (1984)
. The
synthetic flavone 7,8-benzoflavone was most active. Aromatization of
androstenedione was affected by several flavonoids, of which
7-hydroxyflavone and 7,4-dihydroxyflavone were the most potent (Ibrahim
and Abul-Hajj, 1990
). Inhibition by 7-hydroxyflavone was competitive
with respect to the substrate androstenedione. According to Moochhala
et al. (1988)
, flavonoids of the 5,7-dihydroxyflavone series could bind
to the active site human cytochrome P450 aromatase with affinity. The
flavonoid kaempferol inhibited aromatase enzyme activity competitively
in a human Glyoxalase cell culture system (Wang et al., 1994
). Such
results suggest that diets rich in these compounds could contribute to
the control of estrogen-dependent conditions, such as breast cancer.
R. 11-
-Hydroxysteroid Dehydrogenase
This enzyme oxidizes hydrocortisone to inactive cortisone. It is
also a key regulator of renal K+ clearance.
Slight inhibition of enzyme activity was noted with morin and quercetin
(Song et al., 1992
).
S. Catechol-O-methyltransferase
Early studies demonstrated that certain flavonoids have an
epinephrine-sparing action (Clark and Geissman, 1949
) that is probably attributable to inhibition of the catecholamine-metabolizing enzyme catechol-O-methyltransferase (COMT) (Gugler and Dengler,
1973
; Borchardt and Huber, 1975
). Three isoflavone inhibitors of COMT were isolated from a streptomyces culture filtrate (Chimura et al.,
1975
).
T. Aldose Reductase
Lens aldose reductase has been implicated in the pathogenesis of
cataracts in diabetic and galactosemic animals. The enzyme catalyzes
the reduction of glucose and galactose to their polyols, which
accumulate in large quantities in the lens and ultimately lead to
mature lens opacities. Several key bioflavones have activity against
this enzyme (Iwu et al., 1989
). In 1977, Varma et al. found that oral
administration of quercitrin decreased the accumulation of sorbitol in
the lens of the rodent Ocrodon degus; a similar effect was
seen with quercetin in the galactosemic neonatal rat. The accumulation
of lens opacities could be partially abrogated by certain flavonoids.
In a study of 30 flavones, 4 isoflavones and 13 coumarins, many potent
inhibitors were found, but 5,7,3',4'-tetrahydroxy-3,6-dimethoxyflavone and 6,3',4'-trihydroxy-5,7,8-trimethoxyflavone were especially active
(Varma, 1986
). In a subsequent study (Okuda et al., 1984
) of
3',4'-dihydroxyflavones, another potent inhibitor was discovered: 3',4'-dihydroxy-5,6,7,8-tetramethoxyflavone (Okuda et al., 1982
). Aldose reductase inhibition by the compounds was noncompetitive with
respect to both DL-glyceraldehyde and the reduced
form of NADP. Hypoglycemia-inducing effects (rabbits) and inhibition of rat lens aldose reductase activity of a mixture of biflavanones were
reported by Iwu et al. (1989)
.
U. Monoamine Oxidase (FAD-Containing)
Flavones, coumarins (neoflavonoids), and other oxygen-containing
compounds were found to inhibit monoamine oxidases A and B in a
reversible and time-independent manner (Thull and Testa, 1994
).
V. Aldo-Keto-Reductase Family of Enzymes
Carbonyl reduction is a metabolic pathway widely distributed in
nature. Many endogenous substances, such as prostaglandins, biogenic
amines, and steroids, together with xenobiotic chemicals of several
varieties, are transformed to the corresponding alcohols before further
metabolism and elimination. Carbonyl reduction in several continuous
cell lines was investigated using metyrapone as a substrate ketone.
Quercitrin was reported to inhibit carbonyl reductase (Maser and
Netter, 1991
).
W. Hyaluronidase
Hyaluronidases depolymerize hyaluronic acid to oligosaccharides by
breaking glucosaminidic bonds, have been referred to as "spreading
factor", and are possibly involved in tumor cell invasiveness. Rodney
and coworkers (1950)
described the inhibitory effect of a series of
flavonoids on hyaluronidase and some other related enzymes. More
recently, Kuppusamy et al. (1990)
re-examined the effects of 31 flavonoids representing several chemical classes on the activity of
bovine testis hyaluronidase. Kaempferol and silybin were most active.
Kinetic analysis revealed that these compounds acted competitively.
X. Histidine Decarboxylase and DOPA Decarboxylase
Early experiments (Martin et al., 1949
) suggested that histidine
decarboxylase was inhibited by selected flavonoids such as quercetin
and (+)-catechin, whereas the flavonoid glycosides were inactive.
Histamine stimulates gastric acid secretion, making the reported
inhibition of histamine-induced gastric secretion by the synthetic
flavone-6-carboxylic acid of interest (Pfister et al., 1980
). Parmar et
al. (1984)
described the gastric antisecretory activity of the flavan
derivative 3-methoxy-5,7,3',4'-tetrahydroxyflavan, a compound that
appears to be a specific histidine decarboxylase inhibitor in rats and
is as effective as cimetidine in reducing gastric acid secretion. This
flavan also reduced gastric tissue histamine content in rats (Parmar
and Hennings, 1984
; Parmar et al., 1984
). Naringenin, the aglycone of
naringin, was a weak inhibitor of histidine decarboxylase and also
exhibited some gastric antiulcer activity (Parmar, 1983
).
Umezawa et al. (1975)
reported orobol and
3',4',5,7-tetrahydroxy-8-methoxy isoflavone from culture filtrates of
fungi and streptomyces were effective inhibitors of DOPA decarboxylase, and orobol had a significant hypotensive effect in spontaneously hypertensive rats.
Y. Malate Dehydrogenase
Malate dehydrogenase was inhibited by quercetin, which Seddon and
Douglas (1981)
also showed could produce photo-induced covalent labeling of the enzyme.
Z. Lactic Dehydrogenase and Pyruvate Kinase
Grisiola and coworkers (1975)
found that these enzymes were quite
effectively inhibited by quercetin.
AA. Aldehyde and Alcohol Dehydrogenases
An extract of R. puerariae, an herb
long-used in traditional Chinese medicine for alcohol addiction and
intoxication, suppressed the free-choice ethanol intake of
ethanol-preferring Syrian golden hamsters (Keung and Vallee, 1994
). The
isoflavonoids daidzein (4',7-dihydroxyisoflavone) and daidzin
(7-glucoside of daidzein) isolated from the extract (Keung, 1993
) were
shown to account for this effect by inhibiting human alcohol
dehydrogenase. Daidzin and daidzein, at doses that suppressed ethanol
intake, exhibited no effect on overall acetaldehyde and ethanol
metabolism in hamsters, although they inhibited human mitochondrial
aldehyde dehydrogenase and gamma-gamma alcohol dehydrogenase in vitro.
These observations clearly distinguish the action(s) of these
isoflavones from those of the classic, broadly acting inhibitors of
aldehyde dehydrogenase and of class 1 alcohol dehydrogenase enzymes.
Consequently, daidzin and daidzein represent a new class of compounds
offering promise as safe and effective therapeutic agents for alcohol abuse.
BB. Amylase
Rat pancreatic acinar cell amylase secretion stimulated by
cholecystokinin octapeptide, carbachol, or TPA was inhibited by quercetin; however, vasoactive intestinal polypeptide-induced secretion
was unaffected (Lee et al., 1988
).
CC. RNA and DNA Polymerases
The experiments of Nose (1984)
demonstrated that quercetin,
kaempferol, and fisetin inhibited transcription with RNA polymerase II
in permeabilized normal human fibroblasts (Wl-38 cells); flavone and
chrysin exhibited weak activity. Addition of quercetin to an ongoing
transcription reaction arrested it promptly, suggesting that quercetin
was inhibiting the elongation step. The effects of several flavonoids
(quercetin, quercetagetin, myricetin, and baicalein) exhibited complex
interactions with DNA and RNA polymerases, depending on the particular
flavonoid and the enzyme species (Ono and Nakane, 1990
).
DD. Human DNA Ligase I
In an ongoing effort to identify clinically useful anticancer
drugs, Tan et al. (1996)
examined the effect of several natural products for their ability to disrupt the function of human DNA ligase
I, which catalyzes the covalent joining of single-stranded breaks in
double-stranded DNA. Interestingly, a flavonoxanthone glucoside,
swertifrancheside (isolated from Swerua
franchetiana), inhibited enzyme function with
IC50 of 11 µM.
EE. Ribonuclease
Mori and Noguchi (1970)
studied the effects of flavonoids on
bovine pancreatic ribonuclease 1. They found that flavones and flavonols with hydroxy substitutions at positions 7, 3', and 4 dramatically inhibited the activity of ribonuclease 1. A keto group at
position 4 was also important.
FF. Sialidase
Sialidase (neuraminidase) catalyzes the hydrolysis of sialic acid
residues from sialoglycoconjugates and may have an effect on biological
functions such as antigen presentation and receptor function. Mouse
liver sialidase was noncompetitively inhibited by
isoscutellarein-8-O-glucuronide (IC50,
40 µM), while influenza virus sialidase was only weakly inhibited
(Nagai et al., 1989
). Flavanone and chalcone structures essentially
lacked activity against the liver enzyme. In studies of influenza
sialidase, Nagai and coworkers (1990
, 1992
) examined the effect of
other flavonoids derived from Scutellana baicalensis.
5,7,4'-Trihydroxy-8-methoxyflavone proved to be a moderately active
compound among 103 tested. Since binding of influenza virus to target
cells takes place via sialic acid residues in the viral envelope
glycoprotein, it is of interest that 5,7,4'-trihydroxy-8-methoxyflavone
also inhibited infection by influenza virus A/PR/8/34 of Madin-Darby
canine kidney cells and replication of virus in embryonated egg
allantoic sacs.
GG. Cytochrome P450 Systems
Studies on the influence of flavonoids on cytochrome P450 enzymes
are discussed elsewhere. A recent study has examined the relationship
between the electrochemical properties of flavonoids and the influence
on phenol hydroxylase of rat liver microsomes. The effect of flavonoids
on this P450-dependent hydroxylase activity was found to correlate well
with the oxidation potential for flavonoid aglycones (Hendrickson et
al., 1994
). Easily oxidizable flavonoids inhibited microsomal phenol
hydroxylase activity in a dose-dependent manner, with the extent of
inhibition correlating with the ease of oxidation. In contrast,
flavonoids with high oxidation potentials stimulated the hydroxylase
activity in a dose-independent manner. No correlation was apparent
between electrochemical properties and effects on microsomal benzene
hydroxylase activity.
HH. Elastase
A unique flavonoid, 3'-hydroxyfarrerol
(6,8-dimethyl-5,7,3',4'-tetrahydroxyflavanone (also known as IdBl03l),
inhibited human neutrophil elastase, but only weakly
(IC50, approximately 200 µM), acting with a
reversible, noncompetitive mode of inhibition (Meloni et al., 1995
).
Moreover, this compound significantly reduced tumor necrosis factor
(TNF)-
and IL-8 generation in lipopolysaccharide (LPS)-stimulated
peripheral blood mononuclear cells (at 10 µM) (Meloni et al., 1995
).
These properties, together with its ability to inhibit human neutrophil
elastase, make it a possible candidate for pharmacotherapy of chronic
lung disorders characterized by leukocytic infiltration.
II. Nitric-Oxide Synthase
The recently recognized and intriguing chemical mediator, nitric
oxide (NO), possesses many important physiological activities, e.g.,
smooth muscle relaxation, tumor cell lysis and destruction of
microorganisms, among many others (Lowenstein and Snyder, 1992
; Nathan,
1992
; Moncada and Higgs, 1993
). Its synthesis from arginine is
catalyzed by an inducible enzyme, nitric oxide synthase (iNOS). Of
great interest is the observation that genistein and two other PTK
inhibitors (herbimycin and tyrphostin) inhibited the generation of NO and the induction of iNOS in murine macrophages (Dong et al.,
1993
). Both LPS- and cytokine-dependent inducible NO synthase were
blocked by genistein in C6 glioma cells (Feinstein et al., 1994
).
Several dietary polyphenolic compounds were shown to attenuate NO
production in C6 astrocyte cell cultures. Active flavonoid compounds
included quercetin, epigallocatechin gallate, morin, apigenin,
taxifolin, fisetin, and catechin (Soliman and Mazzio, 1998
). Chiesi and
Schwaller (1995)
found tannin and quercetin to inhibit NO synthase
activity of three isoforms of the enzyme.
It is hard to speculate on the broad ability of flavonoids to inhibit the activity of so many different enzyme systems. The apparent requirement of a C2-C3 double bond and hydroxylation of the B ring points toward some stereospecific interaction, especially as it concerns the competitive interferences