I. General Aspects

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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.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Chemical structures of the most common flavonoid subclasses. The lower part of the figure shows the generic structure of flavon-3-ols and some representative compounds where the hydroxyl groups of ring B are shown.

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).

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.

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.

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.

	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.

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 p56^lck 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.

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).

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).

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).

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.

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.

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.

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.

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.

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).

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).

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).

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).

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).

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).

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.

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.

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).

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).

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).

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).

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).

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.

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.

Malate dehydrogenase was inhibited by quercetin, which Seddon and Douglas (1981) also showed could produce photo-induced covalent labeling of the enzyme.

Grisiola and coworkers (1975) found that these enzymes were quite effectively inhibited by quercetin.

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.

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).

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).

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.

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.

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.

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.

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.

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