Bone and Mineral, 19 (Suppl.) (1992) S3-S 14 0169-6009/92/$05.00 0 1992 Elsevier Science Publishers B.V. All rights reserved.
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BAM 00483 This paper was presented at a satellite symposium at the XIth international Conference on Calcium Regulating Hormones, held April 24-29, 1992, Florence, Italy.
Flavonoids: biochemical effects and therapeutic applications
Maria Luisa Brandi Endocrine Unit, Department of Clinical Physiopathology,
University of Florence, Florence, Italy
Key words: Flavonoids
Historical background
Bioflavonoids are benzo-gamma-pyrone derivatives which are ubiquitous in photosynthesizing cells. Over 4000 heavebeen identified from both higher and lower plants and the list constantly expands. This multiplicity is not surprising in view of the structural diversity of flavonoids. Through their food, all plant-eating animals are influenced by the flavonoids. Indeed, the average daily human intake of these compounds in western countries has been estimated to be 1 g or more. Therefore, the importance of flavonoids lies in their widespread occurrence in human foods. Moreover, they are used in more purified forms as drugs and food supplements. Flavonoids first interested pharmacologists when they were shown to possess vitamin-like properties, In the early 1930sthe discovery of their vitamin C-sparing activity led to the short-lived proposal of “vitamin P” [l]. Flavonoids have been shown to be the main conseituents of folk remedies used for the treatment of thyroid and other hormonal disorders [2]. They also possess alAoxidant and antimicrobial properties and are of particular interest because several of them have been shown to be antimutagenic and anticarcinogenic [3].
Correspondence to: Prof. Maria Luisa Brandi, Endocrine Unit, Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy.
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FLAVONE SKELETON Fig. 1, Basicstructurulfeatureof flavonoids.A and B: benzenerings;C: pyrane ring,
Biochemistry of flavonoids
The biosynthesis of flavonoids in plants has been elucidated. It commences with phenylalanine and proceeds through transcinnamic acid and p-cumarie acid. The basic structural feature of flavonoid compounds is the flavone nucleus made up of two benzene rings (A and B) linked through a heterocyclic pyrane C ring (Fig, 1). The position of the benzenoid B ring divides the flavonoid class into flavonoids (Zposition) and isoflavonoids (3-position). Flavonoids are often hydroxylated in position 3, 5, 7, 3’, 4’ and 5’. The presence or absence of a hydroxyl group at position 3 results in the generation of the two main subgroups of flavonoids: 3-hydroxyflavonoids (flavonols, flavanols or catechins) and 3desoxyflavonoids (flavones and flavanones). When glycosides are formed, the glycosidiclinkage is normally located in position 3 or 7 and the carbohydrate can be L-rhamnose, D-glucose,glucorhamnose, galactose or arabinose. Flavonoids are easily oxidized at the B-ring which leads to opening of this ring at the oxygen atom. Based on just a few backbone structures, such as various hydroxylation, methoxylation, sulfation and glycosilation patterns, there are at least 20736000 possible members of the flavonoid family. Indeed, the structural diversity of flavonoids appears to determine their activity.
Biochemicaleffects of flavonoids
Numerous reports described the inhibition by flavonoids of a perplexing number and variety of enzymes. When one examines these enzymes, which are all influenced by a group of compounds of a rather homogeneous structure, one notices that they seem to have little in common. Therefore, they apparently interact with different parts of the flavonoid molecule. Flavonoids are powerful inhibitors of both S-lipoxygenase and cycloxygenase, interfering therefore with the metabolism of arachidonic acid [4,5]. Hyaluronidases are enzymes which depolymerize hyaluronic acid [6], claimed to be involved in allergic effects [7], migration of cancer cells [8]and permeability of the vascular system [9].Flavonoids have been shown to inhibit in a competitive manner this enzymatic activity [lo]. Flavonoids have been found to exhibit strong antioxidative properties, provid-
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ing direct scavenging activity by preventing the oxygen-related Fenton reaction. The lipid peroxidation might play an important role in cell ageing and oxygen toxicity in general. Flavonoids inhibit non-enzymatic lipid peroxidation and lipid peroxidation induced by NADPH in vitro [I 1,121. Both the propensity of flavonoids for electron transfer and the capability of binding of heavy metal ions may explain their interference with the appearance of free radicals. Flavonoids inhibit CAMP phosphodiesterase from different species and can, therefore, mimic CAMP effects [ 13,14,15]. DNA topoisomerases I and II are enzymes that catalyse the concerted breaking and rejoining of DNA strands, thereby controlling the topological states of DNA. Flavonoids are inducers of DNA-topoir.omerase II cleavable complex which upon exposure to denaturing agents results in the induction of DNA cleavage, with production of bulky DNA adducts t at can lead to cell death [ 16,171. Tyrosine protein kinase is known to be associated with oncogene products of retroviral genes, being correlated with the ability of retroviruses to transform cells [l&19]. Similar kinase activities are also associated with growth factor receptors [20-231.It is supposed, therefore, that tyrosine phasphorylation plays an important role for diverse cell functions. Indeed, flavonoids have been reported to inhibit tyrosine protein kinase activity and growth of Rous sarcoma virustransformed cells [24]. Flavonoids inhibit diverse reverse transcriptases and, therefore, they should be further studied for their potential as anti-retroviral agents [25]. Flavonoids have also potent protein kinase C and proteolytic activities inhibitors [26,27]. Early studies indicated that natural flavonoids are able to induce goiter [28]. Together with this increase in the weight of the thyroid, a decrease in iodine organification and decrease in the thyroxine concentration of the gland were found. All these effects were suppressed by adding small amounts of iodide to the diet, suggesting a flavonoid inhibition of iodide organification [28].Additionally, flavonoids have been found to inhibit iodothyronine deiodinase [29]. Indeed, a synthetic flavonoid, EDM 21388, is the most potent flavonoid inhibitor of Sdeiodinase in liver [30]. Furthermore, EDM 21388 exhibits a markedly higher affinity for binding to transthyretin than T4 and T3 both in vivo and in vitro, but it does not inhibit binding of the latter to either thyroxin-binding globulin or albumin [31,32]. The estrogenic effect of flavonoids was noticed when sheep ate a particular flavonoid-rich vegetation. It may be rationalized by reference to the spatial relationship between the phenolic hydroxyl groups of estradiol and certain flavonoids. The close agreement between these configurations and between the associated chemical properties suggests that flavonoids after binding to the cytosolic estrogen receptor can activate the same genes as true estrogens. Screening of specific hormone-receptor-binding inhibitors showed that flavanones isolated from soil samples inhibit estrogen binding to their receptors 1331.Some of the flavonoids were found to have estrogenic or antiestrogenic activities [34,35], as well as aromatase inhibitory activities [36].It has been reported that quercetin
S6 binds to the so-called type II estrogen-binding sites originally described by Clark et al. in rat uterus [37].Although these sites display the same steroid and tissue specificity shown by the true estrogen receptor, they are distinct from the latter, occurring at higher concentrations and exhibiting a lower apparent affinity for estrogen receptor than estradiol. Moreover, their presence has been described in many primary tumors 138-401.In rat uterus type II estrogen-binding sites are occupied in vivo by flavonoid-like ligand. with growth inhibitory activity [41]. Furthermore, it has recently been shown that in the human breast cancer cell line MCF7, flavonoids bind to type II estrogen-binding sites and inhibits cell growth by mimicking the endogenous ligand [42].Similarly to estrogen, some:flavonoids are able to induce transcription. Indeed, in plants genistein, daidzein and genistin are able to induce nodulation genes, probably acting at&he transcriptional level where nodulation proteins activate the nodulation genes. The identification of certain domains in the primary structure of nodulation proteins responsible for flavonoid recognition would point to a direct interaction between the two [43].
Pllannacologlcal proprtics
The flavonoids are one of the most common plant metabolites, occurring almost
ubiquitously in the fruits, vegetables and beverages in our diet as well as in several important medicinal plants [44,45].Owing to their importance, flavonoids have been extensively tested for the genotoxic and carcinogenic potential [46]. The use of flavonoids in the treatment of diseases is to a large extent based on empirism since this praxis is much older than the science of chemistry. Until very recently, our knowledge of the biochemistry of flavonoids was not sufficient for rational medical application of these substances. The controlled clinical experiments on the use of flavonoids by well-defined illness were hitherto few and the flavonoids used were those which were readily available as pure substances or in mixtures, like Propolis preparation. Most of the studies have been performed in small groups, without quantification of the absorption. Their structural similarity to nucleosides, isoalloxazine and folic acid is the basis of many of the current hypotheses of their physiological actions. Some of the diseases on which therapeutic attempts have been made with flavonoids are listed in Table 1. The in vivo antiinflammatory effect of flavonoids can be explained on the basis of their inhibitory effect on arachidonic acid metabolism [47]. Inflammation is known to be accompanied by the release of prostaglandins which by chemotaxis attract leukocytes to the point of invasion, create local pain and raise the body temperature. The known inhibition of prostaglandin cycloxigenase and lipoxigenase would, therefore, lead to the observed local pain relief and antipyretic effect. Moreover, flavonoids interfere with histamine metabolism [48]and inhibit migration of murine neutrophils in vitro [49]. There is a relative paucity of information on the effects of flavonoid substances on the central nervous system. Only few results showed an analgesic activity for certain flavonoids with no involvement of the opiate receptors, probably via the
s7 Table 1
Biochemical effects and therapeutic applications of flavonoids Target
Disorder
Prostaglandin synthesis
Anti-inflammatory Analgesic Anti-allergic Anti-atherosclerotic Anti-proliferative Anti-proliferative Anti-retroviral Anti-metastatic Anti-viral Anti-proliferative Bone anabolic Bone anabolic
Hyaluronidases Lipid peroxidation Topoisomerases I and II Tyrosine protein kinase Reverse transcriptases Matrix proteolysis Lysosomal enzymes Estrogenic properties Proline hydroxylase _^_..__.
inhibition of prostaglandin synthesis [50]. With headache, a plausible reason would be the relaxation of smooth muscles, which have been seized by cramps, through the action of prostaglandins and leukotrienes. Flavonoids also relieve the pain in stomach and duodenal ulcer, paradentosis, insect bites and inflamed joints, without causing side effects. The inhibition of induction of catabolic hydrolases by prostaglandins would offer a plausible explanation for the effect of the compounds. Quercetin and kaempferol are inhibitors of erythrophagocytosis in monocytesmacrophages by the inhibition of stress proteins and heme oxygenase synthesis [51]. Flavonoids also inhibit LDL oxidative modifications by macrophages, raising the possibility that they may protect to a certain extent against atherosclerosis [52]. The flavonoid quercetin exerts powerful growth-inhibitory activity of human leukemic cells [53] and on several human cancer cell lines [38,41,42,54,55]. Moreover, quercetin and certain related flavonoids may be inhibitors of experimental skin carcinogenesis [56]. Moreover, flavone-8-acetic acid is a flavonoid drug that augments mouse natural killer cell activity, induces cytokine gene expression and synergizes with recombinant interleukin 2 for the treatment of murine renal cancer [57]. Flavonoids also inhibit invasion of a number of cell. types via different mechanisms, whose the best characterized is the inhibition of proteolysis of extracellular matrix [27]. Recently, in vivo experiments in animal models have shown that ipriflavone, an isoflavone derivative, is active in preventing the decrease of bone mass in several models of experimental osteoporosis [58-601.Moreover, autoradiography studies showed distribution of the compound in bone tissue [61].The mechanism through which ipriflavone exerts its protective effect on the skeleton could be explained on the basis of an inhibitory effect of the compound on bone resorption, as confirmed also by histopathologic studies [62,63]. Indeed, in parathyroid tramplanted parietal bones of rats ipriflavone significantly reduces the numbers of
S8 HEYATOPOIETIC STEM CELL
0 I
IPRIFLAVONE
0+COUAQEN SYNTHESIS %&~:TASE
m
OSTEOBLASTS
Pig. 2. Mechanisms of action of ipriflavone on the bone remodelling process.
tartrate-resistant acid-phosphatase-positive polynucleated osteoclasts and mononucleated cells, suggesting a role of the compound in controlling preosteoclast recruitment (Fig. 2; [64]). In addition, ipriflavone stimulates collagen synthesis in human auditory ossicle samples from whole organ cultures [62,63] and in clonal osteoblast-like cells [65]. This anabolic effect on bone collagen synthesis may probably be related toYhe activation of the proline hydroxylase [66]. Interestingly, high doses of ipriflavone significantly inhibit the response of osteoblastic cells to parathyroid hormone (PTH) (Fig. 2; [65]). The possibility that the inhibitory eff&ztof ipriflavone on bone resorption observed in the parathyroid transplanted animals is mediated through an inhibition of the PTIG responsiveness of osteoblastic cells needs to be tested in co-culture models of osteoblasts and osteoclast precursors.
Structure-activityrelationships The analysis of a structure-activity relationship is critical for the construction of even better flavonoids. A good example comes from the vegetable kingdom. Flavonoids have at least two distinct functions in the interactions of leguminous plants with microorganisms. Phytoalexins are synthesized in response to pathogen attacks and have antimicrobial properties. While, other flavonoids act as transcriptional activators of nodulation (nod) genes in the soil bacteria Rhizobiu and Brudyrhizobia leading to the establishment of a Nz-fixing association [67]. These different biological properties could be referred to the difference in the flavonoid end products. The genotoxicity of flavonoids in human lymphocytes in vitro seems to be connected with the planarity of the molecule. Only the planar flavone or flavonol
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derivatives showed activity, whereas the unplanar flavanols were inactive [68]. Some flavonoids were found to be selective on lipoxygenase inhibitors, some as cycle-oxigenase inhibitors, some inhibited both enzymes and some were very poorly active against either enzyme [4]. These selectivities suggested that different structure-dependent mechanisms determine inhibition of the two enzymes by the flavonoids. Flavonoids possessing hydroxyl groups inhibit P-450 enzymatic activity, whereas those lacking hydroxyl groups stimulate activity [69]. Similar structure-activity relationships exist for the effect of Ravones on ethoxycoumarin deethylase activity in rat hepatic microsomes [70]. Furthermore, Friedman et al, have shown that various flavonoids differ in their inhibitory effects on constitutive and induced aryl hydrocarbon hydroxylase activity in rat liver [71]. Also the hydroxylation and glycosylation of flavone modify the potency of these compounds [5,72].
Pharmacodynamics
Flavonoids are metabolized by animal cells, expecially those of the liver and excreted in the urines, without accumulation in the body. The toxicity of flavonoids is very low in animals. Only a few data are available on the pharmacokinetics of flavonoids. It is known that bacterial glycosidases are able of liberating flavonoid aglycons. Larger amounts of orally taken flavonoids must, therefore, be expected to be largely present as aglycons in the intestine and to become absorbed with micelles of bile acids into the epithelium and then into the blood. Through the portal vein, the major part of the flavonoids would probably be delivered more or less directly to the liver, which decomposes them. Indeed, the lack of success of flavonoid treatment may often be ascribed to the failure of the compound to reach its target. So far little is known about the affinity of flavonoids to blood plasma proteins. But due to the low polarity of many aglycons they would probably be bound to serum albumin. Interestingly, an extensive characterization of the pharmacokinetics of ipriflavone in humans revealed that the compound is metabolized into seven metabolites (Fig. 3; [73,74]).The main metabolites in humans are metabolites I, II, and V. Metabolites I and II circulate mainly as conjugated forms, but metabolite V, like ipriflavone, is recovered in unconjugated form in the human body, suggesting that the latter metabolite should greatly contribute to the action of ipriflavone. Indeed, in vitro studies revealed differential effects of ipriflavone and its metabolites both on bone resorption [62] and bone formation [65]. Therefore, ipriflavone metabolism may contribute both to the inactivation as well as to the induction of the osteotrophic properties of the compound.
SIO
Fig. 3. Structuralskeletonsof ipritlavone and its metr.bolites.
Flavonoids represent a large area of still open investigation. Indeed, the information we have is rarely supported by controlled clinical studies in large numbers of patients. Areas, like the antiproliferative effect of genistein on cancer cells and the
Sll anabolic action of ipriflavone on the skeleton, need to be analyzed in detail. These models offer, in fact, unique possibilities both for understanding the molecular mechanisms which underlie the effects of these compounds on target organs and for characterizing their therapeutic applications in clinical disorders. Indeed, from studies performed with cancer cells it appears that bioflavonoids may inhibit cell proliferation through an interaction with type II binding sites [75,76]. Moreover, ipriflavone appears to interact with the estrogen receptors and to possess unique binding sites in bone-derived cells (ML Brandi, unpublished data). The results obtained from in vitro and in vivo studies will constitute the basis for the construction of better flavonoid compounds.
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BAM 00483 This paper was presented at a satellite symposium at the XIth international Conference on Calcium Regulating Hormones, held April 24-29, 1992, Florence, Italy.
Flavonoids: biochemical effects and therapeutic applications
Maria Luisa Brandi Endocrine Unit, Department of Clinical Physiopathology,
University of Florence, Florence, Italy
Key words: Flavonoids
Historical background
Bioflavonoids are benzo-gamma-pyrone derivatives which are ubiquitous in photosynthesizing cells. Over 4000 heavebeen identified from both higher and lower plants and the list constantly expands. This multiplicity is not surprising in view of the structural diversity of flavonoids. Through their food, all plant-eating animals are influenced by the flavonoids. Indeed, the average daily human intake of these compounds in western countries has been estimated to be 1 g or more. Therefore, the importance of flavonoids lies in their widespread occurrence in human foods. Moreover, they are used in more purified forms as drugs and food supplements. Flavonoids first interested pharmacologists when they were shown to possess vitamin-like properties, In the early 1930sthe discovery of their vitamin C-sparing activity led to the short-lived proposal of “vitamin P” [l]. Flavonoids have been shown to be the main conseituents of folk remedies used for the treatment of thyroid and other hormonal disorders [2]. They also possess alAoxidant and antimicrobial properties and are of particular interest because several of them have been shown to be antimutagenic and anticarcinogenic [3].
Correspondence to: Prof. Maria Luisa Brandi, Endocrine Unit, Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy.
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FLAVONE SKELETON Fig. 1, Basicstructurulfeatureof flavonoids.A and B: benzenerings;C: pyrane ring,
Biochemistry of flavonoids
The biosynthesis of flavonoids in plants has been elucidated. It commences with phenylalanine and proceeds through transcinnamic acid and p-cumarie acid. The basic structural feature of flavonoid compounds is the flavone nucleus made up of two benzene rings (A and B) linked through a heterocyclic pyrane C ring (Fig, 1). The position of the benzenoid B ring divides the flavonoid class into flavonoids (Zposition) and isoflavonoids (3-position). Flavonoids are often hydroxylated in position 3, 5, 7, 3’, 4’ and 5’. The presence or absence of a hydroxyl group at position 3 results in the generation of the two main subgroups of flavonoids: 3-hydroxyflavonoids (flavonols, flavanols or catechins) and 3desoxyflavonoids (flavones and flavanones). When glycosides are formed, the glycosidiclinkage is normally located in position 3 or 7 and the carbohydrate can be L-rhamnose, D-glucose,glucorhamnose, galactose or arabinose. Flavonoids are easily oxidized at the B-ring which leads to opening of this ring at the oxygen atom. Based on just a few backbone structures, such as various hydroxylation, methoxylation, sulfation and glycosilation patterns, there are at least 20736000 possible members of the flavonoid family. Indeed, the structural diversity of flavonoids appears to determine their activity.
Biochemicaleffects of flavonoids
Numerous reports described the inhibition by flavonoids of a perplexing number and variety of enzymes. When one examines these enzymes, which are all influenced by a group of compounds of a rather homogeneous structure, one notices that they seem to have little in common. Therefore, they apparently interact with different parts of the flavonoid molecule. Flavonoids are powerful inhibitors of both S-lipoxygenase and cycloxygenase, interfering therefore with the metabolism of arachidonic acid [4,5]. Hyaluronidases are enzymes which depolymerize hyaluronic acid [6], claimed to be involved in allergic effects [7], migration of cancer cells [8]and permeability of the vascular system [9].Flavonoids have been shown to inhibit in a competitive manner this enzymatic activity [lo]. Flavonoids have been found to exhibit strong antioxidative properties, provid-
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ing direct scavenging activity by preventing the oxygen-related Fenton reaction. The lipid peroxidation might play an important role in cell ageing and oxygen toxicity in general. Flavonoids inhibit non-enzymatic lipid peroxidation and lipid peroxidation induced by NADPH in vitro [I 1,121. Both the propensity of flavonoids for electron transfer and the capability of binding of heavy metal ions may explain their interference with the appearance of free radicals. Flavonoids inhibit CAMP phosphodiesterase from different species and can, therefore, mimic CAMP effects [ 13,14,15]. DNA topoisomerases I and II are enzymes that catalyse the concerted breaking and rejoining of DNA strands, thereby controlling the topological states of DNA. Flavonoids are inducers of DNA-topoir.omerase II cleavable complex which upon exposure to denaturing agents results in the induction of DNA cleavage, with production of bulky DNA adducts t at can lead to cell death [ 16,171. Tyrosine protein kinase is known to be associated with oncogene products of retroviral genes, being correlated with the ability of retroviruses to transform cells [l&19]. Similar kinase activities are also associated with growth factor receptors [20-231.It is supposed, therefore, that tyrosine phasphorylation plays an important role for diverse cell functions. Indeed, flavonoids have been reported to inhibit tyrosine protein kinase activity and growth of Rous sarcoma virustransformed cells [24]. Flavonoids inhibit diverse reverse transcriptases and, therefore, they should be further studied for their potential as anti-retroviral agents [25]. Flavonoids have also potent protein kinase C and proteolytic activities inhibitors [26,27]. Early studies indicated that natural flavonoids are able to induce goiter [28]. Together with this increase in the weight of the thyroid, a decrease in iodine organification and decrease in the thyroxine concentration of the gland were found. All these effects were suppressed by adding small amounts of iodide to the diet, suggesting a flavonoid inhibition of iodide organification [28].Additionally, flavonoids have been found to inhibit iodothyronine deiodinase [29]. Indeed, a synthetic flavonoid, EDM 21388, is the most potent flavonoid inhibitor of Sdeiodinase in liver [30]. Furthermore, EDM 21388 exhibits a markedly higher affinity for binding to transthyretin than T4 and T3 both in vivo and in vitro, but it does not inhibit binding of the latter to either thyroxin-binding globulin or albumin [31,32]. The estrogenic effect of flavonoids was noticed when sheep ate a particular flavonoid-rich vegetation. It may be rationalized by reference to the spatial relationship between the phenolic hydroxyl groups of estradiol and certain flavonoids. The close agreement between these configurations and between the associated chemical properties suggests that flavonoids after binding to the cytosolic estrogen receptor can activate the same genes as true estrogens. Screening of specific hormone-receptor-binding inhibitors showed that flavanones isolated from soil samples inhibit estrogen binding to their receptors 1331.Some of the flavonoids were found to have estrogenic or antiestrogenic activities [34,35], as well as aromatase inhibitory activities [36].It has been reported that quercetin
S6 binds to the so-called type II estrogen-binding sites originally described by Clark et al. in rat uterus [37].Although these sites display the same steroid and tissue specificity shown by the true estrogen receptor, they are distinct from the latter, occurring at higher concentrations and exhibiting a lower apparent affinity for estrogen receptor than estradiol. Moreover, their presence has been described in many primary tumors 138-401.In rat uterus type II estrogen-binding sites are occupied in vivo by flavonoid-like ligand. with growth inhibitory activity [41]. Furthermore, it has recently been shown that in the human breast cancer cell line MCF7, flavonoids bind to type II estrogen-binding sites and inhibits cell growth by mimicking the endogenous ligand [42].Similarly to estrogen, some:flavonoids are able to induce transcription. Indeed, in plants genistein, daidzein and genistin are able to induce nodulation genes, probably acting at&he transcriptional level where nodulation proteins activate the nodulation genes. The identification of certain domains in the primary structure of nodulation proteins responsible for flavonoid recognition would point to a direct interaction between the two [43].
Pllannacologlcal proprtics
The flavonoids are one of the most common plant metabolites, occurring almost
ubiquitously in the fruits, vegetables and beverages in our diet as well as in several important medicinal plants [44,45].Owing to their importance, flavonoids have been extensively tested for the genotoxic and carcinogenic potential [46]. The use of flavonoids in the treatment of diseases is to a large extent based on empirism since this praxis is much older than the science of chemistry. Until very recently, our knowledge of the biochemistry of flavonoids was not sufficient for rational medical application of these substances. The controlled clinical experiments on the use of flavonoids by well-defined illness were hitherto few and the flavonoids used were those which were readily available as pure substances or in mixtures, like Propolis preparation. Most of the studies have been performed in small groups, without quantification of the absorption. Their structural similarity to nucleosides, isoalloxazine and folic acid is the basis of many of the current hypotheses of their physiological actions. Some of the diseases on which therapeutic attempts have been made with flavonoids are listed in Table 1. The in vivo antiinflammatory effect of flavonoids can be explained on the basis of their inhibitory effect on arachidonic acid metabolism [47]. Inflammation is known to be accompanied by the release of prostaglandins which by chemotaxis attract leukocytes to the point of invasion, create local pain and raise the body temperature. The known inhibition of prostaglandin cycloxigenase and lipoxigenase would, therefore, lead to the observed local pain relief and antipyretic effect. Moreover, flavonoids interfere with histamine metabolism [48]and inhibit migration of murine neutrophils in vitro [49]. There is a relative paucity of information on the effects of flavonoid substances on the central nervous system. Only few results showed an analgesic activity for certain flavonoids with no involvement of the opiate receptors, probably via the
s7 Table 1
Biochemical effects and therapeutic applications of flavonoids Target
Disorder
Prostaglandin synthesis
Anti-inflammatory Analgesic Anti-allergic Anti-atherosclerotic Anti-proliferative Anti-proliferative Anti-retroviral Anti-metastatic Anti-viral Anti-proliferative Bone anabolic Bone anabolic
Hyaluronidases Lipid peroxidation Topoisomerases I and II Tyrosine protein kinase Reverse transcriptases Matrix proteolysis Lysosomal enzymes Estrogenic properties Proline hydroxylase _^_..__.
inhibition of prostaglandin synthesis [50]. With headache, a plausible reason would be the relaxation of smooth muscles, which have been seized by cramps, through the action of prostaglandins and leukotrienes. Flavonoids also relieve the pain in stomach and duodenal ulcer, paradentosis, insect bites and inflamed joints, without causing side effects. The inhibition of induction of catabolic hydrolases by prostaglandins would offer a plausible explanation for the effect of the compounds. Quercetin and kaempferol are inhibitors of erythrophagocytosis in monocytesmacrophages by the inhibition of stress proteins and heme oxygenase synthesis [51]. Flavonoids also inhibit LDL oxidative modifications by macrophages, raising the possibility that they may protect to a certain extent against atherosclerosis [52]. The flavonoid quercetin exerts powerful growth-inhibitory activity of human leukemic cells [53] and on several human cancer cell lines [38,41,42,54,55]. Moreover, quercetin and certain related flavonoids may be inhibitors of experimental skin carcinogenesis [56]. Moreover, flavone-8-acetic acid is a flavonoid drug that augments mouse natural killer cell activity, induces cytokine gene expression and synergizes with recombinant interleukin 2 for the treatment of murine renal cancer [57]. Flavonoids also inhibit invasion of a number of cell. types via different mechanisms, whose the best characterized is the inhibition of proteolysis of extracellular matrix [27]. Recently, in vivo experiments in animal models have shown that ipriflavone, an isoflavone derivative, is active in preventing the decrease of bone mass in several models of experimental osteoporosis [58-601.Moreover, autoradiography studies showed distribution of the compound in bone tissue [61].The mechanism through which ipriflavone exerts its protective effect on the skeleton could be explained on the basis of an inhibitory effect of the compound on bone resorption, as confirmed also by histopathologic studies [62,63]. Indeed, in parathyroid tramplanted parietal bones of rats ipriflavone significantly reduces the numbers of
S8 HEYATOPOIETIC STEM CELL
0 I
IPRIFLAVONE
0+COUAQEN SYNTHESIS %&~:TASE
m
OSTEOBLASTS
Pig. 2. Mechanisms of action of ipriflavone on the bone remodelling process.
tartrate-resistant acid-phosphatase-positive polynucleated osteoclasts and mononucleated cells, suggesting a role of the compound in controlling preosteoclast recruitment (Fig. 2; [64]). In addition, ipriflavone stimulates collagen synthesis in human auditory ossicle samples from whole organ cultures [62,63] and in clonal osteoblast-like cells [65]. This anabolic effect on bone collagen synthesis may probably be related toYhe activation of the proline hydroxylase [66]. Interestingly, high doses of ipriflavone significantly inhibit the response of osteoblastic cells to parathyroid hormone (PTH) (Fig. 2; [65]). The possibility that the inhibitory eff&ztof ipriflavone on bone resorption observed in the parathyroid transplanted animals is mediated through an inhibition of the PTIG responsiveness of osteoblastic cells needs to be tested in co-culture models of osteoblasts and osteoclast precursors.
Structure-activityrelationships The analysis of a structure-activity relationship is critical for the construction of even better flavonoids. A good example comes from the vegetable kingdom. Flavonoids have at least two distinct functions in the interactions of leguminous plants with microorganisms. Phytoalexins are synthesized in response to pathogen attacks and have antimicrobial properties. While, other flavonoids act as transcriptional activators of nodulation (nod) genes in the soil bacteria Rhizobiu and Brudyrhizobia leading to the establishment of a Nz-fixing association [67]. These different biological properties could be referred to the difference in the flavonoid end products. The genotoxicity of flavonoids in human lymphocytes in vitro seems to be connected with the planarity of the molecule. Only the planar flavone or flavonol
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derivatives showed activity, whereas the unplanar flavanols were inactive [68]. Some flavonoids were found to be selective on lipoxygenase inhibitors, some as cycle-oxigenase inhibitors, some inhibited both enzymes and some were very poorly active against either enzyme [4]. These selectivities suggested that different structure-dependent mechanisms determine inhibition of the two enzymes by the flavonoids. Flavonoids possessing hydroxyl groups inhibit P-450 enzymatic activity, whereas those lacking hydroxyl groups stimulate activity [69]. Similar structure-activity relationships exist for the effect of Ravones on ethoxycoumarin deethylase activity in rat hepatic microsomes [70]. Furthermore, Friedman et al, have shown that various flavonoids differ in their inhibitory effects on constitutive and induced aryl hydrocarbon hydroxylase activity in rat liver [71]. Also the hydroxylation and glycosylation of flavone modify the potency of these compounds [5,72].
Pharmacodynamics
Flavonoids are metabolized by animal cells, expecially those of the liver and excreted in the urines, without accumulation in the body. The toxicity of flavonoids is very low in animals. Only a few data are available on the pharmacokinetics of flavonoids. It is known that bacterial glycosidases are able of liberating flavonoid aglycons. Larger amounts of orally taken flavonoids must, therefore, be expected to be largely present as aglycons in the intestine and to become absorbed with micelles of bile acids into the epithelium and then into the blood. Through the portal vein, the major part of the flavonoids would probably be delivered more or less directly to the liver, which decomposes them. Indeed, the lack of success of flavonoid treatment may often be ascribed to the failure of the compound to reach its target. So far little is known about the affinity of flavonoids to blood plasma proteins. But due to the low polarity of many aglycons they would probably be bound to serum albumin. Interestingly, an extensive characterization of the pharmacokinetics of ipriflavone in humans revealed that the compound is metabolized into seven metabolites (Fig. 3; [73,74]).The main metabolites in humans are metabolites I, II, and V. Metabolites I and II circulate mainly as conjugated forms, but metabolite V, like ipriflavone, is recovered in unconjugated form in the human body, suggesting that the latter metabolite should greatly contribute to the action of ipriflavone. Indeed, in vitro studies revealed differential effects of ipriflavone and its metabolites both on bone resorption [62] and bone formation [65]. Therefore, ipriflavone metabolism may contribute both to the inactivation as well as to the induction of the osteotrophic properties of the compound.
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Fig. 3. Structuralskeletonsof ipritlavone and its metr.bolites.
Flavonoids represent a large area of still open investigation. Indeed, the information we have is rarely supported by controlled clinical studies in large numbers of patients. Areas, like the antiproliferative effect of genistein on cancer cells and the
Sll anabolic action of ipriflavone on the skeleton, need to be analyzed in detail. These models offer, in fact, unique possibilities both for understanding the molecular mechanisms which underlie the effects of these compounds on target organs and for characterizing their therapeutic applications in clinical disorders. Indeed, from studies performed with cancer cells it appears that bioflavonoids may inhibit cell proliferation through an interaction with type II binding sites [75,76]. Moreover, ipriflavone appears to interact with the estrogen receptors and to possess unique binding sites in bone-derived cells (ML Brandi, unpublished data). The results obtained from in vitro and in vivo studies will constitute the basis for the construction of better flavonoid compounds.
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