**5.2 LOX inhibition in cancer**

112 Recent Trends for Enhancing the Diversity and Quality of Soybean Products

Recent reviews describe the role of lipoxygenase in cancer (Bhattacharya et al., 2009; Pidgeon et al., 2007; Moreno, 2009), inflammation (Duroudier et al., 2009; Hersberger, 2010) and vascular biology (Chawengsub et al., 2009; Mochizuki & Kwon, 2008) and for an extensive presentation of the role of eicosanoids in prevention and management of diseases

**5. Interaction of lipoxygenase with inhibitors as theoretical approach for food** 

In terms of the structure and function, LOXs are unique, because their metal cofactor is a single ion bound by the side chains of the surrounding amino acids and the carboxylic group of the C-terminus, and their inhibitors bind to or near the Fe co-factor (Skrzypczak-Jankun et al., 2007). Lipoxygenases are inhibited by a large number of chemicals, some of

Besides their physiological role, plant lipoxygenases are of significant importance to the food industry, since these enzymes have been implicated in the generation of the flavour and aroma in many plant products. For instance, they are responsible for the undesirable 'beany', 'green' and 'grassy' flavours produced during processing and storage of protein products derived from legume seeds (Fukushima, 1994; Robinson et al., 1995) and the development of the stale flavour in beer during storage (Kobayashi et al., 1993). Lipoxygenases also play an important role in the baking industry. They are quite effective as bleaching agents, increase mixing tolerance and improve dough rheology (Nicolas & Potus,

Freshly refined soybean oil is practically odourless and bland, but "green, grassy, fishy" offflavors may develop quickly if the oil is heated or stored under conditions that expose it to light and oxygen or by contamination with pro-oxidant metals such as copper and iron (Berk, 1992). "Beany" flavour is the principal inconvenience of traditional soymilk and its products (e.g., tofu) and is caused by some ketones and aldehydes, particularly hexanals

Fish lipids are susceptible to oxidation owing to the high levels of polyunsaturated fatty acids (PUFA), even in frozen storage, and this can affect the flavour, texture, taste, aroma and shelf life of fish (Ke & Ackman, 1976). Since the direct interaction between oxygen and highly unsaturated lipids is kinetically hindered (Kanner et al., 1987), the enzymatic initiation of oxidation by enzymes such as lipoxygenase, peroxidases and microsomal

Green tea glazing was shown to improve the storage quality of frozen bonito fillets (Lin & Lin, 2005). In addition, hot water tea extract was shown to suppress the pro-oxidant activities of the dark meat and skin of blue sprat (Seto et al., 2005). Banerjee (2006) proposes that the improvement in the shelf life of fish by green tea polyphenols is at least in part due to inhibition of LOX resulting in delaying oxidation of fish lipids and because of that impregnation of muscle fillets in tea extract by itself or in combination with other natural

Besides its function of oxidizing the polyunsaturated fatty acids (linoleic, linolenic and arachidonic), the enzyme may also catalyse the co-oxidation of carotenoids, resulting in the loss of natural colorants and essential nutrients (Robinson et al., 1995). LOX have been

the reader is referred to the review of Szefel et al. (2011).

**industry and medical applications** 

enzymes has been gaining favour.

which also serve as co-substrates (Kulkarni, 2001).

**5.1 Importance of lipoxygenase inhibition for food industry** 

1994; Larreta-Garde, 1995; Cumbee et al., 1997; Borellib et al., 1999).

and heptanals, produced through LOX catalyzed oxidation (Berk, 1992).

inhibitors may improve the shelf-life and storage quality of fish fillets.

Molecular studies of the well-known relationship between polyunsaturated fatty acid metabolism and carcinogenesis have revealed novel molecular targets for cancer chemoprevention and treatment (Lipkin et al., 1999; Willett, 1997; Klurfeld & Bull, 1997; Guthrie & Carroll, 1999).

The role of lipoxygenase in the development and progression of cancer is complex due to the variety of lipoxygenase genes that have been identified in humans, in addition to different profiles of lipoxygenase observed between studies on human tumor biopsies and experimentally induced animal tumor models (Pidgeon et al., 2007). The literature emerging on the role of lipoxygenases in tumor growth, for the most part, suggests that distinct lipoxygenase isoforms, whose expression are lost during the progression of cancer, may exhibit anti-tumor activity, while other isoforms may exert pro-tumorigenic effects and are preferentially expressed during the development of various cancers.

The involvement of 5-lipoxygenase and 12-lipoxygenase in human cancer progression is now supported by a growing body of literature. The involvement of 15-lipoxygenase-1 in colorectal cancer involves its implication in carcinogenesis having pro-carcinogenic as well as anti-carcinogenic roles (Bhattacharya et al., 2009). The co-localization of these enzymes and the similarities of their bioactions on cancer cell growth suggest that the simultaneous inhibition of these enzymes may represent novel and promising therapeutic approaches in selected cancer types (Pidgeon et al., 2007). Therefore, when targeting the regulation of arachidonic acid metabolism, blocking 5-lipoxygenase, 12-lipoxygenase and 15 lipoxygenase-1 without altering the expression of the anti-carcinogenic 15-lipoxygenase-2 may be the most effective, however at present no drug recapitulates these capabilities (Pidgeon et al., 2007).

#### **5.3 Mechanisms of lipoxygenase inhibition**

In general, lipoxygenase inhibitors can bind covalently to iron or form the molecular complexes blocking access to iron (Skrzypczak-Jankun et al., 2007). It was pointed out by Walther et al., that a course of inhibition, by the drug ebselen, (noncompetitive vs competitive) and its reversibility depend on the oxidation state of iron, i.e. whether the enzyme is catalytically silent with Fe2+ when it binds covalently, causing irreversible

Inhibition of Soybean Lipoxygenases – Structural

potential ligands (Pham et al., 1998).

over the years (Barlow, 1990).

C ring (Beecher, 2003).

Fig. 3. The basic structure of flavonoids.

undergoes degradation (Borbulevych et al., 2004).

and Activity Models for the Lipoxygenase Isoenzymes Family 115

Genistein is neither consumed nor does state change during the course of the reaction of lipoxygenase (Mahesha et al., 2007), while quercetin entrapped within lipoxygenase

Gillmor et al. (1997) obtained the structure of the rabbit reticulocyte enzyme as a complex with the inhibitor RS75091. Located in one of the hydrophobic channels of the enzyme, the inhibitor was found to be close but not biding the iron atom of the catalytic situs. These observations provided the first indications of how the native enzyme can interact with

Natural flavonoids don't affect only the lipoxygenase oxidation of its classical substrates but also the co-oxidation of xenobiotics by this enzyme. Epigallocatechin-gallate, quercetin and rutin proved to reduce the co-oxidation rate of guaiacol, benzidine, paraphenylenediamine and dimethoxybenzidine by soybean lipoxygenase-1 (Hu et al., 2006). This data suggest that flavonoids may have anticarcinogenic and antitoxic effect through inhibition of oxidative activation generated by lipoxygenase (Hu et al., 2006). Green tea polyphenols have potent free radical quenching and antioxidant activities (Wiseman et al., 1997) and have structural features that may specifically interfere with the arachidonic acid cascade, including the lipoxygenase pathway (Hong & Yang, 2003; Hussain et al., 2005). In addition, with growing concerns regarding the safety of synthetic antioxidants such as BHT and BHA, alternative mechanisms of antioxidant protection by the use of natural antioxidants have been in review

Polyphenols, mainly flavonoids and phenolic acids, are abundant in a number of dietary sources such as certain cocoas, tea, wine, fruits and vegetables. More than 8000 different flavonoids of natural origin are known (Schewe & Sies, 2003). The flavonoids exist in nature as aglycons (free form) or conjugated (with O-glucosides or methylated). The aglycons can be subdivided in different subclasses (flavanols, flavanones, flavones, izoflavones, flavonols, anthocyanidines, aurones, chalcones) in function of how the B ring from their structure is linked to the heterocycle C, of the oxidation state and of the functional groups linked to the

The basic structure of flavonoids is represented by the flavan nucleus containing 15 carbons structured in 2 benzene rings, named A and B and linked by a C3 unit, which together with an oxygen atom forms the γ-pyronic or γ-pyranic ring, named the C ring as shown in Fig.3. A number of *in vitro* and *in vivo* studies as well as clinical trials suggest beneficial effects of flavonoids for health, counteracting the development of cardiovascular diseases, cancer and obesity. Bors et al. (1990) were the first to claim three partial structures contributing to the

**5.4 Inhibition of soybean lipoxygenase by different classes of polyphenols** 

inhibition or preoxidized and active with Fe3+ in the presence of the fatty acid substrate (Walther et al., 1999). In both cases the enzyme's performance can be illustrated by a classic Lineweaver-Burk plot. Many inhibitors do not follow such a linear relation between velocity and the inhibitor's concentration showing a hyperbolic curve instead as observed by Skrzypczak-Jankun et al. (2002) for polyphenolic inhibitors (curcumin, quercetin, epigallocatechin gallate and epigallocatechin) interacting with soybean lipoxygenase-3. In general, the kinetic data are seldom reported (Skrzypczak-Jankun et al., 2007). Xenobiotic oxidation by soy lipoxygenase has been investigated and described, while human enzymes lack such thorough studies (Skrzypczak-Jankun et al., 2007). The in vivo susceptibility of lipoxygenases' inhibitors may depend not only on the source of lipoxygenase and its isozyme (Pham et al., 1998; Schewe et al., 1986) but also on the oxidation state of iron and the competition between peroxidase and co-oxidase activities of enzyme (Borbulevych et al., 2004).

The first mode to inhibit the lipoxygenase would be a direct reduction of iron to its inactive form. For soybean lipoxygenase, it has been demonstrated that nordihydroguaiaretic acid rapidly reduces the active ferric species of the enzyme to its inactive ferrous form, thus causing interruption of the catalytic cycle (Kemal et al., 1987). For the polyphenol inhibition of lipoxygenase it was firstly suggested that this molecules strongly complex of the ferric iron moiety of the lipoxygenase, thus preventing its reduction *via* the catalytic cycle as proposed for the action of 4-nitrocatechol on the soybean lipoxygenase-1 (Spaapen et al., 1980). The second observation that complexation of the flavanols with Fe3+ did not abolish the inhibitory effect may rule out a direct complexation of the iron moiety in ferric lipoxygenase by these catechol compounds. The X-ray analysis shows 4-nitrocatechol near iron with partial occupancy, blocking access to Fe but not covalently bound to it (Skrzypczak-Jankun et al., 2004). If a similar mode of action holds for the interaction of flavanols with mammalian lipoxygenases, the corresponding iron polyphenol complexes may retain their lipoxygenase-inhibitory effect.

A third conceivable mode of action of polyphenols is the effective reduction of hydroperoxides that are essential activators of lipoxygenase *via* conversion of the enzymatically silent ferrous species to the active ferric form. The observation of Schewe et al. (2001) that lowering of the hydroperoxide tone by glutathione plus glutathione peroxidase did not modulate the inhibitory effects of flavanols on 15-lipoxygenase-1 does not support the latter possibility.

In case of carotenoids, more specifically, β-carotene, the lipoxygenase was inhibited by keeping it in the inactive form of Fe(II) (Serpen & Gökmen, 2006). These authors suggest that β-carotene reacts with linoleyl radical (L•) at the beginning of the chain reaction, so it prevents the accumulation of conjugated diene forms (LOO•, LOO− and LOOH). Since L• transforms back to its original form of LH, the enzyme cannot complete the chain reaction and thus remains in the inactive Fe(II) form, which is not capable of catalyzing linoleic acid hydroperoxidation (Serpen & Gökmen, 2006).

Wu et al. (1999) have reported that β-carotene scavenges the linoleyl peroxy radical (LOO•) by a hydrogen transfer mechanism and the oxidation of β-carotene occurs during this action. In these conditions, it is absolutely clear that the amount of inactivated enzyme depends on the concentration of β-carotene present in the medium (Serpen & Gökmen, 2006).

According to Mahesha et al. (2007) the lipoxygenase inhibition by isoflavones follows the next mechanism: an electron donated by isoflavones is accepted by the ferric form (Fe3+) of lipoxygenase, which is reduced to resting ferrous form (Fe2+), thus inhibiting lipoxygenase.

inhibition or preoxidized and active with Fe3+ in the presence of the fatty acid substrate (Walther et al., 1999). In both cases the enzyme's performance can be illustrated by a classic Lineweaver-Burk plot. Many inhibitors do not follow such a linear relation between velocity and the inhibitor's concentration showing a hyperbolic curve instead as observed by Skrzypczak-Jankun et al. (2002) for polyphenolic inhibitors (curcumin, quercetin, epigallocatechin gallate and epigallocatechin) interacting with soybean lipoxygenase-3. In general, the kinetic data are seldom reported (Skrzypczak-Jankun et al., 2007). Xenobiotic oxidation by soy lipoxygenase has been investigated and described, while human enzymes lack such thorough studies (Skrzypczak-Jankun et al., 2007). The in vivo susceptibility of lipoxygenases' inhibitors may depend not only on the source of lipoxygenase and its isozyme (Pham et al., 1998; Schewe et al., 1986) but also on the oxidation state of iron and the competition between peroxidase and co-oxidase activities of enzyme (Borbulevych et al.,

The first mode to inhibit the lipoxygenase would be a direct reduction of iron to its inactive form. For soybean lipoxygenase, it has been demonstrated that nordihydroguaiaretic acid rapidly reduces the active ferric species of the enzyme to its inactive ferrous form, thus causing interruption of the catalytic cycle (Kemal et al., 1987). For the polyphenol inhibition of lipoxygenase it was firstly suggested that this molecules strongly complex of the ferric iron moiety of the lipoxygenase, thus preventing its reduction *via* the catalytic cycle as proposed for the action of 4-nitrocatechol on the soybean lipoxygenase-1 (Spaapen et al., 1980). The second observation that complexation of the flavanols with Fe3+ did not abolish the inhibitory effect may rule out a direct complexation of the iron moiety in ferric lipoxygenase by these catechol compounds. The X-ray analysis shows 4-nitrocatechol near iron with partial occupancy, blocking access to Fe but not covalently bound to it (Skrzypczak-Jankun et al., 2004). If a similar mode of action holds for the interaction of flavanols with mammalian lipoxygenases, the corresponding iron polyphenol complexes

A third conceivable mode of action of polyphenols is the effective reduction of hydroperoxides that are essential activators of lipoxygenase *via* conversion of the enzymatically silent ferrous species to the active ferric form. The observation of Schewe et al. (2001) that lowering of the hydroperoxide tone by glutathione plus glutathione peroxidase did not modulate the inhibitory effects of flavanols on 15-lipoxygenase-1 does

In case of carotenoids, more specifically, β-carotene, the lipoxygenase was inhibited by keeping it in the inactive form of Fe(II) (Serpen & Gökmen, 2006). These authors suggest that β-carotene reacts with linoleyl radical (L•) at the beginning of the chain reaction, so it prevents the accumulation of conjugated diene forms (LOO•, LOO− and LOOH). Since L• transforms back to its original form of LH, the enzyme cannot complete the chain reaction and thus remains in the inactive Fe(II) form, which is not capable of catalyzing linoleic acid

Wu et al. (1999) have reported that β-carotene scavenges the linoleyl peroxy radical (LOO•) by a hydrogen transfer mechanism and the oxidation of β-carotene occurs during this action. In these conditions, it is absolutely clear that the amount of inactivated enzyme depends on

According to Mahesha et al. (2007) the lipoxygenase inhibition by isoflavones follows the next mechanism: an electron donated by isoflavones is accepted by the ferric form (Fe3+) of lipoxygenase, which is reduced to resting ferrous form (Fe2+), thus inhibiting lipoxygenase.

the concentration of β-carotene present in the medium (Serpen & Gökmen, 2006).

2004).

may retain their lipoxygenase-inhibitory effect.

hydroperoxidation (Serpen & Gökmen, 2006).

not support the latter possibility.

Genistein is neither consumed nor does state change during the course of the reaction of lipoxygenase (Mahesha et al., 2007), while quercetin entrapped within lipoxygenase undergoes degradation (Borbulevych et al., 2004).

#### **5.4 Inhibition of soybean lipoxygenase by different classes of polyphenols**

Gillmor et al. (1997) obtained the structure of the rabbit reticulocyte enzyme as a complex with the inhibitor RS75091. Located in one of the hydrophobic channels of the enzyme, the inhibitor was found to be close but not biding the iron atom of the catalytic situs. These observations provided the first indications of how the native enzyme can interact with potential ligands (Pham et al., 1998).

Natural flavonoids don't affect only the lipoxygenase oxidation of its classical substrates but also the co-oxidation of xenobiotics by this enzyme. Epigallocatechin-gallate, quercetin and rutin proved to reduce the co-oxidation rate of guaiacol, benzidine, paraphenylenediamine and dimethoxybenzidine by soybean lipoxygenase-1 (Hu et al., 2006). This data suggest that flavonoids may have anticarcinogenic and antitoxic effect through inhibition of oxidative activation generated by lipoxygenase (Hu et al., 2006). Green tea polyphenols have potent free radical quenching and antioxidant activities (Wiseman et al., 1997) and have structural features that may specifically interfere with the arachidonic acid cascade, including the lipoxygenase pathway (Hong & Yang, 2003; Hussain et al., 2005). In addition, with growing concerns regarding the safety of synthetic antioxidants such as BHT and BHA, alternative mechanisms of antioxidant protection by the use of natural antioxidants have been in review over the years (Barlow, 1990).

Polyphenols, mainly flavonoids and phenolic acids, are abundant in a number of dietary sources such as certain cocoas, tea, wine, fruits and vegetables. More than 8000 different flavonoids of natural origin are known (Schewe & Sies, 2003). The flavonoids exist in nature as aglycons (free form) or conjugated (with O-glucosides or methylated). The aglycons can be subdivided in different subclasses (flavanols, flavanones, flavones, izoflavones, flavonols, anthocyanidines, aurones, chalcones) in function of how the B ring from their structure is linked to the heterocycle C, of the oxidation state and of the functional groups linked to the C ring (Beecher, 2003).

Fig. 3. The basic structure of flavonoids.

The basic structure of flavonoids is represented by the flavan nucleus containing 15 carbons structured in 2 benzene rings, named A and B and linked by a C3 unit, which together with an oxygen atom forms the γ-pyronic or γ-pyranic ring, named the C ring as shown in Fig.3. A number of *in vitro* and *in vivo* studies as well as clinical trials suggest beneficial effects of flavonoids for health, counteracting the development of cardiovascular diseases, cancer and obesity. Bors et al. (1990) were the first to claim three partial structures contributing to the

Inhibition of Soybean Lipoxygenases – Structural

order to interact with LOX better (Chedea et al., 2006).

**5.6 Inhibition of soybean lipoxygenase by epigallocatechin gallate** 

with a potential to be utilized in disease prevention and treatment.

(Fiorucci et al., 2008).

and Activity Models for the Lipoxygenase Isoenzymes Family 117

Structural analysis reveals that quercetin entrapped within LOX undergoes degradation and the resulting compound has been identified by X-ray analysis as protocatechuic acid (3,4-

dihydroxybenzoic acid) positioned near the iron site (Borbulevych et al., 2004).

Fig. 4. Product of quercetin degradation by soybean LOX-3 (Borbulevych et al., 2004).

We demonstrated that pH values may influence the molecular interactions between soybean LOX-1 and quercetin, and especially the alcaline pH favours the ionic display of quercetin in

Quercetin inhibited the 12 (S)-hydroxytetraenoic acid production at concentrations below those necessary for growth inhibition in colorectal cancer cells overexpressing the enzyme 12(S)-lipoxygenase with an IC50 of 1μM (Bednar et al., 2007). The finding that LOX can turn different compounds into simple catechol derivatives (with one aromatic ring only) might be of importance as an additional small piece of a "jigsaw puzzle" in the much bigger picture of drug metabolism (Borbulevych et al., 2004). Their interactions with LOX can be more complicated than simply blocking the access to the enzyme's active site. The studies on LOX and quercetin contribute to the understanding of biocatalytic properties of this enzyme and its role in the metabolism of this popular (as a medicinal remedy) flavonol and possibly other, similar compounds (Borbulevych et al., 2004). Acting both as a substrate and a source of inhibition, quercetin seems to play an antinomic role (Fiorucci et al., 2008). But this could be explained as quercetin, one of the most representative flavonoids, is a highly functionalized substrate and can thus be activated and degraded following several ways

Flavanols (or flavan-3-ols or catechins) are a class of flavonoids that include the catechins and the catechin gallates. Catechins are described as colorless, astringent, water-soluble polyphenols found in many fruits and grains, such as coffee, red grapes, prunes and raisins. Their main source however comes from a beverage made from tea leaves of *Camellia sinensis*. (-)-Epigallocatechin gallate (EGCG) together with other galloylated catechins constitute more than 90% of the total catechin content in green tea (Lekli et al., 2010). Laboratory studies strongly indicate that tea inhibits certain cancers, and there is a multitude of evidence confirming the antioncogenic properties of the individual catechins. For instance: EGCG alone shows anticancer effectiveness against carcinogen-induced skin, lung, forestomach, esophagus, duodeum, liver and colon tumors in rodents. It was found to cause apoptosis and/or cell cycle arrest in human carcinoma cells of skin and prostate cancers (Zimeri & Tong, 1999). Catechins have also known inhibitory activity toward dioxygenases

radical-scavenging activity of flavonoids: (a) an o-dihydroxyl structure in the B ring (catechol structure) as a radical target site providing good electron delocalization and stabilization of the phenoxy radical; (b) a 2,3-double bond with conjugation to the 4-oxo group which is necessary for delocalization of an unpaired electron from the B ring, (c) hydroxyl groups at the 3-and 5-positions, which are necessary for enhancement of radical scavenging activity, increasing the delocalisation of electrons across the flavonoid scaffold. The catechol group is essential for the radical-scavenging activity of flavan-3-ols and flavanones lacking 2,3-double bonds (Bors et al., 1990).

In parallel to the free radical-scavenging properties the following structural features were found to enhance the inhibitory potency: (i) presence of a catechol arrangement in the B or A ring, (ii) a carbonyl group together with a 2,3-double bond in the C ring (Schewe & Sies, 2003). Other structural features were opposite to the free radical scavenging potencies: (i) presence of a 3-OH group in the C ring diminished than reinforced the inhibition of lipoxygenases, (ii) in the absence of a catechol arrangement there was an inverse correlation to the total number of OH groups in the flavonoid molecule (Schewe & Sies, 2003). Although either reducing or ferric iron chelating properties are prerequisites for a lipoxygenase- inhibitory compound, and both of them are also inherent to flavonoids, the inhibitory effects cannot be ascribed solely to one of these mechanisms (Schewe & Sies, 2003). The inhibition of lipoxygenases by flavonoids appears to be of more complex nature (Schewe & Sies, 2003).

Inhibitors studies of lipoxygenase from pea showed that phenolic antioxidant components were effective and can be used to protect food lipids against oxidation (Szymanowska et al., 2009). The conducted research proved that activity of lipoxygenase from pea seeds could be effectively inhibit by some phenolic compounds. The most effective inhibitor is caffeic acid (about 57% of inhibition). Flavonoids like catechin and quercetin considerably inhibit the lipoxygenase activity. Inhibitors used for investigation in this study were placed in the following order: caffeic acid > quercetin > catechin > benzoic acid > ferulic acid > kaempferol (Szymanowska et al., 2009).

#### **5.5 Lipoxygenase inhibition by quercetin**

Quercetin is the most abundant among the flavonoid molecules and can be found in the fruits, vegetables, seeds, nuts, and flowers of many plants. Its documented impact on human health includes cardiovascular protection, anticancer, antiviral, anti-inflammatory activities, antiulcer effects and cataract prevention. Like other flavonoids, quercetin appears to combine both lipoxygenase-inhibitory activities and free radical-scavenging properties in one agent and thus belongs to a family of very effective natural antioxidants (Sadik et al., 2003). Quercetin is a flavonol that can be easily oxidized in an aqueous environment, and in the presence of iron and hydroxyl free radicals (Borbulevych et al., 2004).

The inhibition of rabbit 15-lipoxygenase-1 and of soybean lipoxygenase-1 by quercetin was studied in detail (Sadik et al., 2003). Quercetin modulates the time course of the lipoxygenase reaction in a complex manner by exerting three distinct effects: (i) prolongation of the kinetic lag period, (ii) instant decrease in the initial rate after the lag phase being overcome, (iii) time-dependent inactivation of the enzyme during reaction, but not in the absence of substrate (Schewe & Sies, 2003). The literature data obviously indicate that quercetin represents one of the most potent inhibitors of different LOXs (Schneider & Bucara, 2005; Schneider & Bucarb, 2005).

radical-scavenging activity of flavonoids: (a) an o-dihydroxyl structure in the B ring (catechol structure) as a radical target site providing good electron delocalization and stabilization of the phenoxy radical; (b) a 2,3-double bond with conjugation to the 4-oxo group which is necessary for delocalization of an unpaired electron from the B ring, (c) hydroxyl groups at the 3-and 5-positions, which are necessary for enhancement of radical scavenging activity, increasing the delocalisation of electrons across the flavonoid scaffold. The catechol group is essential for the radical-scavenging activity of flavan-3-ols and

In parallel to the free radical-scavenging properties the following structural features were found to enhance the inhibitory potency: (i) presence of a catechol arrangement in the B or A ring, (ii) a carbonyl group together with a 2,3-double bond in the C ring (Schewe & Sies, 2003). Other structural features were opposite to the free radical scavenging potencies: (i) presence of a 3-OH group in the C ring diminished than reinforced the inhibition of lipoxygenases, (ii) in the absence of a catechol arrangement there was an inverse correlation to the total number of OH groups in the flavonoid molecule (Schewe & Sies, 2003). Although either reducing or ferric iron chelating properties are prerequisites for a lipoxygenase- inhibitory compound, and both of them are also inherent to flavonoids, the inhibitory effects cannot be ascribed solely to one of these mechanisms (Schewe & Sies, 2003). The inhibition of lipoxygenases by flavonoids appears to be of

Inhibitors studies of lipoxygenase from pea showed that phenolic antioxidant components were effective and can be used to protect food lipids against oxidation (Szymanowska et al., 2009). The conducted research proved that activity of lipoxygenase from pea seeds could be effectively inhibit by some phenolic compounds. The most effective inhibitor is caffeic acid (about 57% of inhibition). Flavonoids like catechin and quercetin considerably inhibit the lipoxygenase activity. Inhibitors used for investigation in this study were placed in the following order: caffeic acid > quercetin > catechin > benzoic acid > ferulic acid >

Quercetin is the most abundant among the flavonoid molecules and can be found in the fruits, vegetables, seeds, nuts, and flowers of many plants. Its documented impact on human health includes cardiovascular protection, anticancer, antiviral, anti-inflammatory activities, antiulcer effects and cataract prevention. Like other flavonoids, quercetin appears to combine both lipoxygenase-inhibitory activities and free radical-scavenging properties in one agent and thus belongs to a family of very effective natural antioxidants (Sadik et al., 2003). Quercetin is a flavonol that can be easily oxidized in an aqueous environment, and in

The inhibition of rabbit 15-lipoxygenase-1 and of soybean lipoxygenase-1 by quercetin was studied in detail (Sadik et al., 2003). Quercetin modulates the time course of the lipoxygenase reaction in a complex manner by exerting three distinct effects: (i) prolongation of the kinetic lag period, (ii) instant decrease in the initial rate after the lag phase being overcome, (iii) time-dependent inactivation of the enzyme during reaction, but not in the absence of substrate (Schewe & Sies, 2003). The literature data obviously indicate that quercetin represents one of the most potent inhibitors of different LOXs (Schneider &

the presence of iron and hydroxyl free radicals (Borbulevych et al., 2004).

flavanones lacking 2,3-double bonds (Bors et al., 1990).

more complex nature (Schewe & Sies, 2003).

kaempferol (Szymanowska et al., 2009).

Bucara, 2005; Schneider & Bucarb, 2005).

**5.5 Lipoxygenase inhibition by quercetin** 

Structural analysis reveals that quercetin entrapped within LOX undergoes degradation and the resulting compound has been identified by X-ray analysis as protocatechuic acid (3,4 dihydroxybenzoic acid) positioned near the iron site (Borbulevych et al., 2004).

Fig. 4. Product of quercetin degradation by soybean LOX-3 (Borbulevych et al., 2004).

We demonstrated that pH values may influence the molecular interactions between soybean LOX-1 and quercetin, and especially the alcaline pH favours the ionic display of quercetin in order to interact with LOX better (Chedea et al., 2006).

Quercetin inhibited the 12 (S)-hydroxytetraenoic acid production at concentrations below those necessary for growth inhibition in colorectal cancer cells overexpressing the enzyme 12(S)-lipoxygenase with an IC50 of 1μM (Bednar et al., 2007). The finding that LOX can turn different compounds into simple catechol derivatives (with one aromatic ring only) might be of importance as an additional small piece of a "jigsaw puzzle" in the much bigger picture of drug metabolism (Borbulevych et al., 2004). Their interactions with LOX can be more complicated than simply blocking the access to the enzyme's active site. The studies on LOX and quercetin contribute to the understanding of biocatalytic properties of this enzyme and its role in the metabolism of this popular (as a medicinal remedy) flavonol and possibly other, similar compounds (Borbulevych et al., 2004). Acting both as a substrate and a source of inhibition, quercetin seems to play an antinomic role (Fiorucci et al., 2008). But this could be explained as quercetin, one of the most representative flavonoids, is a highly functionalized substrate and can thus be activated and degraded following several ways (Fiorucci et al., 2008).
