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

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

Inhibition of Soybean Lipoxygenases – Structural

Fig. 6. Dietary isoflavones inhibitors of lipoxygenase.

(2) (Mahesha et al., 2007).

2011).

and osteoporosis (Ma et al., 2008), and heart disease ( Xiao, 2008).

**5.8 Inhibition of soybean lipoxygenase by carotenoids** 

contain a polyene chains, with or without cyclisation at the ends.

and Activity Models for the Lipoxygenase Isoenzymes Family 119

The impact of dietary isoflavones, daidzein and genistein, on the health of adults and infants is well documented, an increasing interest for these compounds being registered due to their biological effects including: estrogen-like activity, prevention of breast (Warri et al., 2008), prostate (Matsumura et al., 2008) and colon cancer (Mac Donald et al., 2005), antioxidant activity (Malencić et al., 2007; Sakthivelu et al., 2008), prevention of menopausal symptoms

In the work of Mahesha et al. (2007) the inhibition of soy lipoxygenase-1 and 5-lipoxygenase from human polymorph nuclear lymphocyte by isoflavones, genistein and daidzein as glycosilated and unglycosilated compounds was studied. Soybean isoflavones inhibit LOX either as aglycons, or as glucosides. Isoflavones exert combined dual actions as inhibitors: they compete with the hydroperoxide formation to prevent the generation of LOX active ferric state (1) and also are capable of reducing the ferric enzyme to its inactive ferrous form

Vicaş et al. (2011) showed that genistein was almost twice more potent inhibitor than daidzein at similar concentration with concentration that induces 50% soybean lipoxygenase-1 inhibition values of 5.33 mM versus 11.53 mM. Genistein and daidzein proved to be noncompetitive with inhibition constants Ki of 33.65 and 43.45 mM, respectively (Vicaş et al., 2011). The inhibitory efficiency of the genistein and daidzein depended both on their concentration and on the substrate's concentration (Vicaş et al.,

There are over 600 fully characterized, naturally occurring molecular species belonging to the class of carotenoids. In humans, some carotenoids (the provitamin A carotenoids: αcarotene β-carotene, γ-carotene and the xanthophyll, β-cryptoxanthin) are best known for converting enzymatically into vitamin A; diseases resulting from vitamin A deficiency remain among the most significant nutritional challenges worldwide. Also, the role that carotenoids play in protecting those tissues that are the most heavily exposed to light (e.g. photo protection of the skin, protection of the central retina) is perhaps most evident, while other potential roles for carotenoids in the prevention of chronic diseases (cancer, cardiovascular disease) are still being investigated. Because carotenoids are widely consumed and their consumption is a modifiable health behaviour (via diets or supplements), health benefits for chronic disease prevention, if real, could be very significant for public health (Mayne, 2010). Carotenoids are isoprenoid molecules which

The study of Banerjee (2006) shows that green tea polyphenols are very potent inhibitors of mackerel muscle LOX, with EGCG (epigallocatechin gallate) as the most effective inhibitor (IC50 0.13 nM) followed by ECG (epicatechin gallate) (IC50 0.8 nM), EC (epicatechin) (IC50 6.0 nM), EGC (epigallocatechin) (IC50 9.0 nM) and C (catechin) (IC50 22.4 nM). Chocolate and cocoa are also sources of catechins. Epigallocatechin gallate isolated from the seeds of *Theobroma cacao* had the best inhibitory activity on rabit 15-lipoxygenase-1, with an IC50=4M, epicatechin gallate had IC50=5M and epicatechin an IC50=60M (Schewe et al., 2002). A better inhibition of epicatechin (IC50 approx. 15M) was registered in the case of recombinant human platelet 12-lipoxygenase.

Obtained from X-ray analysis, the 3D structure of the resulting complex of (-) epigallocatechin gallate (EGCG) interacting with soybean lipoxygenase-3 reveals the inhibitor depicting (-)-epigallocatechin that lacks the galloyl moiety (Skrzypczak-Jankun et al., 2003). The A-ring is near the iron co-factor, attached by the hydrogen bond to the Cterminus of the enzyme, and the B-ring hydroxyl groups participate in the hydrogen bonds and the van der Waals interactions formed by the surrounding amino acids and water molecules (Skrzypczak-Jancun et al., 2003).

Fig. 5. Lipoxygenase-3 in complex with epigallocatechin gallate as an inhibitor determines the degradation of natural flavonoid to epigallocatechin (Skrzypczak-Jancun et al., 2003).

X-ray analysis of soybean lipoxygenase-3 crystals soaked with EGCG shows the molecular complex of LOX-3 with (-)epigallocatechin molecule.

#### **5.7 Inhibitory effects of soybean isoflavones on lipoxygenase activity**

Soybeans are important sources of isoflavone levels (Song et al., 1998), present as 12 derivatives, including free genistin, daidzin, glycitin and their acetyl, malonyl or glycosilated forms. Isoflavones are composed of 2 benzene rings (A and B) linked through a heterocyclic pyrane C ring. The position of the B ring discriminate flavonoid flavones (C2 position) from isoflavones (C3-position).

The study of Banerjee (2006) shows that green tea polyphenols are very potent inhibitors of mackerel muscle LOX, with EGCG (epigallocatechin gallate) as the most effective inhibitor (IC50 0.13 nM) followed by ECG (epicatechin gallate) (IC50 0.8 nM), EC (epicatechin) (IC50 6.0 nM), EGC (epigallocatechin) (IC50 9.0 nM) and C (catechin) (IC50 22.4 nM). Chocolate and cocoa are also sources of catechins. Epigallocatechin gallate isolated from the seeds of *Theobroma cacao* had the best inhibitory activity on rabit 15-lipoxygenase-1, with an IC50=4M, epicatechin gallate had IC50=5M and epicatechin an IC50=60M (Schewe et al., 2002). A better inhibition of epicatechin (IC50 approx. 15M) was registered in the case of recombinant

Obtained from X-ray analysis, the 3D structure of the resulting complex of (-) epigallocatechin gallate (EGCG) interacting with soybean lipoxygenase-3 reveals the inhibitor depicting (-)-epigallocatechin that lacks the galloyl moiety (Skrzypczak-Jankun et al., 2003). The A-ring is near the iron co-factor, attached by the hydrogen bond to the Cterminus of the enzyme, and the B-ring hydroxyl groups participate in the hydrogen bonds and the van der Waals interactions formed by the surrounding amino acids and water

Fig. 5. Lipoxygenase-3 in complex with epigallocatechin gallate as an inhibitor determines the degradation of natural flavonoid to epigallocatechin (Skrzypczak-Jancun et al., 2003).

X-ray analysis of soybean lipoxygenase-3 crystals soaked with EGCG shows the molecular

Soybeans are important sources of isoflavone levels (Song et al., 1998), present as 12 derivatives, including free genistin, daidzin, glycitin and their acetyl, malonyl or glycosilated forms. Isoflavones are composed of 2 benzene rings (A and B) linked through a heterocyclic pyrane C ring. The position of the B ring discriminate flavonoid flavones (C2-

**5.7 Inhibitory effects of soybean isoflavones on lipoxygenase activity** 

human platelet 12-lipoxygenase.

molecules (Skrzypczak-Jancun et al., 2003).

complex of LOX-3 with (-)epigallocatechin molecule.

position) from isoflavones (C3-position).

Fig. 6. Dietary isoflavones inhibitors of lipoxygenase.

The impact of dietary isoflavones, daidzein and genistein, on the health of adults and infants is well documented, an increasing interest for these compounds being registered due to their biological effects including: estrogen-like activity, prevention of breast (Warri et al., 2008), prostate (Matsumura et al., 2008) and colon cancer (Mac Donald et al., 2005), antioxidant activity (Malencić et al., 2007; Sakthivelu et al., 2008), prevention of menopausal symptoms and osteoporosis (Ma et al., 2008), and heart disease ( Xiao, 2008).

In the work of Mahesha et al. (2007) the inhibition of soy lipoxygenase-1 and 5-lipoxygenase from human polymorph nuclear lymphocyte by isoflavones, genistein and daidzein as glycosilated and unglycosilated compounds was studied. Soybean isoflavones inhibit LOX either as aglycons, or as glucosides. Isoflavones exert combined dual actions as inhibitors: they compete with the hydroperoxide formation to prevent the generation of LOX active ferric state (1) and also are capable of reducing the ferric enzyme to its inactive ferrous form (2) (Mahesha et al., 2007).

Vicaş et al. (2011) showed that genistein was almost twice more potent inhibitor than daidzein at similar concentration with concentration that induces 50% soybean lipoxygenase-1 inhibition values of 5.33 mM versus 11.53 mM. Genistein and daidzein proved to be noncompetitive with inhibition constants Ki of 33.65 and 43.45 mM, respectively (Vicaş et al., 2011). The inhibitory efficiency of the genistein and daidzein depended both on their concentration and on the substrate's concentration (Vicaş et al., 2011).

#### **5.8 Inhibition of soybean lipoxygenase by carotenoids**

There are over 600 fully characterized, naturally occurring molecular species belonging to the class of carotenoids. In humans, some carotenoids (the provitamin A carotenoids: αcarotene β-carotene, γ-carotene and the xanthophyll, β-cryptoxanthin) are best known for converting enzymatically into vitamin A; diseases resulting from vitamin A deficiency remain among the most significant nutritional challenges worldwide. Also, the role that carotenoids play in protecting those tissues that are the most heavily exposed to light (e.g. photo protection of the skin, protection of the central retina) is perhaps most evident, while other potential roles for carotenoids in the prevention of chronic diseases (cancer, cardiovascular disease) are still being investigated. Because carotenoids are widely consumed and their consumption is a modifiable health behaviour (via diets or supplements), health benefits for chronic disease prevention, if real, could be very significant for public health (Mayne, 2010). Carotenoids are isoprenoid molecules which contain a polyene chains, with or without cyclisation at the ends.

Inhibition of Soybean Lipoxygenases – Structural

the dial metabolite.

and Activity Models for the Lipoxygenase Isoenzymes Family 121

by LOX-mediated hydroperoxidation reactions, inhibition of LOX activity takes place also (Lomnitski et al., 1993; Trono et al., 1999; Pastore et al., 2000). The activity of soybean lipoxygenase-1 was inhibited by β-carotene which breaks the chain reaction at the beginning stage of linoleic acid hydroperoxidation (Serpen & Gökmen, 2006). Besides soybean lipoxygenase (Ikedioby & Snyder, 1977; Hildebrand & Hymowitz, 1982) carotene oxidation during lipoxygenase-mediated linoleic acid oxidation has been reported in various studies for the enzymes extracted from potato (Aziz et al., 1999), pea (Yoon & Klein, 1979; Gökmen et al., 2002), wheat (Pastore et al., 2000), olive (Jaren-Galan et al., 1999) and pepper (Jaren-Galan & Minguez-Mosquera, 1997). Soybean lipoxygenase-1 and recombinant pea lipoxygenase-2 and lipoxygenase-3, oxidizing β-carotene, yield apocarotenal,

Through molecular modeling Hazai et al. (2006) predicted that lycopene and lycophyll bind with high affinity in the superficial cleft at the interface of the β-barrel and the catalytic domain of 5-LOX (the "cleavage site") suggesting potential direct competitive inhibition of 5-LOX activity by these molecules after in vivo supplementation, particularly in the case of

**5.9 Quinone and semiquinone formation during the lipoxygenase inhibition reaction**  In his excellent review from 2001, Kulkarni presents the studies up to that date indicating the semiquinone and quinone formation in different lipoxygenase catalyzed reactions of xenobiotics oxidation. Diethylstilbestrol (DES) is a human transplacental carcinogen. DESquinone, one of the metabolites of DES, binds to DNA and is presumed to be the ultimate toxicant. Although DES-quinone formation by human tissue lipoxygenase has yet to be examined, soybean lipoxygenase has been shown to initiate one-electron oxidation to DES semiquinone in the presence of H2O2 (Nunez-Delicado et al., 1997). Subsequent dismutation of two molecules of DES semiquinone yields one molecule each of DES-quinone and DES. Although phenol is oxidized slowly by different lipoxygenase isoenzymes, potato 5 lipoxygenase (Cucurou et al., 1991) and soybean lipoxygenase-1 (Cucurou et al., 1991; Mansuy et al., 1988), substituted phenols and catechols undergo extensive one-electron oxidation and yield the corresponding reactive phenoxyl radicals or semiquinones. These

However, it seems that quercetin may act, in most of cases, after being metabolically activated (Metodiewa et al., 1999), and despite a constant increase of knowledge on both positive and negative biological effects of this natural product, it remains often unclear which activated form should play a role in a given process (Fiorucci et al., 2007). Indeed, semiquinone and quinone forms of quercetin, deriving from the abstraction of respectively one or two H•, are involved in many oxidative processes (Metodiewa et al., 1999; Gliszczynska-Swiglo et al., 2003; Hirakawa et al., 2002). For instance, quercetin reduces peroxyl radicals involved in lipid peroxidation, and through this reaction, a semiquinone species is produced, which then undergoes a disproportionation to generate a quinone form (Fiorucci et al., 2007). Three semiquinone forms for quercetin have been considered by Fiorucci et al. (2008) in order to study the quercetin binding to lipoxygenase-3 by molecular modeling simulations. In the case of lipoxygenase–catechol complexes, the formation of the catechol-iron(III) complex of soybean lipoxygenase 1 gradually results in reduction of the cofactor and release of the semiquinone but no evidence of quinone formation in the UVvisible spectra of samples of the native enzyme treated with catechol was obtained (Spaapen

epoxycarotenal, apocarotenone and epoxycarotenone (Wu et al., 1999).

free radicals polymerize to yield a mixture of complex metabolites.

et al., 1980; Nelson, 1988; Pham et al., 1998).

Fig. 7. Carotenoids serving as lipoxygenase co-substrates

The existence of an enzyme "carotene oxidase" in soybeans, which catalyzes the oxidative destruction of carotene was reported by Bohn and Haas in 1928 (Bohn & Haas, 1928). Four years later, Andre and Hou found that soybeans contained an enzyme, lipoxygenase (linoleate oxygen oxidoreductase), which they termed *"lipoxidase",* catalyzing the peroxidation of certain unsaturated fatty acids (Andre & Hou, 1932).

In 1940 the observation that "lipoxydase" is identical to "carotene oxidase" was published (Sumner & Sumner, 1940). These early findings of lipoxygenase peroxidizing the unsaturated fats and bleaches the carotene were reported as the result of studies on the oxidation of crystalline carotene or carotene dissolved in unsaturated oil. Surprisingly it was found that the carotene oxidase had an almost negligible bleaching action upon the crystalline carotene. On the contrary, when one employs carotene dissolved in a small quantity of fat, the bleaching is extremely rapid. With excessive quantities of fat, the rate of bleaching of the carotene diminishes, and it was concluded that the effect of added fat upon the rate of bleaching of carotene is probably due to a coupled oxidation (Sumner & Sumner, 1940).

Studying the soya-lipoxygenase-catalyzed degradation of carotenoids from tomato Biacs and Daood (2000) found that β-carotene was the most sensitive component, followed by lycoxanthin and lycopene. Their results also implied that β-carotene can actively perform its antioxidant function during the course of lipid oxidation. It seems that oxidative degradation and, accordingly, antioxidant activity of each carotenoid depends on the rate of its interaction with the peroxyl radical produced through the lipoxygenase pathway (Biacs & Daood, 2000) and thus is able to inhibit lipoxygenase. The inhibition of the hydroperoxide formation by carotenoids has been attributed to their lipid peroxyl radical-trapping ability (Burton & Ingold, 1984).

In vitro, lycopene is a substrate of soybean lipoxygenase. The presence of this enzyme also significantly increased the production of lycopene oxidative metabolites (dos Anjos Ferreira et al., 2004; Biacs & Daood, 2000). It was reported that during the co-oxidation of β-carotene

The existence of an enzyme "carotene oxidase" in soybeans, which catalyzes the oxidative destruction of carotene was reported by Bohn and Haas in 1928 (Bohn & Haas, 1928). Four years later, Andre and Hou found that soybeans contained an enzyme, lipoxygenase (linoleate oxygen oxidoreductase), which they termed *"lipoxidase",* catalyzing the

In 1940 the observation that "lipoxydase" is identical to "carotene oxidase" was published (Sumner & Sumner, 1940). These early findings of lipoxygenase peroxidizing the unsaturated fats and bleaches the carotene were reported as the result of studies on the oxidation of crystalline carotene or carotene dissolved in unsaturated oil. Surprisingly it was found that the carotene oxidase had an almost negligible bleaching action upon the crystalline carotene. On the contrary, when one employs carotene dissolved in a small quantity of fat, the bleaching is extremely rapid. With excessive quantities of fat, the rate of bleaching of the carotene diminishes, and it was concluded that the effect of added fat upon the rate of bleaching of carotene is probably due to a coupled oxidation (Sumner & Sumner,

Studying the soya-lipoxygenase-catalyzed degradation of carotenoids from tomato Biacs and Daood (2000) found that β-carotene was the most sensitive component, followed by lycoxanthin and lycopene. Their results also implied that β-carotene can actively perform its antioxidant function during the course of lipid oxidation. It seems that oxidative degradation and, accordingly, antioxidant activity of each carotenoid depends on the rate of its interaction with the peroxyl radical produced through the lipoxygenase pathway (Biacs & Daood, 2000) and thus is able to inhibit lipoxygenase. The inhibition of the hydroperoxide formation by carotenoids has been attributed to their lipid peroxyl radical-trapping ability

In vitro, lycopene is a substrate of soybean lipoxygenase. The presence of this enzyme also significantly increased the production of lycopene oxidative metabolites (dos Anjos Ferreira et al., 2004; Biacs & Daood, 2000). It was reported that during the co-oxidation of β-carotene

Fig. 7. Carotenoids serving as lipoxygenase co-substrates

1940).

(Burton & Ingold, 1984).

peroxidation of certain unsaturated fatty acids (Andre & Hou, 1932).

by LOX-mediated hydroperoxidation reactions, inhibition of LOX activity takes place also (Lomnitski et al., 1993; Trono et al., 1999; Pastore et al., 2000). The activity of soybean lipoxygenase-1 was inhibited by β-carotene which breaks the chain reaction at the beginning stage of linoleic acid hydroperoxidation (Serpen & Gökmen, 2006). Besides soybean lipoxygenase (Ikedioby & Snyder, 1977; Hildebrand & Hymowitz, 1982) carotene oxidation during lipoxygenase-mediated linoleic acid oxidation has been reported in various studies for the enzymes extracted from potato (Aziz et al., 1999), pea (Yoon & Klein, 1979; Gökmen et al., 2002), wheat (Pastore et al., 2000), olive (Jaren-Galan et al., 1999) and pepper (Jaren-Galan & Minguez-Mosquera, 1997). Soybean lipoxygenase-1 and recombinant pea lipoxygenase-2 and lipoxygenase-3, oxidizing β-carotene, yield apocarotenal, epoxycarotenal, apocarotenone and epoxycarotenone (Wu et al., 1999).

Through molecular modeling Hazai et al. (2006) predicted that lycopene and lycophyll bind with high affinity in the superficial cleft at the interface of the β-barrel and the catalytic domain of 5-LOX (the "cleavage site") suggesting potential direct competitive inhibition of 5-LOX activity by these molecules after in vivo supplementation, particularly in the case of the dial metabolite.

#### **5.9 Quinone and semiquinone formation during the lipoxygenase inhibition reaction**

In his excellent review from 2001, Kulkarni presents the studies up to that date indicating the semiquinone and quinone formation in different lipoxygenase catalyzed reactions of xenobiotics oxidation. Diethylstilbestrol (DES) is a human transplacental carcinogen. DESquinone, one of the metabolites of DES, binds to DNA and is presumed to be the ultimate toxicant. Although DES-quinone formation by human tissue lipoxygenase has yet to be examined, soybean lipoxygenase has been shown to initiate one-electron oxidation to DES semiquinone in the presence of H2O2 (Nunez-Delicado et al., 1997). Subsequent dismutation of two molecules of DES semiquinone yields one molecule each of DES-quinone and DES.

Although phenol is oxidized slowly by different lipoxygenase isoenzymes, potato 5 lipoxygenase (Cucurou et al., 1991) and soybean lipoxygenase-1 (Cucurou et al., 1991; Mansuy et al., 1988), substituted phenols and catechols undergo extensive one-electron oxidation and yield the corresponding reactive phenoxyl radicals or semiquinones. These free radicals polymerize to yield a mixture of complex metabolites.

However, it seems that quercetin may act, in most of cases, after being metabolically activated (Metodiewa et al., 1999), and despite a constant increase of knowledge on both positive and negative biological effects of this natural product, it remains often unclear which activated form should play a role in a given process (Fiorucci et al., 2007). Indeed, semiquinone and quinone forms of quercetin, deriving from the abstraction of respectively one or two H•, are involved in many oxidative processes (Metodiewa et al., 1999; Gliszczynska-Swiglo et al., 2003; Hirakawa et al., 2002). For instance, quercetin reduces peroxyl radicals involved in lipid peroxidation, and through this reaction, a semiquinone species is produced, which then undergoes a disproportionation to generate a quinone form (Fiorucci et al., 2007). Three semiquinone forms for quercetin have been considered by Fiorucci et al. (2008) in order to study the quercetin binding to lipoxygenase-3 by molecular modeling simulations. In the case of lipoxygenase–catechol complexes, the formation of the catechol-iron(III) complex of soybean lipoxygenase 1 gradually results in reduction of the cofactor and release of the semiquinone but no evidence of quinone formation in the UVvisible spectra of samples of the native enzyme treated with catechol was obtained (Spaapen et al., 1980; Nelson, 1988; Pham et al., 1998).

Inhibition of Soybean Lipoxygenases – Structural

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Besides their antioxidant properties, catechins have been described to display pro-oxidant activity having the potential to oxidize the quinones or semiquinones resulting in redox cycling and reactive oxygen species production as well as in thiol, DNA and protein alkylation (Galati & O'Brien, 2004; van der Woude et al., 2006).

Our previous study shows that the oxidation products of catechins are formed within the cellular matrix but also in the extracellular medium (Chedea et al., 2010). We have demonstrated by UV-Vis spectroscopy, that the quinones are involved in the modulation of lipoxygenase activity in the presence of catechins within the cells (Chedea et al., 2010). This conclusion is in agreement with that of Sadik et al. (2003) and Banerjee (2006). An irreversible covalent modification of soybean LOX by flavonoids has been suggested by Sadik et al. (2003) whereby during the formation of fatty acid peroxyl radical in the LOX pathway, the flavonoids are co-oxidized to a semi-quinone or quinone, which in turn may bind to sulfhydryl or amino groups of the enzyme causing inhibition (Banerjee, 2006).
