Tricin—a potential multifunctional nutraceutical

Phytochem Rev (2010) 9:413–424 DOI 10.1007/s11101-009-9161-5

Tricin—a potential multifunctional nutraceutical Jian-Min Zhou • Ragai K. Ibrahim

Received: 13 October 2009 / Accepted: 8 December 2009 / Published online: 29 December 2009 Ó Springer Science+Business Media B.V. 2009

Abstract This review throws light on the natural occurrence and distribution of tricin (5,7,40 -trihydroxy-30 ,50 -dimethoxyflavone) and its conjugated forms, as more common natural plant constituents than previously known. It examines the current literature dealing with its biosynthesis, regulation, biological significance, pharmacological effects, and potential role as a chemopreventive and anticancer agent. Because of its common occurrence in cereal grain plants and the wide spectrum of its health promoting effects, a metabolic engineering strategy is proposed to produce tricin in sufficient amounts for further experimentation, and increase its accumulation in wheat grain endosperm as a nutraceutical. Keywords Tricin  Natural occurrence  Biology  Metabolic engineering  Nutraceutical

Introduction Flavonoids constitute one of the largest groups of naturally occurring plant secondary metabolites that

J.-M. Zhou  R. K. Ibrahim (&) Plant Biochemistry Laboratory and Centre for Structural and Functional Genomics, Biology Department, Concordia University, 7141 Sherbrooke Street W., Montreal, QC H4B 1R6, Canada e-mail: [email protected]

originate from the phenylpropanoid and polyketide pathways. There are more than 8,000 different flavonoid compounds reported so far from vascular plants and bryophytes (Anderson and Markham 2006). According to the oxidation level of ring C, flavonoids are grouped into different classes that include the chalcones, flavanones, flavones, isoflavones, flavonols, proanthocyanidins and anthocyanidins. They are widely distributed in foods, such as fruits and vegetables, and beverages of plant origin. In addition, they are considered an important part of the human diet and act as the active principles of many medicinal plants. Consumption of flavonoids is usually associated with health benefits due to their antioxidant, antiviral, antitumorigenic, and cancerpreventive activities (Kuo 1997). Several plant flavonoids are epidemiologically correlated with apoptotic pathways leading to lower cancer incidence, and to cancer chemoprevention that yielded encouraging results both in vitro and in vivo (reviewed in Ramos 2007). Within the subgroups of yellow pigments, the flavonol quercetin is the most frequently occurring compound in foods; although kaempferol, myricetin, and the flavones apigenin, luteolin and chrysoeriol are also common. While most reviews on seed flavonoids (e.g. Lepinie`c et al. 2006) deal mainly with flavonols, proanthocyanidins and anthocyanidins, this brief review is intended to draw attention to the flavone, tricin (5,7,40 trihydroxy-30 ,50 -dimethoxyflavone, Fig. 1). Tricin accumulates in most cereal crop plants, and this review

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summarizes our current knowledge of its natural occurrence, biosynthesis, biological activities and potential uses. Wheat is one of the most economically important cereal crops worldwide; it is valued not only as a food source, but also as a potential source of natural products with nutraceutical and pharmaceutical importance. Recent studies seem to indicate that tricin commands several biological properties that are superior to other flavonoids and polyphenols in terms of agricultural, nutraceutical and pharmaceutical values. The term ‘nutraceutical’ was originally coined by Stephen DeFelice (1992) to define a food, or a part thereof, that provides medicinal or health benefits to humans, including the prevention and/or treatment of disease. In this regard, it is interesting to note that flavonoids have been considered the major active nutraceutical ingredients in plants.

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Natural occurrence and distribution Tricin was first isolated in the free form from rustinfected wheat (Triticum dicoccum L.) leaves (Anderson and Perkin 1931), although its accumulation in the plant may have been the result of biotic stress caused by rust infection. It was later found to be typically distributed in grasses, sedges and palms (Harborne and Hall 1964; Harborne 1975; Harborne and Williams 1976), including many important cereal crop plants, such as wheat, rice, barley, sorghum, oat and maize. A recent survey (Wollenweber and Do¨rr 2008) indicates that the pentahydroxyflavone, tricetin, and its methyl ether derivatives (Fig. 1) are more widespread than was previously known; having been detected in several plant species belonging to unrelated families, such as Phoenix formosana (Lin et al. 2009), Ficus sp. (Zhang et al. 2008a, b), bamboo

Fig. 1 Proposed pathway for the biosynthesis of the flavone tricin. F3H, flavanone 3-hydroxylase; FNS, flavone synthase; F30 H and F30 50 H, flavonoid 30 - and 30 , 50 -hydroxylases; FOMT, flavone O-methyltransferase

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leaves (Jiao et al. 2007), sugar cane (Saccharum officinarum) juice (Duarte-Almeida et al. 2007; Colombo et al. 2006), Sasa borealis (Poaceae) (Park et al. 2007), Sorghum bicolor (Kwon and Kim 2003), Japanese millet (Watanabe 1999), Medicago sativa (Stochmal et al. 2001) and Trigonelle foenumgraecum (Shang et al. 1998), the higher plant parasite, Orobanche ramosa (Melek et al. 1992), the traditional herbal medicine, Pyrethrum tatsienense (Yang et al. 2006) and Lycopodium japonicum (Yan et al. 2005), among others. Tricin is also reported to occur naturally in various conjugated forms, mostly as 7-O-b-D-glucopyranoside (e.g. Mabry et al. 1984; Lin et al. 2009; Park et al. 2007; Wang et al. 2004; Zhang et al. 2008a, b), and less often as the 5-glucoside (Wallace 1974; AdjeiAfriyie et al. 2000a, b), although tricin 5-glucoside and tricin 5-diglucoside and tricin 5,7-glucoside were also reported to occur in M. flacata (Polyakova 1992); 40 -glucoside (Hasegawa et al. 2008) and 40 -apioside (Syrchina et al. 1992); 7-glucuronide (Harborne and Hall 1964; Markham and Porter 1973); 7-diglucuronide (Timoteo et al. 2008); 7-glucuronide sulphate (Williams et al. 1983) as well as several tricin glycosides conjugated with sulphate (Harborne and Williams 1976), 7-(200 -rhamnosyl)-a-galacturonide (Mabry et al. 1984); 7-glucuronide-40 -glucoside (Stochmal et al. 2001; Kowalska et al. 2007); 7-diglucuronide (Timoteo et al. 2008) and 7-neohesperidoside (Colombo et al. 2006, 2008) derivatives, to mention only a few. Other recently reported conjugated forms of tricin include the flavonolignan derivatives: 7-b-(600 -methoxycinnamic)-glucoside (Duarte-Almeida et al. 2007); 40 -b-guaiacylglyceryl (Bouaziz et al. 2002; Lei et al. 2007); 40 -(b-guaiacylglyceryl)-7-b-glucoside (Yang et al. 2006); six new guaiacylglyceryl derivatives, considered as three pairs of stereoisomers (Nakajima et al. 2003); 7-[20 -pcoumaroyl]-b-triglucuronide and the feruloyl diglucuronide derivative (Kowalska et al. 2007); as well as two other related conjugates, one with a coniferyl moiety linked to tricin by an ether bond and the other linked by a C–C bond (Wenzig et al. 2005). Another class of conjugated compounds includes the tricin C-glycosides: 6-C-xyloside-8-C-hexoside, 6,8-di-Cglycoside, as well as tricetin 6,8-di-C-glucoside (Theodor et al. 1980; 1981). It is interesting to note that tricin may also undergo further in vivo O-methylation, as evidenced by the natural occurrence

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of its 7- (Shao et al. 2008; Yamazaki et al. 2007; Zhao et al. 2007) and 40 - (Yang et al. 2006; Shao et al. 2008;) methyl ether derivatives. In fact, the latter (5,7-dihydroxy-30 ,40 ,50 -trimethoxyflavone) was earlier identified as a natural constituent in 18 graminaceous species (Kaneta and Sugiyama 1973). In most cases, both free tricin and/or its conjugated forms occur naturally in association with a variety of C-glycoflavones and flavonol derivatives (Harborne and Hall 1964; Cavalie`re et al. 2005) that are stored in the vacuoles (Joseph and Grotewold 2004). Glycosylation and/or methylation of tricin renders it less reactive and more water soluble, although tricin is also known to accumulate as a free lipophilic aglycone on the surfaces of plant leaves, flowers and other tissues (Wollenweber and Do¨rr 2008 and refs therein). Lipophilicity of the aglycone allows tricin to pass readily through cell membranes and act more effectively against herbivores and pathogens. However, its parent aglycone, tricetin, which is structurally similar to the widely distributed anthocyandin delphinidin and the flavonol myricetin, was only reported as a glycoside in 1986, and as an aglycone in 1997, but was later reported from the pollen and honey of several myrtaceous species (Wollenweber and Do¨rr 2008 and refs therein). The scarcity of tricetin occurrence in plants may be ascribed to its potential cytotoxicity and the requisite inactivation of its reactive hydroxyl groups by further glycosylation and/or methylation.

Phytochemical aspects Tricin is considered a dominant flavone in cereal crop plants, and is mainly detected in leaves and stems, but rarely in roots (Zhang et al. 2008a, b). In the wheat grain, tricin is mainly found as an aglycone in the outer layers: husk, pericarp and aleurone (Naczk and Shahidi 2006 and refs. therein). It has been isolated, as aglycone or a glycoside, in variable amounts from different sources, ranging from minor amounts in the leaves Triticum spp. 1.3 g of the pure aglycone from ca. 5.8 kg of Khapli wheat leaves (Anderson and Perkin 1931); 45 mg from 9 kg of dry tops of Spartina cynosuroides (Miles et al. 1983); 13 lg of an acylated glucoside per 100 g of dry sugarcane juice (Duarte-Almeida et al. 2007); 3.09 g of HPLCpurified tricin from ca. 5 kg of bamboo shoot

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(Jiao et al. 2007); 100 mg each of tricin and its 7-glucoside per kg of dry Desmostachia bipinnata (Graminaceae) leaves (Awaad et al. 2008). Being the dominant flavone pigment in whole wheat meal, prompted the elaboration of a method for tricin isolation and quantification, based on ultrasonic-assisted extraction and ultra-performance HPLC purification, and guided by electrospray ionization MS in negative ion mode with primary transitions monitoring of m/z 329.4/314.3 and secondary transition monitoring of m/z 329.4/299.3 with a detection limit of 1.2 lg/kg tissue (Zhao et al. 2009). A simple method for tricin isolation from the pressed juice of Medicago sativa was earlier described (Bichoff et al. 1964), and a recent HPLC method for its separation (Estiarte et al. 1997) and quantification was established (Li et al. 2006). The latter is based on its detection at 349 nm and elution from a C18 column with MeOH–water (65:35) as the mobile phase, with linear calibration curves and 97–100% recoveries of three different concentrations. Unlike most flavonoid compounds, tricin is not readily available commercially and its isolation from native plants is often limited by its low abundance and unstable resources. The only alternative to making it available in suitable quantities for experimentation and pharmacological testing is through chemical synthetic methods (Gulati and Venkataraman 1933; Mentzer and Pillon 1953; Owada and Mieno 1970; Laas and Eicher 1989; Nagarathnam and Cushman 1991; Xu et al. 1996; Shong et al. 1999). Most of these methods are based on direct condensation of 2,4,6-trihydroxyacetophenone and 4-hydroxy-3,5-dimethoxybenzaldehyde to give rise to the corresponding flavanone, followed by dehydrogenation with iodine and sodium acetate. These methods are laborious and suffer from difficulties in product purification and low yield. A short and facile synthetic route (Nagarathnam and Cushman 1991) employs the reaction of lithium polyanions generated from the appropriately substituted o-hydroxyacetophenones with O-silyloxylated benzoates, followed by treatment of the product with acetic acid containing 0.5% H2SO4 at 95–100°C, thus affording a 72–92% yield of the target flavone. In spite of their limited distribution, the glycosides of tricetin methyl ethers have proven useful as chemotaxonomic markers in a Stachys subgenus (Marin et al. 2004) and as indicators of parentage in

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six ornamental Fuchsia spp. and their hybrids (Williams et al. 1983). It was also shown that the relative amounts of glycosylflavones and four tricin glycosides, in seven Triticum species, could be used as indicators of the three more likely diploid ancestors to the hexaploid T. aestivum than the other four spp. (Harborne et al. 1986). Finally, it is worth mentioning that the dyeing properties of leaf extracts of a number of wheat varieties, including var. Khapli and var. Marquis, are mainly due to tricin glycosides and their parent aglycone tricetin (Anderson 1932), which dates back to the use of wheat extracts to create the ochre colours used by the Pharaohs for cloth dyeing and tomb-wall paintings.

Biosynthesis of tricin There is a serious lack of information as to the nature and sequence of the enzymatic steps leading to the production of flavones in general (Schijlen et al. 2004) and particularly those involved in biosynthesis of the tricin precursor, tricetin, as well as the genes encoding their enzymes. This may be ascribed to the absence of flavone synthase genes in the genome of Arabidopsis that is consistent with the near absence of flavones in the Brassicaceae (Martens and Mitho¨fer 2005). However, as is known for other flavonoids, the early steps of flavonoid biosynthesis (Forkmann and Heller 1999) involve the stepwise addition of malonyl CoA (polyketide pathway) and p-coumaroyl CoA (phenylpropanoid pathway), mediated by the sequential action of chalcone synthase and chalcone isomerase, to give rise to naringenin chalcone and the flavanone, naringenin, respectively (Fig. 1). As depicted in most recent reviews (Winkel 2006; Schijlen et al. 2004; Lepinie`c et al. 2006), it is reasonable to assume that both the flavanone 30 -hydroxylase (F30 H) and flavonoid 30 ,50 -hydroxylase (F30 ,50 H) are involved in B-ring hydroxylation to give rise to their respective products, eriodictyol and 50 -hydroxyeriodictyol. This is presumably followed by the action of flavone synthase (FNS) that introduces a double bond between C-2 and C-3, and ultimately gives rise to tricetin. FNS I is a cytosolic, 2-oxoglutarate-dependent dioxygenase that has been identified only in members of the Apiacea family, whereas the membrane-associated, cytochrome P450dependent FNS II is of widespread occurrence in

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other plants (Fokmann and Heller 1999). Both F30 H and F30 ,50 H have been shown to hydroxylate various flavonoid intermediates and end products in vitro; thus, affording B-ring hydroxylation at the 30 - and 50 -positions. It is not known whether both F30 H and F30 ,50 H precede the action of FNS (I/II) in wheat, or rice (Shi et al. 2008), since most recent reviews focused mainly on the biosynthesis of proanthocyanidins and anthocyanidins, or did not consider B-ring hydroxylation in reviewing flavone biosynthesis (Martens and Mitho¨fer 2005). It has recently been suggested that the reduced accumulation of tricin in light-induced rice seedlings could be explained by the competing flavone and anthocyanidin pathways (Shi et al. 2008). However, it is surprising to note that rice FNS was cloned by a reverse transcription-polymerase chain reaction, expressed in E. coli and characterized as a dioxygenase gene (Kim et al. 2008). This enzyme was shown to catalyze the synthesis of apigenin from naringenin, thus suggesting that rice FNS is a cytosolic enzyme whose action precedes F30 H and F30 50 H in the biosynthesis of tricetin. Therefore, if this gene is to be used for metabolic engineering experiments (see later), this controversy calls for the cloning of wheat FNS (I/II) and characterization of its gene product as to its nature and substrate specificity. However, both FNS (I/II) and F30 ,50 H genes have been cloned and characterized from various sources (Martens and Mitho¨fer 2005; Ayabe and Akashi 2006; Zhang et al. 2007; Seitz et al. 2006). The later step in tricin biosynthesis involves the sequential O-methylation of tricetin to its 30 -monomethyl-(selgin) and 30 ,50 -dimethyl-(tricin), with small amounts of 30 ,40 ,50 -trimethyl ether derivatives (Fig. 1). Our earlier studies have indicated that the stepwise methylation of tricetin is catalyzed by flavone O-methyltransferase (TaOMT2) as a single gene product. The TaOMT2 gene was recently cloned and characterized from wheat, Triticum aestivum L. (Zhou et al. 2006a). Mutational analysis of the structurally-guided active site residues allowed for the identification of those involved in binding and catalysis, as well as a single residue, Val309, that defines the preference of TaOMT2 for tricetin as the substrate (unpublished data). Phylogenetic analysis indicates that a number of flavone OMT homologous genes occur in most cereal plants, such as barley, rice, maize and sorghum, as well as in ryegrass and

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sugarcane, indicating a plant flavone OMT gene superfamily (Zhou et al. 2006b). Flavone OMT genes are often mis-annotated as caffeic acid/5-hydroxyferulic acid 3/5-OMTs (COMTs), possibly because of their high amino acid sequence similarity/identity to COMTs as well as the structural similarity between the phenylpropanoid moiety and the flavonoid-B-ring with its 3-C side chain (Zhou et al. 2008, 2009). Recently, the putative wheat COMT, TaCOMT1, (Jang et al. 2005) was biochemically characterized as a wheat flavone OMT, TaOMT1 (Zhou et al. 2009). Based on sequence similarity and in vitro enzymatic activity, TaOMT1 and TaOMT2 seem to belong to the same gene family, and they both accept tricetin as their preferred substrate to produce tricin. The TaOMT1 gene was shown to be expressed in all tissues of wheat (Jung et al. 2008) and its gene product accumulated in significant amounts in the leaves and roots treated with MeJA, ethylene, ABA, wounding, PEG, UV-B irradiation, as well as after infection with the Hessian fly larvae (Jang et al. 2005). Promoter analysis of the TaOMT1 gene identified sets of hormone and abiotic stress responsive regulatory elements. Taken together, these data clearly indicate that tricin and its biosynthetic genes play an important role in plant-environmental biotic/ abiotic stress interactions.

Regulation of tricin biosynthesis In contrast with the accumulated wealth of knowledge on the regulation of biosynthesis of proanthocyanidins and anthocyanidins in flowers (Schijlen et al. 2004; Winkel-Shirley 2001a, b; Winkel 2006) and seeds/grains (Himi and Noda 2005; Lepinie`c et al. 2006), there is a conspicuous lack of information on the regulation of flavone biosynthesis, or the use of the latter in the metabolic engineering of crop plants (Schijlen et al. 2004). In fact, very little has yet been published on the flavonoid pathways in rice, wheat or barley (Winkel-Shirley 2001b). However, it seems reasonable to assume that flavanone 3-hydroxylase (F3H; Fig. 1), which results in the formation of dihydroflavonols, constitutes an key step that regulates the ratio between 3-hydroxyflavonoid (anthocyanidins) and 3-deoxyflavonoid (C-glycoflavones, 3-deoxyanthocyanidins and phlobaphene pigments) synthesis in a given plant (Schijlen et al. 2004),

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especially given that the F3H gene has been shown to be coordinately expressed with chalcone synthase and chalcone isomerase in Arabidopsis (Pelletier and Shirley 1996) and rice (Shi et al. 2008) seedlings. In addition, various regulatory elements, including the R2R3-MYB transcription factor PAP1 (Mehrtens et al. 2005) have been implicated in the regulation of flavonoid synthesis (reviewed by Quattrocchio et al. 2006). Advances in plant genomics and systems biology, including the availability of the complete genome sequences of both Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), have offered an unprecedented opportunity to identify regulatory genes and networks that control these important traits. Because transcription factors naturally act as master regulators of cellular processes, they are expected to be excellent candidates for modifying complex traits in crop plants, and transcription factors-based technologies are likely to constitute a prominent part of the next generation of successful biotechnology crops (Century et al. 2008), including tricin-enriched wheat.

Biological significance of tricin Flavonoid compounds are of ubiquitous occurrence in vascular plants, but are rare in bryophytes (Markham and Porter 1973; Theodor et al. 1980; 1981) and algae. Generally, flavonoids function as antioxidants, antimicrobial/antiviral agents, allelochemicals, photoreceptors, visual attractors, and signaling molecules that are involved in plant growth and development, as well as their interactions with the environment. Among the various groups of flavonoid compounds, flavones have been shown to possess a higher fungicidal activity against 34 different fungal species, known to cause detrimental effects to stored seeds and pods, than flavanones (Weidenborner and Iha 1997). This is in agreement with the fact that the outer layers of cereal grains usually contain the highest flavonoid content (Fig. 2, unpublished data). Structural analyses indicated that methylated flavones and many lipophilic flavonoids were more effective in inhibiting mycelial growth of Pyricularia oryzae, Rhyzoctinia solani and killing many bacteria (Laks and Pruner 1989; Zhang et al. 1996; Padmavati et al. 1997; Almada-Ruiz and Martinez-Tellez 2003; Pretorius 2003; Kong et al. 2004).

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Fig. 2 A cross section of wheat kernel stained with 5 mM diphenyl boric acid 2-aminoethyl ester showing greenish fluorescence of flavonoids in the outer layers of the grain, as observed in a Zeiss LSM 510 META (405 nm) diode laser confocal microscope and post-processing software (LSM5 Macro). Scale bar = 100 lM

Tricin was first isolated from rust infected wheat leaves (Anderson and Perkin 1931), together with small amounts of its parent aglycone, tricetin (Anderson 1932), and was later shown be an active agent involved in plant defensive reactions against weeds, bacteria and fungi (Bylka et al. 2004; Kong et al. 2004; Tsuchida 2008). Furthermore, tricin was shown to possess potential herbicidal activity (Chung et al. 2005) and its novel flavonolignan acts as a germination inhibitor (Cooper et al. 1977). A recent study of the effects of the herbicide safener on wheat seedlings revealed not only the depletion in the levels of apigenin-, luteolin- and isorhamnetin C-glycosides, but also the selective accumulation of tricin and ferulic acid; thus causing a shift in the metabolism of endogenous phenolics (Cummins et al. 2006). Tricin was shown to be a highly efficient rice vir-gene expression factor (Xu et al. 1996; Shong et al. 1999), as detected by Agrobacterium::lacZ fusion genes; which may explain the difficulty in transforming monocotyledonous plants in the absence of vir gene required for induction of Agrobacterium. Tricin was also reported to be involved in plant-insect interactions (Adjei-Afriyie et al. 2000a, b; Simmonds 2003; Ling et al. 2007), acting as an anti-feedant against boll weevil (Miles et al. 1993) and an aphidfeeding deterrent in wheat (Dreyer and Jones 1981).

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Health-promoting effects and putative mechanism of tricin action Flavonoids constitute an important part of the human diet and many studies have strongly suggested that they may be involved in cardioprotection, neuroprotection and chemoprevention. Traditionally, flavonoid-mediated health effects were attributed to their antioxidant and free radical-scavenging abilities observed in vitro. However, recent studies indicate that flavonoids strongly influence cell signalling pathways and gene expression via interactions with specific target proteins, rather than through their antioxidant activities (Williams et al. 2004). Flavones and flavonols can interact with proteins, as a substrate or inhibitor, as they possess some structural similarities to the adenine nucleus (Shimmyo et al. 2008; Steffen et al. 2007). Other studies have also shown that intracellular metabolism (conjugation and oxidation) strongly affects the bioavailability (the amount that can be absorbed from the lumen) of flavonoids, thus regulates the biological activities of flavonoids in vivo (Williams et al. 2004; Holst and Williamson 2008). Methoxylated flavones are considered to represent a superior anti-cancer flavonoid subclass due to their easy access to the target cells (lipophilicity) and large bioavailability (masking of reactive hydroxyl group) (Deng et al. 2006; Arroo et al. 2009; Walle 2007a, b). Based on these data, tricin appears to be an attractive candidate for pharmacological and medicinal studies although more and detailed human clinical trials as well as the studies of metabolism and bioavailability of tricin in vivo are needed. Tricin has long been credited for its healthbeneficial effects: as an antioxidant (e.g. Watanabe 1999; Kwon et al. 2002; Kwon and Kim 2003; Lu et al. 2006; Maurı´cio Duarte-Almeida et al. 2006; Hasegawa et al. 2008; Mu et al. 2008) due to its potent inhibition of lipoperoxidation and its sparing effect on vitamin E in erythrocyte membranes Rice-Evans et al. 1997; Pietta 2000); as an antiviral (Li et al. 2005; Sakai et al. 2008); antihistaminic Kuwabara et al. 2003); and in immunomodulatory (Liang et al. 1997; Wang et al. 2004) and antitubercular (Gu et al. 2004) activities. The promising antiulcerogenic activity of tricin and its 7-glucoside was recently reported with a curative ratio of 77 and 79%, respectively (Awaad et al. 2008). In addition, tricin

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isolated from alfalfa (Medicago sativa), was first reported to cause smooth muscle relaxation in intestinal tissue, and was shown to be a powerful antioxidant with a slight estrogenic activity (Bichoff et al. 1964). One of the most prominent and well documented features of tricin is its potential antitumor/anticancer activity (Lee et al. 1981; Miles et al. 1983; Hudson et al. 2000, 2004; Cai et al. 2004, 2005, 2009; Yan et al. 2005) and inhibition of lymphocyte proliferation in normal mice (Xiong et al. 2006). In fact, tricin extracted from rice bran has been shown to inhibit the growth of human malignant breast tumor cells and colon cancer cells (Hudson et al. 2000; Cai et al. 2004). Tricin was also shown to interfere with murine gastrointestinal tract carcinogenesis and was considered safe for clinical development as a cancer chemopreventive agent (Verschoyle et al. 2006) when compared with the flavonol, quercetin. An acylated tricin glycoside from sugarcane (Saccharum officinarum) juice has been shown to have antiproliferative activity in vitro against several human cancer cell lines, and a higher selectivity toward cells of the breast resistant NIC/ADR line (DuarteAlmeida et al. 2007). Based on a study of structure– activity relationships of flavonoid-induced cytotoxicity on human leukemia cells, tricin is considered one of the most potent anti-cancer agents tested thus far (Plochmann et al. 2007) and this is most probably due to the stability of its structure, since it was earlier detected undegraded in the feces of tricin-fed rats, but not quercetin-fed rats (Stelzig and Ribeiro 1972). This is in contrast with most other flavonoids with free 40 -OH groups which undergo degradation by intestinal microflora and loss of the degradation products via urinary excretion (Griffiths and Smith 1972). In addition, tricin, being a competitive inhibitor of cytosolic sulfotransferases (Harris and Waring 2008), which reduce sulfonation of flavonoids, is thus more available than apigenin in blood and tissues (Cai et al. 2007) and may be more useful as a cancer chemoprotectant, since methylated flavonoids exhibit a higher oral bioavailability (Wen and Walle 2006). Although not fractionated, a wheat germ flavonoid extract has been reported to cause a dose- and timedependent growth inhibition of colony proliferation and 3H-thymidine incorporation into DNA, in the human breast cancer cell, BCap-37, in vitro (Xu et al. 1999).

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Fig. 3 HPLC profile of the culture medium extract showing tricin production in a biotransformation process. Biotransformation was carried out using the wheat flavone TaOMT2 gene subcloned in E. coli, and subjected to a fermentation system in the presence of substrate tricetin. The small scale experiment demonstrated a high yield (ca. 90%) of almost pure tricin that can be scaled up to a commercial scale. HPLC analysis was carried out with a Millennium HPLC System (Waters, Milford,

MA). Separation of the enzyme reaction products was performed on a Waters YMC-Pack Pro C18 column (150 9 4.6 mm I.D., S-5 lM, 12 nm), and eluted using a linear gradient consisting of 40–90% MeOH in 1% acetic acid. Identity of the reaction products was confirmed by comparing their retention times (Rt) and their uv absorption maxima with those of reference compounds

Finally, a recent comparative study of flavones as colorectal cancer preventive agents, using APC10.1 mouse adenoma cells, indicates that the rank order of cancer chemopreventive efficacy is pentamethoxyflavone [ tricin [ apigenin, thus supporting the notion that methylation of flavones promotes gastrointestinal chemopreventive efficacy (Cai et al. 2009).

better candidate than other flavonoids for human disease prevention and protection. The isolation of tricin from native plants is often limited by its low abundance, whereas chemical synthesis is commercially expensive, and its purification is cumbersome and often results in low yields. In view of the health promoting effects of tricin and its potential use as a nutraceutical, we propose the use of metabolic engineering strategies that aim at increasing the levels of tricin in wheat, and direct its production into the endosperm tissue, the latter usually being flavonoid-deficient. Our preliminary analyses indicate that tricin accumulates mainly in the grain bran (25 lg/g) and husks (49.5 lg/g), but only in minute amounts (1.4 lg/g) in both the germ and endosperm tissues (unpublished data). Over-expression of specific structural genes involved in tricin biosynthesis and down-regulation

Towards metabolic engineering of tricin production Cereals constitute an important staple diet for the world’s population. However, the major cereal crop plants consumed for nutrition often lack tricin or contain only small amounts in the relevant tissues, especially the endosperm, which restricts its benefit. Several studies have demonstrated that tricin is a

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of competing pathways, as well as manipulation of transcription factor genes have been successfully used in metabolic engineering strategies targeting a wide range of flavonoid compounds in transgenic plants (for reviews see Dixon and Steele 1999; Forkmann and Martens 2001; Schijlen et al. 2004). In the case of tricin, down-regulation of the F3H gene would divert naringenin towards flavone synthesis, rather than the competing anthocyanidin pathway. Furthermore over-expression of F30 H, F30 ,50 H and TaOMT2 genes would assure maximum synthesis of tricin. Ectopic expression of foreign genes has been achieved in several crop plants with the aim of increasing the content of value-added products, or conferring resistance to microbial attack (Leckband and Lo¨rz 1998). On the other hand, the reconstruction of a biosynthesis pathway branch (an artificial biosynthetic gene cluster) in a heterologous microorganism system may offer another alternative for the scalable production of tricin. A biotransformation system using the wheat flavone TaOMT2 gene subcloned in E. coli in the presence of tricetin as a substrate, resulted in a high (ca. 90%) yield of tricin (Fig. 3, unpublished data), which can be upscaled for use in further experimentation and clinical testing. Recent advances in in vitro production of natural products offer a great promise for this approach (Chemier and Koffas 2008 and refs. therein). Acknowledgments We wish to thank the Natural Sciences and Engineering Research Council (NSERC) of Canada for financial support of the work cited from the authors’ laboratory.

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