Phloridzin: Biosynthesis, distribution and physiological relevance in plants

Phytochemistry 71 (2010) 838–843

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Molecules of Interest

Phloridzin: Biosynthesis, distribution and physiological relevance in plants Christian Gosch, Heidi Halbwirth, Karl Stich * Vienna University of Technology, Institute of Chemical Engineering, Getreidemarkt 9, 1060 Vienna, Austria

a r t i c l e

i n f o

Article history: Received 26 January 2010 Received in revised form 1 March 2010 Available online 29 March 2010 Keywords: Malus  domestica Flavonoid biosynthesis Plant physiology Polyphenol Dihydrochalcone Phloridzin NADPH-dependent dehydrogenase Chalcone synthase UDP-glucose:phloretin 20 -Oglycosyltransferase

a b s t r a c t The phenolic compound phloridzin (phloretin 20 -O-glucoside, phlorizin, phlorrhizin, phlorhizin or phlorizoside) is a prominent member of the chemical class of dihydrochalcones, which are phenylpropanoids. The apple tree (Malus sp.) accumulates high amounts of phloridzin, whereas few other species contain this compound only in low amounts. Additionally, Malus sp. show a species- and tissue-specific distribution of phloridzin and its derivatives. Whereas the physiological role of phloridzin in planta is not fully understood, the effect on human health – especially diabetes – and membrane permeability is well documented. The biosynthesis of phloridzin was investigated only recently with recombinant enzymes and plant protein extracts and involved a NADPH-dependent dehydrogenase, chalcone synthase and UDPglucose:phloretin 20 -O-glycosyltransferase. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction De Koninck (1835a,b) isolated and described a bitter tasting substance with antipyretic effects from the bark of the apple tree. He found that this new compound was more prevalent in root bark than in stem bark and suggested calling it phloridzin (ukoió1: bark, qífa: root). Phloridzin is sometimes also referred to as phlorizin, phlorrhizin, phlorhizin or phlorizoside. Phloridzin belongs to the chemical class of dihydrochalcones, phenylpropanoids with structures closely related to those of the immediate flavonoid precursors, the chalcones. It consists of a C6–C3–C6 skeleton structure (two aromatic rings connected by a C3 chain) with a b-D-glucopyranose moiety attached at position 20 (Fig. 1). More than 700 peerreviewed articles dealing with phloridzin or its derivatives have been published since 2000. In more than 170 years since its discovery, phloridzin and its derivatives have been widely used in human medicine and for physiological studies on biological membranes. Most studies relate to diabetes, obesity, stress hyperglycemia, antioxidative activity, membrane permeability and longevityextending agents in foods, beverages, food additives, pharmaceuticals and cosmetics (Ehrenkranz, 2006, 2005; Gaudout et al., 2006; Rezk et al., 2002; Sukhorukov et al., 2001; Valenta et al., 2001). In particular, the effect of phloridzin on glucose uptake and diabetes has been intensively investigated and was reviewed by Ehrenkranz

et al. (2005). In contrast, knowledge about the physiological relevance of phloridzin in planta is limited. The biosynthetic steps leading to phloridzin were recently investigated with recombinant enzymes (Gosch et al., 2010; Jugdé et al., 2008) and plant protein extracts (Gosch et al., 2009). 2. Biosynthesis of phloridzin 2.1. Precursors Whereas the biosynthesis of chalcones and flavonoids is well understood at the enzyme and gene level, the biosynthesis of closely related dihydrochalcones such as phloridzin has only recently been elucidated. The biosynthetic pathway leading to phloridzin is shown in Fig. 2. Malonyl-CoA (1) is synthesized from acetyl-CoA, whereas p-coumaroyl-CoA (2) originates from phenylalanine, which is produced via the shikimate pathway. Phenylalanine ammonia-lyase catalyzes the formation of cinnamate from phenylalanine and marks the branching point between primary metabolism and cinnamate-related polyphenolic compounds. Cinnamate is further hydroxylated by the cinnamate 4-hydroxylase and activated by the hydroxycinnamate:CoA ligase, resulting in p-coumaroyl-CoA (4-hydroxycinnamoyl-CoA). 2.2. Formation of p-dihydrocoumaroyl-CoA from p-coumaroyl-CoA

* Corresponding author. Tel.: +43 1 58801 17320; fax: +43 1 58801 17399. E-mail address: [email protected] (K. Stich). URL: http://www.vt.tuwien.ac.at (K. Stich). 0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2010.03.003

Whereas p-coumaroyl-CoA is the precursor for the naringenin chalcone (3) and further flavonoid formation (Fig. 2),

C. Gosch et al. / Phytochemistry 71 (2010) 838–843

OH 4

HO

OH 6´

4´

O

HOH2C

2´

O

O

OH

HO HO

Fig. 1. The dihydrochalcone phloridzin consists of a C6–C3–C6 skeleton structure with a b-D-glucopyranose moiety attached at position 20 .

DH

OH

OH

NADPH NADP+, FAD

CoA-S

CoA-S

(2) O

3x

(4)

O

O S-CoA

CHS

O OH

HO

CHS

OH

(1)

OH

HO

OH

OH

(5)

(3) OH

OH

O

O

P2´GT + UDP-glucose

OH

839

a hydroxycinnamate:CoA ligase and a chalcone synthase were fed p-dihydrocoumaric acid. This implies the existence of a dehydrogenase in E. coli. Single transgenic E. coli (empty plasmid vector or overexpressing a hydroxycinnamate:CoA ligase or a chalcone synthase) revealed that this unknown dehydrogenase is active on the level of the CoA ester. Similar results were obtained by Jiang et al. (2005), who used Saccharomyces cerevisiae overexpressing a phenylalanine ammonia lyase, a hydroxycinnamate:CoA ligase and a chalcone synthase. After feeding either phenylalanine or tyrosine, naringenin and phloretin were detected. A similar dehydrogenase reaction was involved in the proposed degradation pathway of naringenin by Eubacterium ramulus with a naringenin chalcone, phloretin and p-dihydrocoumaric acid as intermediates (Herles et al., 2004; Schneider and Blaut, 2000). Since the dehydrogenase activity does not appear to be specific to the dihydrochalcone biosynthesis, Gosch et al. (2009) screened different enzymes, which catalyze similar dehydrogenase reaction steps and use structurally related native substrates for their ability to interconvert p-coumaroyl-CoA and p-dihydrocoumaroyl-CoA. A recombinant human medium chain acyl-CoA dehydrogenase, which actually catalyzes the FAD-dependent oxidation of octanoyl-CoA to octenoyl-CoA, could convert p-dihydrocoumaroyl-CoA to p-coumaroyl-CoA under certain conditions, but no homologous gene from apple was found. Another candidate, a recombinant enoylACP (acyl carrier protein) reductase, which actually catalyzed crotonyl-ACP to butyryl-ACP, could not interconvert the CoA esters. In summary, the dehydrogenase activity involved in the formation of dihydrochalcones in apple is characterized at the enzymatic level. Some potential candidate genes can be excluded, but the identity of the dehydrogenase remains unclear. 2.3. Formation of phloretin from 4-hydroxydihydrocinnamoyl-CoA and malonyl-CoA

flavonoids HO

OH

(6) O-Glc O Fig. 2. Biosynthetic pathway of phloridzin and flavonoids. CHS, chalcone synthase; DH, dehydrogenase; P20 GT, UDP-glucose:phloretin 20 -O-glycosyltransferase.

p-dihydrocoumaroyl-CoA (4-hydroxydihydrocinnamoyl-CoA; 4) is required for the biosynthesis of dihydrochalcones such as phloretin (5). Avadhani and Towers (1961) showed that young apple leaves fed with radiolabeled phenylalanine or cinnamic acid form radiolabeled phloridzin via p-coumaric acid, but that p-dihydrocoumaric acid was not detected as an intermediate. Therefore, the authors assumed that p-dihydrocoumaric acid was a breakdown product after fungal attack, for example, rather than a precursor during phloridzin formation. Yamazaki et al. (2001) showed with recombinant enzymes from Psilotum nudum that the CoA ester (pdihydrocoumaroyl-CoA) can act as a precursor of phloretin. Gosch et al. (2009) showed the formation of phloretin when the CoA ester of p-coumaric acid, radiolabeled malonyl-CoA and NADPH were incubated with protein extracts of apple leaves. With naringenin as a substrate or without the cofactor NADPH no phloretin formation was detected. Therefore, it is assumed that p-dihydrocoumaroyl-CoA is formed from p-coumaroyl-CoA by a NADPHdependent dehydrogenase (NADPH:p-coumaroyl-CoA oxidoreductase; Gosch et al., 2009). In general, the dehydrogenase catalyzing the interconversion of the CoA esters p-coumaroyl-CoA and pdihydrocoumaroyl-CoA does not appear to be plant-specific. Watts et al. (2004) also detected the formation of naringenin besides phloretin when double transgenic Escherichia coli overexpressing

The high similarity of p-coumaroyl-CoA and p-dihydrocoumaroyl-CoA led to the assumption that the chalcone synthase could utilize both substrates with three molecules of malonyl-CoA to form a naringenin chalcone or phloretin, respectively. This was supported by studies of recombinant chalcone synthases from plants, which are not known to accumulate dihydrochalcones such as Sinapis alba (Tropf et al., 1994), Arabidopsis thaliana (Watts et al., 2004), P. nudum (Yamazaki et al., 2001) or Pyrus communis (Gosch et al., 2009). Studies of chalcone synthases from M.  domestica (Gosch et al., 2009) finally confirmed this hypothesis. No substrate preference was found – at least for different recombinant chalcone synthases from apple – suggesting that the formation of dihydrochalcones is catalyzed by the common chalcone synthase and not a specialized enzyme with distinct substrate specificity. 2.4. Glycosylation of phloretin to phloridzin The attachment of a glucose moiety to phloretin at position 20 is the final step in the formation of phloridzin (phloretin 20 -O-b-Dglucopyranoside, 6). A cDNA clone from M.  domestica encoding a UDP-glucose:phloretin 20 -O-glycosyltransferase was recently isolated (Jugdé et al., 2008). Among several substrates tested, the recombinant enzyme accepted only phloretin as a substrate. Gosch et al. (2010) showed that several unspecific glycosyltransferases from M.  domestica with low sequence homology to one another could glycosylate phloretin to phloridzin. In addition, other phloretin glycosides (phloretin 4-O-glucoside and phloretin 4’-O-glucoside) were formed in vitro as byproducts in low amounts. In contrast to Jugdé et al. (2008), these recombinant glycosyltransferases showed a broader substrate acceptance. Therefore, more than one specific glycosyltransferase could be involved in the formation of phloridzin in apple. Interestingly, these glycosyltransferases are

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also present in the pear (P. communis), leading to the assumption that the absence of phloridzin in this closely related genus could be because of its lack of ability to catalyze the conversion of p-coumaroyl-CoA to p-dihydrocoumaroyl-CoA (Gosch et al., 2009). Similar to the glycosyltransferase of pear, Werner and Morgan (2009) recently reported a glycosyltransferase from Dianthus, which converts phloretin to phloridzin, although phloretin is not present in this plant species. 3. Distribution of phloridzin in the plant kingdom Beside neohesperidin dihydrochalcone (a semisynthetic sweetener derived from neohesperidin), phloridzin is the most prominent of the approximately 230 dihydrochalcones described to date (Veitch and Grayer, 2006, 2008). Since phloridzin is the predominant phenolic compound in Malus species, it is mostly discussed in this context. Phloridzin has been used for the chemotaxonomic differentiation of the rosaceous plant species (Challice, 1981) and identification of fraudulent admixtures of apple juice to other fruit juices (Wald and Galensa, 1989). Beside apple, also few other plant species are described to contain phloridzin (Table 1), which belong mainly to the Rosaceae and Ericaceae families. However, in species other than M.  domestica only low amounts of phloridzin are detected. 4. Phloretin derivatives in Malus species Ten phloretin derivatives are described from Malus species (Fig. 3). These are phloretin (a), phloridzin (b), trilobatin (c), 3hydroxyphloretin (d), 3-hydroxyphloridzin (e), sieboldin (f) (Williams, 1961), phloretin 20 -O-xyloglucoside (g) (Williams, 1966), phloretin 20 -O-xylogalactoside (h) (Burda et al., 1990), 4-O-transp-coumaroyl-phloridzin (i) and 4-O-cis-p-coumaroyl-phloridzin

Table 1 Phloridzin containing plant species. Taxonomy according to the Integrated Taxonomic Information System (www.itis.gov, 2010-02-18). Plant species

References

Asteraceae Ericaceae

Lactuca sativa Kalmia latifolia Pieris japonica Vaccinium macrocarpon Flemingia strobilifera Lithocarpus litseifolius Lithocarpus polystachyus Hemerocallis x hybrida Punica granatum Adenostoma fasciculatum Fragaria x ananassa Malus sp. Rosa canina Lithofragma affine Symplocos lancifolia Camellia japonica Lippia graveolens

Altunkaya and Gökmen (2009) Williams (1964) Williams (1964) Turner et al. (2005) Saxena et al. (1976) Rui-Lin et al. (1982) Dong et al. (2007) Cichewicz and Nair (2002) Poyrazog˘lu et al. (2002) McPherson et al. (1971) Hilt et al. (2003) De Koninck (1835a) Hvattum (2002) Nicholls and Bohm (1984) Tanaka et al. (1980) Cho et al. (2008) Lin et al., 2007

Liliaceae Punicaceae Rosaceae

Saxifragaceae Symplocaceae Theaceae Verbenaceae

R1 R2 R4

OH A

R3

O

B

(a) (b) (c) (d) (e) (f) (g) (h) (i),( j)

5. Physiological relevance of phloridzin in planta Knowledge about the physiological relevance of phloridzin in planta is limited. Since the apple plant accumulates high amounts of this compound, research has concentrated on this genus. 5.1. Resistance against plant diseases

Plant family

Fabaceae Fagaceae

(j) (Römmelt et al., 2003). The latter two were only detected after treatment with the dioxygenase inhibitor prohexadione-Ca. The interglycosidic linkage of (g) and (h) is only confirmed for the xyloglucoside (Lu and Foo, 1997; Huemmer, 2009), whereas a structure elucidation of the xylogalactoside is lacking. Moreover, phloretin 20 ,40 -O-diglucoside and 3-hydroxyphloretin 20 -O-xyloglucoside were found but only in apple juice extracts (Huemmer, 2009). Phloridzin is present in most Malus species but is replaced completely by trilobatin in Malus trilobata, whereas species of the Sieboldiana group (e.g. Malus sieboldii and Malus floribunda) also contain sieboldin besides phloridzin (Williams, 1966). Trilobatin and sieboldin were not found together in the same species. In some progeny of M.  domestica crossed with M. trilobata, phloridzin and trilobatin can be detected (Hunter, 1975). The different glucose moiety attachment sites of phloridzin and sieboldin seem to be determined by the ortho-dihydroxyl structure at the B-ring of sieboldin. Influences of the ortho-dihydroxyl structure at the B-ring on the sugar attachment site were also found for some flavonoid glycosyltransferases (Isayenkova et al., 2006; Vogt et al., 1999). Within M.  domestica, the quantity of dihydrochalcones can vary depending on the tissue (Table 2), variety (e.g. Łata et al., 2009), developmental stage (e.g. Zhang et al., 2007), sampling time (e.g. Mikulic-Petkovšek et al., 2009) and external factors like pathogen attack (e.g. Mikulic-Petkovšek et al., 2008). In seeds (66%; Guyot et al., 1998), bark (70–80%; Mornau, 2004) and leaves (80–90%; Mayr et al., 1997; Pontais et al., 2008) dihydrochalcones represent the majority of the total phenolic compounds. In fruits, only 2–6% of the phenolic compounds are phloridzin, whereas flavanols and proanthocyanidins dominate with more than 90% (Guyot et al., 1998; Tsao et al., 2003; Vrhovsek et al., 2004).

R1 H H H OH OH OH H H H

R2 OH OH OH OH OH OH OH OH O-coumaroyl

Phloridzin has commonly been proposed to be involved in disease resistance of apple against various pathogens. However, the involvement of phloridzin and its derivatives in pathogen resistance is controversial because its content does not always correlate with the degree of resistance of different apple cultivars. Holowczak et al. (1962) showed in vitro that the aglycone phloretin inhibits the growth of Venturia inaequalis (apple scab). Alt and Schmidle (1980) found strong fungitoxic effects of phloridzin and phloretin on the mycelial growth of Phytophthora cactorum (crown rot of apple). An accumulation of phloridzin was detected in apple bark infected with P. cactorum (Mornau, 2004) or leaves infected with V. inaequalis (Mikulic-Petkovšek et al., 2008). Römmelt et al. (2003) found an antimicrobial effect of phloretin and 4-O-cis-p-coumaroyl-phloridzin against the fire blight bacterium Erwinia amylovora. Pontais et al. (2008) also reported an antimicrobial effect of phlo-

R3 OH Glc OH OH Glc OH O-Glc-Xyl O-Gal-Xyl* Glc

R4 OH OH Glc OH OH Glc OH OH OH

Phloretin Phloretin 2´-O-glucoside (phloridzin) Phloretin 4´-O-glucoside (trilobatin) 3-Hydroxyphloretin 3-Hydroxyphloretin 2´-O-glucoside 3-Hydroxyphloretin 4´-O-glucoside (sieboldin) Phloretin 2´-O-xyloglucoside Phloretin 2´-O-xylogalactoside 4-O-cis/trans-p-coumaroyl-phloretin 2´-O-glucoside

Fig. 3. Phloretin derivatives described from Malus species. *The confirmation of the interglycosidic linkage is lacking.

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Table 2 Tissue-specific and quantitative distribution of dihydrochalcones in M.  domestica. All values except those in parentheses are the percentage of dry matter. Values in square brackets were calculated from the original values (in parentheses, mg/kg fresh weight) to percentage of dry matter using the factors 1.67  104 (for peel with 40% water content) or 6.67  104 (pitted fruits, flesh and peel or flesh with 85% water content). Compound

Minimum

Maximum

Tissue

References

Phloretin

0 0.031 2.2 2.49 5.41 6.07 4 n.s. 6.0 5.8 5.5 0.42 0 [0.017] (100) [0.002] (12) 0.079 0.108 [0.0063] (37.6) [0.0003] (1.9) [0.0048] (29.15) 0.071 [0.0067] (10) [0.0027] (4) 0.008 [0.0053] (8.0) [0.00017] (0.25) [0.0034] (5.12) [0.00096] (1.44) [0.0066] (10) [0.0043] (6.4) 0.011 0.741 0.24 (0) [0.00038] (2.3) 0.011 [0.00027] (0.4) [0.001] (1.72) [0.007] (11) [0.0045] (6.8) [0.01] (60) [0.0067] (10)

0.12 0.109 5.65 10.87 14.00 13.94 18 >12 9.5 8.5 8 10.00 0.82 [0.025] (150) [0.069] (418) n.s. 0.269 [0.029] (172.0) n.s. [0.026] (153.61) 0.242 n.s. [0.013] (20) n.s. [0.016] (24.6) n.s. [0.0088] (13.27) [0.0019] (2.80) [0.105] (158) [0.061] (91.1) 0.043 n.s. 6.21 [0.0019] (2.8) n.s. 0.056

Leaf Leaf Leaf Leaf Leaf Leaf Leaf Bark Bark Bark Bark Bark Bud Peel Peel Peel Peel Peel Peel Peel Peel Flesh Flesh Flesh Flesh Flesh Flesh Pitted fruits Flesh and peel Flesh and peel Flesh and peel Seed Seed coat Flesh and peel Peel Peel Flesh Pitted fruits Flesh and peel Flesh and peel Peel Flesh

Hunter and Hull (1993) Picinelli et al. (1995) Hunter and Hull (1993) Jham (1996) Picinelli et al. (1995) Mikulic-Petkovšek et al. (2008) Mikulic-Petkovšek et al. (2009) Williams (1966) Martin and Williams (1967) Alt and Schmidle (1980) Mornau (2004) Zhang et al. (2007) Zhang et al. (2007) Burda et al. (1990) Escarpa and Gonzáles (1998) Awad et al. (2000) Treutter (2001) Tsao et al. (2003) ´ Abrosca et al. (2007) D Mikulic-Petkovšek et al. (2007) Łata et al. (2009) Burda et al. (1990) Escarpa and Gonzáles (1998) Awad et al. (2000) Tsao et al. (2003) ´ Abrosca et al. (2007) D Mikulic-Petkovšek et al. (2007) Lee et al. (2003) Wald and Galensa (1989) Vrhovsek et al. (2004) Łata et al. (2009) Awad et al. (2000) Jham (1996) Vrhovsek et al. (2004) ´ Abrosca et al. (2007) D Treutter (2001) ´ Abrosca et al. (2007) D Lee et al. (2003) Wald and Galensa (1989) Vrhovsek et al. (2004) Burda et al. (1990) Burda et al. (1990)

Phloridzin

3-Hydroxyphloretin Phloretin 20 -O-xyloglucoside

Phloretin 20 -O-xylogalactoside

[0.0039] (5.88) [0.15] (230) [0.041] (61.4) [0.038] (230) [0.02] (30)

n.s., not specified.

ridzin against E. amylovora, but apple leaf extracts of the cultivars tested contained slightly less than the in vitro minimal inhibitory concentration. Muthuswamy and Rupasinghe (2007) showed the antimicrobial effects of phloridzin against various pathogenic bacteria. In contrast, Hunter (1975), Noveroske et al. (1964a) and Raa (1968) found no correlation between phloridzin content and resistance against the apple scab. Picinelli et al. (1995) found the phloridzin/flavanol ratio to be decisive for scab resistance, which was to some extent confirmed by results of Mikulic-Petkovšek et al. (2007). A higher phloridzin/flavanol ratio was detected in susceptible apple cultivars. In contrast to Hunter (1975), both Noveroske et al. (1964a,b) and Raa (1968) suggested oxidation products of phloridzin as antifungal. Phloridzin and phloretin were shown to inhibit spore germination of postharvest apple fruit rot Phlyctaena vagabunda when co-incubated with polyphenol oxidase (Lattanzio et al., 2001). The oxidation process of phloridzin, which leads to toxic compounds, is proposed as follows. Cellular decompartmentalization leads to the subsequent hydrolysis by specific glucosidases to phloretin. Phloretin, and also phloridzin, can undergo a peroxidase and/or polyphenol oxidase mediated oxidation resulting in o-diphenols and toxic, highly reactive o-quinones, which can react with the NH2- or SH-groups of proteins (Elstner et al., 1996; Noveroske et al., 1964a,b; Raa, 1968; Raa and Overeem,

1968). Since all apple cultivars accumulate high amounts of phloridzin, differences in disease resistance could be determined by the speed of the oxidation cascade (hypersensitive reaction) after pathogen attack rather than the actual amount of phloridzin present in the plant. Slatnar et al. (in press) found a clear decrease of UDP-glucose:phloretin 20 -O-glycosyltransferase activity in scab spots on apple leaves compared to healthy tissue, which contrasted with the observed increase of other flavonoid pathway enzymes. This supports the hypothesis that the aglycone rather than the 2’-O-glucoside is involved in the pathogen defense. Recently, Dugé de Bernonville et al. (2010) provided evidence for a bioactivity of dihydrochalcones – especially the complementary effects of sieboldin and phloridzin – as functional antioxidants by lowering the oxidative stress of apple leaves. This could maybe have implications for plant resistance during the oxidative burst of hypersensitive reactions (Venisse et al., 2001). 5.2. Further physiological effects of phloridzin Besides this beneficial role, phloridzin and its degradation products can exhibit negative effects on plants. Necrotic symptoms after infection of apple with the fungus Valsa ceratosperma are caused by degradation products of phloridzin (Natsume et al.,

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1982). Hancock et al. (1961) found an inhibition of phloridzin in the wheat coleoptile straight-growth test and Lockard and Schneider (1981) in lettuce hypocotyls. In contrast, phloridzin and its degradation products were found to stimulate apple shoot growth (Jones, 1976). Rooting of mung bean cuttings was increased by reaction products of phloridzin and polyphenol oxidase (Bassuk et al., 1981). Recent investigations identified phloridzin as a potential polyphenol marker of the phase change between the juvenile and adult phase in apple buds (Zhang et al., 2007). Hofmann et al. (2009) assume that phloridzin in apple root exudates could be a host recognition signal for microorganisms, which may cause apple replant disease. Phloridzin, as a competitive inhibitor of distinct carbohydrate carriers, reduced the uptake of glucose, 3-Omethylglucose and sucrose into broad bean cells (Lemoine and Delrot, 1987). Some aphids seem to use phloridzin as a recognition signal for distinguishing apple leaves from non-host plants (Klingauf, 1971). 6. Outlook Further investigations are necessary to clarify questions relating to the physiological role of phloridzin, especially in apple, which accumulates a high amount of phloridzin and its derivatives. A big step forward is expected from the molecular identification of the unknown dehydrogenase catalyzing the conversion of p-coumaroylCoA to p-dihydrocoumaroyl-CoA. This would also provide insight into the metabolic grid of the complex biosynthesis of polyphenols in apple. The biosynthesis of other dihydrochalcones in different plant species could also be enlightened. Identification of the dehydrogenase could further lead to a sequence-based identification of genes for similar biosynthetic steps such as the benzalacetone reductase, which catalyzes the reduction of p-hydroxyphenylbut3-ene-2-one to the raspberry aroma compound p-hydroxyphenylbutan-2-one (Borejsza-Wysocki and Hrazdina, 1994). Other examples are the biosynthesis of dihydrocinnamic acids involved in the formation of mesembrine alkaloids (Jeffs et al., 1978) or phenylphenalenones (Schmitt and Schneider, 1999). Finally, since activity of the unknown dehydrogenase seems to be the crucial point of phloridzin formation, identifying the unknown dehydrogenase could allow the establishment of various transgenic crop plants containing phloridzin, whether for nutrition physiological aims for the consumer or positive effects for plant disease resistance. References Alt, D., Schmidle, A., 1980. Untersuchungen über mögliche Resistenzfaktoren des Apfels gegen. Phytophthora cactorum (Leb. et Cohn) Schroet. Angew. Bot. 54, 139–156. Altunkaya, A., Gökmen, V., 2009. Effect of various anti-browning agents on phenolic compounds profile of fresh lettuce (L. sativa). Food Chem. 117, 122–126. Avadhani, P.N., Towers, G.H.N., 1961. Fate of phenylalanine-C14 and cinnamic acidC14 in Malus in relation to phloridzin synthesis. Can. J. Biochem. Physiol. 39, 1605–1616. Awad, M.A., de Jager, A., van Westing, L.M., 2000. Flavonoid and chlorogenic acid levels in apple fruit: characterisation of variation. Sci. Hortic. (Amsterdam) 83, 249–263. Bassuk, N.L., Hunter, L.D., Howard, B.H., 1981. The apparent involvement of polyphenol oxidase and phloridzin in the production of apple rooting cofactors. J. Hortic. Sci. 56, 313–322. Borejsza-Wysocki, W., Hrazdina, G., 1994. Biosynthesis of p-hydroxy-phenylbutan2-one in raspberry fruits and tissue cultures. Phytochemistry 35, 623–628. Burda, S., Oleszek, W., Lee, C.-Y., 1990. Phenolic compounds and their changes in apples during maturation and cold storage. J. Agric. Food Chem. 38, 945–948. Challice, J., 1981. Chemotaxonomic studies in the family Rosaceae and the evolutionary origins of the subfamily Maloideae. Preslia 53, 289–301. Cho, J.-Y., Ji, S.-H., Moon, J.-H., Lee, K.-H., Jung, K.-H., Park, K.-H., 2008. A novel benzoyl glucoside and phenolic compounds from the leaves of Camellia japonica. Food Sci. Biotechnol. 17, 1060–1065. Cichewicz, R.H., Nair, M.G., 2002. Isolation and characterization of stelladerol, a new antioxidant naphthalene glycoside, and other antioxidant glycosides from edible daylily (Hemerocallis) flowers. J. Agric. Food Chem. 50, 87–91.

´ Abrosca, B., Pacifico, S., Cefarelli, G., Mastellone, C., Fiorentiono, A., 2007. D Limoncella´ apple, an Italian apple cultivar: phenolic and flavonoid contents ´ and antioxidant activity. Food Chem. 104, 1333–1337. De Koninck, L., 1835a. Ueber das Phloridzin (Phlorrhizin). In: Geiger, L.P., Liebig, J., Trommsdorff, J.B. (Eds.), Annalen der Pharmacie, Band XV, UniversitätsBuchhandlung von C. F. Winter, Heidelberg, pp. 75–77. De Koninck, L., 1835b. Weitere Notiz über das Phloridzin. In: Geiger, L.P., Liebig, J., Trommsdorff, J.B. (Eds.), Annalen der Pharmacie, Band XV, UniversitätsBuchhandlung von C. F. Winter, Heidelberg, pp. 258–263. Dong, H., Ning, Z., Yu, L., Li, L., Lin, L., Huang, J., 2007. Preparative separation and identification of the flavonoid phlorhizin from the crude extract of Lithocarpus polystachyus Rehd. Molecules 12, 552–562. Dugé de Bernonville, T., Guyot, S., Paulin, J.-P., Gaucher, M., Loufrani, L., Henrion, D., Derbré, S., Guilet, D., Richomme, P., Dat, J.F., Brisset, M.-N., 2010. Dihydrochalcones: implication in resistance to oxidative stress and bioactivities against advanced glycation end-product and vasoconstriction. Phytochemistry 71, 443–452. Ehrenkranz, J.R.L., Lewis, N.G., Kahn, C.R., Roth, J., 2005. Phlorizin: a review. Diabetes Metab. Res. Rev. 21, 31–38. Ehrenkranz, J.R.L., 2006. Compositions containing botanical extracts rich in phlorizin and methods for using such compositions in blood glucose modification and to affect aging. Patent Pub. No. WO2006078848. Elstner, E.F., Oßwald, W., Schneider, I., 1996. Phytopathologie. Allgemeine und biochemische Grundlagen. Spektrum Akademischer Verlag, Heidelberg, Berlin, Oxford. Escarpa, A., Gonzáles, M.C., 1998. High-performance liquid chromatography with diode-array detection for the determination of phenolic compounds in peel and pulp from different apple varieties. J. Chromatogr. A 823, 331–337. Gaudout, D., Megard, D., Inisan, C., Esteve, C., Lejard, F., 2006. Phloridzin-rich phenolic fraction and use thereof as a cosmetic, dietary or nutraceutical agent. Patent Pub. No. US2006073223. Gosch, C., Halbwirth, H., Kuhn, J., Miosic, S., Stich, K., 2009. Biosynthesis of phloridzin in apple (Malus domestica Borkh.). Plant Sci. 176, 223–231. Gosch, C., Stich, K., Halbwirth, H., 2010. Cloning and heterologous expression of glycosyltransferases from Malus x domestica and Pyrus communis, which convert phloretin to phloretin 20 -O-glucoside (phloridzin). Plant Sci. 178, 299–306. Guyot, S., Marnet, N., Laraba, D., Sanoner, P., Drilleau, J.-F., 1998. Reversed-phase HPLC following thiolysis for quantitative estimation and characterization of the four main classes of phenolic compounds in different tissue zones of a French cider apple variety (Malus domestica var. Kermerrien). J. Agric. Food Chem. 46, 1698–1705. Hancock, C.R., Barlow, H.W.B., Lacey, H.J., 1961. The behaviour of phloridzin in the coleoptile straight-growth test. J. Exp. Bot. 12, 401–408. Herles, C., Braune, A., Blaut, M., 2004. First bacterial chalcone isomerase isolated from Eubacterium ramulus. Arch. Microbiol. 181, 428–434. Hilt, P., Schieber, A., Yildirim, C., Arnold, G., Klaiber, I., Conrad, J., Beifuss, U., Carle, R., 2003. Detection of phloridzin in strawberries (Fragaria x ananassa Duch.) by HPLC-PDA-MS/MS and NMR spectroscopy. J. Agric. Food Chem. 51, 2896–2899. Hofmann, A., Wittenmayer, L., Arnold, G., Schieber, A., Merbach, W., 2009. Root exudation of phloridzin by apple seedlings (Malus x domestica Borkh.) with symptoms of apple replant disease. J. Appl. Bot. Food Qual. 82, 193–198. Holowczak, J., Kuc´, J., Williams, E.B., 1962. Metabolism in vitro of phloridzin and other compounds by Venturia inaequalis. Phytopathology 52, 1019–1023. Huemmer, W., 2009. Analyse potentiell chemopräventiv wirksamer Inhaltsstoffe von Apfelsaft. Ph.D. thesis. Julius-Maximilians-Universität Würzburg. Hunter, L.D., 1975. Phloridzin and apple scab. Phytochemistry 14, 1519–1522. Hunter, L.D., Hull, L.A., 1993. Variation in concentrations of phloridzin and phloretin in apple foliage. Phytochemistry 34, 1251–1254. Hvattum, E., 2002. Determination of phenolic compounds in rose hip (Rosa canina) using liquid chromatography coupled to electrospray ionisation tandem mass spectrometry and diode-array detection. Rapid Commun. Mass Spectrom. 16, 655–662. Isayenkova, J., Wray, V., Nimtz, M., Strack, D., Vogt, T., 2006. Cloning and functional characterisation of two regioselective flavonoid glucosyltransferases from Beta vulgaris. Phytochemistry 67, 1598–1612. Jeffs, P.W., Karle, J.M., Martin, N.H., 1978. Cinnamic acid intermediates as precursors to mesembrine and some observations on the late stages in the biosynthesis of the mesembrine alkaloids. Phytochemistry 17, 719–728. Jham, G.N., 1996. High-performance liquid chromatographic quantification of phloridzin in apple seed, leaf and callus. J. Chromatogr. A 719, 444–449. Jiang, H., Wood, K.V., Morgan, J.A., 2005. Metabolic engineering of the phenylpropanoid pathway in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 71, 2962–2969. Jones, O.P., 1976. Effect of phloridzin and phloroglucinol on apple shoots. Nature 262, 392–393. Jugdé, H., Nguy, D., Moller, I., Cooney, J.M., Atkinson, R.G., 2008. Isolation and characterization of a novel glycosyltransferase that converts phloretin to phlorizin, a potent antioxidant in apple. FEBS J. 275, 3804–3814. Klingauf, F., 1971. Die Wirkung des Glucosids Phlorizin auf das Wirtswahlverhalten von Rhopalosiphum insertum (Walk.) und Aphis pomi De Geer (Homoptera: Aphididae). Z. Angew. Entomol. 68, 41–55. Łata, B., Trampczynska, A., Paczesna, J., 2009. Cultivar variation in apple peel and whole fruit phenolic composition. Sci. Hortic. (Amsterdam) 121, 176–181. Lattanzio, V., Di Venere, D., Linsalata, V., Bertolini, P., Ippolito, A., Salerno, M., 2001. Low temperature metabolism of apple phenolics and quiescence of Phlyctaena vagabunda. J. Agric. Food Chem. 49, 5817–5821.

C. Gosch et al. / Phytochemistry 71 (2010) 838–843 Lee, K.W., Kim, Y.J., Kim, D.O., Lee, H.J., Lee, C.Y., 2003. Major phenolics in apple and their contribution to the total antioxidant capacity. J. Agric. Food Chem. 51, 6516–6520. Lemoine, R., Delrot, S., 1987. Recognition of phlorizin by the carriers of sucrose and hexose in broad bean leaves. Physiol. Plantarum 69, 639–644. Lin, L.-Z., Mukhopadhyay, S., Robbins, R.J., Harnly, J.M., 2007. Identification and quantification of flavonoids of Mexican oregano (Lippia graveolens) by LC–DAD– ESI/MS analysis. J. Food Compost. Anal. 20, 361–369. Lockard, R.G., Schneider, G.W., 1981. Phenols and the dwarfing mechanism in apple rootstocks. Acta Horticulturae 120, 107–111. Lu, Y., Foo, Y., 1997. Identification and quantification of major polyphenols in apple pomace. Food Chem. 59, 187–194. Martin, G.C., Williams, M.W., 1967. Comparison of some biochemical constituents of EM IX and XVI bark. HortScience 2, 154. Mayr, U., Michalek, S., Treutter, D., Feucht, W., 1997. Phenolic compounds of apple and their relationship to scab resistance. J. Phytopathol. 145, 69–75. McPherson, J.K., Chou, C.-H., Muller, C.H., 1971. Allelopathic constituents of the chaparral shrub Adenostoma fasciculatum. Phytochemistry 10, 2925–2933. Mikulic-Petkovšek, M., Stampar, F., Veberic, R., 2007. Parameters of inner quality of the apple scab resistant and susceptible apple cultivars (Malus domestica Borkh.). Sci. Hortic. (Amsterdam) 114, 37–44. Mikulic-Petkovšek, M., Stampar, F., Veberic, R., 2008. Increased phenolic content in apple leaves infected with the apple scab pathogen. J. Plant Pathol. 90, 49–55. Mikulic-Petkovšek, M., Stampar, F., Veberic, R., 2009. Seasonal changes in phenolic compounds in the leaves of scab-resistant and susceptible apple cultivars. Can. J. Plant Sci. 89, 745–753. Mornau, M., 2004. Beteiligung von Phenylpropanoiden der Apfelrinde an der WirtParasit-Interaktion von Malus domestica (Borkh.) und Phytophthora cactorum (Lebert & Cohn) Schröter unter veränderter Ressourcenverfügbarkeit. Ph.D thesis. Technische Universität München, Wissenschaftszentrum Weihenstephan, Fachgebiet Obstbau. Muthuswamy, S., Rupasinghe, H.P.V., 2007. Fruit phenolics as natural antimicrobial agents: selective antimicrobial activity of catechin, chlorogenic acid and phloridzin. J. Food Agric. Environ. 5, 81–85. Natsume, H., Seto, H., Otake, N., 1982. Studies on apple canker disease. The necrotic toxins produced by Valsa ceratosperma. Agric. Biol. Chem. 46, 2101–2108. Nicholls, K.W., Bohm, B.A., 1984. The flavonoids of Lithophragma (Saxifragaceae). Can. J. Bot. 62, 1636–1639. Noveroske, R.L., Kuc´, J., Williams, E.B., 1964a. Oxidation of phloridzin and phloretin related to resistance of Malus to Venturia inaequalis. Phytopathology 54, 92–97. Noveroske, R.L., Williams, E.B., Kuc´, J., 1964b. ß-Glycosidase and phenoloxidase in apple leaves and their possible relation to resistance to Venturia inaequalis. Phytopathology 54, 98–103. Picinelli, A., Dapena, E., Mangas, J.J., 1995. Polyphenolic pattern in apple tree leaves in relation to scab resistance. A preliminary study. J. Agric. Food Chem. 43, 2273–2278. Pontais, I., Treutter, D., Paulin, J.P., Brisset, M.N., 2008. Erwinia amylovora modifies phenolic profiles of susceptible and resistant apple through its type III secretion system. Physiol. Plantarum 132, 262–271. Poyrazog˘lu, E., Gökmen, V., Artik, N., 2002. Organic acids and phenolic compounds in pomegranates (Punica granatum L.) grown in Turkey. J. Food Compost. Anal. 15, 567–575. Raa, J., 1968. Polyphenols and natural resistance of apple leaves against Venturia inaequalis. Neth. J. Plant Pathol. 74 (Suppl. 1), 37–45. Raa, J., Overeem, J.C., 1968. Transformation reactions of phloridzin in the presence of apple leaf enzymes. Phytochemistry 7, 721–731. Rezk, B.M., Haenen, G.R.M.M., van der Vijgh, W.J.F., Bast, A., 2002. The antioxidant activity of phloretin: the disclosure of a new antioxidant pharmacophore in flavonoids. Biochem. Biophys. Res. Commun. 295, 9–13. Römmelt, S., Fischer, T.C., Halbwirth, H., Peterek, S., Schlangen, K., Speakman, J.B., Treutter, D., Forkmann, G., Stich, K., 2003. Effect of dioxygenase inhibitors on the resistance-related flavonoid metabolism of apple and pears: chemical, biochemical and molecular biological aspects. Eur. J. Hort. Sci. 68, 129–136. Rui-Lin, N., Tanaka, T., Zhou, J., Tanaka, O., 1982. Phlorizin and trilobatin, sweet dihydrochalcone-glucosides from leaves of Lithocarpus litseifolius (Hance) Rehd. (Fagaceae). Agric. Biol. Chem. 46, 1933–1934.

843

Saxena, V.K., Nigam, S.S., Singh, R.B., 1976. Glycosidic principles from the leaves of Flemengia strobilifera. Planta Med. 29, 94–97. Schmitt, B., Schneider, B., 1999. Dihydrocinnamic acids are involved in the biosynthesis of phenylphenalenones in Anigozanthos preisii. Phytochemistry 52, 45–53. Schneider, H., Blaut, M., 2000. Anaerobic degradation of flavonoids by Eubacterium ramulus. Arch. Microbiol. 173, 71–75. Slatnar, A., Mikulic-Petkovšek, M., Halbwirth, H., Stampar, F., Stich, K., Veberic, R., in press. Enzyme activity of the phenylpropanoid pathway as a response to apple scab (Venturia inaequalis (Cooke) G. Wint.) infection. Ann. Appl. Biol. Sukhorukov, V.L., Kürschner, M., Dilsky, S., Lisec, T., Wagner, B., Schenk, W.A., Benz, R., Zimmermann, U., 2001. Phloretin-induced changes of lipophilic ion transport across the plasma membrane of mammalian cells. Biophys. J. 81, 1006–1013. Tanaka, T., Yamasaki, K., Kohda, H., Tanaka, O., Mahato, S.B., 1980. Dihydrochalconeglucosides as sweet principles of Symplocos ssp. Planta Med. 40, 81–83. Treutter, D., 2001. Biosynthesis of phenolic compounds and its regulation in apple. Plant Growth Regul. 34, 71–89. Tropf, S., Lanz, T., Rensing, S.A., Schröder, J., Schröder, G., 1994. Evidence that stilbene synthases have developed from chalcone synthases several times in the course of evolution. J. Mol. Evol. 38, 610–618. Tsao, R., Yang, R., Young, J.C., Zhu, H., 2003. Polyphenolic profiles in eight apple cultivars using high-performance liquid chromatography (HPLC). J. Agric. Food Chem. 51, 6347–6353. Turner, A., Chen, S.N., Joike, M.K., Pendland, S.L., Pauli, G.F., Farnsworth, N.R., 2005. Inhibition of uropathogenic Escherichia coli by cranberry juice: a new antiadherence assay. J. Agric. Food Chem. 53, 8940–8947. ´ Shea, P., Hadgraft, J., 2001. Effect of phloretin on the Valenta, C., Cladera, J., O percutaneous absorption of lignocaine across human skin. J. Pharm. Sci. 90, 485–492. Veitch, N.C., Grayer, R.J., 2006. Chalcones, dihydrochalcones and aurones. In: Andersen, Ø.M., Markham, K.R. (Eds.), Flavonoids: Chemistry, Biochemistry and Applications. Taylor & Francis Group, USA, pp. 1033–1071. Veitch, N.C., Grayer, R.J., 2008. Flavonoids and their glycosides, including anthocyanins. Nat. Prod. Rep. 25, 555–611. Venisse, J.-S., Gullner, G., Brisset, M.-N., 2001. Evidence for the involvement of an oxidative stress in the initiation of infection of pear by Erwinia amylovora. Plant Physiol. 125, 2164–2172. Vogt, T., Grimm, R., Strack, D., 1999. Cloning and expression of a cDNA encoding betanidin 5-O-glucosyltransferase, a betanidin- and flavonoid-specific enzyme with high homology to inducible glucosyltransferases from the Solanaceae. Plant J. 19, 509–519. Vrhovsek, U., Rigo, A., Tonon, D., Mattivi, F., 2004. Quantification of polyphenols in different apple varieties. J. Agric. Food Chem. 52, 6532–6538. Wald, B., Galensa, R., 1989. Nachweis von Fruchtsaftmanipulationen bei Apfel- und Birnensaft. Z. Lebensmitteluntersuchung und -forschung 188, 107–114. Watts, K.T., Lee, P.C., Schmidt-Dannert, C., 2004. Exploring recombinant flavonoid biosynthesis in metabolically engineered Escherichia coli. ChemBioChem 5, 500– 507. Werner, S.R., Morgan, J.A., 2009. Expression of a Dianthus flavonoid glucosyltransferase in Saccharomyces cerevisiae for whole-cell biocatalysis. J. Biotechnol. 142, 233–241. Williams, A.H., 1961. Dihydrochalcones of Malus species. J. Chem. Soc. 155, 4133– 4136. Williams, A.H., 1964. Dihydrochalcones: their occurrence and use as indicators in chemical plant taxonomy. Nature 202, 824–825. Williams, A.H., 1966. Dihydrochalcones. In: Swain (Ed.), Comparative Biochemistry. Academic Press, London, New York, pp. 297–307. Yamazaki, Y., Suh, D.Y., Sitthithaworn, W., Ishiguro, K., Kobayashi, Y., Shibuya, M., Ebizuka, Y., Sankawa, U., 2001. Diverse chalcone synthase superfamily enzymes from the most primitive vascular plant, Psilotum nudum. Planta 214, 75–84. Zhang, X.Z., Zhao, Y.B., Li, C.M., Chen, D.M., Wang, G.P., Chang, R.F., Shu, H.R., 2007. Potential polyphenol markers of phase change in apple (Malus domestica). J. Plant Physiol. 64, 574–580.