Apple Polyphenols, Phloretin and Phloridzin: New Trapping Agents of

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Apple Polyphenols, Phloretin and Phloridzin: New Trapping Agents of Reactive Dicarbonyl Species Xi Shao,†,‡,§ Naisheng Bai,| Kan He,| Chi-Tang Ho,§ Chung S. Yang,† and Shengmin Sang*,†,‡,⊥ Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers UniVersity, 164 Frelinghuysen Road, Piscataway, NJ 08854-8020; Human Nutrition research program, Julius L. Chambers Biomedical/ Biotechnology Research Institute, North Carolina Central UniVersity, North Carolina Research Campus, 500 Laureate Way, Kannapolis, NC 28081; Department of Food Science, Rutgers UniVersity, 65 Dudley Road, New Brunswick, NJ 08901-8520; Naturex, 375 Huyler Street, South Hackensack, NJ 07606; and Department of Chemistry, North Carolina Central UniVersity, 1801 FayetteVille Street, Durham, NC 27707 ReceiVed June 20, 2008

Reactive dicarbonyl species, such as methylglyoxal (MGO) and glyoxal (GO), have received extensive attention recently due to their high reactivity and ability to form advanced glycation end products (AGEs) with biological substances such as proteins, phospholipids, and DNA. In the present study, we found that both phloretin and its glucoside, phloridzin, the major bioactive apple polyphenols, could efficiently trap reactive MGO or GO to form mono- and di-MGO or GO adducts under physiological conditions (pH 7.4, 37 °C). More than 80% MGO was trapped within 10 min, and 68% GO was trapped within 24 h by phloretin. Phloridzin also had strong trapping efficiency by quenching more than 70% MGO and 60% GO within 24 h. The glucosylation of the hydroxyl group at position 2 could significantly slow down the trapping rate and the formation of MGO or GO adducts. The products formed from phloretin (or phloridzin) and MGO (or GO), combined at different ratios, were analyzed using LC/MS. We successfully purified the major mono-MGO adduct of phloridzin and found that it was a mixture of tautomers based on the one- and two-dimensional NMR spectra. Our LC/MS and NMR data showed that positions 3 and 5 of the phloretin or phloridzin A ring were the major active sites for trapping reactive dicarbonyl species. We also found that phloretin was more reactive than lysine and arginine in terms of trapping reactive dicarbonyl species, MGO or GO. Our results suggest that dietary flavonoids that have the same A ring structure as phloretin may have the potential to trap reactive dicarbonyl species and therefore inhibit the formation of AGEs. Introduction Increasing evidence has identified the formation of advanced glycation end products (AGEs)1 as a major pathogenic link between hyperglycemia and diabetes-related complications (1). It has been well-documented that AGEs progressively accumulate in the tissues and organs to develop chronic complications of diabetes mellitus, such as retinopathy, nephropathy, neuropathy, and macrovascular disease (2-5). R-Oxoaldehydes such as methylglyoxal (MGO) and glyoxal (GO), the reactive dicarbonyl intermediates generated during the nonenzymatic glycation between reducing sugars and amino groups of proteins, lipids, and DNA, are precursors of AGEs and exert direct toxicity to cells and tissues. It has been shown that dicarbonyl compounds are more reactive in the glycation reaction than reducing sugars and play an important role for cross-linking proteins in the glycation process (3, 6, 7). Plasma levels of MGO and GO are reportedly elevated in diabetes, dialysis, and chronic * To whom correspondence should be addressed. E-mail: ssang@ nccu.edu. † Ernest Mario School of Pharmacy, Rutgers University. ‡ Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University. § Department of Food Science, Rutgers University. | Naturex. ⊥ Department of Chemistry, North Carolina Central University. 1 Abbreviations: AGEs, advanced glycation end products; DB, 1,2diaminobenzene; EGCG, (-)-epigallocatechin-3-gallate; GO, glyoxal; MGO, methylglyoxal; PZMGO, mono-MGO adduct of phloridzin.

kidney disease patients. Trapping of reactive dicarbonyl compounds by several pharmaceutical agents, such as aminoguanidine, metformin, and pyridoxamine, has been shown to be a useful strategy for inhibiting the formation of AGEs and thus inhibiting or delaying diabetic complications (8-13). Many studies have shown that dietary flavonoids, which are widely distributed in fruits, vegetables, grains, and beverages, are strong antioxidants and may prevent diabetes and its complications. However, the mechanisms by which dietary flavonoids prevent the development of diabetic complications are unclear. It has been observed in vitro that dietary flavonoids may inhibit the formation of AGEs by trapping reactive dicarbonyl compounds. A recent study found that dietary flavonoids, luteolin, rutin, (-)-epigallocatechin-3-gallate (EGCG), and quercetin, exhibited significant inhibitory effects on MGOmediated AGEs formation by 82.2, 77.7, 69.1, and 65.3%, respectively, at a concentration of 100 µM (14). A more recent study found that flavonoids containing vicinyl dihydroxyl groups, such as quercetin and myricetin, could significantly decrease the level of GO during the auto-oxidation of glucose in vitro (15). These authors also found that rutin and its metabolite, quercetin, could effectively inhibit GO-derived AGEs formation during glycation of collagen I by glucose (15). Although the authors did not discuss the mechanism involved, it appears that flavonoids could trap reactive dicarbonyl species (MGO and GO) and therefore inhibited the formation of AGEs. To further investigate whether dietary flavonoids can directly

10.1021/tx800227v CCC: $40.75  2008 American Chemical Society Published on Web 09/06/2008

Phloretin and Phloridzin Trap Dicarbonyl Species

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Figure 1. Chemical structures of phloretin, phloridzin, and the mono-MGO conjugated phloridzin adducts, PZMGO-A and PZMGO-B.

trap reactive dicarbonyl species, we studied the trapping efficacy of EGCG, the major bioactive green tea polyphenol, under physiological conditions (pH 7.4, 37 °C) (16). We found that EGCG could efficiently trap reactive dicarbonyl compounds (MGO or GO) to form mono- and di- MGO or GO adducts. Our LC/MS and NMR data showed that positions 6 and 8 of the EGCG A ring were the major active sites for trapping reactive dicarbonyl compounds. To investigate whether this finding applies to other type of flavonoids and to gain more understanding of the structure-activity relationship, we further studied the trapping efficacy of another type of important dietary flavonoid, chalcone, using phloretin and phloridzin as the representative compounds. Phloretin is a flavonoid found exclusively in apples and in apple-derived products where it is present as free and its glucosidic form, phloridzin (phloretin 2′-O-glucose) (17-19) (Figure 1). It was reported that phloridzin could be hydrolyzed to phloretin by lactase-phloridzin hydrolase in the small intestine (20). A more recent study showed that urinary phloretin could be used as the exposure biomarker of apple consumption (21). In addition, several studies have shown that both phloretin and phloridzin have antidiabetic effects by inhibiting intestinal glucose absorption (22, 23). In this paper, we describe our findings on the trapping of reactive dicarbonyl compounds, MGO and GO, by the major bioactive apple polyphenols, phloretin and phloridzin, under physiological conditions. Adducts formed with phloretin or phloridzin and MGO or GO at different ratios were analyzed using LC/MS/MS. The monoMGO adduct of phloridzin (PZMGO) was purified through column chromatography and identified based on the one- (1D) and two-dimensional (2D) NMR spectra.

Materials and Methods Materials. Phloretin, phloridzin, MGO (40% in water), GO (40% in water), quinoxaline, 2-methylquinoxaline, 1,2-diaminobenzene (DB), CD3OD, RP C-18 silica gel, Sephadex LH20 gel, and TLC plates (250 µm thickness, 2-25 µm particle size) were purchased from Sigma (St. Louis, MO). HPLC-grade solvents and other reagents were obtained from VWR Scientific (South Plainfield, NJ). HPLC-grade water was prepared using a Millipore Milli-Q purification system (Bedford, MA).

Kinetic Study of the Trapping of MGO or GO by Phloretin or Phloridzin under Physiological Conditions. MGO (0.33 mM) or GO (0.33 mM) was incubated with 1 mM phloretin or phloridzin in a pH 7.4 phosphate buffer solution (100 mM) at 37 °C and shaken at 40 rpm for 0, 10, 30, 60, 120, 240, 480, or 1440 min. Then, to each triplicated vial at each time point, 1 µL of acetic acid was added to stop the reaction, and 100 mM DB was added next to derivatize the remaining MGO or GO using our previous method (16). Trapping of MGO by Phloretin under Acidic Conditions. MGO (0.33 mM) was incubated with 1 mM phloretin in a pH 4.0 phosphate buffer solution (100 mM) at 37 °C and shaken at 40 rpm for 0, 10, 30, 60, 120, 240, 480, or 1440 min. Then, to each triplicated vial at each time point, 1 µL of acetic acid was added to stop the reaction, and 100 mM DB was added next to derivatize the remaining MGO using the method detailed above. HPLC Analysis. The levels of methylquinoxaline and quinoxaline were analyzed using our previous HPLC method with slight modification (16). In brief, the HPLC system consisted of a Waters 717 refrigerated autosampler, a HITACHI L-6200 Inteligent pump, and a Waters 490E programmable multiwavelength UV-vis detector. A Supelcosil C18 reversedphase column (150 mm × 4.6 mm inner diameter; Supelco Co., Bellefonte, PA) was used with a flow rate of 1 mL/min. The binary mobile phase system consisted of 5% aqueous acetonitrile with 0.2% acetic acid as A and 95% aqueous acetonitrile with 0.2% acetic acid as B. The column was eluted with a binary gradient system: 100 to 65% A from 0 to 5 min; 65 to 60% A from 5 to 15 min; 60 to 0% A from 15 to 20 min; then 100% A from 21 to 30 min. The injection volume was 50 µL for each sample. The wavelength of the UV detector was set at 280 nm for methylquinoxaline and 313 nm for quinoxaline with ∼100 ng/mL as the limit of detection and 1 µg/mL as the limit of quantification for both methylquinoxaline and quinoxaline. LC/MS Analysis. A Finnigan LC/ESI-MS System equipped with a Surveyou MS pump, a Surveyou refrigerated autosampler, and a LTQ linear ion trap mass detector (ThermoFinigan, San Jose, CA) incorporated with an electrospray ionization (ESI) interface was used. A Gemini C18 column (50 mm × 2.0 mm i.d., 3 µm, Phenomenex) was used for the analysis of the reaction products with a flow rate of 0.2 mL/min. The binary mobile

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phase system consisted of 5% aqueous methanol with 0.2% acetic acid as A and 95% aqueous methanol with 0.2% acetic acid as B. The column was eluted with isocratic phase A for 5 min followed by the gradient progress: 100 to 70% A from 5 to 20 min; 70 to 0% A from 20 to 40 min; then 100% A from 41 min for 10 min. The injection volume was 10 µL for each sample. The column temperature was maintained at 20 °C. The LC elute was introduced into the ESI interface. The negative ion polarity mode was set for ESI ion source with the voltage on the ESI interface maintained at approximately 5 kV. Nitrogen gas was used as the sheath gas at a flow rate of 30 arb units and the auxiliary gas at 5 arb units, respectively. The structural information of phloretin, phloridzin, and the major MGO or GO adducts was obtained by tandem mass spectrometry (MS/ MS) through collision-induced dissociation (CID) with a relative collision energy setting of 35%. Studying the Formation of MGO or GO Adducts of Phloretin or Phloridzin Using LC/MS. Phloretin or phloridzin (1.0 mM) was incubated with different concentrations of MGO or GO (0.33, 1.0, 3.0, or 100.0 mM) in a pH 7.4 phosphate buffer solution at 37 °C for 60 min, respectively. Then, 10 µL samples were taken and transferred to vials containing 190 µL of a solution consisting of 0.2% ascorbic acid and 0.05% EDTA to stabilize phloretin or phloridzin and related MGO or GO adducts. These samples were immediately analyzed or stored at -80 °C before analyzing by LC/MS. NMR Analysis. 1H (400 MHz), 13C (100 MHz), and all 2D NMR spectra were acquired on a Varian Inova 400 instrument (Varian Inc., Palo Alto, CA) at 25 °C. The DEPT-135 13C NMR spectra were obtained on a Bruker DRX 500 NMR instrument at 25 °C using a Nalorac 3 mm 1H-13C dual probe. Compounds were analyzed in CD3OD in a 3 mm i.d. tube. 1H-13C HMQC (heteronuclear multiple quantum correlation) and HMBC (heteronuclear multiple band correlation) experiments were performed as described previously (24). The 1H and 13C chemical shift assignments were referenced internally to TMS at 0 ppm. Purification of the Major PZMGO. Phloridzin (0.5 g, 1.06 mmol) and MGO (4.132 g, 57.34 mmol) were dissolved in 300 mL of phosphate buffer (100 mmol/L, pH 7.4) and then kept at 37 °C for 60 min. After extraction with ethyl acetate and the evaporation of the solvent in vacuum, the residue was loaded into a Sephadex LH-20 column eluted with ethanol to obtain mono-MGO conjugated phloridzin (130 mg). 1H and 13C NMR data of phloridzin and mono-MGO conjugated phloridzin are listed in Table 1.

Results Trapping of MGO or GO by Phloretin and Phloridzin under Physiological Conditions. We found that both phloretin and phloridzin could effectively trap MGO or GO under physiological conditions. More than 80% MGO was trapped within 10 min, and 68% GO was trapped within 24 h by phloretin (Figure 2). Phloridzin also had strong trapping efficiency by quenching more than 70% MGO and 60% GO within 24 h (Figure 2). In addition, we found that phloretin could not trap reactive MGO under acidic conditions (pH 4) (Figure 2). Studying the Formation of MGO and GO Adducts of Phloretin by LC/MS. The reaction mixtures of phloretin with MGO or GO under four different ratios (3:1, 1:1, 1:3, and 1:100) were analyzed by LC/MS (Figures 3 and 4). The structural information of these products was obtained using LC/MS/MS analysis under selective ion monitoring (SIM) mode (Figures 5 and 6).

Shao et al. Table 1. δH (400 MHz) and δC (100 MHz) NMR Spectra Data of Phloridzin and Mono-MGO Conjugated Phloridzin (PZMGO) (CD3OD) (δ in ppm, J in Hz) phloridzin position 1 2 3 4 5 6 7 8 9 10-A 10-B 11-A 11-B 12-A 12-B 1′ 2′ 3′ 4′ 5′ 6′ 1′′ 2′′ 3′′ 4′′ 5′′ 6′′

δH

6.15 d, J 2.0 5.93 d, J 2.0 3.42 m 2.84 t, J 7.6

PZMGO δC 102.0 s 167.5 s 98.3 d 165.9 s 95.4 d 162.3 s 206.5 s 46.9 t 30.8 t

δH

6.24 s

3.42 m 2.83 t, J 7.2 5.32 s 3.91 m 2.17 s 1.14 d, 6.1

7.03 d, J 8.8 6.66 d, J 8.8 6.66 7.03 5.10 3.44 3.42 3.40 3.42 3.87 3.68

d, J 8.8 d, J 8.8 d, J 6.8 m m m m brd, J 12.0 dd, J 5.2, 12.0

133.8 s 130.4 d 116.0 d 156.3 s 116.0 d 130.4 d 102.0 d 74.6 d 78.4 d 71.0 d 78.3 d 62.3 t

7.02 d, J 8.4 6.64 d, J 8.4 6.64 7.02 5.10 3.44 3.42 3.40 3.42 3.90 3.68

d, J 8.4 d, J 8.4 d, J 6.8 m m m m brd, J 12.4 dd, J 5.6, 12.4

δC 108.6 162.2 94.3 d 164.8 106.7 s 165.9 206.9 47.0 30.7 71.5 d 206.2 s 206.5 s 64.7 d 25.5 t 24.7 t 133.7 130.4 116.0 156.3 116.0 130.4 102.0 74.6 78.4 71.0 78.4 62.4

Figure 2. Trapping of MGO and GO by phloretin and phloridzin in phosphate buffer (pH 7.4 for all of the compounds and pH 4.0 for trapping of MGO by phloretin only, 37 °C). MGO (0.33 mM) or GO (0.33 mM) was incubated with 1 mM phloretin and phloridzin in pH 7.4 (or pH 4.0 for trapping of MGO by phloretin) phosphate buffer solutions at 37 °C for 10, 30, 60, 120, 240, 480, and 1440 min, respectively.

After 1 h of incubation of phloretin with MGO (3:1 ratio), two major new peaks appeared in the LC chromatogram (Figure 3). One had the molecular ion m/z 345 ([M - H]-; RT, 21.97 min), and the other had the molecular ion 417 ([M - H]-; RT, 20.51 min) (Figures 3 and 5A). The peak at 21.97 min had the fragment ion m/z 273 [M - 72 - H]-, which lost one MGO (m/z 72) molecule, and the MS/MS spectrum of this daughter ion (MS3 273/345) was identical to the MS/MS spectrum of the standard phloretin (MS2 273) (Figure 5A). All of these features indicated that this product was the mono-MGO conjugate of phloretin. The MS/MS spectrum of the most abundant daughter ion m/z 327 [M - 18]- (MS3 327/345) of the mono-MGO adduct had the typical loss of the B ring unit (m/z 106) (Figure 7) to generate the fragment ion 221 [M - 18 - 106]- indicating that the MGO conjugated at the A ring of phloretin (Figure 5A). The peak at 20.51 min had the fragment ion m/z 345 [M - 72 - H]-, which lost one MGO (m/z 72) molecule, and the MS/MS spectrum of this daughter ion (MS3

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Figure 3. LC chromatograms of phloretin after incubation with different ratios of (A) MGO (3:1, 1:1, 1:3, and 1:100) and (B) GO (3:1, 1:1, 1:3, and 1:100) for 60 min, respectively.

Figure 4. LC chromatograms of phloridzin after incubation with different ratios of (A) MGO (3:1, 1:1, 1:3, and 1:100) and (B) GO (3:1, 1:1, 1:3, and 1:100) for 60 min, respectively.

345/417) was identical to the MS/MS spectrum of the monoMGO adduct (MS2 345) (Figure 5A,B). Therefore, the peak at 20.51 min was identified as the di-MGO adduct of phloretin. The MS3 spectrum of the most abundant daughter ion of this

product (m/z 399 [M - 18]-) also had the fragment ion that lost the typical B ring unit (m/z 293 [M - 18 - 106]-) (Figure 7), indicating that both MGO units conjugated at the A ring, one at position 3 and the other at position 5.

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Figure 5. Tandem MS/MS spectra of (A) mono-MGO adducts of phloretin and phloretin standard and (B) di-MGO adducts of phloretin.

Figure 6. Tandem MS/MS spectra of (A) mono-GO and (B) di-GO adducts of phloretin.

When phloretin and MGO at a 1:1 ratio were incubated at 37 °C for 1 h, it was observed that the level of di-MGO adduct was higher than that from the reaction at a 3:1 ratio, both monoand di-MGO adducts were the major products, and the amount of unreacted phloretin dramatically decreased (Figure 3A). At a 1:3 ratio, we found that the amount of mono-MGO adduct decreased and di-MGO adduct became the dominant product

(Figure 3A). Increasing the amount of MGO to 100-fold of the amount of phloretin did not change the product profile in comparison to the 1:3 ratio (phloretin: MGO), and there was no detectable tri-MGO adduct in this sample. In the reaction between phloretin and GO, we found that mono-GO adduct was the major product at ratios 3:1, 1:1, and 1:3. Di-MGO adducts were not detectable in the samples from

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Figure 7. Formation pathway of the major fragments of phloretin.

Figure 8. Tandem MS/MS spectra of (A) mono-MGO and (B) di-MGO adducts of phloridzin.

3:1 and 1:1 reactions, and only tiny amounts of di-MGO adducts were detected from the 1:3 reaction mixture after 1 h of incubation at 37 °C (Figure 3B). At a 1:100 ratio, the amount of mono-GO adduct dramatically decreased and di-GO adducts became the dominant products (Figure 3B). Similar to the identification of mono-MGO adduct of phloretin, we found that the mono-GO adduct (m/z 331 [M - H]-) had the fragment ion m/z 273 [M - 58 - H]-, which lost one GO (m/z 58) molecule, and the MS/MS spectrum of this daughter ion (MS3 273/331) was identical to the MS/MS spectrum of the standard phloretin (MS2 273) (Figure 6A). At a 1:100 ratio, there were two di-GO adducts formed (m/z 389 [M - H]-). They had identical MS2 spectra (data not shown), and both of them had the fragment ion m/z 331 [M -58 - H]-, which lost one GO (m/z 58) molecule, and the MS/MS spectrum of this daughter ion (MS3 331/389) was identical to the MS/MS spectrum of the mono-GO adduct (MS2 331) (Figure 6A,B). In addition, the MS3 spectrum of the most abundant daughter ion (m/z 313 [M - 18]-) of the mono-GO adduct (m/z 331 [M - H]-) had the fragment ion m/z 207 [M - 18 - 106]- that lost the typical B ring unit (Figures 6A and 7), and the MS/MS spectrum of the most abundant daughter ion m/z 371 [M - 18]- of the diGO adduct (MS3 371/389) also had the typical loss of the B ring unit to generate the fragment ion 207 [M - 18 - 106]-

(Figures 6B and 7). Therefore, positions 3 and 5 at the A ring of phloretin were also the major active sites for trapping GO. Studying the Formation of MGO and GO Adducts of Phloridzin by LC/MS. Phloridzin is the monoglucoside of phloretin (Figure 1). To understand whether glucosylation affects the trapping efficacy, we conducted similar studies using phloridzin under four different ratios (3:1, 1:1, 1:3, and 1:100). We found that the trapping efficacies of phloridzin for both MGO and GO were much lower than those of phloretin, especially for short-term reactions (Figure 2). Mono-MGO adduct was the major product in the reaction between phloridzin and MGO. This product was barely detectable at a 3:1 ratio after 1 h of incubation of phloridzin and MGO (Figure 4A). The amount of this product was slightly increased at 1:1 and 1:3 ratios and became the dominant product only at a 1:100 ratio (Figure 4A). Di-MGO adduct was not detectable at 3:1, 1:1, and 3:1 ratios, and only tiny amounts of di-MGO adduct were formed at a 1:100 ratio (Figure 4A). The MS2 spectrum of the PZMGO (m/z 507 [M - H]-) had the fragment ion m/z 345 [M - 162 - H]-, which lost one glucose unit, and the MS/MS spectrum of this daughter ion (MS3 345/507) was identical to the MS/MS spectrum of the mono-MGO adduct of phloretin (MS2 345) (Figures 5A and 8A), indicating that this peak was the PZMGO. The MS2 spectrum of the di-MGO

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Figure 9. Tandem MS/MS spectra of mono-GO adduct of phloridzin.

adduct of phloridzin (m/z 579 [M - H]-) had the fragment ion m/z 507 [M - 58 - H]-, which lost one MGO (m/z 72) molecule, and the MS/MS spectrum of this daughter ion (MS3 507/579) was identical to the MS/MS spectrum of the monoMGO adduct (MS2 507) (Figure 8A,B). In the reaction between phloridzin and GO, we found that phloridzin could not trap GO after 1 h of incubation at 3:1, 1:1, and 1:3 ratios, and no product was detectable. At a 1:100 ratio, two mono-GO adducts (m/z 493 [M - H]-) were formed. They had identical MS/MS spectra, and both of them had the daughter ion 331 [M - 162 - H]-, which lost one glucose unit, and the MS/MS spectrum of this daughter ion (MS3 331/ 493) was identical to the MS/MS spectrum of the mono-GO adduct of phloretin (MS2 331) (Figures 5B and 9), indicating that these two products were the mono-GO adducts of phloridzin. Purification and Structure Elucidation of the Major PZMGO. The PZMGO was purified from the reaction between phloridzin and MGO in a 1:50 ratio using a Sephadex LH-20 column eluted with ethanol. Its structure was established by analyzing the 1H, 13C, DEPT-135, and 2D NMR (1H-1H COSY, HMQC, and HMBC) as well as the MS/MS spectra. PZMGO was assigned the molecular formula C24H28O12 based on negative-ion ESI-MS (- at m/z 507) and the 13C and DEPT-135 NMR data. The molecular weight of PZMGO was 72 (MW of MGO, 72) mass units higher than that of phloridzin, indicating that PZMGO was a PZMGO, which was further confirmed by the observation of the fragment ion m/z 435 [M - H - 72]-. Its 1H and 13C NMR spectra showed very similar patterns to the spectra of phloridzin (Table 1) (25). The 1H NMR spectrum of PZMGO showed two distinct sets of aromatic ring proton signals: an AA′BB′ system for four protons, two at δ 6.64 and two at δ 7.02 (J ) 8.4 Hz), indicating a para-substituted benzene ring, which was similar to those of phloridzin; and one singlet signal for one proton (δ 6.24 s), instead of the two mutually coupled doublets (2H) at δ 5.93 and δ 6.15 (J ) 2.0 Hz) in the 1H NMR spectrum of phloridzin, suggesting MGO conjugated with phloridzin at position 3 or 5 of the A ring. However, there were two sets of proton signals for the MGO

group (δH 5.32 s, 1H, and 2.17 s, 3H; δH 3.91 m, 1H, and 1.14 d, J ) 6.1 Hz, 3H) instead of one set as expected in the 1H NMR spectrum of PZMGO. Its 13C spectra also showed two sets of carbon signals for the MGO group (δC 25.5 t, 71.5 d, and 206.2 s; and 24.7 t, 64.7 d, and 206.5 s). All of these features indicate that this compound is a mixture of two tautomers (Figure 1). In comparison with the 13C spectrum of phloridzin, PZMGO had a quaternary carbon at δC 105.3 in its 13C spectrum in lieu of an unsubstituted aromatic carbon from the A ring of phloridzin. In addition, the MS/MS spectrum of its daughter ion m/z 327 [M - 180]- (MS3 327/507) had the typical loss of the B ring unit (m/z 106) (Figure 7) to generate the fragment ion m/z 221 [M - 180 - 106]- indicating that the MGO conjugated at the C-3 or C-5 position of the A ring (Figure 8A). All of these spectral features supported the presence of a MGO group in PZMGO at either the C-3 or the C-5 position of the A -ring. The HMBC spectrum showed correlation between δC 162.2 and H-1′′ (δH 5.10 d, J ) 6.8 Hz) of the glucose group indicating that δC 162.2 was C-2 of the A ring. In addition, the only proton on A ring (δH 6.24 s) showed correlation with δC 162.2, suggesting that this proton was H-3 and the MGO group was located at the C-5 position of the A ring. Thus, the structure of PZMGO was identified as shown in Figure 1.

Discussion In this study, we found that phloretin could rapidly trap MGO under neutral or alkaline conditions. Similar to the phenomenon that we observed for EGCG, the trapping efficacy of phloretin for GO was much lower than that for MGO (16). This was because the hydrated monomer, dimer, and trimer were the main forms of GO in aqueous solution, and the trapping reaction between phloretin and GO was slowed down by the transformation from the hydrated monomer, dimer, and trimer to free GO (26). The trapping rates of phloridzin for both MGO and GO were much slower than those of phloretin, indicating the hydroxyl group at position 2 of the A ring played significant role on the trapping of reactive dicarbonyl species. In addition,

Phloretin and Phloridzin Trap Dicarbonyl Species

Figure 10. Formation pathway of mono- and di-MGO adducts of dietary flavonoids under neutral or base conditions.

our data showed that both phloretin and phloridzin were more reactive than lysine, phloretin was also more active than arginine, and phloridzin was as effective as arginine in terms of trapping MGO or GO (16). All of these results indicated that both phloretin and phloridzin had the potential to compete with lysine and arginine in vivo and therefore prevent the formation of AGEs. To understand the mechanism by which phloretin and phloridzin could trap reactive dicarbonyl species, we studied the formation of MGO or GO adducts of phloretin and phloridzin using LC/MS. Our results indicated that positions 3 and 5 of the A ring of phloretin and phloridzin were the major active sites for trapping both MGO and GO. Glucosylation of the hydroxyl group at position 2 could significantly slow down the trapping rate and the formation of MGO or GO adducts. To further confirm that the A ring was the major active site, we purified the major product from the reaction between phloridzin and MGO at a 1:50 mol ratio. Its structure was identified as the PZMGO with the MGO conjugated at position 5 of the A ring. Therefore, our results clearly indicated that the two unsubstituted carbons at the A ring, positions 3 and 5, were the major active sites for chalcone type of compounds, which have the same A ring as phloretin, to trap reactive dicarbonyl species. This was consistent with our previous finding that the two unsubstituted carbons at the A ring of EGCG (positions 6 and 8 for flavanol type of compounds) were the major active sites to trap reactive dicarbonyl species and form mono- and di-MGO or GO adducts. On the basis of our observations on two different types of flavonoids, chalcone and flavanol, we predict that flavonoids that have the same A ring structure as EGCG or phloretin can efficiently trap reactive dicarbonyl species to form mono- and di-MGO or GO adducts, and glycosylation can significantly decrease the trapping efficacy. Therefore, we propose a mechanism of how flavonoids trap MGO to form mono- and di-MGO adducts (Figure 10). The slightly alkaline pH can increase the nucleophilicity of the unsubstituted carbons at the A ring and facilitate the addition of MGO at these two positions to form mono- and di-MGO adducts. It is worthwhile to further study whether this observation applies to simple phenols or polymers of flavonoids, such as proanthocyanidins. Many studies in humans, animal models, and cell lines suggest potential health benefits from the consumption of apple, including prevention of cancer, heart disease, and diabetes

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(27, 28). It has been reported that whole apple and cereal bran consumption significantly decreased plasma glucose level in noninsulin-dependent diabetics (29). Partial replacement of bread with the same unit of apple at breakfast could reduce the glucose response in type 2 diabetic subjects. A more recent study showed that women consuming g1 apple/day showed a significant 28% reduced risk of type 2 diabetes as compared with those who consumed no apples (30). Apples are one of the major sources for polyphenols in human diets, which contain a variety of polyphenols, such as catechin, epicatechin (EC), procyanidins, quercetin glycosides, chlorogenic acid, phloretin, and phloridzin (27, 28). On the basis of our results and other reports (14, 31), polyphenols in apple may have the capability to trap reactive dicarbonyl species, inhibit the formation of AGEs, and therefore prevent the development of diabetic complications. In summary, we found that phloretin was a very efficient trapping agent of reactive dicarbonyl species under in vitro conditions (pH 7.4, 37 °C). The glucosylation of the hydroxyl group at position 2 significantly decreased the trapping efficiency. Both phloretin and its glucoside, phloridzin, could react with MGO or GO to form related adducts, and the major active sites were positions 3 and 5 of the A ring. We also found that phloretin was more reactive than lysine and arginine in terms of trapping reactive dicarbonyl species, MGO, or GO. The structural information of the MGO or GO adducts of phloretin and phloridzin obtained from LC/MS and NMR analysis will help us further understand the mechanisms by which dietary flavonoids can trap reactive dicarbonyl species and whether they can trap reactive dicarbonyl species through the same mechanism and therefore inhibit the formation of AGEs in vivo. Acknowledgment. We gratefully acknowledge the financial support by the Apple Products Research and Education Council and the U.S. Apple Association.

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