Trapping of Phenylacetaldehyde as a Key Mechanism Responsible for

Aug 15, 2008 - Qin Zhu , Zong-Ping Zheng , Ka-Wing Cheng , Jia-Jun Wu , Shuo Zhang , Yun Sang Tang , Kong-Hung Sze , Jie Chen , Feng Chen and Mingfu ...
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Chem. Res. Toxicol. 2008, 21, 2026–2034

Trapping of Phenylacetaldehyde as a Key Mechanism Responsible for Naringenin’s Inhibitory Activity in Mutagenic 2-Amino-1-methyl-6-phenylimidazo [4,5-b]Pyridine Formation Ka-Wing Cheng,† Chi Chun Wong,† Chi Kong Cho,‡ Ivan K. Chu,‡ Kong Hung Sze,‡ Clive Lo,† Feng Chen,† and Mingfu Wang*,† School of Biological Sciences and Department of Chemistry, The UniVersity of Hong Kong, Pokfulam Road, Hong Kong 123456, People’s Republic of China ReceiVed June 18, 2008

Chemical model reactions were carried out to investigate the mechanism of inhibition by a citrus flavonoid, naringenin, on the formation of 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), the most abundant mutagenic heterocyclic amine found in foods. GC-MS showed that naringenin dose dependently reduced the level of phenylacetaldehyde, a key intermediate on the pathway to the formation of PhIP. Subsequent LC-MS analyses of samples from a wide range of model systems consisting of PhIP precursors, including phenylalanine, glucose, and creatinine, suggested that naringenin scavenged phenylacetaldehyde via adduct formation. An isotope-labeling study showed that the postulated adducts contain fragment(s) of phenylalanine origin. Direct reaction employing phenylacetaldehyde and naringenin further confirmed the capability of naringenin to form adducts with phenylacetaldehyde, thus reducing its availability for PhIP formation. Two of the adducts were subsequently isolated and purified. Their structure was elucidated by one- and two-dimensional NMR spectroscopy as 8-C-(E-phenylethenyl)naringenin (1) and 6-C-(E-phenylethenyl)naringenin (2), respectively, suggesting that C-6 and C-8 are two of the active sites of naringenin in adduct formation. These two adducts were also identified from thermally processed beef models, highlighting phenylacetaldehyde trapping as a key mechanism of naringenin to inhibit PhIP formation. Introduction During cooking or processing of muscle foods, a group of potent mutagens are produced (1, 2). These are collectively called heterocyclic amines (HAs). Many HAs have demonstrated carcinogenic activity in rodents (3, 4), and one of them, 2-amino3-methylimidazo[4,5-f]quinoline, induced tumors in nonhuman primates (5). Recently, research focus has been on 2-amino-1methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), the most abundant mutagenic HA found in foods (6, 7). It has caused tumors in animal models (8), and exposure to PhIP has been shown to increase the risk of mammary carcinogenesis in the second generation, probably via a transplacental route or via its secretion into the milk (9). The liver, mammary gland, colon, and prostate have been identified as major target organs of carcinogenesis of HAs in animal studies (10-14). Very recent studies have reported the detection of PhIP-DNA adducts in these tissues in humans (15-17). In addition, epidemiological evidence has revealed a positive correlation between red meat consumption, PhIP intake, and cancer of these tissues (18-21). It is recommended that one minimizes dietary exposure to HAs, and a probable way would be through preventing their formation. In concert with the international effort to minimize HA-associated health risks, our group has performed a series of experiments to search for effective strategies to reduce HA formation. Notably, we found that phenolics such as procyanidins, flavanones, and flavan-3-ols significantly decreased the * To whom correspondence should be addressed. Tel: 852-22990338. Fax: 852-22990347. E-mail: [email protected]. † School of Biological Sciences. ‡ Department of Chemistry.

formation of HAs, including PhIP, in both model systems and real meat samples (22, 23). We suggested that these inhibitors would be relevant to mitigation of HA-associated health risks since they are all of natural origins, and more importantly, many extracts containing these inhibitors have a long consumption history and can be easily incorporated into the daily cuisine of many cultural groups. One of the inhibitors, naringenin, which widely occurs in citrus fruits and tomato (24), is of particular interest. Naringenin is a bioactive flavanone with diverse health effects on human diseases as demonstrated by its ability to inhibit the multiple stages of carcinogenesis (25-29), suppress inflammation (30), and confer a hypocholesterolemic effect in in vitro and animal studies (31, 32). It has also been suggested to possess hepatoprotective (33) and neuroprotective (34) activities. However, little has been done to characterize its roles in the Maillard reaction, and our group was the first to recognize its value as a potent inhibitor against the formation of mutagenic/ carcinogenic Maillard reaction products, especially PhIP, in both model systems and real meat samples (22). It is thus of interest to further examine the mechanism of action responsible for the inhibitory effect of naringenin on PhIP formation. For more than a decade, no significant breakthrough has been made in understanding the mechanism of action of phytochemicals on HA formation. So far, most previous mechanistic studies have ascribed the inhibitory effect of polyphenols on HA formation to their free radical scavenging activity (35, 36). Yet, there is still not a clear-cut mechanistic link between these two property parameters, and recent studies have reported a lack of significant positive correlations between radical scavenging capacity and HA inhibitory activity (22, 37). This implies that mechanism(s) other than antioxidant activity could be respon-

10.1021/tx800220h CCC: $40.75  2008 American Chemical Society Published on Web 08/15/2008

Trapping of Phenylacetaldehyde

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Table 1. Aqueous and Di(ethylene) Glycol Chemical Model Reactionsa reactant concentration (mM) reactant

A

Phe glucose creatinine naringenin [13C2]Phe PheAcet

20

B

5

C

D

E

F

G

20 10

20 10 20

20 10

20 10 20 5

20

5

H

I

J

10 10

5

5

5 10

5 20

a

Model reactions A-I were carried out in phosphate buffer (0.1 M, pH 7.0) and heated at 128 °C for 30 min; model reaction J was in di(ethylene) glycol and heated at 128 °C for 30 min.

sible for some of the negative influences of polyphenols on HA formation. In view of the fact that naringenin, a very weak (22, 38) radical scavenger, showed especially potent inhibitory activity against the formation of PhIP (22), the antioxidant activity was considered insufficient in itself to account for the inhibitory effect observed. Consequently, a shift in thinking was stimulated toward the reactive carbonyl-scavenging pathway since, apart from radicals, Maillard reaction products including reactive carbonyl species (RCS) arising from thermal and/or Strecker degradation reactions are also proposed as key intermediates in the formation of HAs (35, 39-41). The efficient conversion of reactive HA intermediate(s) into stable reaction products could therefore divert them from pathways that lead to the formation of mutagenic HAs. However, it should be noted that identification of such reaction products from food samples would remain a major technical challenge. The reasons are 3-fold: occurrence of the reaction products in trace quantities, an enormous number of concurring closely related reactions, and complexity of the matrix in which the reaction products are embraced. The aim of the present study was to unravel the inhibitory mechanism of naringenin on the formation of mutagenic PhIP through systematic chemical model investigations. The probable mechanism(s) of action, hypothetically, the scavenging of phenylacetaldehyde, which is a key intermediate RCS of PhIP (42), was subsequently elucidated via identification and structural characterization of the final reaction products formed. Finally, the relevance of the proposed mechanism of action to practical applications was assessed through identification of these adducts in food systems.

Experimental Procedures Solvents and Reagents. Phenylalanine, glucose, phenylacetaldehyde, di(ethylene) glycol, diethyl ether, and ammonium acetate were purchased from Sigma-Aldrich Co. (St. Louis, MO). Disodium hydrogen phosphate was from BDH Chemicals Ltd. (Poole England). Naringenin was purchased from SigmaAldrich Co. Fresh lean beef was purchased from a local beef vender. All solvents used were of analytical grade and were obtained from BDH laboratory Supplies (Poole, United Kingdom). The Reacti-Therm III heating module (model 18840) and the screw cap Tuf-Bond Teflon fitted glass reaction vials were purchased from Pierce (Rockford, IL). Model Maillard Reactions. The role of naringenin in PhIP formation was investigated in chemical model systems. Compositions of the models are listed in Table 1. The reaction mixtures were dissolved in phosphate buffer (0.1 M, pH 7.0, 20 mL) in crew cap Tuf-Bond Teflon fitted glass reaction vials (40 mL capacity) and heated in a Reacti-Therm III heating module at 128 °C for 30 min. The reaction mixtures were

subsequently cooled under room conditions for 1 h and then prepared for GC- or LC-MS analysis. For the investigation of the direct reaction between naringenin and phenylacetaldehyde, di(ethylene) glycol was used as the reaction medium because phenylacetaldehyde is insoluble in aqueous solvent systems. All other parameters were identical to those in the amino acid-glucose models. Sample Preparation. For analysis of volatile compounds, 15 out of 20 mL of selected reaction mixtures was extracted with diethyl ether (8 mL) (spiked with 0.02 µL of n-dodecane/8 mL, internal standard) under vortex for 1 min. The ether extract was dried with anhydrous sodium sulfate, filtered, and then subjected to GC-MS analysis. For the identification of adducts, reaction mixtures from the amino acid-glucose models were subjected to LC-MS analysis after a single syringe-driven filtering step without further sample processing. Samples from the phenylacetaldehyde models were diluted (10×) in methanol before subjecting them to LC-MS analysis. Gas Chromatography-Mass Spectrometry. The diethyl ether extracts were analyzed by an Agilent gas chromatograph (6890N) equipped with an autosampler (G2614A) and coupled to an Agilent mass spectrometer (5973N, EI mode). Separation was performed on a DB-Wax capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness). Analyses were carried out using the following parameters: One microliter of sample injected in splitless mode; inlet temperature, 200 °C; column flow, 1 mL/min (He); temperature program, 40 °C for 4 min, ramp at 5 to 230 °C and hold for 5 min; and MS temperature, 230 °C. Identification of phenylacetaldehyde was by comparing with mass spectrum and linear retention index of an authentic standard analyzed under identifical conditions; percent inhibition of phenylacetaldehyde formation ) TIC peak area of naringenin treatment/TIC peak area of control × 100. The peak area was adjusted relative to the peak area of the internal standard (ndodecane). Liquid Chromatography-Mass Spectrometry. All filtrates were analyzed on an LC-MS instrument equipped with an electrospray ionization (ESI) source interfaced to a Finnigan LCQ-Deca XP mass spectrometer. Liquid chromatography was run on an Agillent HPLC system with a degasser (G1379A), a quaternary pump (G1311A), a thermostatted autosampler (G1329A), and a diode array detector (G1315B). Separation of Maillard reaction products was carried out on an YMC-Pack ODS C-18 column (5 µm, 150 mm × 4.6 mm). The mobile phase composed of 10 mM ammonium acetate aqueous solution (solvent A) and acetonitrile (B) of the following gradients: 0 min, 5% B:95% A; 35 min, 80% B:20% A; 37 min, 5% B:95% A; and 50 min, 5% B:95% A. Effluent from the UV detector was split 4:1 with one part (200 µL/min) directed to the MS for spectrometric analysis and the remaining to waste. The MS conditions were as follows: negative ion mode; spray voltage, 3.5 kV; scan range, 120-1000 Da; and capillary temperature, 300 °C. Isolation, Purification, and Structural Elucidation of Polyphenol-Phenylacetaldehyde Adducts. Naringenin and phenylacetaldehyde in a molar ratio of 1:5 were first dissolved in di(ethylene) glycol to a final concentration of 125 and 25 mM, respectively. The total reaction time was 4 h. At the 4 h time point, the reaction vials were inserted into an ice-water mixture to stop further heat treatment. To facilitate the subsequent isolation process, liquid-liquid extraction was used both for removing di(ethylene) glycol and for obtaining a concentrated sample. The extraction process was monitored

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using HPLC-PDA. Ethyl acetate-hexane (2:1, v/v) was determined to be the solvent system of choice for extraction. It was found that the addition of water in an amount equal to the volume of the sample dramatically improved the extraction efficiency to over 90%. Hence, the system was composed of sample-H2O-ethyl acetate-hexane in a ratio of 1:1:2:1 (v/v). The solvent mixture was vortex-extracted for 2 min and then centrifuged for 10 min (8000g). After that, the clear supernatant was pooled and concentrated on a rotary evaporator under vacuum. The extract thus obtained was redissolved in methanol and then loaded onto a Sephadex LH-20 column (40 cm × 4 cm). Elution was performed with methanol, and eluate was collected using an automatic fraction collector. The profile of the fractions was checked on a Shimadzu HPLC system with a separation module (LC-20AT), an autosampler (SIL-20A), a degasser (DGU-20A3) and a photodiode array detector (SPDM20A). Similar fractions were combined. This open-column chromatographic process eventually led to two fractions with enhanced content of the target adducts and much reduced level of interfering compounds. These two fractions were finally separated by semipreparative HPLC on a Waters HPLC system to obtain the two adducts in high purity (>95% by HPLC-PDA). The structure of the adducts was determined by one- (1D) and two-dimensional (2D) NMR spectroscopy on a 600 MHz spectrometer (Bruker, AVANCE 600). Ground Beef Model Reaction. Ground beef models were designed to simulate heat treatment under low and high moisture contents, respectively. For the former purpose, lean beef was homogenized to a paste. Forty grams of the resulting paste was then weighed into a screw cap Tuf-Bond Teflon fitted glass reaction vial. A small hole was made in the middle of the cap septum. Duplicate samples were prepared for both the control and the naringenin treatment. Naringenin (80 mg) was first dissolved in di(ethylene) glycol (20 mL), and the test solution (10 mL) was then thoroughly mixed with the beef. For the control, di(ethylene) glycol (10 mL) was added. Heat treatment was conducted on a Reacti-Therm III heating module. The heater was preheated to 160 °C, and the duration of heating was 40 min. After heating, the two samples for each treatment were combined and homogenized in MilliQ water (100 mL) for 2 min. The cloudy suspensions were centrifuged (5 min, 6000g). The supernatant (∼100 mL) was transferred to a Nalgene bottle and then extracted with ethyl acetate (150 mL, 3×). The extracts were combined and evaporated to a minimum volume on a rotary evaporator at 38 °C under vacuum. Four milliliters of methanol was added to the resulting concentrated extract, which was then sonicated and filtered prior to subjecting it to LC-MS analysis. To simulate cooking under high-moisture conditions, 300 g of lean beef was homogenized in phosphate buffer (pH 7, 400 mL) to a paste. Duplicate samples were taken for each treatment, each with 25 g of beef paste added with 40 mg of naringenin, which was predissolved in di(ethylene) glycol (3 mL). Subsequent steps were the same as those applied for the low-moisture model, except that heat treatment was done at 125 °C for 1 h and extraction with ethyl acetate was repeated five times. The mobile phase for LC-MS analysis was composed of 10 mM ammonium acetate aqueous solution (solvent A) and acetonitrile (B) of the following gradients: 0 min, 10% B:90% A; 40 min, 80% B:20% A; 42 min, 10% B:90% A; and 52 min, 10% B:90% A. Other parameters were identical to those applied for analysis of chemical model reaction samples, except that SIM at m/z 373 and CRM at m/z 253 were used for MS analysis.

Cheng et al.

Spectral Data. Compound 1. 1H NMR (600 MHz, CD3OD, 25 °C, TMS): δ ) 7.45 (d, J ) 16.8 Hz, 1H; H-2′′), 7.41 (d, J ) 8.4 Hz, 2H; H-2′ and H-6′), 7.31 (m, 2H; H-4′′ and H-8′′), 7.29 (d, J ) 16.8 Hz, 1H; H-1′′), 7.25 (m, 2H; H-5′′ and H-7′′), 7.13 (t, J ) 7.3 Hz, 1H; H-6′′), 6.87 (d, J ) 8.4 Hz, 2H; H-3′ and H-5′), 6.10 (s, 1H; H-6), 5.49 (dd, J ) 3.0 Hz and J ) 13.0 Hz, 1H; H-2), 3.19 (dd, J ) 17.0 Hz and J ) 13.0 Hz, 1H; H-3a), 2.80 ppm (dd, J ) 17.0 Hz and J ) 3.0 Hz, 1H; H-3b). 13C NMR (600 MHz, CD3OD, 25 °C, TMS): δ ) 198.4 (s, C-4), 166.5 (s, C-7), 164.1 (s, C-5), 162.3 (s, C-9), 159.1 (s, C-4′), 140.6 (s, C-3′′), 131.4 (d, C-1′′), 131.1 (s, C-1′), 129.6 (d, C-2′ and C-6′), 129.0 (d, C-5′′ and C-7′′), 127.8 (d, C-6′′), 126.9 (d, C-4′′ and C-8′′), 119.6 (d, C-2′′), 116.5 (d, C-3′ and C-5′), 106.8 (s, C-8), 103.5 (s, C-10), 96.1 (d, C-6), 80.7 (d, C-2), 43.7 ppm (t, C-3). Compound 2. 1H NMR (600 MHz, CD3OD, 25 °C, TMS): δ ) 7.58 (d, J ) 16.8 Hz, 1H; H-2′′), 7.46 (d, J ) 7.6 Hz, 2H; H-4′′ and H-8′′), 7.35 (d, J ) 16.8 Hz, 1H; H-1′′), 7.33 (d, J ) 8.4 Hz, 1H; H-2′ and H-6′), 7.30 (m, 2H; H-5′′ and H-7′′), 7.17 (t, J ) 7.2 Hz, 1H; H-6′′), 6.82 (d, J ) 8.4 Hz, 2H; H-3′ and H-5′), 6.10 (s, 1H; H-8), 5.37 (dd, J ) 3.0 Hz and J ) 13.0 Hz, 1H; H-2), 3.17 (dd, J ) 17.0 Hz and J ) 13.0 Hz, 1H; H-3a), 2.74 ppm (dd, J ) 17.0 Hz and J ) 3.0 Hz, 1H; H-3b). 13 C NMR (600 MHz, CD3OD, 25 °C, TMS): δ ) 198.1 (s, C-4), 166.5 (s, C-7), 163.6 (s, C-5), 163.2 (s, C-9), 159.1 (s, C-4′), 140.8 (s, C-3′′), 131.3 (d, C-1′′), 131.0 (s, C-1′), 129.6 (d, C-2′ and C-6′), 129.1 (d, C-5′′ and C-7′′), 127.7 (d, C-6′′), 127.0 (d, C-4′′ and C-8′′), 119.6 (d, C-2′′), 116.3 (d, C-3′ and C-5′), 107.3 (s, C-6), 103.1 (s, C-10), 95.9 (s, C-8), 80.5 (d, C-2), 44.1 ppm (t, C-3).

Results and Discussion Effect of Naringenin on Maillard Products Generation in PhIP-Producing Models. In our previous study (22), we found that naringenin, a flavonoid, which widely occurs in citrus fruits, had strong inhibitory activity against the formation of HAs. The effect was particularly prominent with regard to PhIP formation. Previously free radical scavenging activities of flavonoids were considered of key importance for their inhibitory effects on the formation of mutagenic HAs. The free radical scavenging activity of flavonoids is ascribed to hydrogen- and electron-donating capacities of their hydroxyl substituents (38, 43). From a kinetic point of view, it is suggested that flavonoids with a single 4′-OH substitution in the B ring tend to have low reactivity with radicals, but the coexistence of a 3-OH in the C ring would enhance such reaction probably by increasing the stability of the flavonoid radical (38). As a flavanone, naringenin is thus considered a rather weak radical scavenger based on both structural consideration (38) and various in vitro radical scavenging assays (22, 38). Therefore, the possibility of alternative mechanism(s) leading to naringenin’s potent negative influence on PhIP formation was explored. With the reaction conditions and sample preparation method adopted in the current study, phenylacetaldehyde was identified by GC-MS as the chief volatile compound formed. No other volatile related to the formation of PhIP such as phenylethylamine (42, 44) was found. Quantitative analysis showed that the addition of naringenin effectively reduced the concentration of phenylacetaldehyde dose dependently in PhIP-producing model systems (Figure 1). The level of reduction of phenylacetaldehyde agreed well with that found for PhIP in our previous study (22). This suggested that inhibition of phenylacetaldehyde formation from phenylalanine and/or scavenging/trapping of

Trapping of Phenylacetaldehyde

Chem. Res. Toxicol., Vol. 21, No. 10, 2008 2029 Table 2. Major LC-MS Analytes [(M - H)]- from Model G model composition naringenin + phenylalanine

Figure 1. Effect of different concentrations of naringenin on the inhibition of the formation of phenylacetaldehyde in an aqueous model system containing phenylalanine, glucose, and creatinine at pH 7 and 128 °C for 30 min. *% inhibition ) TIC peak area of naringenin treatment/TIC peak area of control × 100; the peak area was adjusted by internal standard (n-dodecane).

Figure 2. MS/MS chromatograms showing relative abundance of selected analytes (m/z 373) in chemical models subjected to heat treatment at 128 °C for 30 (A), 120 (B), 240 (C), and 360 (D) min, respectively.

phenylacetaldehyde after it was formed may be the dominant mechanism responsible for naringenin’s inhibition of PhIP formation. Identification of Reaction Products Formed from Naringenin and Phenylalanine Degradation Products in Chemical Model Systems. To examine the roles of naringenin

m/z [(M - H)]- value and proposed molecular composition 271: naringenin 373: naringenin + phenylacetaldehyde - H2O 391: naringenin + phenylacetaldehyde 645: 2 × naringenin + phenylacetaldehyde - H2O

in phenylalanine-Maillard models, LC-ESIMSn analyses were performed to identify reaction products that may arise from reactions between naringenin and phenylalanine or its degradation products. Reaction mixtures were only processed by a single syringe-driven filtering step prior to LC-MS analysis. LC-MS results indicated that this sample preparation procedure was adequate for achieving our analytical goals. Close examination and comparison of the LC-UV and MS TIC chromatograms revealed that models containing phenylalanine plus naringenin with or without glucose or creatinine (models E-G) all contained reaction products with molecular weight corresponding to adducts formed between naringenin and phenylacetaldehyde (Table 2). That is, the predicted molecular weight of these adducts was larger than that of naringenin but not equal to that of naringenin dimer or oligomer or phenylacetaldehyde oligomer. These adducts were formed in significant quantities as evidenced by their appearance as distinct peaks in the TIC chromatograms of samples from the above models. These characteristic reaction products were not detected in models containing phenylalanine or naringenin alone (models A and B). They were also absent in models containing phenylalanine and glucose (model C), phenylalanine, glucose, and creatinine (model D) or naringenin and glucose (model H). In other words, they were generated only in model systems where phenylalanine was present together with naringenin. Most of the potential adducts have predicted molecular weight of 374 (m/z [M H]- 373), corresponding to an electrophilic substitution product between naringenin and phenylacetaldehyde followed by elimination of a water molecule to form phenylethenyl naringenin. In HA formation, creatinine contributes to the aminomethylimidazo part of the HA molecule (45). It has been proposed to undergo aldol addition with phenylacetaldehyde in the formation of PhIP (42). Surprisingly, creatinine did not demonstrate an obvious impact on the generation of the above-mentioned compounds. It is worth pointing out that the initial molar concentration of creatinine in these models was much higher (4:1) than that of naringenin. Hence, from a kinetic point of view, it was likely that under such reaction conditions, reactivity of naringenin toward phenylacetaldehyde was much higher than that of creatinine, whose reaction with phenylacetaldehyde would thus be interrupted. Alternatively, the depletion of this RCS intermediate would subsequently reduce its availability for PhIP formation. The above findings tend to support the central role of phenylalanine/phenylacetaldehyde in the formation of adducts with naringenin. Subsequently, an isotope-labeling ([13C2]Phe) study (model I) was performed to acquire more solid evidence. Each of the adducts mentioned above displayed two isotopomers with equal ion intensity but differed by 1 Da in their molecular mass. This suggested that both the labeled and the unlabeled phenylalanine fragments were equally incorporated into the respective reaction products and that phenylalanine gave rise to reactive fragment(s) that could form adducts with naringenin.

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Figure 3. Structure of proposed naringenin-phenylacetaldehyde adducts (1 and 2) showing significant HMBC correlations.

Figure 4. Postulated pathways for naringenin’s inhibitory activity in PhIP formation.

Considering the predicted molecular weight of the above reaction products, the most important phenylalanine-derived fragment taking part in the proposed adduct formation reaction was likely phenylacetaldehyde. To further explore the capability of naringenin in direct reaction with phenylacetaldehyde, a model reaction (model H) employing these two postulated reactants was performed. Chromatograms of analytes of m/z 373 showed only two major ion peaks whose retention time and UV-absorption spectra completely matched with two of the adducts identified from models containing phenylalanine in place of phenylacetaldehyde. Collisioninduced dissociation (CID) of these two m/z 373 adduct ions gave m/z 253 and 185 fragment ions, in agreement with Retro-Diels-Alder degradation of the C ring, followed by further losses of CO and CCO fragments. This fragmentation behavior was also found for the corresponding adducts in phenylalanine-containing models. In contrast, in addition to reaction products of m/z 373, several other adducts of m/z 391 and 645 were also generated from model reactions starting with the parent amino acid, phenylalanine (Table 2). m/z 391 may result from the combination between a naringenin molecule and a phenylacetaldehyde molecule, while m/z 645 may be composed of two naringenin molecules and a phenylacetaldehyde with elimination of a water molecule. Apparently, these two reaction pathways did not proceed to detectable extents in the phenylacetaldehyde-containing model with di(ethylene) glycol as the reaction medium where reactions leading to adducts of molecular weights of 374 (m/z 373) dominated.

Characterization of the Structure of Naringenin-Phenylacetaldehyde Adducts. To better understand the chemistry of the proposed reaction of adduct formation, two of the reaction products were isolated and purified from the naringenin-phenylacetaldehyde model, and their structure was characterized by 1D and 2D NMR spectroscopy. The reaction time was identified as an important factor for optimizing the yield of the target adducts. Among the time frames investigated (15, 30, 60, 120, 240, 360, and 720 min), 240 min was determined to be optimal both in terms of the yield of the adducts formed and the level of interfering compounds generated in the systems (Figure 2). After a series of solvent extraction and chromatographic processes, two compounds corresponding to the two peaks displayed in Figure 2 with retention times of 26.3 (compound 1) and 29.6 min (compound 2) were successfully purified. Compound 1 was a yellow solid, and compound 2 was a golden yellow solid. Their 1H and 13C NMR spectra showed the same pattern of carbon and hydrogen atoms. Comparison with reported data suggested the presence of a naringenin substructure (46). The 13C NMR spectrum of 1 and 2 established the presence of 21 sp2 carbons, which matched well with a structure derived from substitution of a phenylethylene on naringenin (Figure 4). DEPT spectra revealed the presence of a single methylene group in both 1 and 2. The appearance of dd splitting at δH 2.80 and δH 3.19 in 1H spectrum of 1 supported that the CH2 group at the 3-position of naringenin substructure remained intact. The same splitting patterns were also observed for 2 but at slightly different chemical shifts

Trapping of Phenylacetaldehyde

Figure 5. SIM (m/z 373) chromatograms of thermally processed beef samples (A) and compound 1 (B) and compound 2 (C), respectively.

(δH 2.74 and δH 3.17, respectively). In their 2D 1H-1H COSY spectra, signals at these two chemical shifts in 1 and 2 showed cross-peaks to the signal at δH 5.49 (1) and 5.37 (2), respectively. These data therefore largely precluded the possibility of a substitution at the C-3 position. The occurrence of an aromatic singlet at δH 6.10 (1H) in the 1H spectrum of both compounds suggested substitution at C-6 or C-8. Literature data reported very close chemical shifts between C-6 and C-8 of naringenin with C-8 at a slightly lower field than C-6 (46). 13C NMR spectra showed that C-8 (1) or C-6 (2) was shifted by about 10 ppm downfield, consistent with the substitution of a downfield-shifting group at the corresponding carbon atom. Further evidence for the assignment of H-6 and H-8 was provided by HMBC experiments (Figure 3). For 1, the HMBC spectrum revealed three-bond couplings of H-1′′ to C-7 and C-9 and that of H-2′′ to C-8, while for 2, couplings were observed for H-1′′ to C-5 and C-7 and H-2′′ to C-6. Two-bond coupling was also observed for H-1′′ to C-8 and H-1′′ to C-6 in the HMBC spectrum of 1 and 2, respectively. On the basis of the 3J (16.8 Hz) between H-1′′ and H-2′′, both compounds were suggested to have a trans configuration. The two adducts are therefore established as 8-C-(E-phenylethenyl)naringenin (1) and 6-C-(E-phenylethenyl)naringenin (2), respectively. It is noted that in 1, H-2′′ can be associated with the oxygen atom at position 1 via hydrogen bonding, giving the molecule extra stability. On the basis of the elucidated structures, reaction between naringenin and phenylacetaldehyde is proposed to proceed via an electrophilic substitution mechanism at C-6 or C-8 on naringenin. A schematic illustration of the proposed reactions is given in Figure 4. This is in good agreement with previous reports, which identified C-6 and C-8 of the A ring of flavonoids like epicatechin and epigallocatechin gallate as active sites of RCS trapping (47, 48). Theoretically, simultaneous substitution could have occurred at both C-6 and C-8 on naringenin. Nevertheless, no analyte with a mass corresponding to such a molecular composition was identified,

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implying that this reaction was less favored as compared with monosubstitution at either C-6 or C-8 alone. Identification of Adducts Formed from Naringenin and Phenylalanine Degradation Products from Ground Beef Models. Although chemical model systems composed of pure PhIP precursors are considered good surrogates for studying different aspects of this potent mutagen (41, 49), to test the relevance of the aforementioned mechanism of action (phenylacetaldehyde scavenging) to applications in real food systems, two ground beef models were developed to simulate heat processing under low and high moisture content, respectively. Identification of adducts of interest from the complex matrix, however, represents a rigorous challenge. A series of selective solvent extraction and concentration steps led to clean samples that have gotten rid of major impurities that would otherwise hinder proper chromatographic and spectroscopic analyses. In addition, as our preliminary LC-MS analysis showed poor spectral features for analytes of interest, LC conditions applied for analyses of samples prepared from chemical model reactions were slightly modified as described in the Experimental Procedures. SIM and CRM were used for identification of target analytes, and the two purified adducts (1 and 2) were used as reference standards. It was found that beef samples treated with naringenin under the two different heating conditions both contained analytes whose LC behavior and MS spectral characteristics completely matched with those of the two standard compounds (Figure 5). Figure 6 illustrates a proposed fragmentation pathway leading to the generation of characteristic fragment ions. These pointed out the occurrence of the aforeproposed adduct formation (electrophilic substitution) reactions in the beef models employed in the current study and emphasized the importance of the reaction pathways leading to the formation of these two adducts among many other possible analogues identified in chemical model systems. Moreover, the beef models developed in the present study together with the simple sample preparation methods may facilitate further research on flavonoids-RCS interaction in food matrices. Certain flavonoids, especially green tea catechins, have been shown to quench RCS such as formaldehyde and acetaldehyde in wine (50, 51) and glucose-derived C2-C6 fragments in glucose-glycine models (48). These RCS have beenproposedtoparticipateintheformationofimidazole-quinoline and imidazo-quinoxaline type HAs (40, 41). Their scavenging may likely be part of the inhibitory mechanism of some flavoniods in the formation of the above HAs. The exact role of such mechanism of action, however, is yet to be verified considering the fact that model reactions used in these previous studies were mostly not satisfactory surrogates for HA investigation in terms of both the precursors composition and the precursors ratio (47, 48, 52). With the wide range of model system reactions, the current study has been, to the best of our knowledge, the first to report scavenging of amino acid-derived RCS as a key mechanism of action against the formation of mutagenic HA (i.e., PhIP).

Conclusion In conclusion, naringenin reduced the content of phenylacetaldehyde in PhIP-producing models dose dependently. An isotope-labeling study and LC-MS analyses have offered strong evidence that naringenin is capable of forming many adducts with phenylalanine or phenylalanine degradation

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Figure 6. CRM MS3 fragmentation of compounds 1 and 2.

products. The scavenging of phenylacetaldehyde, a key PhIP intermediate, may thus lead to significant inhibition of PhIP formation. Two of such adducts (1 and 2) have been purified through a series of reaction optimization, solvent extraction, and column chromatographic processes. Characterization of their structure revealed C-6 and C-8 of the A ring as two of the active sites of naringenin in forming adducts with phenylacetaldehyde. The availability of these two pure adducts has facilitated further identification of naringenin adducts in food systems (i.e., ground beef), which represent a much more complex sample matrix. Although free radical scavenging and/or metal chelation have been proposed to modulate HA formation by phenolic antioxidants, information about strong inhibitory activity of week antioxidants is currently lacking. The results of the present study may help fill the gap in our understanding of the lack of significant correlations between antioxidant and HA-inhibitory activity of polyphenols. A new chapter in HA chemistry has thus been started, and more studies in this area are definitely called for. Given the reactivity of naringenin toward phenylacetal-

dehyde, findings of the present study could have implications beyond HA-associated health risk alone. As is now widely recognized, RCS play a central role in a number of health disorders, in particular diabetes complications. Recently, safety issues concerning RCS generated in foods during various processing procedures have also gained increasing attention. Theoretically, phenylacetaldehyde is likely less reactive than many other known RCS, such as formaldehyde and acetaldehyde, and dicarbonyls, such as glyoxal and methyl glyoxal. Hence, naringenin may be anticipated to possess scavenging activity against a wide spectrum of RCS, and this merits further investigation. Although naringenin mainly occurs in conjugated forms, especially glycosides in citrus fruits and some vegetables, the conjugates could be easily converted into naringenin via treatment (e.g., acid or enzymatic hydrolysis) of crude extracts of its natural sources. Moreover, crude natural extracts could be fractionated to obtain naringnin-rich extracts, which could thus be incorporated into diets and daily cuisine to benefit the general public.

Trapping of Phenylacetaldehyde

Acknowledgment. We thank the HKSAR Research Grand Council for financial support (Project HKU 7778/07M to M.W.).

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