ARTICLE pubs.acs.org/crt
Genistein Inhibits Advanced Glycation End Product Formation by Trapping Methylglyoxal Lishuang Lv,†,‡ Xi Shao,†,|| Huadong Chen,† Chi-Tang Ho,§ and Shengmin Sang*,† †
Center for Excellence in Post-Harvest Technologies, North Carolina Agricultural and Technical State University, North Carolina Research Campus, 500 Laureate Way, Kannapolis, North Carolina 28081, United States ‡ Department of Food Science and Technology, Ginling College, Nanjing Normal University, 122# Ninghai Road, Nanjing, 210097, P. R. China § Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, New Jersey 08901, United States ABSTRACT:
Methylglyoxal (MGO) is a highly reactive endogenous metabolite derived from several nonenzymatic and enzymatic reactions, and identified as a well-known precursor of advanced glycation end products (AGEs). In the present study, genistein, a naturally occurring isoflavone derived from soy products, demonstrated significant trapping effects of MGO and consequently formed monoand di-MGO adducts under physiological conditions (pH 7.4, 37 °C). More than 80.0% of MGO was trapped within 4 h, and the trapping efficiency could be up to 97.7% at 24 h. The reaction adducts formed from genistein and MGO under different ratios were analyzed using LC/MS. We also successfully purified and identified the major mono- and di-MGO conjugated adducts of genistein. The NMR data showed that positions 6 and 8 of the A ring of genistein were the major active sites for trapping MGO. We further demonstrated that genistein could effectively inhibit the formation of AGEs in the human serum albumin (HSA)-MGO assay. Two mono-MGO adducts and one di-MGO adduct of genistein were detected in this assay using LC/MS. The di-MGO adduct of genistein became the dominant reaction product during prolonged incubation. Results from this study, as well as our previous findings on (-)-epigallocatechin 3-gallate (EGCG), phloridzin and phloretin, indicate that dietary flavonoids that have the same A ring structure as genistein, EGCG, phloridzin, and phloretin may have the potential to inhibit the formation of AGEs by trapping reactive dicarbonyl species.
’ INTRODUCTION Recent progress in the understanding of diabetes has confirmed that advanced glycation end products (AGEs) play a significant pathogenic role in the development of diabetic complications. In particular, diseases such as atherosclerosis, retinopathy, nephropathy, and cataract formation are suggested to be either caused or promoted by AGEs.1 In patients with diabetes mellitus, increased concentrations of glycated proteins have been found in various tissues.2,3 Because of the abilities of altering enzymatic activity and immunogenicity, lowering the ligand binding, and modifying protein half-life, accumulation of AGEs in tissues have been implicated in several diabetic complications.4 Among the many reactive dicarbonyl compounds and AGEs precursors, methylglyoxal (MGO) is believed to contribute significantly to intracellular AGE formation due not only to its high reactivity but also to its multiple origins under in vivo conditions. The degradation of triosephosphates,5 peroxidation of lipids, and side reactions from glucose-mediated glycation are considered as three major resources associated with the formation of MGO.6,7 Moreover, deamination of aminoacetone by semicarbazide-sensir 2011 American Chemical Society
tive amine oxidase (SSAO) also leads to the production of MGO.8 MGO could readily react with arginine, lysine, and cysteine residues of proteins, modify proteins, including bovine serum albumin, collagen, ribonuclease A, cytosolic aspartate aminotransferase,9-11 and damage DNA to exhibit cellular toxicity.12 In patients with both insulin-dependent and noninsulin-dependent diabetes mellitus, the concentration of MGO was found to be increased 2-6-fold.13 Several pharmacological reagents, such as aminoguanidine,14,15 tenilsetam,16,17 carnosine,18 metformin,19,20 and pyridoxamine,21,22 have been investigated for inhibiting the formation of AGEs and the development of diabetic complications by trapping reactive dicarbonyl species. However, all of these pharmaceutical agents have serious side effects. For example, the nucleophilic hydrazine compound aminoguanidine failed in phase III clinical trials because of the high toxicity in diabetic patients.23 Therefore, it is critical to develop effective and safe agents to protect diabetics from complications. We have previously demonstrated that dietary flavonoids, such as (-)-epigallocatechin 3-gallate (EGCG) from tea and Received: December 27, 2010 Published: February 23, 2011 579
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Figure 1. Chemical structures of genistein, mono-, and di-MGO adducts of genistein, and significant HMBC (HfC) correlations of mono- and di-MGO adducts of genistein.
phloridzin and phloretin from apples, could efficiently trap reactive dicarbonyl compounds to form mono- and di-MGO adducts.24,25 It has been reported that genistein protects MGO-induced oxidative DNA damage and cell injury in human mononuclear cells.26 Whether dietary flavonoids can inhibit MGO-induced AGEs formation, oxidative DNA damage, and cell injury through this trapping mechanism is still unclear. In the present study, we investigate whether genistein (5,7,40 -trihydroxyisoflavone, Figure1), one of the major dietary isoflavones present at a relatively high level (ca. 3 mg/g) in soybeans and plant-derived foods,27 can trap MGO and therefore inhibit the formation of AGEs.
Kinetic Study of the Trapping of MGO under Physiological Conditions. MGO (0.33 mM) was incubated with 1 mM genistein in a
pH 7.4 phosphate buffer solution (100 mM) at 37 °C and shaken at 40 rpm speed for 0, 10, 30, 60, 120, 240, 480, and 1440 min. Then, 200 μL of reacted mixtures were collected at each time point, 1 μL of acetic acid was added to stop the reaction, and 100 mM DB was used for the derivatization of the remaining MGO according to our previous method.24 HPLC Analysis. The levels of methylquinoxaline were analyzed using our previous HPLC method.24 Briefly, a Waters ACQUITY UPLC system coupled with a PDA detector (Waters, Milford, MA) was used. A Waters BEH RP-C18 column (50 mm 2.1 mm inner diameter, 1.7 μm) was used with a flow rate of 0.5 mL/min. For binary gradient elution, mobile phase A (90% water, 10% methanol, and 0.2% acetic acid) and B (100% methanol with 0.2% acetic acid) were used. The mobile phase began with 100% A, followed by progressive linear increases in B to 20% at 1.5 min, 35% at 2.5 min, and 100% at 4.5 min. Then, the mobile phase was maintained at 100% B until 5.5 min and reequilibrated to 100% A from 5.6 to 7.0 min. The injection volume was 5 μL for each sample. The wavelength of the UV detector was set at 280 nm with 100 ng/mL as the limit of detection and 1 μg/mL as the limit of quantification for methylquinoxaline.
’ MATERIALS AND METHODS Materials. Geinstein, methylglyoxal (40% in water), 1,2-diaminobenzene (DB), methylquinoxaline, human serum albumin (HSA), DMSO, and preparative TLC plates (2000 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). 580
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Table 1. δH (700 MHz) and δC (175 MHz) NMR Spectra Data of Genistein and the Mono-MGO Adduct of Genistein (MM-1) (DMSO, δ in ppm and J in Hz) genistein δH
MM-1A
tandem mass spectrometry (MS/MS) through collision-induced dissociation (CID) with a relative collision energy setting of 35%. Data acquisition was performed with Xcalibur version 2.0 (Thermo Electron, San Jose, CA, USA).
Purification of the Major MGO Adducts of Genistein.
MM-1B
δC
δH
δC
δH
δC
154.4 d
8.28 s
153.5 d
8.28 s
153.5 d
Genistein (97.3 mg, 2 mM) and MGO (216 mg, 200 mM) were dissolved in phosphate buffer (100 mM, pH 7.4) and then kept at 37 °C for 24 h. The reaction mixture was loaded into a reverse phase C18 open column eluted with 40% aqueous methanol to remove the remaining genistein. The mixture of the MGO adducts was then applied to preparative TLC plates and eluted with chloroform/methanol (15:1, v/ v) to obtain the mono-MGO adduct (MM-1, 12 mg) and di- MGO adduct (20 mg). NMR Analysis. 1H (700 MHz), 13C (175 MHz), 1H-13C HMQC (heteronuclear multiple quantum correlation)/ and HMBC (heteronuclear multiple band correlation) spectra were acquired on a Bruker 700 MHz instrument. All compounds were analyzed in DMSO. 1H and 13C NMR data of genistein and mono- and di-MGO conjugated genistein are listed in Tables 1 and 2.
1 2
8.32 s
3
121.7 s
121.8 s
121.8 s
4
180.7 s
180.2 s
180.2 s
5 6
162.4 s 99.4 d
162.3 s 103.4 d
6.20 d,
6.12 s
6.12 s
162.3 s 103.4 d
J 2.1 7
164.8 s
167.0 s
167.0 s
94.1 d
99.5 s
99.5 s
9
158.0 s
155.6 s
155.6 s
10
104.9 s
106.3 s
106.3 s
70.2 d 207.1 s
a
3.50 m
nd 62.5 d
25.9 q
1.05 d,
19.0 q
rent time points (0, 2, 4, 8, 48, and 144 h) from the genistein treated HSA-MGO system and the purified mono- and di-MGO conjugated genistein were analyzed using the LC/MS method described above.
122.5 s
’ RESULTS
8
6.38 d, J 2.1
11 12
5.17 s
13
2.17 s
J 7.80 10 20
122.7 s 7.38 d,
130.6 d
J 14.3 0
3
6.83 d,
115.5 d
6.83 d,
60
7.38 d, J 14.3
7.38 d,
115.5 d
6.81 d,
6.81 d,
130.6 d
7.38 d,
115.5 d
suggested that genistein could effectively trap MGO under physiological conditions (Figure 2A). More than 80.0% of MGO was trapped within 4 h, and the trapping efficiency could be up to 97.7% at 24 h. In addition, genistein significantly inhibited the formation of AGEs in the HSA-MGO assay. The formation of AGEs was inhibited by almost 50% at 408 h (Figure 2B). Studying the Formation of MGO Adducts of Genistein by LC/MS. The reaction mixtures of genistein with MGO under four different ratios (3:1, 1:1, 1:3 and 1:10) were analyzed by LC/MS (Figure 3). The structural information of these products was obtained using LC/MS/MS analysis under selective ion monitoring (SIM) mode (Figure 4). After 24 h of incubation of genistein with MGO, three new major peaks (RT 14.06, 15.97, and 16.74 min) appeared in the LC chromatogram for all ratios (Figure 3). Two of these peaks (15.97 and 16.74 min) had the same molecular ion m/z 343 [M þ H]þ, which is 72 mass units higher than that of genistein (m/z 270) indicating that both of them are the mono-MGO adducts of genistein (molecular weight of MGO is m/z 72) (Figure 4A and B). Both peaks had similar MS/MS fragments (Figure 4A and B). In addition, the peak at 16.74 min had the fragment ion m/z 271 [M - 72 þ H]þ, suggesting it lost one MGO (m/z 72) molecule, further indicating that this product was the mono-MGO adduct of genistein. The third new peak was observed at the RT 14.07 min with the molecular ion m/z 415 [M þ H]þ, which was 72 mass units higher than that of the mono-MGO adduct of genistein, suggesting that this peak is the di-MGO adduct of genistein. This was further confirmed by the observation of the fragment ion m/z 343 [M - 72 þ H]þ and by the MS/MS spectrum of this daughter ion (MS3 343/415) which was almost identical to the MS/MS spectra of the mono-MGO adducts of genistein (Figure 4). When genistein and MGO at a 1:1 ratio were incubated at 37 °C for 24 h, we observed that the levels of mono- and di-MGO
157.8 s
115.5 d
6.81 d,
130.6 d
7.38 d,
J 8.50 J 8.50
Kinetic Study of the Trapping of MGO and the Inhibitory Effects on the Formation of AGEs by Genistein. Our results
J 8.50 157.8 s
115.5 d
130.6 d
J 8.50
J 8.50
J 11.4
a
6.81 d,
157.9 s
50
130.6 d
J 8.50
J 11.4 40
122.5 s 7.38 d,
Determining the Formation of MGO Adducts of Genistein in the HSA-MGO System Using LC/MS. Samples collected at diffe-
115.5 d
J 8.50 130.6 d
J 8.50
nd: not detected.
Kinetic Study of the Inhibitory Effects on the Formation of AGEs by Genistein. HSA (1.4 mg/mL) was incubated with MGO
(500 μM) in the presence or absence of genistein (1.5 mM) in PBS buffer, pH 7.4, at 37 °C. Streptomycin and penicillin mixed solution (0.3 mL) was added before incubation to prevent the growth of bacteria. The reaction mixture (400 μL) was collected and frozen at different time points (0, 2, 4, 8, 48, 144, 240, and 408 h). AGEs levels were quantified using fluorescence at an excitation/emission wavelength of 370/440 nm, which is characteristic of AGEs. LC/MS Analysis. LC/MS analysis was carried out with a ThermoFinnigan Spectra System which consisted of an Accela high speed MS pump, an Accela refrigerated autosampler, and an LCQ Fleet ion trap mass detector (Thermo Electron, San Jose, CA, USA) incorporated with electrospray ionization (ESI) interfaces. A 50 2.0 mm i.d., 3 μm Gemini C18 column (Phenomenex, Torrance, CA, USA) was used for separation at a flow rate of 0.2 mL/min. The column was eluted with 100% solvent A (5% aqueous methanol with 0.2% acetic acid) for 5 min, followed by linear increases in B (95% aqueous methanol with 0.2% acetic acid) to 50% from 5 to 10 min, to 65% from 10 to 25 min, to 100% from 25 to 40 min, and then with 100% B from 40 to 45 min. The column was then re-equilibrated with 100% A for 5 min. The LC eluent was introduced into the ESI interface. The positive ion polarity mode was set for the 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. The structural information of genistein and the major MGO adducts was obtained by 581
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Table 2. δH (700 MHz) and δC (175 MHz) NMR Spectra Data of the Di-MGO Adduct of Genistein (DM) (DMSO, δ in ppm and J in Hz) DM-A δH
DM-B δC
δH
DM-C δC
DM-D
δH
δC
δH
δC
1 2
8.00 s
151.60 d
8.00 s
151.60 d
8.00 s
151.60 d
8.00 s
151.60 d
3 4
121.50 s 178.61 s
121.50 s 178.61 s
121.50 s 178.61 s
121.50 s 178.61 s
5
159.34 (159.11) s
159.34 (159.11) s
159.34 (159.11) s
159.34 (159.11) s
6
112.74 (112.79) s
112.74 (112.79) s
112.74 (112.79) s
112.74 (112.79) s
7
175.70 (175.64) s
175.70 (175.64) s
175.70 (175.64) s
175.70 (175.64) s
8
105.41 (105.25) s
105.41 (105.25) s
105.41 (105.25) s
105.41 (105.25) s
9
155.86 (155.72) s
155.86 (155.72) s
155.86 (155.72) s
155.86 (155.72) s
10
105.25 s
105.25 s
105.25 s
105.25 s
11 12
5.16 s
72.42 (72.35) d 210.95 s
5.16 s
13 or 14
2.05 s
26.13 (26.06) q
1.99 s
25.97 (25.83) q
5.14 s
15
72.42 (72.35) d 210.95 s
3.49 m
201.35 s 71.95 d
3.49 m
201.35 s 71.95 d
1.99 s
25.97 (25.83) q
1.25 m
22.54 (22.48, 22.31) q
1.25 m
22.54 (22.48, 22.31) q
2.05 s
26.13 (26.06) q 201.35
5.14 s
72.42 (72.35) d
72.42 (72.35) d
201.35
210.95 s
3.49 m
71.95 d
3.49 m
71.95 d
1.25 m
22.54 (22.48, 22.32) q
1.25 m
22.54 (22.48, 22.32) q
16
2.05 s
26.13 (26.06) q
or
1.99 s
25.97 (25.83) q
10 20
7.34 d, J 11.2
122.57 s 130.64 (130.59) d
7.34 d, J 11.2
122.57 s 130.64 (130.59) d
7.34 d, J 11.2
30
6.81 d, J 12.2
115.45 (115.62) d
6.81 d, J 12.2
115.45 (115.62) d
6.81 d, J 12.2
40
157.51 s
157.51s
210.95 s 1.99 s
25.97 (25.83) q
2.05 s
26.13 (26.06) q
122.57 s 130.64 (130.59) d
7.34 d, J 11.2
122.57 s 130.64 (130.59) d
115.45 (115.62) d
6.81 d, J 12.2
115.45 (115.62) d
157.51 s
157.51 s
50
6.81 d, J 12.2
115.45 (115.62) d
6.81 d, J 12.2
115.45 (115.62) d
6.81 d, J 12.2
115.45 (115.62) d
6.81 d, J 12.2
115.45 (115.62) d
60
7.34 d, J 11.2
130.64 (130.59) d
7.34 d, J 11.2
130.64 (130.59) d
7.34 d, J 11.2
130.64 (130.59) d
7.34 d, J 11.2
130.64 (130.59) d
Figure 2. (A) Trapping of MGO by genistein under physiological conditions (pH 7.4, 37 °C) and (B) inhibitory effect of the formation of AGEs by genistein in the HSA-MGO assay. Data are presented as the means ( SD of three replications.
adducts were higher than those from the reaction at a 3:1 ratio, both mono- and di-MGO adducts were the major products, and that the amount of unreacted genistein decreased (Figure 3). Increasing the amount of MGO to 10-fold of the amount of genistein did not significantly change the formation of both the mono- and di-MGO adducts of genistein compared to the reaction at a 1:3 ratio. Purification and Structure Elucidation of the Mono- and Di-MGO Adducts of Genistein. The mono- and di-MGO adducts were purified from the reaction mixture of genistein and MGO at a ratio of 1:100 using a C18 open column followed by preparative TLC. Their structures were established by analyzing the 1H, 13C, and 2D NMR (HMQC and HMBC)
as well as MS/MS spectra. The mono-MGO adduct (MM-1) was assigned the molecular formula C18H14O7 based on positive-ion ESI-MS at m/z 343 [M þ H]þ and the 1H and 13 C NMR data (Table 1). The molecular weight of MM-1 was 72 mass units higher than that of genistein, indicating that MM-1 was a mono-MGO adduct of genistein. The 1H NMR spectrum of MM-1 showed two distinct sets of aromatic ring proton signals: an AA0 BB0 system for four protons, two at δ 6.81 (J = 8.50 Hz), and two at δ 7.38 (J = 8.50 Hz), indicating a para-substituted benzene ring, which was similar to those of genistein (Table 1); and one singlet signal for one proton (δ 6.12 s), instead of the two proton signals at δ 6.20 (J = 2.10 Hz) and δ 6.38 (J = 2.10 Hz) in the 1H NMR spectrum of 582
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Figure 3. LC chromatogram of genistein after incubation with MGO (3:1, 1:1, 1: 3, 1:10) for 24 h (MM-1, mono-MGO-1; MM-2, mono-MGO-2; and DM, di-MGO).
Figure 4. Tandem MS/MS spectra of mono (A, MM-1, and B, MM-2)- and di-MGO (C, DM) adducts of genistein.
1H, and 2.17 s, 3H; δH 3.50 m, 1H, and 1.05 d, J = 7.80 Hz, 3H) instead of one set as expected in the 1H NMR spectrum of the mono-MGO adduct. Its 13C NMR spectra also showed two sets of carbon signals for the MGO group (δC 25.9 t, 70.2 d, and 207.1 s; 19.0 t and 62.5 d). All of these features indicate that MM-1 is a mixture of two tautomers (MM-1A and MM1B), and their structures were identified as shown in Figure 1.
genistein, suggesting that MGO conjugated with genistein at position 6 or 8 of the A ring. The HMBC spectrum showed that the only proton on the A ring (δH 6.12 s) was correlated with δC 167.0 (C-7), 99.5 (C-8), 162.3 (C-5), and 106.3 (C-10), indicating that this proton was H-6 and that the MGO group was located at the C-8 position of the A ring. However, there were two sets of proton signals for the MGO group (δH 5.17 s, 583
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Figure 5. LC chromatogram of genistein and mono- and di-MGO adducts of genistein after incubation of genistein in the HSA-MGO assay for 1 h, 2 h, 4 h, 24 h, 48 h, and 6 days as well as after the incubation of genistein with MGO at a 1:10 ratio.
We failed to obtain the second mono-MGO adduct of genistein, MM-2, due to its instability. The di-MGO adduct of genistein (DM) had the molecular formula C21H18O9 based on the positive-ion ESI-MS at m/z 415 [M þ H]þ and its 1H and 13C NMR data (Table 2). The molecular weight of DM was 72 mass units higher than that of the mono-MGO adduct of genistein, indicating that DM was a diMGO adduct of genistein. The 1H NMR spectrum of DM was similar to that of genistein, except that the two proton signals at δ 6.20 (J = 2.10 Hz) and δ 6.38 (J = 2.10 Hz) in the 1H NMR spectrum of genistein had disappeared, indicating that MGO conjugated with genistein at positions 6 and 8 of the A ring. In addition, there were four sets of proton signals for the MGO group (δH 5.16 s, 1H, and 2.05 s, 3H; δH 3.49 s, 1H, and 1.25 m, 3H; δH 5.13 s, 1H, and 1.99 s, 3H; and δH 3.49 s, 1H, and 1.25 m, 3H) instead of two sets as expected in the 1H NMR spectrum of DM. Its 13C spectrum also showed four sets of carbon signals for the MGO group [δC 26.1 (26.06) t, 72.42 d, and 210.95; 22.54 (22.48) t, 71.95 d, and 201.35; 25.97 (25.83) t, 72.35 d, and 210.95; 22.32 (22.48) t, 71.95 d, and 201.35]. Therefore, DM is a mixture of four tautomers (DM-A, DM-B, DM-C, and DM-D) and their structures were identified as shown in Figure 1. Determining the Formation of MGO Adducts of Genistein in the HSA-MGO System Using LC/MS. In order to further understand whether the inhibition of AGE formation was due to the trapping of MGO by genistein, we determined the existence of the mono- and di-MGO conjugated genistein in the samples collected after the incubation of genistein with HSA and MGO using LC/MS. As the results show in Figure 5, mono-MGO adducts could be detected at 1 h, and di-MGO could be detected at 4 h. They had identical retention times (Figure 5) and MS/MS spectra (data not shown) with those of the mono- and di-MGO
adducts that we purified from the reaction between genistein and MGO. In addition, the di-MGO adducts became the dominant product after 6 days of incubation.
’ DISCUSSION Flavonoids are polyphenolic compounds that are present in fruits, vegetables, and beverages derived from plants (tea and wine), as well as many dietary supplements or herbal remedies. They are categorized, according to chemical structure, into flavanols, flavonols, flavones, flavanones, isoflavones, anthocyanidins and chalcones.28 Several in vivo and in vitro studies have indicated that dietary flavonoids could inhibit the formation of AGEs and prevent diabetes-related complications.29-31 However, the mechanisms by which dietary flavonoids inhibit the formation of AGEs are unclear. Matsuda et al. examined 62 flavonoids for AGE formation inhibitory activity using the glucose-BSA assay in which 7 flavones, 8 flavonols, 3 flavanols, and 4 anthocyanins showed stronger effects than that of a reference compound, aminoguanidine (1.2 mM), and 4 isoflavones weakly inhibited the AGE formation by 25-46% at 200 μM.29 It has been reported that dietary flavonoids, luteolin, rutin, EGCG, and quercetin, showed significant inhibitory effects on MGO-mediated AGE formation.32 It was found that the flavonoids containing vicinyl dihydroxyl groups, such as rutin and its metabolite, quercetin, could effectively inhibit the formation of glyoxal (GO)-derived AGEs during the glycation of collagen I.33 In addition, Wu and co-workers found that coincubation with genistein and MGO could inhibit MGO-induced reactive oxygen species and apoptosis in human mononuclear cells.26 However, none of these studies have elucidated the mechanism of inhibition. 584
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Chemical Research in Toxicology Present Addresses
)
As reactive dicarbonyl intermediates, such as MGO, play an important role in the chemistry of AGEs formation, we have hypothesized that dietary flavonoids can inhibit the formation of AGEs by trapping reactive dicarbonyl intermediates.24,25 Results of our current investigations revealed for the first time that genistein, an important dietary isoflavone, could inhibit the formation of AGEs by trapping MGO under neutral and alkaline conditions in vitro. Two major MGO adducts of genistein were purified and identified. For both compounds, the MGO was conjugated on the A-ring of genistein, the mono-MGO adducts with the MGO conjugated at position 8 of genistein, and the di-MGO adducts with the MGO conjugated at positions 6 and 8 of genistein. This is consistent with our previous finding that the two unsubstituted carbons at the A ring of EGCG (flavanol, positions 6 and 8) and the A ring of phloridzin (chalcone, positions 3 and 5) were the active sites to trap reactive dicarbonyl species and form mono- and di-MGO adducts.24,25 This is the first report to purify and identify the structures of di-MGO adducts of dietary flavonoids. The formation of di-MGO adducts further confirms that both of the two unsubstituted carbons at the A ring of flavonoids are the active sites to trap reactive dicarbonyl species. Results generated from this study on isoflavone further support our previous conclusion that flavonoids that have the same A ring structure as EGCG or phloridzin and phloretin can efficiently trap reactive dicarbonyl species to form mono- and di-MGO adducts. Although the three different types of flavonoids (flavanol, chalcone, and isoflavone) that we have studied have different C rings, they all can efficiently trap reactive dicarbonyl compounds indicating that the C ring of flavonoids may not play an important role in the trapping of reactive dicarbonyl species. The mechanism of flavonoids trapping MGO to form mono- and di-MGO conjugated adducts was discussed in our previous studies.24,25 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 diMGO adducts. Using the mono- and di-MGO adducts mixture (MM-1 and DM) that we purified from the model reaction between genistein and MGO, we further studied whether genistein can inhibit the formation of AGEs through the trapping of MGO in the HSAMGO assay. Both mono- and di-MGO adducts of genistein were detected in this assay using LC/MS. Our results demonstrated for the first time that genistein can inhibit the formation of MGO-induced AGEs by trapping MGO. This further supports our hypothesis that dietary flavonoids can inhibit the formation of AGEs by trapping reactive dicarbonyl species. In conclusion, the results of the present study indicate that genistein can efficiently trap reactive dicarbonyl species and inhibit the formation of AGEs under in vitro conditions (pH 7.4, 37 °C). Similar to EGCG, phloretin and phloridzin, the two unsubstituted carbons at the A ring of genistein are the major active sites. Our results indicate that dietary flavonoids that have the same A ring structure as EGCG, phloretin, phloridzin, or genistein, such as quercetin, luteolin, and epicatechin, may also efficiently inhibit the formation of AGEs by trapping reactive dicarbonyl species and therefore prevent the development of diabetic complications. Whether dietary flavonoids can inhibit the formation of AGEs in vivo through the same mechanisms is a subject for future study.
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Analytical R&D, Department of Product Development, Sandoz Inc., One Health Plaza, East Hanover, New Jersey 07963, United States. Funding Sources
We gratefully acknowledge the financial support by USDA-NIFA grant 2009-65503-21116 to S.S.
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