Article pubs.acs.org/JAFC
Essential Structural Requirements and Additive Effects for Flavonoids to Scavenge Methylglyoxal Xi Shao,†,‡ Huadong Chen,†,‡ Yingdong Zhu,†,‡ Rashin Sedighi,‡ 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, Rutgers, The State University of New Jersey, 65 Dudley Road, New Brunswick, New Jersey 08901, United States ABSTRACT: Reactive dicarbonyl species, such as methylglyoxal (MGO), are considered as the major precursors of advanced glycation end products (AGEs), which are believed to be one of the physiological causes of diabetes and its complications. Scavenging of reactive dicarbonyl species using naturally occurring flavonoids has been proposed as an effective way to prevent diabetic complications. To elucidate the structural requirements of flavonoids in scavenging MGO, seven flavonoids (quercetin, luteolin, epicatechin, genistein, daidzein, apigenin, and phloretin) and five sub-components of the flavonoids (gallic acid, phloroglucinol, pyrogallol, pyrocatechol, and resorcinol) were examined in this study. Our results showed the following: (1) 1,2,3-trihydroxybenzene (pyrogallol) has higher MGO scavenging activity than 1,3,5-trihydroxybenzene and 1,2- and 1,3dihydroxybenzene, and substitution at position 5 of pyrogallol diminished the scavenging activity, indicating that position 5 is the active site of pyrogallol; (2) the A ring is the active site of flavonoids in contributing the MGO-trapping efficacy, and the hydroxyl group at C-5 on the A ring enhances the trapping efficacy; (3) the double bond between C-2 and C-3 on the C ring could facilitate the trapping efficacy; and (4) the number of hydroxyl groups on the B ring does not significantly influence the trapping efficacy. In addition, we found there is an additive effect in MGO trapping by two common flavonoids, quercetin and phloretin, indicating that flavonoid-enriched foods and beverages hold great promise to prevent the development of diabetic complications. KEYWORDS: flavonoids, MGO trapping, SAR, additive effect, AGEs
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INTRODUCTION Diabetes mellitus commonly affects about 8% of the world population and carries large health and social consequences, and its incidence is increasing at an alarming rate.1 Increasing evidence indicates that hyperglycemia is the initiating cause of the tissue damage occurring in diabetes, largely through the long-term accumulation of glycated biomolecules and advanced glycation end products (AGEs). AGEs represent a heterogeneous group of chemical products resulting from a nonenzymatic reaction between reducing sugars and proteins, lipids, nucleic acids, or a combination of these. 2 αOxoaldehydes, such as methylglyoxal (MGO), the reactive dicarbonyl intermediates generated during the non-enzymatic glycation between reducing sugars and amino groups of proteins, lipids, and DNA, have been reported to be more active than reducing sugars, are precursors of AGEs, and exert direct toxicity to cells and tissues.3 Recently, MGO has been given much attention because of its possible clinical significance in diabetes and its related complications. In healthy humans, plasma levels of MGO are ≤1 μM; however, these levels are elevated 2−4-fold in patients with diabetes. 4 Reactive dicarbonyl-derived AGEs, such as Nε-carboxymethyl-lysine (CML), Nε-carboxyethyl-lysine (CEL), and pentosidine (PENT), have been identified in humans and are often used as the biomarkers of AGEs.5,6 Furthermore, several animal studies have shown MGO as a key mechanism for AGE production. For example, MGO chronic administration (50−75 mg/kg for 3 months) to normal rats mimicked most diabetic alterations and significantly elevated the cardiac level of AGEs.7 © 2014 American Chemical Society
It is also reported that chronic MGO treatment (200 mg/kg for 6 weeks) significantly increased the formation and accumulation of AGEs in the serum and liver of male Balb/C mice.8 Arti Dhar et al. reported that chronic MGO infusion by minipump (2.5 μL/h amounting to 60 mg kg−1 day−1 for 28 days) to Sprague-Dawley (SD) rats caused significantly elevated levels of MGO and glutathione (GSH) in the plasma, pancreas, adipose tissue, and skeletal muscle as well as increased MGOinduced AGE, CML formation in pancreas.9 In addition, Guo et al. reported that, after 4 weeks of MGO treatment (1% in drinking water), the renal CEL level was significantly increased in MGO-treated SD rats compared to non-MGO-treated rats.10 A more recent study showed that, in non-diabetic C57BL/6 mice, knockdown of glyoxalase 1 (Glo1), the enzyme that metabolized MGO to D-lactate in vivo, increases MGO modification of proteins and oxidative stress, causing alterations in kidney morphology, indistinguishable from those caused by diabetes.11 They also found that, in diabetic mice, Glo1 overexpression completely prevents diabetes-induced oxidative stress and kidney pathology, despite unchanged levels of diabetic hyperglycemia. These data clearly indicate that Glo1 activity regulates the sensitivity of the kidney to hyperglycemicinduced renal pathology and that alterations in the rate of MGO detoxification are sufficient to determine the glycemic set Received: Revised: Accepted: Published: 3202
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point at which diabetic nephropathy occurs. All of the above evidence clearly indicate that MGO is a key factor in in vivo AGE production. Thus, scavenging reactive dicarbonyl species has been used as an effective strategy to prevent protein modification and AGE formation.12−14 Driven by this strategy, several pharmacological reagents, such as aminoguanidine,15,16 tenilsetam,17,18 carnosine,19 metformin,20,21 and pyridoxamine,22,23 have been developed for inhibiting the formation of AGEs and preventing the development of diabetic complications. 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.24 Therefore, it is critical to develop effective and safe agents to protect diabetics from complications. Flavonoids are polyphenolic compounds that are ubiquitous in nature, many of which occur in fruits, vegetables, and beverages (tea, coffee, beer, wine, and fruit drinks). Recently, flavonoids have aroused considerable interest because of their potential beneficial effects on human health. They have been reported to reduce the risk of chronic diseases, such as cancer, cardiovascular disease, asthma, and diabetes. Certain flavonoids have shown more effective inhibition of AGE formation than aminoguanidine, a well-known AGE inhibitor.25 For example, luteolin, rutin, (−)-epigallocatechin-3-gallate (EGCG), and quercetin demonstrated significant inhibitory effects on MGO-mediated AGE formation by 82.2, 77.7, 69.1, and 65.5%, respectively.25 Researchers have documented the effects of added natural phenolic compounds on the generation of reactive carbonyl intermediates.26 Previous studies in our lab have shown that EGCG from green tea, phloretin and phloridzin from apple, and genistein from soybean were able to effectively trap MGO to form mono- and di-MGO adducts.27−29 Besides, in a study by Ma et al., phloretin or phloridzin exhibited great inhibitory effects on arginine residue modification by MGO,30,31 which indicated that a direct capture of the reactive α-dicarbonyl species, such as MGO, would effectively inhibit the formation of AGEs and eventually benefit the prevention of diabetic complications. In studying the mechanism that flavonoids can scavenge reactive dicarbonyl species, we revealed that the A ring of EGCG played significant role in trapping of reactive dicarbonyl species.27−29 We also found that the A ring is the active site of phloretin or phloridzin on both MGO and glyoxal (GO) trapping. However, the structure−activity relationship (SAR) of flavonoids in scavenging reactive α-dicarbonyl species was not thoroughly investigated. In addition, the daily intake of individual flavonoids may not be high enough to significantly decrease the levels of reactive dicarbonyls in humans; however, the total intake of flavonoids from a variety of foods can reach the effective doses to trap a significant amount of exogenous and endogenous reactive dicarbonyls. Therefore, it is important to study the potential additive effects of different flavonoids. In the present study, we investigated the reaction of some naturally occurring flavonoids together with their subcomponents with MGO, in an attempt to establish the SAR of flavonoids in scavenging dicarbonyl species. The additive effects of trapping MGO at different concentrations by phloretin and quercetin were also carried out under an in vitro system.
Article
MATERIALS AND METHODS
Materials. Quercetin, luteolin, epicatechin, genistein, daidzein, apigenin, phloretin, gallic acid, phloroglucinol, methylglyoxal (40% in water), 2-methylquinoxaline, and 1,2-diaminobenzene (DB) were purchased from Sigma (St. Louis, MO). Pyrogallol (Pg), pyrocatechol, resorcinol, and liquid chromatrography/mass spectrometry (LC/MS)grade MeOH and water were purchased from Thermo Fisher Scientific (Pittsburgh, PA). High-performance liquid chromatography (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 Trapping MGO by Dietary Flavonoids and Simple Phenols. MGO (0.33 mM) was mixed with 1 mM quercetin, luteolin, epicatechin, genistein, daidzein, apigenin, gallic acid, phloroglucinol, Pg, pyrocatechol, or resorcinol in pH 7.4 phosphatebuffered saline (PBS, 100 mM). The mixed solutions were incubated at 37 °C and shaken at 40 revolutions per minute (rpm) for 0, 10, 30, 60, 120, 240, 480, and 1440 min. Afterward, 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 allowed to react with the remaining MGO for 30 min. The level of methylquinoxaline was determined using the HPLC method described below.28 Trapping of MGO by Phloretin, Quercetin, and the Combination of Phloretin and Quercetin. MGO (300 or 60 μM) was incubated with phloretin (50 or 10 μM), quercetin (50 or 10 μM), or a combination of phloretin and quercetin (50 or 10 μM each) in pH 7.4 PBS (100 mM) at 37 °C and shaken at 40 rpm for 24 h. Then, 400 μL of each reacted mixture was collected, and 2 μL of acetic acid was added to stop the reaction. Half of this solution was used for LC/MS analysis, and the other half was used for the derivatization of the remaining MGO by adding 100 mM DB according to our previous method.28 Trapping of MGO by Pg. Pg (12.6 mg, 0.1 mmol) was dissolved in 10 mL of PBS (100 mM, pH 7.4) and treated with a diluted solution of MGO (0.033 or 0.1 mmol) in PBS (100 mM, pH 7.4). The mixture was incubated at 37 °C for 3 h and then quenched by adding 200 μL of acetic acid. After dilution, 100 times by MeOH, the reaction mixture was then analyzed by LC/MS. HPLC Analysis. The level of methylquinoxaline was determined using our previous HPLC method, with slight modification.28 Briefly, the HPLC system was equipped with a Waters 717 refrigerated autosampler, a HITACHI L-6200 Inteligent pump, and a Waters 490E programmable multi-wavelength ultraviolet−visible (UV−vis) detector. A Supelcosil C18 reversed-phase column (150 × 4.6 mm inner diameter, Supelco Co., Bellefonte, PA) was used with a flow rate of 1 mL/min. Both the column and sample temperatures were set at ambient conditions. 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 eluted with a binary gradient system: 100−65% A from 0 to 5 min, 65−60% A from 5 to 15 min, 60−0% A from 15 to 20 min, and then 100% A from 21 to 30 min. The injection volume was 50 μL. The wavelength of the UV detector was set at 280 nm with 100 ng/mL as the limit of detection (LOD) and 1 μg/mL as the limit of quantification (LOQ). 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 a LCQ Fleet ion trap mass detector (Thermo Electron, San Jose, CA) incorporated with electrospray ionization (ESI) interfaces. For the analysis of Pg or its main MGO adducts, a 150 × 3.0 mm inner diameter, 5 μm Gemini C18 column (Phenomenex, Torrance, CA) was employed with a flow rate of 0.3 mL/min in the current study. The column was eluted with 100% solvent A (100% water with 0.2% acetic acid) for 5 min, followed by linear increases in B (100% methanol with 0.2% acetic acid) to 30% from 5 to 20 min, 80% from 20 to 40 min, 100% from 40 to 50 min, and then 100% from 50 to 55 min. The column was then re-equilibrated with 100% A for 5 min. As far as phloretin, quercetin, or their major MGO adducts were concerned, a 50 × 2.0 mm inner diameter, 3 μm Gemini C18 column (Phenomenex, Torrance, CA) was used for separation at a flow rate of 0.2 mL/min. The column was 3203
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Figure 1. Chemical structures of gallic acid, pytogallol (Pg), phloroglucinol, pyrocatechol, resorcinol, genistein, daidzein, quercetin, luteolin, epicatechin, phloretin, and apigenin.
Figure 2. Trapping of MGO by gallic acid, pytogallol (Pg), phloroglucinol, pyrocatechol, and resorcinol. eluted with 100% solvent A (100% water with 0.2% acetic acid) for 5 min, followed by linear increases in B (95% aqueous methanol with 0.2% acetic acid) to 20% from 5 to 10 min, 50% from 10 to 20 min, 100% from 20 to 28 min, and then 100% from 28 to 33 min. The column was then re-equilibrated with 100% A for 7 min. The LC eluent was introduced into the ESI interface. The negative-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 arbitrary units and the auxiliary gas at 5 arbitrary units. The structural information of phloretin, quercetin, Pg, and the major MGO adducts was obtained by 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).
B, and C (chalcones are ring C cleavage products). To obtain the essential SAR in trapping dicarbonyl species by dietary flavonoids, we selected some typical sub-components of flavonoids to simplify the influential factors. Five simple phenols, gallic acid, Pg, pyrocatechol, resorcinol, and phloroglucinol (Figure 1), were chosen for our current study. Among these compounds, Pg showed the highest scavenging activity by trapping almost 90% MGO within 24 h of incubation, followed by phloroglucinol, which could trap 60.5% MGO (Figure 2). Resorcinol and pyrocatechol showed relatively lower scavenging activity by trapping 31.6 and 21.8% MGO, respectively (Figure 2). In contrast, gallic acid was found to be the least active agent by trapping only 14.9% MGO within 24 h of incubation, even if it has the same vic-trihydroxyl groups as Pg (Figure 2). MGO Adducts of Pg. To further investigate the MGOtrapping mechanism of Pg, the reaction of Pg and MGO was
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RESULTS Trapping of MGO by Simple Phenols. In general, flavonoids are composed of three different small units, rings A, 3204
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Figure 3. LC chromatogram of reaction mixtures of (A) pyrogallol (Pg) (0.1 mmol) with MGO (0.033 mmol) under a ratio of 3:1 and (C) Pg (0.1 mmol) with MGO (0.1 mmol) under a ratio of 1:1 in PBS (100 mM, pH 7.4) at 37 °C for 3 h. (B) MS/MS spectra of MM−Pg and (D) DM−Pg. (E) Chemical reaction equation of Pg and MGO (Pg = pyrogallol).
Figure 4. Trapping of MGO by genistein and daidzein.
(Figure 3A). This new peak had a molecular ion at m/z 197 [M − H]−, which is 72 mass units (molecular weight of MGO of 72) higher than that of Pg, indicating that it is a mono-MGO
tried at two different ratios (3:1 or 1:1 Pg/MGO). After 3 h of incubation (3:1 Pg/MGO), one major new peak [retention time (RT) of 13.04 min] appeared in the LC chromatogram 3205
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Figure 5. Trapping of MGO by (A) apigenin and luteolin and (B) quercetin, luteolin, and epicatechin.
conjugated pyrogallol (MM−Pg). Its MS2 spectrum displayed a predominant dehydration ion at m/z 179 [M − H2O − H]− (Figure 3B), while one more peak (RT of 13.90 min) was observed besides the peak of MM−Pg (RT of 12.83 min) with Pg and MGO at a 1:1 ratio (Figure 3C). This peak had a molecular ion at 269 [M − H]−, which is 72 mass units higher than that of MM−Pg, suggesting that this product is the diMGO conjugated pyrogallol (DM−Pg). Similarly, its MS2 spectrum also displayed a predominant dehydration ion at m/ z 251 [M − H2O − H]− (Figure 3D). Because of the instability of these adducts, we failed to purify either MM−Pg or DM−Pg from the reaction mixture. However, the proton nuclear magnetic resonance (1H NMR) spectrum (600 MHz in CD3OD) of the crude mixture with MM−Pg as the major component showed only singlet signals for the aromatic ring protons (data not shown), suggesting that MGO was located at C-5 of Pg in MM−Pg (Figures 1 and 3B). The formation of mono- and di-MGO conjugated products of Pg was tentatively proposed in Figure 3E. The identification of position 5 as the active site of Pg further explained why gallic acid has almost no trapping effect of MGO and the B or D ring of EGCG is not the active site to trap MGO. Effects of the A Ring on MGO-Trapping Efficacy. We used phloroglucinol and resorcinol to represent the A-ring structures of flavonoids. Our result that phloroglucinol is a better MGO scavenger than resorcinol indicates that the hydroxyl group on C-5 of the A ring will enhance the trapping efficacy of flavonoids. To test this hypothesis, we compared the MGO-trapping effects of genistein and daidzein, which have Aring structures almost identical to phloroglucinol and resorcinol, respectively. In addition, they have the same Band C-ring structures. Similar to our finding on phloroglucinol
and resorcinol, genistein was more effective than daidzein (Figure 4). At 4 h, genistein could trap almost 90% MGO, whereas daidzein could only trap 54.5% MGO. Our results clearly support our hypothesis that the hydroxyl group on C-5 of the A ring is crucial for the trapping of MGO by flavonoids. This is also supported by our previous observation that glycosylation of the hydroxyl group at C-5 of the A ring of phloretin tended to decrease its activity.27 In addition, glycosylation of the 5-hydroxyl group may increase the steric hindrance of position C-6 and, therefore, decrease its activity. Effects of B and C Rings on MGO-Trapping Efficacy. By comparing the trapping of MGO by apigenin and luteolin (Figure 5A), which possess the same A and C rings but a different number of hydroxyl groups on the B ring, we found that the number of hydroxyl groups on the B ring does not play a significant role on the trapping efficacy of flavonoids, albeit that luteolin with two hydroxyl groups at C-2′ and C-3′ positions shows a slightly stronger trapping capacity than apigenin that has only one hydroxyl group on the B ring (Figure 5A). To understand how the C ring of the flavonoids affects the MGO-trapping capacity, we studied the MGOtrapping effects of quercetin, luteolin, and epicatechin, which have identical A and B rings with a different structure of the C ring. Quercetin and luteolin belong to the flavone with a double bond between C-2 and C-3 and one ketone group at C-4 (Figure 5B). Epicatechin belongs to flavan-3-ol with a hydroxy group on C-3 but with no double bond and a ketone group on the C ring. Quercetin showed the best MGO trapping efficacy, followed by luteolin and epicatechin. More than 90% MGO was trapped within 8 h by both quercetin and luteolin. Epicatechin also had strong trapping efficiency by quenching more than 85% MGO within 24 h. Both quercetin and epicatechin could 3206
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(SIM) mode (Figures 8). Three major new peaks appeared in the LC chromatogram of the reaction mixture of quercetin with MGO at a ratio of 1:6 after 24 h of incubation (Figures 7A). Two of the three peaks had the same molecular ion (m/z 373 [M − H]−) and MS/MS fragments but different RTs (18.34 and 18.86 min). Both of the two compounds had the fragment ion 301 ([M − 72 − H]−), showing that they both lost one MGO (m/z 72) molecule (Figure 8A), thus indicating that they are mono-MGO adducts of quercetin. Furthermore, the tandem mass spectrum of product ion 301 (MS3, m/z 301/ 373) of the two compounds (Figure 8A) was almost identical to the MS2 spectrum of authentic quercetin (MS2, m/z 301), suggesting that the two compounds were the mono-MGO adducts of quercetin. The third peak (16.77 min) showed the molecular ion at m/z 445 [M − H]−, which was 72 mass units higher than that of mono-MGO quercetin, indicating that it was the di-MGO quercetin. The tandem mass spectrum of its product ion 373 (MS3, m/z 373/445) was almost identical to the MS2 spectrum of mono-MGO quercetin (MS2, m/z 373), suggesting that this peak was the di-MGO adduct of quercetin.
rapidly trap MGO; almost 50% MGO was scavenged within 11.25 min. Additive MGO-Trapping Effects by Phloretin and Quercetin. To test whether different flavonoids have additive effects in terms of trapping reactive dicarbonyl species, we investigated the MGO-trapping capability of phloretin, quercetin, or the combination of phloretin and quercetin at two different concentrations (50 and 10 μM) under a ratio of each flavonoid/MGO at 1:6 at pH 7.4 and 37 °C. Our results indicated that phloretin and quercetin trapped about 16 and 23% MGO, respectively, at a concentration of 50 μM within 24 h. About 37% MGO was trapped by the combination of phloretin and quercetin (50 μM each) (Figure 6). At a lower
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DISCUSSION Flavonoids are biologically active, polyphenolic constituents of plant foods and are present in many fruits, vegetables, and beverages (tea, coffee, beer, wine, and fruit drinks), as well as dietary supplements and herbal remedies. The flavonoids are further divided into six major subclasses known as flavanols, flavanones, flavonols, flavones, isoflavones, and anthocyanidins on the basis of the connection of the B ring to the C ring, as well as the oxidation state and functional groups of the C ring.32 All of the major classes of flavonoids are comprised of three sixmembered rings: an aromatic A ring fused to a heterocyclic C ring that is attached through a single carbon−carbon bond to an aromatic B ring. Chalcone, an important precursor in the synthesis of anthocyanin pigments and other flavonoids in plants, is another type of important dietary flavonoid. In contrast to other flavonoids, the middle three carbon atoms in chalcones do not form a closed ring. Several in vivo and in vitro studies have indicated that dietary flavonoids could inhibit the formation of AGEs and prevent diabetes-related complications.25,33,34 Previously, we found three different types of flavonoids, flavanol (EGCG), chalcone (phloretin and phloredzin), and isoflavone (genistein), could rapidly trap MGO at pH 7.4 and 37 °C and, therefore, inhibit the formation of AGEs.27−29 We also demonstrated that the two unsubstituted carbons at the A ring of these flavonoids were the active sites for trapping reactive dicarbonyl species and forming mono- and di-MGO adducts. Most of the flavonoids have the same A-ring structure as EGCG, phloretin, phloridzin, or genistein. It is unclear whether they can also efficiently trap reactive dicarbonyl species to form mono- and di-MGO adducts. More importantly, it is largely unknown whether the number of hydroxyl groups on the A ring and the structures of B or C rings will play an important role in the trapping of reactive dicarbonyl species. To tackle these questions, we conducted the SAR studies of flavonoids as the scavengers of reactive dicarbonyl species. We first compared the trapping efficacy of the subcomponents of flavonoids, gallic acid (gallate ring of EGCG), Pg (B ring of flavonoids), pyrocatechol (B ring of flavonoids), resorcinol (A ring of flavonoids), and phloroglucinol (A ring of flavonoids). Our results showed that the MGO scavenging potency of these sub-components is in the order of Pg >
Figure 6. Tapping of MGO (300 or 60 μM) by phloretin (P), quercetin (Q), and the combination of phloretin and quercetin (P + Q, equal molar combination) at different concentrations (50 or 10 μM) in phosphate buffer (pH 7.4 and 37 °C) at 24 h. Bars on left and right sides represent the concentrations of MGO with 300 or 60 μM, respectively. Data are presented as the means ± standard deviation (SD) of three replications.
concentration (10 μM) with the same ratio, both phloretin and quercetin showed similar trapping capability of scavenging nearly 19 and 23% MGO within 24 h, respectively. Almost 35% MGO was trapped by the combination of phloretin and quercetin (10 μM each) (Figure 6). Our results clearly indicate that different flavonoids can additively trap MGO. Studying the Formation of MGO Adducts of Quercetin and Phloretin by LC/MS. To understand the underlying mechanism that different flavonoids can additively trap MGO, we analyzed the formation of MGO adducts under each flavonoid along or the combination of phloretin and quercetin using LC/MS. Figure 7 shows the LC chromatograms of the reaction mixtures of quercetin with MGO (A), phloretin with MGO (B), and the combination of phloretin and quercetin (C). Both mono- and di-MGO adducts of quercetin were the major products under the ratio of quercetin/MGO at 1:6 (Figure 7A). Similar to our previous observation, monoand di-MGO adducts of phloretin were identified as the major products for phloretin (Figure 7B). Under the combination of phloretin and quercetin, the same MGO adducts of each flavonoid were formed, which further confirmed the additive effect of phloretin and quercetin (Figure 7C). We have confirmed the structures of the mono- and di-MGO adducts of phloretin in our previous study.27 The structural information of the MGO adducts of quercetin was obtained using LC/MS/MS analysis under selective ion monitoring 3207
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Figure 7. LC chromatogram of reaction mixtures of (A) quercetin (50 μM) with MGO (300 μM) under a ratio of 1:6 in PBS (100 mM, pH 7.4) at 37 °C for 24 h, (B) phloretin (50 μM) with MGO (300 μM) under a ratio of 1:6 in PBS (100 mM, pH 7.4) at 37 °C for 24 h, and (C) quercetin/ phloretin with MGO under a ratio of 1:1:6.
phloroglucinol > resorinol > pyrocatechol > gallic acid, indicating the pivotal influence of the number and position of the hydroxyl group on the trapping activity. The trihydroxylbenzene (Pg and phloroglucinol) had a higher reactivity than the dihydroxylbenzene (pyrocatechol and resorcinol). In general, the hydroxyl group has electron-donating property. The more hydroxyl groups on the phenyl ring, the stronger the scavenging activity, because the reaction between phenol and MGO is a nucleophilic reaction. Our results also shown that the addition of a carboxyl group to position 5 of Pg almost completely diminished its trapping activity, indicating that position 5 is the active site of Pg. A further study on the trapping mechanism of Pg confirmed that MGO is conjugated at position 5 of Pg as the mono-MGO adduct of Pg, which provides the answer why gallic acid is the least active compound, albeit it has the same vic-trihydroxyl group as that of Pg. This also resolves the puzzle of our previous observation that the major active site of EGCG is at positions 6 and 8 of the A ring, instead of the B ring and the gallate ring, which have the vic-trihydroxyl group and are generally considered as the active sites for its antioxidant activities.28 In addition, the position of the hydroxyl group also plays a significant role in contributing to the trapping capability of these small phenolic compounds. Pg with three ortho-hydroxyl groups demonstrated a much stronger trapping effect on MGO than phloroglucinol, which contains three meta-hydroxyl groups. To extend our observation on the sub-components of flavonoids to the influence of A, B, or C ring of flavonoids on their MGO-trapping effects, we chose flavonoids containing these specific sub-components for our study. To demonstrate how the number of hydroxyl groups on the A ring of flavonoids affects the MGO-trapping efficiency, genistein and daidzein
were chosen as our model compounds, because they possess identical B and C rings but with a different number of hydroxyl groups on the A ring. Genistein contains one more hydroxyl group at position C-5 than daidzein. Our finding that genistein showed stronger capability and efficacy in trapping of MGO than that of daidzein is in line with the conclusion drawn from phloroglucinol and resorcinol, which represent the A-ring structure of genistein and daidzein, respectively. In the same way, the effect of the number of hydroxyl groups on the B ring in the trapping dicarbonyl species by flavonoids was revealed by comparing the MGO-trapping capacity between apigenin and luteolin. A simple comparison among quercetin, luteolin, and epicatechin was carried out to demonstrate how the C ring of flavonoids affects the MGO-trapping ability. Even these simplified comparisons cannot provide the full picture of the essential SAR between dicarbonyl species and flavonoids; our results can still shed light on the following conclusions: (1) 1,2,3-trihydroxybenzene (Pg) has higher MGO scavenging reactivity than 1,2- and 1,3-dihydroxybenzene, and substitution at position 5 of Pg diminished the scavenging activity, indicating that position 5 is the active site of Pg; (2) the A ring is the active site of flavonoids in contributing the MGOtrapping efficacy, and the hydroxyl group at C-5 on the A ring enhances the trapping efficacy; (3) the double bond between C-2 and C-3 on the C ring could facilitate the trapping efficacy; and (4) the number of hydroxyl groups on the B ring does not significantly influence the trapping efficacy. This is the first study to show that quercetin, luteolin, and apigenin can trap MGO at pH 7.4 and 37 °C, indicating flavonol and flavone types of flavonoids can also serve as scavengers of reactive dicarbonyl species. Further studies on the trapping mechanism of quercetin identified mono- and di3208
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Figure 8. MS/MS spectra of (A) mono-MGO conjugated quercetin and authentic quercetin and (B) di-MGO conjugated quercetin.
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MGO adducts as the major products of the reaction between quercetin and MGO. On the basis of our previous studies of EGCG, phloretin, and genistein, we suspect that the MGO conjugation should occur at positions C-6 and C-8 of the A ring. However, this needs to be further confirmed by NMR analysis of the pure mono- and di-MGO quercetin adducts. Our finding that many dietary flavonoids can trap reactive dicarbonyls through the same mechanism indicates that they may have an additive effect if not a synergistic effect. It has been reported that the total intake of flavonoids in the United States is 189.7 mg/day, which is difficult to be achieved for individual flavonoids.35 Therefore, results on the effectiveness of the combination of individual flavonoids can be applied directly to translational studies in humans. To verify this hypothesis, phloretin and quercetin were chosen as the model compounds. Phloretin is a flavonoid found exclusively in apples and in apple-derived products. We previously reported that phloretin can effectively trap MGO.27 Quercetin was selected because of its wide distribution in dietary plants.36 Our results clearly indicated an additive MGO-trapping effect of these two flavonoids in two different concentrations (Figure 6). Thus, flavonoid-enriched foods and beverages as effective dietary strategies hold great promise to prevent the development of diabetic complications and, therefore, reduce the morbidity and mortality in individuals with diabetes. This topic needs to be further studied, especially under in vivo conditions.
AUTHOR INFORMATION
Corresponding Author
*Telephone: 704-250-5710. Fax: 704-250-5709. E-mail: [email protected]. Author Contributions
† Xi Shao, Huadong Chen, and Yingdong Zhu contributed equally to this work.
Funding
The authors gratefully acknowledge the financial support by United States Department of Agriculture (USDA) Grants 2009-65503-21116 and 2012-67017-30175 to Shengmin Sang. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED AGEs, advanced glycation end products; DM−Pg, di-MGO conjugated pyrogallol; HPLC, high-performance liquid chromatography; LC/MS, liquid chromatrography/mass spectrometry; MGO, methylglyoxal; MM−Pg, mono-MGO conjugated pyrogallol; Pg, pyrogallol; SAR, structure−activity relationship; PBS, phosphate-buffered saline
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REFERENCES
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(21) Beisswenger, P. J.; Howell, S. K.; Touchette, A. D.; Lal, S.; Szwergold, B. S. Metformin reduces systemic methylglyoxal levels in type 2 diabetes. Diabetes 1999, 48, 198−202. (22) Nagaraj, R. H.; Sarkar, P.; Mally, A.; Biemel, K. M.; Lederer, M. O.; Padayatti, P. S. Effect of pyridoxamine on chemical modification of proteins by carbonyls in diabetic rats: Characterization of a major product from the reaction of pyridoxamine and methylglyoxal. Arch. Biochem. Biophys. 2002, 402, 110−119. (23) Voziyan, P. A.; Metz, T. O.; Baynes, J. W.; Hudson, B. G. A post-Amadori inhibitor pyridoxamine also inhibits chemical modification of proteins by scavenging carbonyl intermediates of carbohydrate and lipid degradation. J. Biol. Chem. 2002, 277, 3397− 3403. (24) Thornalley, P. J. Use of aminoguanidine (Pimagedine) to prevent the formation of advanced glycation endproducts. Arch. Biochem. Biophys. 2003, 419, 31−40. (25) Wu, C. H.; Yen, G. C. Inhibitory effect of naturally occurring flavonoids on the formation of advanced glycation endproducts. J. Agric. Food Chem. 2005, 53, 3167−3173. (26) Totlani, V. M.; Peterson, D. G. Reactivity of epicatechin in aqueous glycine and glucose maillard reaction models: Quenching of C2, C3, and C4 sugar fragments. J. Agric. Food Chem. 2005, 53, 4130− 4135. (27) Shao, X.; Bai, N.; He, K.; Ho, C. T.; Yang, C. S.; Sang, S. Apple polyphenols, phloretin and phloridzin: New trapping agents of reactive dicarbonyl species. Chem. Res. Toxicol. 2008, 21, 2042−2050. (28) Sang, S.; Shao, X.; Bai, N.; Lo, C. Y.; Yang, C. S.; Ho, C. T. Tea polyphenol (−)-epigallocatechin-3-gallate: A new trapping agent of reactive dicarbonyl species. Chem. Res. Toxicol. 2007, 20, 1862−1870. (29) Lv, L.; Shao, X.; Chen, H.; Ho, C. T.; Sang, S. Genistein inhibits advanced glycation end product formation by trapping methylglyoxal. Chem. Res. Toxicol. 2011, 24, 579−586. (30) Ahmed, M. U.; Brinkmann Frye, E.; Degenhardt, T. P.; Thorpe, S. R.; Baynes, J. W. Nε-(Carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins. Biochem. J. 1997, 324 (Part 2), 565−570. (31) Odani, H.; Shinzato, T.; Usami, J.; Matsumoto, Y.; Brinkmann Frye, E.; Baynes, J. W.; Maeda, K. Imidazolium crosslinks derived from reaction of lysine with glyoxal and methylglyoxal are increased in serum proteins of uremic patients: Evidence for increased oxidative stress in uremia. FEBS Lett. 1998, 427, 381−385. (32) Beecher, G. R. Overview of dietary flavonoids: Nomenclature, occurrence and intake. J. Nutr. 2003, 133, 3248S−3254S. (33) Takasawa, R.; Takahashi, S.; Saeki, K.; Sunaga, S.; Yoshimori, A.; Tanuma, S. Structure−activity relationship of human GLO I inhibitory natural flavonoids and their growth inhibitory effects. Bioorg. Med. Chem. 2008, 16, 3969−3975. (34) Maher, P.; Dargusch, R.; Ehren, J. L.; Okada, S.; Sharma, K.; Schubert, D. Fisetin lowers methylglyoxal dependent protein glycation and limits the complications of diabetes. PloS One 2011, 6, No. e21226. (35) Chun, O. K.; Chung, S. J.; Song, W. O. Estimated dietary flavonoid intake and major food sources of U.S. adults. J. Nutr. 2007, 137, 1244−1252. (36) Sampson, L.; Rimm, E.; Hollman, P. C.; de Vries, J. H.; Katan, M. B. Flavonol and flavone intakes in US health professionals. J. Am. Diet. Assoc. 2002, 102, 1414−1420.
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Essential Structural Requirements and Additive Effects for Flavonoids to Scavenge Methylglyoxal Xi Shao,†,‡ Huadong Chen,†,‡ Yingdong Zhu,†,‡ Rashin Sedighi,‡ 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, Rutgers, The State University of New Jersey, 65 Dudley Road, New Brunswick, New Jersey 08901, United States ABSTRACT: Reactive dicarbonyl species, such as methylglyoxal (MGO), are considered as the major precursors of advanced glycation end products (AGEs), which are believed to be one of the physiological causes of diabetes and its complications. Scavenging of reactive dicarbonyl species using naturally occurring flavonoids has been proposed as an effective way to prevent diabetic complications. To elucidate the structural requirements of flavonoids in scavenging MGO, seven flavonoids (quercetin, luteolin, epicatechin, genistein, daidzein, apigenin, and phloretin) and five sub-components of the flavonoids (gallic acid, phloroglucinol, pyrogallol, pyrocatechol, and resorcinol) were examined in this study. Our results showed the following: (1) 1,2,3-trihydroxybenzene (pyrogallol) has higher MGO scavenging activity than 1,3,5-trihydroxybenzene and 1,2- and 1,3dihydroxybenzene, and substitution at position 5 of pyrogallol diminished the scavenging activity, indicating that position 5 is the active site of pyrogallol; (2) the A ring is the active site of flavonoids in contributing the MGO-trapping efficacy, and the hydroxyl group at C-5 on the A ring enhances the trapping efficacy; (3) the double bond between C-2 and C-3 on the C ring could facilitate the trapping efficacy; and (4) the number of hydroxyl groups on the B ring does not significantly influence the trapping efficacy. In addition, we found there is an additive effect in MGO trapping by two common flavonoids, quercetin and phloretin, indicating that flavonoid-enriched foods and beverages hold great promise to prevent the development of diabetic complications. KEYWORDS: flavonoids, MGO trapping, SAR, additive effect, AGEs
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INTRODUCTION Diabetes mellitus commonly affects about 8% of the world population and carries large health and social consequences, and its incidence is increasing at an alarming rate.1 Increasing evidence indicates that hyperglycemia is the initiating cause of the tissue damage occurring in diabetes, largely through the long-term accumulation of glycated biomolecules and advanced glycation end products (AGEs). AGEs represent a heterogeneous group of chemical products resulting from a nonenzymatic reaction between reducing sugars and proteins, lipids, nucleic acids, or a combination of these. 2 αOxoaldehydes, such as methylglyoxal (MGO), the reactive dicarbonyl intermediates generated during the non-enzymatic glycation between reducing sugars and amino groups of proteins, lipids, and DNA, have been reported to be more active than reducing sugars, are precursors of AGEs, and exert direct toxicity to cells and tissues.3 Recently, MGO has been given much attention because of its possible clinical significance in diabetes and its related complications. In healthy humans, plasma levels of MGO are ≤1 μM; however, these levels are elevated 2−4-fold in patients with diabetes. 4 Reactive dicarbonyl-derived AGEs, such as Nε-carboxymethyl-lysine (CML), Nε-carboxyethyl-lysine (CEL), and pentosidine (PENT), have been identified in humans and are often used as the biomarkers of AGEs.5,6 Furthermore, several animal studies have shown MGO as a key mechanism for AGE production. For example, MGO chronic administration (50−75 mg/kg for 3 months) to normal rats mimicked most diabetic alterations and significantly elevated the cardiac level of AGEs.7 © 2014 American Chemical Society
It is also reported that chronic MGO treatment (200 mg/kg for 6 weeks) significantly increased the formation and accumulation of AGEs in the serum and liver of male Balb/C mice.8 Arti Dhar et al. reported that chronic MGO infusion by minipump (2.5 μL/h amounting to 60 mg kg−1 day−1 for 28 days) to Sprague-Dawley (SD) rats caused significantly elevated levels of MGO and glutathione (GSH) in the plasma, pancreas, adipose tissue, and skeletal muscle as well as increased MGOinduced AGE, CML formation in pancreas.9 In addition, Guo et al. reported that, after 4 weeks of MGO treatment (1% in drinking water), the renal CEL level was significantly increased in MGO-treated SD rats compared to non-MGO-treated rats.10 A more recent study showed that, in non-diabetic C57BL/6 mice, knockdown of glyoxalase 1 (Glo1), the enzyme that metabolized MGO to D-lactate in vivo, increases MGO modification of proteins and oxidative stress, causing alterations in kidney morphology, indistinguishable from those caused by diabetes.11 They also found that, in diabetic mice, Glo1 overexpression completely prevents diabetes-induced oxidative stress and kidney pathology, despite unchanged levels of diabetic hyperglycemia. These data clearly indicate that Glo1 activity regulates the sensitivity of the kidney to hyperglycemicinduced renal pathology and that alterations in the rate of MGO detoxification are sufficient to determine the glycemic set Received: Revised: Accepted: Published: 3202
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point at which diabetic nephropathy occurs. All of the above evidence clearly indicate that MGO is a key factor in in vivo AGE production. Thus, scavenging reactive dicarbonyl species has been used as an effective strategy to prevent protein modification and AGE formation.12−14 Driven by this strategy, several pharmacological reagents, such as aminoguanidine,15,16 tenilsetam,17,18 carnosine,19 metformin,20,21 and pyridoxamine,22,23 have been developed for inhibiting the formation of AGEs and preventing the development of diabetic complications. 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.24 Therefore, it is critical to develop effective and safe agents to protect diabetics from complications. Flavonoids are polyphenolic compounds that are ubiquitous in nature, many of which occur in fruits, vegetables, and beverages (tea, coffee, beer, wine, and fruit drinks). Recently, flavonoids have aroused considerable interest because of their potential beneficial effects on human health. They have been reported to reduce the risk of chronic diseases, such as cancer, cardiovascular disease, asthma, and diabetes. Certain flavonoids have shown more effective inhibition of AGE formation than aminoguanidine, a well-known AGE inhibitor.25 For example, luteolin, rutin, (−)-epigallocatechin-3-gallate (EGCG), and quercetin demonstrated significant inhibitory effects on MGO-mediated AGE formation by 82.2, 77.7, 69.1, and 65.5%, respectively.25 Researchers have documented the effects of added natural phenolic compounds on the generation of reactive carbonyl intermediates.26 Previous studies in our lab have shown that EGCG from green tea, phloretin and phloridzin from apple, and genistein from soybean were able to effectively trap MGO to form mono- and di-MGO adducts.27−29 Besides, in a study by Ma et al., phloretin or phloridzin exhibited great inhibitory effects on arginine residue modification by MGO,30,31 which indicated that a direct capture of the reactive α-dicarbonyl species, such as MGO, would effectively inhibit the formation of AGEs and eventually benefit the prevention of diabetic complications. In studying the mechanism that flavonoids can scavenge reactive dicarbonyl species, we revealed that the A ring of EGCG played significant role in trapping of reactive dicarbonyl species.27−29 We also found that the A ring is the active site of phloretin or phloridzin on both MGO and glyoxal (GO) trapping. However, the structure−activity relationship (SAR) of flavonoids in scavenging reactive α-dicarbonyl species was not thoroughly investigated. In addition, the daily intake of individual flavonoids may not be high enough to significantly decrease the levels of reactive dicarbonyls in humans; however, the total intake of flavonoids from a variety of foods can reach the effective doses to trap a significant amount of exogenous and endogenous reactive dicarbonyls. Therefore, it is important to study the potential additive effects of different flavonoids. In the present study, we investigated the reaction of some naturally occurring flavonoids together with their subcomponents with MGO, in an attempt to establish the SAR of flavonoids in scavenging dicarbonyl species. The additive effects of trapping MGO at different concentrations by phloretin and quercetin were also carried out under an in vitro system.
Article
MATERIALS AND METHODS
Materials. Quercetin, luteolin, epicatechin, genistein, daidzein, apigenin, phloretin, gallic acid, phloroglucinol, methylglyoxal (40% in water), 2-methylquinoxaline, and 1,2-diaminobenzene (DB) were purchased from Sigma (St. Louis, MO). Pyrogallol (Pg), pyrocatechol, resorcinol, and liquid chromatrography/mass spectrometry (LC/MS)grade MeOH and water were purchased from Thermo Fisher Scientific (Pittsburgh, PA). High-performance liquid chromatography (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 Trapping MGO by Dietary Flavonoids and Simple Phenols. MGO (0.33 mM) was mixed with 1 mM quercetin, luteolin, epicatechin, genistein, daidzein, apigenin, gallic acid, phloroglucinol, Pg, pyrocatechol, or resorcinol in pH 7.4 phosphatebuffered saline (PBS, 100 mM). The mixed solutions were incubated at 37 °C and shaken at 40 revolutions per minute (rpm) for 0, 10, 30, 60, 120, 240, 480, and 1440 min. Afterward, 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 allowed to react with the remaining MGO for 30 min. The level of methylquinoxaline was determined using the HPLC method described below.28 Trapping of MGO by Phloretin, Quercetin, and the Combination of Phloretin and Quercetin. MGO (300 or 60 μM) was incubated with phloretin (50 or 10 μM), quercetin (50 or 10 μM), or a combination of phloretin and quercetin (50 or 10 μM each) in pH 7.4 PBS (100 mM) at 37 °C and shaken at 40 rpm for 24 h. Then, 400 μL of each reacted mixture was collected, and 2 μL of acetic acid was added to stop the reaction. Half of this solution was used for LC/MS analysis, and the other half was used for the derivatization of the remaining MGO by adding 100 mM DB according to our previous method.28 Trapping of MGO by Pg. Pg (12.6 mg, 0.1 mmol) was dissolved in 10 mL of PBS (100 mM, pH 7.4) and treated with a diluted solution of MGO (0.033 or 0.1 mmol) in PBS (100 mM, pH 7.4). The mixture was incubated at 37 °C for 3 h and then quenched by adding 200 μL of acetic acid. After dilution, 100 times by MeOH, the reaction mixture was then analyzed by LC/MS. HPLC Analysis. The level of methylquinoxaline was determined using our previous HPLC method, with slight modification.28 Briefly, the HPLC system was equipped with a Waters 717 refrigerated autosampler, a HITACHI L-6200 Inteligent pump, and a Waters 490E programmable multi-wavelength ultraviolet−visible (UV−vis) detector. A Supelcosil C18 reversed-phase column (150 × 4.6 mm inner diameter, Supelco Co., Bellefonte, PA) was used with a flow rate of 1 mL/min. Both the column and sample temperatures were set at ambient conditions. 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 eluted with a binary gradient system: 100−65% A from 0 to 5 min, 65−60% A from 5 to 15 min, 60−0% A from 15 to 20 min, and then 100% A from 21 to 30 min. The injection volume was 50 μL. The wavelength of the UV detector was set at 280 nm with 100 ng/mL as the limit of detection (LOD) and 1 μg/mL as the limit of quantification (LOQ). 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 a LCQ Fleet ion trap mass detector (Thermo Electron, San Jose, CA) incorporated with electrospray ionization (ESI) interfaces. For the analysis of Pg or its main MGO adducts, a 150 × 3.0 mm inner diameter, 5 μm Gemini C18 column (Phenomenex, Torrance, CA) was employed with a flow rate of 0.3 mL/min in the current study. The column was eluted with 100% solvent A (100% water with 0.2% acetic acid) for 5 min, followed by linear increases in B (100% methanol with 0.2% acetic acid) to 30% from 5 to 20 min, 80% from 20 to 40 min, 100% from 40 to 50 min, and then 100% from 50 to 55 min. The column was then re-equilibrated with 100% A for 5 min. As far as phloretin, quercetin, or their major MGO adducts were concerned, a 50 × 2.0 mm inner diameter, 3 μm Gemini C18 column (Phenomenex, Torrance, CA) was used for separation at a flow rate of 0.2 mL/min. The column was 3203
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Figure 1. Chemical structures of gallic acid, pytogallol (Pg), phloroglucinol, pyrocatechol, resorcinol, genistein, daidzein, quercetin, luteolin, epicatechin, phloretin, and apigenin.
Figure 2. Trapping of MGO by gallic acid, pytogallol (Pg), phloroglucinol, pyrocatechol, and resorcinol. eluted with 100% solvent A (100% water with 0.2% acetic acid) for 5 min, followed by linear increases in B (95% aqueous methanol with 0.2% acetic acid) to 20% from 5 to 10 min, 50% from 10 to 20 min, 100% from 20 to 28 min, and then 100% from 28 to 33 min. The column was then re-equilibrated with 100% A for 7 min. The LC eluent was introduced into the ESI interface. The negative-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 arbitrary units and the auxiliary gas at 5 arbitrary units. The structural information of phloretin, quercetin, Pg, and the major MGO adducts was obtained by 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).
B, and C (chalcones are ring C cleavage products). To obtain the essential SAR in trapping dicarbonyl species by dietary flavonoids, we selected some typical sub-components of flavonoids to simplify the influential factors. Five simple phenols, gallic acid, Pg, pyrocatechol, resorcinol, and phloroglucinol (Figure 1), were chosen for our current study. Among these compounds, Pg showed the highest scavenging activity by trapping almost 90% MGO within 24 h of incubation, followed by phloroglucinol, which could trap 60.5% MGO (Figure 2). Resorcinol and pyrocatechol showed relatively lower scavenging activity by trapping 31.6 and 21.8% MGO, respectively (Figure 2). In contrast, gallic acid was found to be the least active agent by trapping only 14.9% MGO within 24 h of incubation, even if it has the same vic-trihydroxyl groups as Pg (Figure 2). MGO Adducts of Pg. To further investigate the MGOtrapping mechanism of Pg, the reaction of Pg and MGO was
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RESULTS Trapping of MGO by Simple Phenols. In general, flavonoids are composed of three different small units, rings A, 3204
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Figure 3. LC chromatogram of reaction mixtures of (A) pyrogallol (Pg) (0.1 mmol) with MGO (0.033 mmol) under a ratio of 3:1 and (C) Pg (0.1 mmol) with MGO (0.1 mmol) under a ratio of 1:1 in PBS (100 mM, pH 7.4) at 37 °C for 3 h. (B) MS/MS spectra of MM−Pg and (D) DM−Pg. (E) Chemical reaction equation of Pg and MGO (Pg = pyrogallol).
Figure 4. Trapping of MGO by genistein and daidzein.
(Figure 3A). This new peak had a molecular ion at m/z 197 [M − H]−, which is 72 mass units (molecular weight of MGO of 72) higher than that of Pg, indicating that it is a mono-MGO
tried at two different ratios (3:1 or 1:1 Pg/MGO). After 3 h of incubation (3:1 Pg/MGO), one major new peak [retention time (RT) of 13.04 min] appeared in the LC chromatogram 3205
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Figure 5. Trapping of MGO by (A) apigenin and luteolin and (B) quercetin, luteolin, and epicatechin.
conjugated pyrogallol (MM−Pg). Its MS2 spectrum displayed a predominant dehydration ion at m/z 179 [M − H2O − H]− (Figure 3B), while one more peak (RT of 13.90 min) was observed besides the peak of MM−Pg (RT of 12.83 min) with Pg and MGO at a 1:1 ratio (Figure 3C). This peak had a molecular ion at 269 [M − H]−, which is 72 mass units higher than that of MM−Pg, suggesting that this product is the diMGO conjugated pyrogallol (DM−Pg). Similarly, its MS2 spectrum also displayed a predominant dehydration ion at m/ z 251 [M − H2O − H]− (Figure 3D). Because of the instability of these adducts, we failed to purify either MM−Pg or DM−Pg from the reaction mixture. However, the proton nuclear magnetic resonance (1H NMR) spectrum (600 MHz in CD3OD) of the crude mixture with MM−Pg as the major component showed only singlet signals for the aromatic ring protons (data not shown), suggesting that MGO was located at C-5 of Pg in MM−Pg (Figures 1 and 3B). The formation of mono- and di-MGO conjugated products of Pg was tentatively proposed in Figure 3E. The identification of position 5 as the active site of Pg further explained why gallic acid has almost no trapping effect of MGO and the B or D ring of EGCG is not the active site to trap MGO. Effects of the A Ring on MGO-Trapping Efficacy. We used phloroglucinol and resorcinol to represent the A-ring structures of flavonoids. Our result that phloroglucinol is a better MGO scavenger than resorcinol indicates that the hydroxyl group on C-5 of the A ring will enhance the trapping efficacy of flavonoids. To test this hypothesis, we compared the MGO-trapping effects of genistein and daidzein, which have Aring structures almost identical to phloroglucinol and resorcinol, respectively. In addition, they have the same Band C-ring structures. Similar to our finding on phloroglucinol
and resorcinol, genistein was more effective than daidzein (Figure 4). At 4 h, genistein could trap almost 90% MGO, whereas daidzein could only trap 54.5% MGO. Our results clearly support our hypothesis that the hydroxyl group on C-5 of the A ring is crucial for the trapping of MGO by flavonoids. This is also supported by our previous observation that glycosylation of the hydroxyl group at C-5 of the A ring of phloretin tended to decrease its activity.27 In addition, glycosylation of the 5-hydroxyl group may increase the steric hindrance of position C-6 and, therefore, decrease its activity. Effects of B and C Rings on MGO-Trapping Efficacy. By comparing the trapping of MGO by apigenin and luteolin (Figure 5A), which possess the same A and C rings but a different number of hydroxyl groups on the B ring, we found that the number of hydroxyl groups on the B ring does not play a significant role on the trapping efficacy of flavonoids, albeit that luteolin with two hydroxyl groups at C-2′ and C-3′ positions shows a slightly stronger trapping capacity than apigenin that has only one hydroxyl group on the B ring (Figure 5A). To understand how the C ring of the flavonoids affects the MGO-trapping capacity, we studied the MGOtrapping effects of quercetin, luteolin, and epicatechin, which have identical A and B rings with a different structure of the C ring. Quercetin and luteolin belong to the flavone with a double bond between C-2 and C-3 and one ketone group at C-4 (Figure 5B). Epicatechin belongs to flavan-3-ol with a hydroxy group on C-3 but with no double bond and a ketone group on the C ring. Quercetin showed the best MGO trapping efficacy, followed by luteolin and epicatechin. More than 90% MGO was trapped within 8 h by both quercetin and luteolin. Epicatechin also had strong trapping efficiency by quenching more than 85% MGO within 24 h. Both quercetin and epicatechin could 3206
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(SIM) mode (Figures 8). Three major new peaks appeared in the LC chromatogram of the reaction mixture of quercetin with MGO at a ratio of 1:6 after 24 h of incubation (Figures 7A). Two of the three peaks had the same molecular ion (m/z 373 [M − H]−) and MS/MS fragments but different RTs (18.34 and 18.86 min). Both of the two compounds had the fragment ion 301 ([M − 72 − H]−), showing that they both lost one MGO (m/z 72) molecule (Figure 8A), thus indicating that they are mono-MGO adducts of quercetin. Furthermore, the tandem mass spectrum of product ion 301 (MS3, m/z 301/ 373) of the two compounds (Figure 8A) was almost identical to the MS2 spectrum of authentic quercetin (MS2, m/z 301), suggesting that the two compounds were the mono-MGO adducts of quercetin. The third peak (16.77 min) showed the molecular ion at m/z 445 [M − H]−, which was 72 mass units higher than that of mono-MGO quercetin, indicating that it was the di-MGO quercetin. The tandem mass spectrum of its product ion 373 (MS3, m/z 373/445) was almost identical to the MS2 spectrum of mono-MGO quercetin (MS2, m/z 373), suggesting that this peak was the di-MGO adduct of quercetin.
rapidly trap MGO; almost 50% MGO was scavenged within 11.25 min. Additive MGO-Trapping Effects by Phloretin and Quercetin. To test whether different flavonoids have additive effects in terms of trapping reactive dicarbonyl species, we investigated the MGO-trapping capability of phloretin, quercetin, or the combination of phloretin and quercetin at two different concentrations (50 and 10 μM) under a ratio of each flavonoid/MGO at 1:6 at pH 7.4 and 37 °C. Our results indicated that phloretin and quercetin trapped about 16 and 23% MGO, respectively, at a concentration of 50 μM within 24 h. About 37% MGO was trapped by the combination of phloretin and quercetin (50 μM each) (Figure 6). At a lower
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DISCUSSION Flavonoids are biologically active, polyphenolic constituents of plant foods and are present in many fruits, vegetables, and beverages (tea, coffee, beer, wine, and fruit drinks), as well as dietary supplements and herbal remedies. The flavonoids are further divided into six major subclasses known as flavanols, flavanones, flavonols, flavones, isoflavones, and anthocyanidins on the basis of the connection of the B ring to the C ring, as well as the oxidation state and functional groups of the C ring.32 All of the major classes of flavonoids are comprised of three sixmembered rings: an aromatic A ring fused to a heterocyclic C ring that is attached through a single carbon−carbon bond to an aromatic B ring. Chalcone, an important precursor in the synthesis of anthocyanin pigments and other flavonoids in plants, is another type of important dietary flavonoid. In contrast to other flavonoids, the middle three carbon atoms in chalcones do not form a closed ring. Several in vivo and in vitro studies have indicated that dietary flavonoids could inhibit the formation of AGEs and prevent diabetes-related complications.25,33,34 Previously, we found three different types of flavonoids, flavanol (EGCG), chalcone (phloretin and phloredzin), and isoflavone (genistein), could rapidly trap MGO at pH 7.4 and 37 °C and, therefore, inhibit the formation of AGEs.27−29 We also demonstrated that the two unsubstituted carbons at the A ring of these flavonoids were the active sites for trapping reactive dicarbonyl species and forming mono- and di-MGO adducts. Most of the flavonoids have the same A-ring structure as EGCG, phloretin, phloridzin, or genistein. It is unclear whether they can also efficiently trap reactive dicarbonyl species to form mono- and di-MGO adducts. More importantly, it is largely unknown whether the number of hydroxyl groups on the A ring and the structures of B or C rings will play an important role in the trapping of reactive dicarbonyl species. To tackle these questions, we conducted the SAR studies of flavonoids as the scavengers of reactive dicarbonyl species. We first compared the trapping efficacy of the subcomponents of flavonoids, gallic acid (gallate ring of EGCG), Pg (B ring of flavonoids), pyrocatechol (B ring of flavonoids), resorcinol (A ring of flavonoids), and phloroglucinol (A ring of flavonoids). Our results showed that the MGO scavenging potency of these sub-components is in the order of Pg >
Figure 6. Tapping of MGO (300 or 60 μM) by phloretin (P), quercetin (Q), and the combination of phloretin and quercetin (P + Q, equal molar combination) at different concentrations (50 or 10 μM) in phosphate buffer (pH 7.4 and 37 °C) at 24 h. Bars on left and right sides represent the concentrations of MGO with 300 or 60 μM, respectively. Data are presented as the means ± standard deviation (SD) of three replications.
concentration (10 μM) with the same ratio, both phloretin and quercetin showed similar trapping capability of scavenging nearly 19 and 23% MGO within 24 h, respectively. Almost 35% MGO was trapped by the combination of phloretin and quercetin (10 μM each) (Figure 6). Our results clearly indicate that different flavonoids can additively trap MGO. Studying the Formation of MGO Adducts of Quercetin and Phloretin by LC/MS. To understand the underlying mechanism that different flavonoids can additively trap MGO, we analyzed the formation of MGO adducts under each flavonoid along or the combination of phloretin and quercetin using LC/MS. Figure 7 shows the LC chromatograms of the reaction mixtures of quercetin with MGO (A), phloretin with MGO (B), and the combination of phloretin and quercetin (C). Both mono- and di-MGO adducts of quercetin were the major products under the ratio of quercetin/MGO at 1:6 (Figure 7A). Similar to our previous observation, monoand di-MGO adducts of phloretin were identified as the major products for phloretin (Figure 7B). Under the combination of phloretin and quercetin, the same MGO adducts of each flavonoid were formed, which further confirmed the additive effect of phloretin and quercetin (Figure 7C). We have confirmed the structures of the mono- and di-MGO adducts of phloretin in our previous study.27 The structural information of the MGO adducts of quercetin was obtained using LC/MS/MS analysis under selective ion monitoring 3207
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Figure 7. LC chromatogram of reaction mixtures of (A) quercetin (50 μM) with MGO (300 μM) under a ratio of 1:6 in PBS (100 mM, pH 7.4) at 37 °C for 24 h, (B) phloretin (50 μM) with MGO (300 μM) under a ratio of 1:6 in PBS (100 mM, pH 7.4) at 37 °C for 24 h, and (C) quercetin/ phloretin with MGO under a ratio of 1:1:6.
phloroglucinol > resorinol > pyrocatechol > gallic acid, indicating the pivotal influence of the number and position of the hydroxyl group on the trapping activity. The trihydroxylbenzene (Pg and phloroglucinol) had a higher reactivity than the dihydroxylbenzene (pyrocatechol and resorcinol). In general, the hydroxyl group has electron-donating property. The more hydroxyl groups on the phenyl ring, the stronger the scavenging activity, because the reaction between phenol and MGO is a nucleophilic reaction. Our results also shown that the addition of a carboxyl group to position 5 of Pg almost completely diminished its trapping activity, indicating that position 5 is the active site of Pg. A further study on the trapping mechanism of Pg confirmed that MGO is conjugated at position 5 of Pg as the mono-MGO adduct of Pg, which provides the answer why gallic acid is the least active compound, albeit it has the same vic-trihydroxyl group as that of Pg. This also resolves the puzzle of our previous observation that the major active site of EGCG is at positions 6 and 8 of the A ring, instead of the B ring and the gallate ring, which have the vic-trihydroxyl group and are generally considered as the active sites for its antioxidant activities.28 In addition, the position of the hydroxyl group also plays a significant role in contributing to the trapping capability of these small phenolic compounds. Pg with three ortho-hydroxyl groups demonstrated a much stronger trapping effect on MGO than phloroglucinol, which contains three meta-hydroxyl groups. To extend our observation on the sub-components of flavonoids to the influence of A, B, or C ring of flavonoids on their MGO-trapping effects, we chose flavonoids containing these specific sub-components for our study. To demonstrate how the number of hydroxyl groups on the A ring of flavonoids affects the MGO-trapping efficiency, genistein and daidzein
were chosen as our model compounds, because they possess identical B and C rings but with a different number of hydroxyl groups on the A ring. Genistein contains one more hydroxyl group at position C-5 than daidzein. Our finding that genistein showed stronger capability and efficacy in trapping of MGO than that of daidzein is in line with the conclusion drawn from phloroglucinol and resorcinol, which represent the A-ring structure of genistein and daidzein, respectively. In the same way, the effect of the number of hydroxyl groups on the B ring in the trapping dicarbonyl species by flavonoids was revealed by comparing the MGO-trapping capacity between apigenin and luteolin. A simple comparison among quercetin, luteolin, and epicatechin was carried out to demonstrate how the C ring of flavonoids affects the MGO-trapping ability. Even these simplified comparisons cannot provide the full picture of the essential SAR between dicarbonyl species and flavonoids; our results can still shed light on the following conclusions: (1) 1,2,3-trihydroxybenzene (Pg) has higher MGO scavenging reactivity than 1,2- and 1,3-dihydroxybenzene, and substitution at position 5 of Pg diminished the scavenging activity, indicating that position 5 is the active site of Pg; (2) the A ring is the active site of flavonoids in contributing the MGOtrapping efficacy, and the hydroxyl group at C-5 on the A ring enhances the trapping efficacy; (3) the double bond between C-2 and C-3 on the C ring could facilitate the trapping efficacy; and (4) the number of hydroxyl groups on the B ring does not significantly influence the trapping efficacy. This is the first study to show that quercetin, luteolin, and apigenin can trap MGO at pH 7.4 and 37 °C, indicating flavonol and flavone types of flavonoids can also serve as scavengers of reactive dicarbonyl species. Further studies on the trapping mechanism of quercetin identified mono- and di3208
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Figure 8. MS/MS spectra of (A) mono-MGO conjugated quercetin and authentic quercetin and (B) di-MGO conjugated quercetin.
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MGO adducts as the major products of the reaction between quercetin and MGO. On the basis of our previous studies of EGCG, phloretin, and genistein, we suspect that the MGO conjugation should occur at positions C-6 and C-8 of the A ring. However, this needs to be further confirmed by NMR analysis of the pure mono- and di-MGO quercetin adducts. Our finding that many dietary flavonoids can trap reactive dicarbonyls through the same mechanism indicates that they may have an additive effect if not a synergistic effect. It has been reported that the total intake of flavonoids in the United States is 189.7 mg/day, which is difficult to be achieved for individual flavonoids.35 Therefore, results on the effectiveness of the combination of individual flavonoids can be applied directly to translational studies in humans. To verify this hypothesis, phloretin and quercetin were chosen as the model compounds. Phloretin is a flavonoid found exclusively in apples and in apple-derived products. We previously reported that phloretin can effectively trap MGO.27 Quercetin was selected because of its wide distribution in dietary plants.36 Our results clearly indicated an additive MGO-trapping effect of these two flavonoids in two different concentrations (Figure 6). Thus, flavonoid-enriched foods and beverages as effective dietary strategies hold great promise to prevent the development of diabetic complications and, therefore, reduce the morbidity and mortality in individuals with diabetes. This topic needs to be further studied, especially under in vivo conditions.
AUTHOR INFORMATION
Corresponding Author
*Telephone: 704-250-5710. Fax: 704-250-5709. E-mail: [email protected]. Author Contributions
† Xi Shao, Huadong Chen, and Yingdong Zhu contributed equally to this work.
Funding
The authors gratefully acknowledge the financial support by United States Department of Agriculture (USDA) Grants 2009-65503-21116 and 2012-67017-30175 to Shengmin Sang. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED AGEs, advanced glycation end products; DM−Pg, di-MGO conjugated pyrogallol; HPLC, high-performance liquid chromatography; LC/MS, liquid chromatrography/mass spectrometry; MGO, methylglyoxal; MM−Pg, mono-MGO conjugated pyrogallol; Pg, pyrogallol; SAR, structure−activity relationship; PBS, phosphate-buffered saline
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