Anti-glycation, Carbonyl Trapping and Anti-inflammatory Activities of Chrysin Derivatives

The aim of this study was searching anti-glycation, carbonyl trapping and anti-inflammatory activities of chrysin derivatives. The inhibitory effect of chrysin on advanced glycation end-products (AGEs) was investigated by trapping methylglyoxal (MGO), and MGO-conjugated adducts of chrysin were analyzed using LC-MS/MS. The mono- or di-MGO-conjugated adducts of chrysin were present at 63.86 and 29.69% upon 48 h of incubation at a chrysin:MGO ratio of 1:10. The MGO adducted positions on chrysin were at carbon 6 or 6 & 8 in the A ring by classic aldol condensation. To provide applicable knowledge for developing chrysin derivatives as AGE inhibitors, we synthesized several O-alkyl or ester derivatives of chrysin and compared their AGE formation inhibitory, anti-inflammatory, and water solubility characteristics. The results showed that 5,7-di-O-acetylchrysin possessed higher AGE inhibitory and water solubility qualities than original chrysin, and retained the anti-inflammation activity. These results suggested that 5,7-di-O-acetylchrysin could be a potent functional food ingredient as an AGE inhibitor and anti-inflammatory agent, and promotes the development of the use of chrysin in functional foods.


Introduction
Protein glycation (PG) is a non-enzymatic reaction that occurs initially from several reactions between a reducing sugar and a free amino group, representing one of the main pathways related to the development and progression of various diabetic complications such as nephropathy, retinopathy, and neuropathy [1]. Advanced glycation end products (AGEs) are irreversibly produced from the glycation process. The resulting products are unstable and can react with other free amino groups, causing protein modifications including alternative protein half-life and altered immune system and enzyme functions, leading to pathophysiological changes [2]. Intracellular AGEs play important roles as stimuli for activating intracellular signaling pathways, as well as for modifying intracellular protein function, altering receptor recognition, and generating oxidative stress and carbonyl stress [3]. Many studies have revealed the vital role of PG in the pathogenesis of age-related diseases such as diabetes, atherosclerosis, end-stage renal disease, and neurodegenerative disease from synthetic and natural sources [4].

Inhibitory Effects of Chrysin on Amadori Compound and AGE Formation
The CS was preliminarily evaluated by examining Amadori compound and AGE formation, and CS inhibited the formation of both these chemical species. CS displayed dose-dependent inhibition rates at different concentrations, on Amadori compound formation, and AGE formation. The IC 50 values of CS were 17.41 and 24.96 µM ( Figure 1A,B) for Amadori compound and AGE formation, respectively.

Identification of the Chrysin MGO-conjugated Adducts by LC-MS/MS
The CS MGO-conjugated adducts were analyzed from an incubated mixture (48 h) of CS and MGO at a ratio of 1:10. After incubating, two high polarity peaks were detected at 25

Structural Elucidation of the Chrysin Mono-and di-MGO Adducts by NMR
Positions of the CS MGO-conjugated adducts were not confirmed by LC-MS. Therefore, the CS MGO-conjugated adducts were subjected to recycle HPLC with H2O-MeOH (0-25%) as the eluent to give mono-and di-MGO from the incubation mixture (48 h) of CS and MGO at a ratio of 1:10. We analyzed the molecular structure of purified MGO-conjugated adducts using 1 H and 13 C-NMR including HMBC. The 1 H-NMR spectrum of the -mono MGO adduct showed two singlet signals for two protons instead of the three proton signals which were observed in the 1 H-NMR spectrum of -

Identification of the Chrysin MGO-conjugated Adducts by LC-MS/MS
The CS MGO-conjugated adducts were analyzed from an incubated mixture (48 h) of CS and MGO at a ratio of 1:10. After incubating, two high polarity peaks were detected at 25

Structural Elucidation of the Chrysin Mono-and di-MGO Adducts by NMR
Positions of the CS MGO-conjugated adducts were not confirmed by LC-MS. Therefore, the CS MGO-conjugated adducts were subjected to recycle HPLC with H 2 O-MeOH (0-25%) as the eluent to give mono-and di-MGO from the incubation mixture (48 h) of CS and MGO at a ratio of 1:10. We analyzed the molecular structure of purified MGO-conjugated adducts using 1 H and 13 C-NMR including HMBC. The 1 H-NMR spectrum of the -mono MGO adduct showed two singlet signals for two protons instead of the three proton signals which were observed in the 1 H-NMR spectrum of -mono MGO adduct with signals of the MGO group, suggesting that MGO-conjugated with CS at position 8 of the A ring. The 13 C and HMBC spectra were utilized to identify the position of the -mono MGO adduct; a long-range correlation between H-11/C-8 confirmed the attachment of the -mono MGO adduct at C-8 (97.3 ppm). Other useful correlations between H-11/C-7, 9, 12, 13, H-13/C-12, and H-6/C-5, 7 confirmed the position of the attachment (Table 1). The 1 H and 13 C-NMR data confirmed the di-MGO adduct. The data indicated the presence of three proton signals in CS; however, only a single signal proton signal at δH 6.86 (1H, s, H-3) ppm in di-MGO adduct was observed. Likewise, di-MGO adduct proton signals did not affect the five proton of C ring, suggesting that di-MGO-conjugated with CS at position 8 and 6 of the A ring. The di-MGO adducts were assigned the long-range correlation between at δH5.18 (1H, s, H-11)-δC 103.7 (C-8) ppm and δH 5.17 (1H, s, H-14)-δC 103.2 (C-6) ppm in the 8 and 6 positions in A ring by HMBC, respectively. Additionally, a long-range correlation between H-11/C-7, 9, 12, H-13, 16/C-12, and H-14/C-5, 7, 15, 16 reconfirmed the attachment at positions 8 and 6 ( Table 1).

Kinetic Study on the Trapping of the MGO-conjugated Adducts on the Formation of AGEs by Chrysin
Next, we conducted a kinetic study on the trapping of the CS MGO-conjugated adducts using HPLC after incubation of CS with MGO at five different ratios and times. The CS-mono MGO adduct displayed high adduct formation, with ratios of 8% at 1:0.1, 21% at 1:0.5, 30% at 1:1, and 36% at 1:5 within 12 h (Table 2A). a Percentage (%) = peak area of MGO-conjugated adducts produced at various incubation ratios and times by HPLC.
The CS-di MGO adduct increased in a low dose-dependent manner between 0.2-3% with ratios shown in Table 2B. However, the mono-MGO and di-MGO adducts were trapped high 42 and 6% within 12 h in 10 mM MGO, and the trapping efficiency increased to 64 and 30%, respectively, when incubated for 48 h. CS appeared to trap MGO much more efficiently than low MGO concentration when high MGO concentrations occurred in the same system.

Amadori Compound Formation
We compared the abilities of CS and its derivatives to inhibit Amadori compound formation. Among the six compounds, CS showed the strongest inhibitory activity on Amadori compound formation, which was 6.30-fold higher than that of Aminoguanidine hydrochloride (AG) (IC 50 = 109.74 µM). In addition, the inhibitory activity of 7-O-A and 5,7-O-DM on early stage was 4.66and 13.92-fold higher than that of the positive control (Table 3).

AGE Formation
The inhibitory activities of CS and derivatives were assayed against AGE formation, and the results are presented in Table 3. The known AGE inhibitor AG (IC 50 = 136.79 µM) was used as a positive control; CS exhibited 5.48-fold higher activity than that of AG. Among the compounds examined, 7-O-A, 5,7-O-DA, and 5,7-O-DM exhibited strong activity with IC 50 values of 21.61, 0.91, and 41.78 µM, respectively. Especially, 5,7-O-DA showed inhibitory activity at low concentrations (Table 3). In the same manner, we examined the effects of 7-O-M and 7-O-P on AGE formation, however 7-O-M and 7-O-P showed low inhibitory activity on AGE formation (i.e., their IC 50 values exceeded 200 µM).

AGEs Cross-Linking
As shown in Table 3

Anti-Inflammatory Effect of Chrysin Derivatives on RAW 264.7 Cells
The effect of CS and its derivatives on LPS-induced inflammation in RAW 264.7 cells was also investigated, and NO concentration was used as a biomarker to indicate the degree of cellular inflammation. Figure

AGE Formation
The inhibitory activities of CS and derivatives were assayed against AGE formation, and the results are presented in Table 3. The known AGE inhibitor AG (IC50 = 136.79 μM) was used as a positive control; CS exhibited 5.48-fold higher activity than that of AG. Among the compounds examined, 7-O-A, 5,7-O-DA, and 5,7-O-DM exhibited strong activity with IC50 values of 21.61, 0.91, and 41.78 μM, respectively. Especially, 5,7-O-DA showed inhibitory activity at low concentrations (Table 3). In the same manner, we examined the effects of 7-O-M and 7-O-P on AGE formation, however 7-O-M and 7-O-P showed low inhibitory activity on AGE formation (i.e., their IC50 values exceeded 200 μM).

AGEs Cross-Linking
As shown in Table 3

Anti-Inflammatory Effect of Chrysin Derivatives on RAW 264.7 Cells
The effect of CS and its derivatives on LPS-induced inflammation in RAW 264.7 cells was also investigated, and NO concentration was used as a biomarker to indicate the degree of cellular inflammation. Figure

Discussion
Formation of AGEs can be inhibited by interfering with the initial attachment between reducing sugars and amino groups through trapping the carbonyls and radicals formed from the glycation process, or by preventing the formation of intermediate Amadori products and blocking the formation of AGEs at the late stage of glycation [18]. Polyphenols such as stilbenes, anthocyanins, coumarins, and phenolics composed of various structures mainly scavenge MGO by trapping MGO at the active site of the aromatic ring or other positions [19][20][21][22]. Navarro & Morales (2015) reported that the α-carbon of the carbonyl group in the side chain of [6]-shogaol and [6]-gingerol is the major active site for trapping MGO [21]. Stilbenes and phenolics were detected as mono MGO adducts in the aromatic ring [20,21]. However, flavonoid MGO-conjugated adducts form by trapping MGO at the active site in the A ring of the flavonoid skeleton such as luteolin, apigenin, quercetin, kaempferol, and genistein [11,16,23,24].
It was reported that, at a slightly alkaline pH, reactive dicarbonyl intermediates such as MGO can act as nucleophilic chelaters, trappers, and conjugators [25]. Wu and Yen (2005) demonstrated that the inhibition of free radicals generation derived from glycation process was one of the mechanisms of the anti-glycation effect [26]. And Lo et al. (2011) reported the efficiency of phenols for trapping MGO [27]. Therefore, the formation of MGO-conjugated adducts might be promoted at the 6 or 6 & 8 positions as sites for nucleophilic substitutions (A ring) of flavonoids [28]. CS is reactive at the 6 and 8 positions, due to the build-up of electron density at these sites as a result of the presence of the phenol group, as illustrated by the CS-mono adduct in Figure 3.

Discussion
Formation of AGEs can be inhibited by interfering with the initial attachment between reducing sugars and amino groups through trapping the carbonyls and radicals formed from the glycation process, or by preventing the formation of intermediate Amadori products and blocking the formation of AGEs at the late stage of glycation [18]. Polyphenols such as stilbenes, anthocyanins, coumarins, and phenolics composed of various structures mainly scavenge MGO by trapping MGO at the active site of the aromatic ring or other positions [19][20][21][22]. Navarro & Morales (2015) reported that the α-carbon of the carbonyl group in the side chain of [6]-shogaol and [6]-gingerol is the major active site for trapping MGO [21]. Stilbenes and phenolics were detected as mono MGO adducts in the aromatic ring [20,21]. However, flavonoid MGO-conjugated adducts form by trapping MGO at the active site in the A ring of the flavonoid skeleton such as luteolin, apigenin, quercetin, kaempferol, and genistein [11,16,23,24].
It was reported that, at a slightly alkaline pH, reactive dicarbonyl intermediates such as MGO can act as nucleophilic chelaters, trappers, and conjugators [25]. Wu and Yen (2005) demonstrated that the inhibition of free radicals generation derived from glycation process was one of the mechanisms of the anti-glycation effect [26]. And Lo et al. (2011) reported the efficiency of phenols for trapping MGO [27]. Therefore, the formation of MGO-conjugated adducts might be promoted at the 6 or 6 & 8 positions as sites for nucleophilic substitutions (A ring) of flavonoids [28]. CS is reactive at the 6 and 8 positions, due to the build-up of electron density at these sites as a result of the presence of the phenol group, as illustrated by the CS-mono adduct in Figure 3. These nucleophilic sites can attack the aldehyde group of MGO in the system to produce mono-MGO, the target molecule [22,28]. As mentioned above, we demonstrated that the intermediate formation of AGEs could be suppressed by MGO conjugation at the 6 or 6 & 8 active site of the A ring in CS. Consequently, CS in food sources may effectively trap the highly-reactive MGO by forming MGO-conjugated adducts [29].
The low solubility of flavonoids restricts their application in medicine. Therefore, many researchers have sought new methods of modifying their structures to improve their physical and physiological activities [30]. In the present study, we  2010) reported that tetra-acetyl-luteolin exhibits increased precursor content in blood compared to that of luteolin observed upon oral administration [31]. Also, the bioavailability of acetyl-L-carnitine is 43% higher after oral administration of its precursor (L-carnitine (14-18%)) [32]. Especially, CS is quickly metabolized to CS-glucuronide by UDP-glucuronosyltransferase (Phase 2 enzyme), whereas 5,7-O-DA is able to increase bioavailability and can be metabolized slowly by phase 1 enzymes from human liver microsomes [33].
Many AGE inhibitors inhibit AGE formation at different stages of glycation. For example, aspirin (acetylsalicylic acid) can block the attachment between reducing sugars and amino groups by acetylating free amino groups of a protein at the early stage of the glycation process [34]. Vitamin B1 and B6 derivatives are known to scavenge reactive carbonyl compounds, and penicillamine reduces AGE yield by decreasing the formation of Amadori products [34].  These nucleophilic sites can attack the aldehyde group of MGO in the system to produce mono-MGO, the target molecule [22,28]. As mentioned above, we demonstrated that the intermediate formation of AGEs could be suppressed by MGO conjugation at the 6 or 6 & 8 active site of the A ring in CS. Consequently, CS in food sources may effectively trap the highly-reactive MGO by forming MGO-conjugated adducts [29].
The low solubility of flavonoids restricts their application in medicine. Therefore, many researchers have sought new methods of modifying their structures to improve their physical and physiological activities [30]. In the present study, we  (2010) reported that tetra-acetyl-luteolin exhibits increased precursor content in blood compared to that of luteolin observed upon oral administration [31]. Also, the bioavailability of acetyl-L-carnitine is 43% higher after oral administration of its precursor (L-carnitine (14-18%)) [32]. Especially, CS is quickly metabolized to CS-glucuronide by UDP-glucuronosyltransferase (Phase 2 enzyme), whereas 5,7-O-DA is able to increase bioavailability and can be metabolized slowly by phase 1 enzymes from human liver microsomes [33].
Many AGE inhibitors inhibit AGE formation at different stages of glycation. For example, aspirin (acetylsalicylic acid) can block the attachment between reducing sugars and amino groups by acetylating free amino groups of a protein at the early stage of the glycation process [34]. Vitamin B 1 and B 6 derivatives are known to scavenge reactive carbonyl compounds, and penicillamine reduces AGE yield by decreasing the formation of Amadori products [34]. was more effective than CS and other derivatives at interfering with the initial attachment between reducing sugars and amino groups, trapping reactive carbonyl intermediates, and blocking the formation of AGEs. The di-O-acetylation at the same position elevated its inhibitory potency significantly, suggesting that the AGE inhibitory activity of CS is strongly related to the number of O-acetyl moieties. Similarly, 5,7-O-DA was more effective than CS and other derivatives at inhibiting AGE formation, and seemed to have more potential as a glycation inhibitor than AG. Additionally, flavonoids are unstable in the body since they are easily degraded by chemical and enzymatic oxidation. Therefore, it seems that O-acetylation increases the stability of CS by protecting it from chemical and enzymatic oxidation in cells, which may enhance its inhibitory effect on AGE production. In our study, the O-acetylated CS showed a similar, more effective inhibition of NO production compared to CS, which supports our hypothesis. Our data revealed that 5,7-O-DA had excellent AGE inhibition and high solubility, and it may serve as a potent mediator for regulating inflammation in the body. The data suggested that the di-O-acetylation of CS could be effective for preventing AGE formation, and therefore may help prevent and treat diabetes complications.

Determination of MGO Trapping Capacity by HPLC
CS (10 mM) was incubated with MGO (1, 5, 10, 50, or 100 mM) in a PBS buffer (pH 7.4, 10 mL) at 37 • C for 1, 2, 6, 12, 36, or 48 h. The incubated mixture was filtered using a Microcon YM-10 centrifugal filter unit by centrifugation at 5167× g for 30 min at 4 • C. The filtrate was subsequently analyzed by high-performance liquid chromatography (HPLC) using the methods mentioned in the HPLC analysis section. The samples were then stored at −80 • C for further use.

Isolation and Identification of Chrysin MGO-conjugated Adducts Using LC-MS/MS and NMR
MGO-conjugated adducts of chrysin were purified by using a recycle HPLC with a gradient system (0-25%, (MeOH)) as the eluent to obtain CS-mono-MGO adduct (5.14 mg) and CS-di-MGO adduct (4.83 mg). Additionally, isolated MGO-conjugated adducts of chrysin were identified as follows: (1) Liquid chromatography mass spectrometry (LC-MS/MS): The LC eluent was introduced into the ESI interface. The positive ion polarity mode was utilized for the ESI ion source. LC-MS/MS spectrum obtained using a QTRAP 4500 system (AB SCIEX, Darmstadt, Germany) with curtain gas 35 psi, ion spray voltage 5500 volts, source temperature 650 • C, nebulizer gas 55 psi, heater gas 55 psi, and scan range of 100-500 Da; (2) Nuclear magnetic resonance (NMR): Approximately 3.0-5.0 mg of each compound was dissolved in 600 µL of dimethyl sulfoxide (DMSO)-d6 and distributed in 3-mm NMR tubes. 1 H and 13 C-NMR spectra and correlation NMR spectra were obtained using an Avance DPX 400 spectrometer (Bruker, Billerica, MA, USA). Spectra were obtained at operating frequencies of 400 ( 1 H) and 100 MHz ( 13 C) with DMSO-d6, and tetramethylsilane was used as an internal standard.

Hemoglobin-δ-gluconolactone Assay of Amadori Compound Formation
Evaluation of initial stage of PG was determined changing the method described by Hwang et al [18]. Briefly, bovine serum albumin (50 mg/mL) was incubated with glucose (144 mg/mL) in phosphate buffer (pH 7.4) containing 0.2 g/L NaN 3 under sterile conditions in the dark at 37 • C for 48 h. This test was used to evaluate the ability of samples to inhibit cross-linking of the GK peptide in the presence of D-ribose using the method described by Hwang et al [18]. The GK peptide (26.7 mg/mL) was incubated with D-ribose (200 mg/mL) in sodium phosphate buffer (0.5 M, pH 7.4) containing 0.2 g/L NaN 3 under sterile conditions at 37 • C for 72 h. The DMSO used for dissolving samples was found to have no effect on the reaction. All reagents and samples were sterilized by filtration through 0.2 mm membrane filters. The fluorescence intensity was measured at an excitation wavelength of 355 nm and an emission wavelength of 460 nm with a Luminescence spectrometer LS50B (Perkin-Elmer Ltd., Buckinghamshire, England). AG was used as a positive control. The concentration of each test sample exhibiting 50% inhibition of activity (IC 50 ) was estimated from the least squares regression line of the logarithmic concentration plotted against the remaining activity.

Determination of NO Generation and Cell Viability in RAW 264.7 Cells
The cytotoxicity of CS and derivatives on RAW 264.7 cells was examined using the MTS assay kit. Cells (1.6 × 10 4 /well) were cultured in 96-well plates and treated with samples (10, 25, and 100 µM) for 12, 24, 48, and 72 h. After incubation, 20 µL/well of MTS solution was incubated for 90 min at 37 • C in a humidified 5% CO 2 atmosphere. The optical density at 490 nm was measured three times using an EL-800 Universal microplate reader (Bio-Tek Instrument Inc., Winooski, VT, USA). Cell viability of the untreated group was set to 100%. RAW 264.7 cells were seeded into 12-well plates at 4 × 10 5 -cells/well, and then incubated with LPS (1 µg/mL) and various concentrations of samples for 24 h. The concentration of nitric oxide (NO) in the medium was measured using the Griess reagent system, as described by the manufacturer. The production of NO was measured at 570 nm using an EL-800 Universal microplate reader (Bio-Tek Instrument Inc., Winooski, VT, USA), and was compared with a sodium nitrite standard calibration curve [30].

Solubility Analysis
CS and derivatives were dissolved in distilled water and incubated at 37 • C with sonication for 1 h to maximize solubility. After sonication, undissolved samples were eliminated by centrifugation (7000× g, 37 • C, 5 min). The supernatants were diluted in methanol and filtered through a 0.45 µm disposable syringe filter (Advantec, Dublin, CA, USA) to analyze the concentration of the samples using HPLC analysis [30].

Conclusions
In summary, we evaluated the AGE formation inhibitory activities of CS and generated MGO-conjugated adducts by incubating a mixture of CS and MGO. We observed the trapping of reactive carbonyls formed during glycation by LC-MS/MS and NMR spectroscopy. The results showed that CS could efficiently inhibit the initial attachment between reducing sugars and amino groups, as well as suppress reactive carbonyl compound-induced PG. In addition, among the five CS derivatives examined, 5,7-O-DA showed the strongest inhibition of AGE formation at three stages, and exhibited increased water solubility than CS while retaining anti-inflammatory activity. Increased water solubility and inhibitory effects of 5,7-O-DA may provide applicable knowledge for developing inhibitors of AGE formation, and can contribute to the development of functional food sources and beneficial materials from CS. However, physiological studies will be needed for drug development and to validate the use of these compounds as functional food sources. Moreover, research on the AGE inhibitory mechanisms of 5,7-O-DA is needed in the future, and it would be worthwhile to further study whether 5,7-O-DA can decrease the levels of reactive dicarbonyl compounds.
Supplementary Materials: The following are available online. Figure S1: Solubility of chrysin derivatives. Table S1: Dose-dependent inhibition rate of CS.