This article has Open Peer Review reports available.
Isoferulic acid prevents methylglyoxal-induced protein glycation and DNA damage by free radical scavenging activity
© Meeprom et al. 2015
Received: 2 July 2015
Accepted: 23 September 2015
Published: 5 October 2015
Isoferulic acid (IFA), a naturally occurring cinnamic acid derivative, is a main active ingredient of the rhizoma of Cimicifuga dahurica. It has been shown various pharmacological activities. The aim of the study was to investigate the effect of IFA against MG-induced protein glycation and oxidative DNA damage. Free radical scavenging activity and the MGO-trapping abilities of IFA were also investigated.
The fluorescent MG-derived AGEs and non-fluorescent Nε-(carboxymethyl) lysine (Nε-CML) was measured using a spectrofluorometer and an enzyme linked immunosorbant assay (ELISA). Protein carbonyl content was used to detect protein oxidation. Gel electrophoresis was used to determine DNA damage. Superoxide anion radicals and hydroxyl radicals were determined using cytochrome c reduction assay and thiobarbituric acid reactive 2-deoxy-D-ribose oxidation products, respectively. The MG-trapping capacity was performed by HPLC.
IFA (1.25–5 mM) inhibited the formation of fluorescent MG-derived AGEs, and Nε-CML, and protein carbonyl in bovine serum albumin. In addition, IFA (0.1–1 mM) also prevented MG/lysine-mediated oxidative DNA damage in the presence and absence of copper ion. The protective ability of IFA was directly correlated to inhibition of hydroxyl and superoxide anion radical generation during the reaction of MG and lysine. Most notably, IFA had no the directly trapping ability to MG.
The present results highlighted that free radical scavenging activity, but not the MG-trapping ability, is the mechanism of IFA for preventing MG-induced protein glycation and DNA damage.
Methylglyoxal (MG), a highly reactive α-oxalaldehyde metabolite, is formed endogenously during glucose, protein and fatty acid metabolism. Other sources of MG, which are formed during industrial processing and long-term storage, are in sugar-containing foods and beverages, such as bread, coffee, honey, wine, and beer . Increased MG levels are possible causal factors for development and progression of diabetes and its complications . MG readily reacts with lysine and arginine residues of protein to produce non-enzymatic protein glycation and subsequent formation of advanced glycation end-products (AGEs), crosslinks like methylglyoxal-lysine dimers, and Nε-(carboxymethyl) lysine (Nε-CML) . The consequences of these reactions alter the characteristics of proteins and their physiochemical and biochemical properties. In vitro experiments have recently shown that reactive oxygen species (ROS) are also generated during the glycation reaction of protein with MG. This results in depletion of thiol-containing protein and an increase in protein carbonyl formation . Besides direct glycation damage to protein, MG reacting with lysine may contribute to oxidative DNA damage, strand breakage and cell apoptosis [5, 6]. Moreover, Cu2+ enhances MG-lysine mediated DNA damage, participating in a Fenton’s-type reaction to produce hydroxyl radicals . ROS-induced oxidative DNA damage has been causally associated with the mechanism of mutagenesis . In this regards, application of AGE inhibitors has emerged as a new strategy to reduce the occurrence of AGE-associated diseases. Recent attention has focused on identification of AGE inhibitors from phytochemical compounds that act as antioxidants, chelate metal ions, or directly trap MG .
Cinnamic acid and its derivatives are widely distributed among fruits and vegetables in the human diet. They exert many biological effects such as anti-inflammatory , anti-oxidation , and anti-hyperglycemic activities . Isoferulic acid (IFA), a naturally occurring cinnamic acid derivative, is a main active ingredient of the rhizoma of Cimicifuga dahurica , which targets multiple pathways associated with antihyperglycemic activity. In vitro and in vivo studies demonstrate that IFA has a plasma glucose-lowering effect in streptozotocin-induced diabetic rats . The mechanism of its action involves activation of α1-adrenoceptors to enhance the secretion of β-endorphin, which can stimulate the opioid μ-receptors [15, 16]. The action leads to increased glucose utilization and reduced hepatic gluconeogenesis. In addition, IFA is the most inhibitor against intestinal α-glucosidase among 11 cinnamic acid derivatives . Most interestingly, IFA acts as an anti-glycating agent against fructose- and glucose-induced protein glycation and oxidation-dependent damage to protein . However, no information exists on the abilities of IFA to inhibit MG-induced protein glycation and DNA damage.
The aim of the present work was to investigate the inhibitory effect of IFA on MG-induced protein glycation and oxidative damage using bovine serum albumin (BSA). Moreover, a glycation model system consisting of lysine and MG together with Cu2+ was created to investigate the ability of IFA to prevent oxidative DNA damage. Furthermore, IFA was evaluated for its free radical scavenging activity in the model of lysine/MG and the capacity in direct trapping of MG using HPLC.
Chemicals and reagents
Methylglyoxal (40 % in water), isoferulic acid (IFA, 3-hydroxy-4-methoxycinnamic acid), bovine serum albumin (BSA, fraction V), aminoguanidine, 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB), 2-deoxy-D-ribose, 2-methylquinoxaline, 5-methylquinoxaline, o-phenylenediamine, thiobarbituric acid and cupric sulfate (CuSO4) were purchased from Sigma-Aldrich (St.Louis, MO, USA). L-lysine, 2,4-dinitrophenyl hydrazine (DNPH) and guanidine hydrochloride were obtained from Himedia (Mumbai, India), Ajax Finechem (Taren Point, Australia) and Fluka (Steinheim, Germany), respectively. OxiSelect™ Nε-(carboxymethyl) lysine (CML) ELISA kit was acquired from Cell Biolabs (San Diego, CA, USA). QIAprep Spin Miniprep kit was obtained from Qiagen (Venlo, Netherlands) and cytochrome c was purchased from Affymetrix (Santa Clara, CA, USA). All other chemicals used were of analytical grade.
Glycation of bovine serum albumin (BSA) by methylglyoxal
The glycated BSA formation assay was modified according to a previously published method . The reaction mixtures (1 mL per reaction) containing 460 μL of methylglyoxal (MG, at final concentration of 1 mM), 500 μL of 20 mg/mL BSA (final concentration: 10 mg/mL) in 0.1 M phosphate buffered saline (PBS, pH 7.4) and 40 μL of IFA at various concentrations (final concentrations: 1.25, 2.5 and 5 mM) or aminoguanidine (AG, final concentration: 1.25 mM) were incubated at 37 °C for 2 weeks. All glycated samples were taken for analysis of fluorescent MG-derived AGEs, non-fluorescent Nε-CML, and carbonyl content.
Measurement of fluorescent MG-derived AGEs
Where FC and FCB were the fluorescent intensity of control with MG and blank of control without MG, FS and FSB were the fluorescent intensity of IFA with MG and blank of IFA without MG.
Measurement of non-fluorescent Nε-CML
Non-fluorescent Nε-(carboxymethyl) lysine (Nε-CML) was measured using an enzyme linked immunosorbant assay (ELISA) kit according to the manufacturer’s instruction. The absorbance of samples was measured immediately at 450 nm and compared with the absorbance of CML-BSA standard provided in the assay kit.
Determination of protein carbonyl content
The carbonyl content in glycated BSA was determined according to a previously published method with slight modifications . Briefly, 10 mM DNPH in 2.5 M HCl (400 μL) was added to 100 μL of glycated samples and incubated for 1 h in the dark. Thereafter, 500 μL of 20 % (w/v) trichloroacetic acid (TCA) was added to precipitate protein for 5 min on ice and then centrifuged at 10,000 g for 10 min at 4 °C. The protein pellet was washed with 1:1 (v/v) ethanol/ethyl acetate mixture three times and resuspended in 250 μL of 6 M guanidine hydrochloride. The absorbance was read at 370 nm. The carbonyl content of each sample was calculated based on the molar extinction coefficient for DNPH (ε = 22,000 M−1 cm−1) and final results were expressed as nmol carbonyl/mg protein.
The pUC19 plasmid was purified from competent Escherichia coli by using QIAprep Spin Miniprep kit according to the manufacturer’s protocol and measured DNA concentration using the NanoDrop-1000 spectrophotometer (Thermo Scientific, MA, USA). The plasmid was kept at −20 °C until use.
DNA strand breakage
Determination of superoxide anion
Superoxide anion was determined by using cytochrome c reduction assay with minor modifications . The reaction mixtures with equal volumes (200 μL) of 50 mM lysine and 50 mM MG (final concentration: 10 mM) with or without 100 μL of IFA (final concentrations: 0.1, 0.25, 0.5 and 1 mM) were adjusted to a total volume of 900 μL before adding 100 μL 300 μM cytochrome c (final concentration: 30 μM) and monitoring the production of superoxide anion by measuring reduced cytochrome c at a wavelength of 550 nm every 10 min until 180 min. The concentration of reduced cytochrome c at each time point was calculated using its molar extinction coefficient (27,700 M−1cm−1) and then subtracting baseline (at 0 min). The results were expressed as nmol/mL.
Determination of hydroxyl radical
Hydroxyl radical was determined by measuring thiobarbituric acid reactive 2-deoxy-D-ribose oxidation products (TBARS) according to a previously published method with minor modifications . The reaction contained equal volumes (20 μL) of 50 mM lysine, 50 mM MG (final concentration: 10 mM) and 100 mM 2-deoxy-D-ribose (final concentration: 20 mM) with or without 20 μL of IFA (final concentrations: 0.1, 0.25, 0.5 and 1 mM). The volume was adjusted to 100 μL using 10 mM PBS before incubating at 37 °C. After 3 h of incubation, the mixture was added to an equal volume of 10 mM PBS (100 μL), 2.8 % (w/v) TCA and 1 % (w/v) thiobarbituric acid (TBA), followed by heating at 100 °C for 10 min, then cooling to room temperature. The degradation of 2-deoxy-D-ribose was measured using a spectrophotometer at a wavelength of 532 nm. The concentration of TBARS was calculated from malondialdehyde (MDA) standard and the results were expressed as nmol/mL.
Determination of the MG-trapping capacity by HPLC
All data are presented as means ± SEM. In the experiment of MG-derived AGEs, two-way ANOVA was evaluated for the significant differences among groups. Other experiments were analyzed the significant differences by one-way ANOVA. Duncan’s post-hoc test was used to examine differences among groups. A p-value < 0.05 was considered statistically significant.
Effect of IFA on the formation of fluorescent MG-derived AGEs and protein oxidation
Effect of IFA on the formation of non-fluorescent Nε-CML and carbonyl content in BSA incubated with MG at week 2
2.97 ± 0.21
0.14 ± 0.02
BSA / MG
5.75 ± 0.50a
1.89 ± 0.11a
BSA / MG / IFA (1.25 mM)
4.03 ± 0.44b
1.34 ± 0.40b
BSA / MG / IFA (2.5 mM)
3.93 ± 0.27b
0.97 ± 0.08b
BSA / MG / IFA (5 mM)
3.37 ± 0.44b
0.90 ± 0.11b
BSA / MG / AG (1.25 mM)
3.62 ± 0.40b
0.41 ± 0.08b
Effect of IFA on MG/lysine-induced DNA strand breakage
Effect of IFA on MG/lysine-induced production of superoxide anion and hydroxyl radical
Effect of IFA on the production of superoxide anion and hydroxyl radical in lysine/MG glycation at the time point of 180 min
Reduced cytochrome c
Lysine / MG
13.79 ± 0.40
0.96 ± 0.03
Lysine / MG / IFA (0.1 mM)
13.10 ± 0.33
0.71 ± 0.03a
Lysine / MG / IFA (0.25 mM)
12.66 ± 0.41
0.58 ± 0.03a
Lysine / MG / IFA (0.5 mM)
11.88 ± 0.49a
0.56 ± 0.03a
Lysine / MG / IFA (1 mM)
10.34 ± 0.30a
0.53 ± 0.04a
Effect of IFA on MG-trapping capacity
The formation of AGEs is classified into three stages: early, intermediate, and late . The reaction between protein and reducing sugars (glucose and fructose) results in Schiff base formation followed by rearrangement to an Amadori product, referred to as the initial stage of glycation. In the intermediate stage, reactive dicarbonyls, particularly 3-deoxyglucosone and methylglyoxal are generated from autoxidation of glucose and the degradation of Amadori products. In the late stage of glycation, irreversible compounds called AGEs are formed through various chemical reactions including direct degradation of Amadori products or Schiff bases, protein modification by dicarbonyl compounds and reactions between Amadori products and AGE precursors. Methylglyoxal (MG) is commonly recognized as the most reactive glycating agent and irreversibly reacts with lysine residues in proteins to form fluorescent crosslinking and non-fluorescent crosslinking AGEs in the last stage of glycation [22, 23]. Our previous findings showed that IFA prevented glucose- and fructose-induced formation of AGE in BSA at the initial stage of glycation resulting in reduced conversion of the initial glycated product to AGEs . In the present study, MG-induced formation of AGEs was also attenuated by IFA at the intermediate stage of glycation. These findings, taken together, suggest that IFA can protect from the initial and intermediate stages of glycation, thus leading to inhibition of the formation of AGEs in the late stage.
Several lines of evidence show that superoxide and hydroxyl radicals can be generated from the reaction between lysine and MG . It has been reported that MG-induced albumin modification generates the cross-linked methylglyoxal dialkylimine radical cation and the enediol radical anion of methylglyoxal during the glycation process [24, 25]. The formation of these intermediates leads to protein cross-linking and formation of radical cation sites on the cross-linked proteins. The presence of trace metal ions (copper and iron ions) enhances hydroxyl radical generation by reacting with hydrogen peroxide (H2O2) through the Fenton reaction . ROS generated from this reaction contributes oxidative modification of protein and DNA . In the present study, evidence of ROS-induced oxidative modifications included the significant increase of protein carbonyl in BSA as well as DNA damage. In addition, the formation of superoxide anion and hydroxyl radicals generated from lysine and MG was confirmed by the observed increase in reduced cytochrome c and TBARS, which was consistent with previous studies [7, 25]. However, when the MG and lysine was incubated with IFA, the increased cytochrome c reduction and TBARS level was attenuated suggesting that IFA scavenges ROS. Considerable interest has been devoted to phytochemical compounds due to their ability to prevent lysine/MG-induced protein glycation and DNA damage by acting as free radical scavengers  and, our present findings indicate that IFA also acts in this manner. Other mechanisms related to the ability to trap MG have been proposed [27, 28] but these results clearly demonstrated that IFA did not directly react with MG, suggesting that carbonyl scavenging activity is not the antiglycation mechanism of IFA. Further experiments are required to investigate the effect of IFA on MG-induced cell toxicity.
The results suggest that the mechanism of IFA for the inhibition of MG-induced protein glycation and DNA damage is free radical scavenging of superoxide anion and hydroxyl radical activity without the MG-trapping ability.
The authors would like to thank the RGJ-PhD program (PHD53K011) of the Thailand Research Fund (TRF) and Chulalongkorn University Fund, and the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphisek Somphot Endowment Fund). This research was supported by National Research University Project, Office of Higher Education Commission (WCU009-HR57) and Ratchadapisek Somphot Fund for Postdoctoral Fellowship, Chulalongkorn University.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Nemet I, Varga-Defterdarović L, Turk Z. Methylglyoxal in food and living organisms. Mol Nutr Food Res. 2006;50(12):1105–17.View ArticlePubMedGoogle Scholar
- Vander Jagt DL. Methylglyoxal, diabetes mellitus and diabetic complications. Drug Metabol Drug Interact. 2008;23(1–2):93–124.PubMedGoogle Scholar
- Poulsen MW, Hedegaard RV, Andersen JM, de Courten B, Bügel S, Nielsen J, et al. Advanced glycation endproducts in food and their effects on health. Food Chem Toxicol. 2013;60:10–37.View ArticlePubMedGoogle Scholar
- Guerin-Dubourg A, Catan A, Bourdon E, Rondeau P. Structural modifications of human albumin in diabetes. Diabetes Metab. 2012;38(2):171–8.View ArticlePubMedGoogle Scholar
- Wu HJ, Chan WH. Genistein protects methylglyoxal-induced oxidative DNA damage and cell injury in human mononuclear cells. Toxicol In Vitro. 2007;21(3):335–42.View ArticlePubMedGoogle Scholar
- Tatone C, Heizenrieder T, Di Emidio G, Treffon P, Amicarelli F, Seidel T, et al. Evidence that carbonyl stress by methylglyoxal exposure induces DNA damage and spindle aberrations, affects mitochondrial integrity in mammalian oocytes and contributes to oocyte ageing. Hum Reprod. 2011;26(7):1843–59.View ArticlePubMedGoogle Scholar
- Kang JH. Oxidative damage of DNA induced by methylglyoxal in vitro. Toxicol Lett. 2003;145(2):181–7.View ArticlePubMedGoogle Scholar
- Tamae D, Lim P, Wuenschell GE, Termini J. Mutagenesis and repair induced by the DNA advanced glycation end product N2-1-(carboxyethyl)-2’-deoxyguanosine in human cells. Biochemistry. 2011;50(12):2321–9.PubMed CentralView ArticlePubMedGoogle Scholar
- Peng X, Ma J, Chen F, Wang M. Naturally occurring inhibitors against the formation of advanced glycation end-products. Food Funct. 2011;2(6):289–301.View ArticlePubMedGoogle Scholar
- Kim EO, Min KJ, Kwon TK, Um BH, Moreau RA, Choi SW. Anti-inflammatory activity of hydroxycinnamic acid derivatives isolated from corn bran in lipopolysaccharide-stimulated Raw 264.7 macrophages. Food Chem Toxicol. 2012;50(5):1309–16.View ArticlePubMedGoogle Scholar
- Natella F, Nardini M, Di Felice M, Scaccini C. Benzoic and cinnamic acid derivatives as antioxidants: structure-activity relation. J Agric Food Chem. 1999;47(4):1453–9.View ArticlePubMedGoogle Scholar
- Choi R, Kim BH, Naowaboot J, Lee MY, Hyun MR, Cho EJ, et al. Effects of ferulic acid on diabetic nephropathy in a rat model of type 2 diabetes. Exp Mol Med. 2011;43(12):676–83.PubMed CentralView ArticlePubMedGoogle Scholar
- Liu IM, Chi TC, Hsu FL, Chen CF, Cheng JT. Isoferulic acid as active principle from the rhizoma of Cimicifuga dahurica to lower plasma glucose in diabetic rats. Planta Med. 1999;65(8):712–4.View ArticlePubMedGoogle Scholar
- Liu IM, Hsu FL, Chen CF, Cheng JT. Antihyperglycemic action of isoferulic acid in streptozotocin-induced diabetic rats. Br J Pharmacol. 2000;129(4):631–6.PubMed CentralView ArticlePubMedGoogle Scholar
- Liu IM, Tsai CC, Lai TY, Cheng JT. Stimulatory effect of isoferulic acid on alpha1A-adrenoceptor to increase glucose uptake into cultured myoblast C2C12 cell of mice. Auton Neurosci. 2001;88(3):175–80.View ArticlePubMedGoogle Scholar
- Liu IM, Chen WC, Cheng JT. Mediation of beta-endorphin by isoferulic acid to lower plasma glucose in streptozotocin-induced diabetic rats. J Pharmacol Exp Ther. 2003;307(3):1196–204.View ArticlePubMedGoogle Scholar
- Adisakwattana S, Chantarasinlapin P, Thammarat H, Yibchok-Anun S. A series of cinnamic acid derivatives and their inhibitory activity on intestinal alpha-glucosidase. J Enzyme Inhib Med Chem. 2009;24(5):1194–200.View ArticlePubMedGoogle Scholar
- Meeprom A, Sompong W, Chan CB, Adisakwattana S. Isoferulic acid, a new anti-glycation agent, inhibits fructose- and glucose-mediated protein glycation in vitro. Molecules. 2013;18(6):6439–54.View ArticlePubMedGoogle Scholar
- Sadowska-Bartosz I, Galiniak S, Bartosz G. Kinetics of glycoxidation of bovine serum albumin by methylglyoxal and glyoxal and its prevention by various compounds. Molecules. 2014;19(4):4880–96.View ArticlePubMedGoogle Scholar
- Sang S, Shao X, Bai N, Lo CY, Yang CS, Ho CT. Tea polyphenol (−)-epigallocatechin-3-gallate: a new trapping agent of reactive dicarbonyl species. Chem Res Toxicol. 2007;20(12):1862–70.View ArticlePubMedGoogle Scholar
- Singh VP, Bali A, Singh N, Jaggi AS. Advanced glycation end products and diabetic complications. Korean J Physiol Pharmacol. 2014;18(1):1–14.PubMed CentralView ArticlePubMedGoogle Scholar
- Li Y, Dutta U, Cohenford MA, Dain JA. Nonenzymatic glycation of guanosine 5’-triphosphate by glyceraldehyde: an in vitro study of AGE formation. Bioorg Chem. 2007;35(6):417–29.View ArticlePubMedGoogle Scholar
- Li Y, Cohenford MA, Dutta U, Dain JA. The structural modification of DNA nucleosides by nonenzymatic glycation: an in vitro study based on the reactions of glyoxal and methylglyoxal with 2’-deoxyguanosine. Anal Bioanal Chem. 2008;390(2):679–88.View ArticlePubMedGoogle Scholar
- Yim HS, Kang SO, Hah YC, Chock PB, Yim MB. Free radicals generated during the glycation reaction of amino acids by methylglyoxal. A model study of protein-cross-linked free radicals. J Biol Chem. 1995;270(47):28228–33.View ArticlePubMedGoogle Scholar
- Suji G, Sivakami S. DNA damage during glycation of lysine by methylglyoxal: assessment of vitamins in preventing damage. Amino Acids. 2007;33(4):615–21.View ArticlePubMedGoogle Scholar
- Wu CH, Yen GC. Inhibitory effect of naturally occurring flavonoids on the formation of advanced glycation endproducts. J Agric Food Chem. 2005;53(8):3167–73.View ArticlePubMedGoogle Scholar
- Shao X, Bai N, He K, Ho CT, Yang CS, Sang S. Apple polyphenols, phloretin and phloridzin: new trapping agents of reactive dicarbonyl species. Chem Res Toxicol. 2008;21(10):2042–50.View ArticlePubMedGoogle Scholar
- Lv L, Shao X, Chen H, Ho CT, Sang S. Genistein inhibits advanced glycation end product formation by trapping methylglyoxal. Chem Res Toxicol. 2011;24(4):579–86.View ArticlePubMedGoogle Scholar