Isoferulic acid prevents methylglyoxal-induced protein glycation and DNA damage by free radical scavenging activity

Abstract

Background: 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.

Methods: The fluorescent MG-derived AGEs and non-fluorescent N(ε)-(carboxymethyl) lysine (N(ε)-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.

Results: IFA (1.25-5 mM) inhibited the formation of fluorescent MG-derived AGEs, and N(ε)-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.

Conclusions: 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.

Fig. 1

Effect of IFA on fluorescent MG-derived AGEs at week 1 and 2. The excitation and emission wavelengths were at 355 and 460 nm, respectively. The results are presented as mean ± SEM (n = 3). ^a p <0.05 when compared to BSA at the same week; ^b p <0.05 when compared to BSA/MG at the same week

Fig. 2

Effect of IFA on lysine/MG-induced DNA strand breakage in the absence or presence of Cu²⁺. Gel images express the intensity of open circular (OC) and supercoiled (SC) forms of plasmid DNA after pUC19 (0.25 μg) was incubated with the following treatments: (a) 50 mM lysine, 50 mM MG, 300 μM CuSO₄ or IFA (0.1 and 1 mM). (b) Lysine and MG in the absence or presence of various IFA concentrations (0.1–1 mM) without addition of CuSO₄. (c) Lysine and MG in the absence or presence of various IFA concentrations (0.1–1 mM) co-incubating with CuSO₄. The percentage of OC (% OC, see calculation method in Methods section) in each treatment was subtracted by % OC of untreated DNA before expressing in (d) and (e) which represents data from gel (b) and (c), respectively. The results are presented as mean ± SEM (n = 3). ^a p <0.05 when compared to lysine/MG; ^b p <0.05 when compared to lysine/MG/Cu²⁺

Fig. 3

Effect of IFA on the production of superoxide anion in lysine/MG-induced glycation as measured by cytochrome c reduction within 180 min. The results are presented as mean ± SEM (n = 3)

Fig. 4

The HPLC chromatogram of MG after reaction with IFA or AG at 1 h (a) and 24 h (b). MG was detected as 2-methylquinoxaline (2-MQ) after derivatization using o-phenylenediamine at 315 nm. 5-methylquinoxaline (5-MQ) was used as the internal standard