Trapping of Methylglyoxal by Curcumin in Cell-Free Systems and in

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Trapping of methylglyoxal by curcumin in cell-free systems and in human umbilical vein endothelial cells Te-Yu Hu, Cheng-Ling Liu, Charng-Cherng Chyau, and Miao-Lin Hu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf302188a • Publication Date (Web): 31 Jul 2012 Downloaded from http://pubs.acs.org on August 6, 2012

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Journal of Agricultural and Food Chemistry

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Trapping of methylglyoxal by curcumin in cell-free systems and in human

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umbilical vein endothelial cells

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Te-Yu Hua§, Cheng-Ling Liua§, Charng-Cherng Chyaub,# and Miao-Lin Hua,c,*

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a

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University,Taichung, Taiwan 402

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b

Department of Biotechnology, Hungkuang University, Taichung, Taiwan 433

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c

Agricultural Biotechnology Center, National Chung Hsing University, Taichung,

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Taiwan 402

Department of Food Science and Biotechnology, National Chung Hsing

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§

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#

Te-Yu Hu and Cheng-Ling Liu contributed equally to this work Charng-Cherng Chyau contributed equally as the corresponding author

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* Corresponding author: Miao-Lin Hu, Ph.D., Department of Food Science and

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Biotechnology, National Chung Hsing University, 250 Kuo Kuang Road, Taichung,

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Taiwan, 402 ROC

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Tel: +886-4-2281-2363; Fax: +886-4-2281-2363

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E-mail address: [email protected]

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Or corresponding author: Charng-Cherng Chyau, Research Institute of Biotechnology,

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Hungkuang University, 34, Chung-Chie Rd., Shalu County, Taichung, Taiwan, 433

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ROC

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Tel.: +886-4-2631-8652 x5602

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E-mail address: [email protected]

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Key words: Methylglyoxal, Advanced glycation end products, Curcumin, HUVECs.

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ACS Paragon Plus Environment


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Abstract Curcumin, the most active compound of curcuminoids, has been shown to inhibit

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formation of advanced glycation end products (AGEs) in streptozotocin-induced

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diabetic rats.

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(MGO), a major reactive dicarbonyl compound, to inhibit AGE formation. We

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found that one molecule of curcumin effectively trapped one molecule of MGO at a

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1:3 ratio at 24 h of incubation under physiological conditions (pH 7.4, 37°C).

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Curcumin decreased Nε-(1-carboxyethyl) lysine (CML) expression in human

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umbilical vein endothelial cells (HUVECs).

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analogues, dimethoxycurcumin (DIMC) and ferulic acid, to investigate the possible

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MGO-trapping mechanism of curcumin. Results reveal that DIMC, but not ferulic

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acid, exhibited MGO-trapping capacity, indicating curcumin trap MGO at the

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electron-dense carbon atom (C10) between the two keto carbons groups. Thus,

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curcumin may prevent MGO-induced endothelial dysfunction by directly trapping

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MGO.

However, little is known whether curcumin may trap methylglyoxal

We further used two curcumin

42 43 44 45 46 47 48 49 50 51 52 2


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Introduction Epidemiological studies have indicated that hyperglycemia is the most important

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factor in the diabetic complications, especially in type 2 diabetes mellitus1.

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Hyperglycemia-induced formation of advanced glycation end products (AGEs) is

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considered the main cause of diabetes-related complications, including retinopathy,

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nephropathy, neuropathy and atherosclerosis 2.

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non-enzymatic glycation reaction, also called Maillard reaction, in which the free

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amino group of the protein is irreversibly modified by the carbonyl group of the

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reducing sugars3, followed by an Amadori rearrangement to from the stable Amadori

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product 4.

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important sources of reactive dicarbonyl species (RCOS) 5, 6, which are the pivotal

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intermediates in the formation of AGEs in vivo 7.

AGEs are formed through the

In addition, the autoxidation of glucose and lipid peroxidation are also

A major RCOS in human body is methylglyoxal (MGO), a precursor of AGEs.

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Plasma levels of MGO are elevated in diabetes, dialysis, and chronic kidney disease

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of patients.

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2.2-3.8 µM, whereas the concentration of MGO in healthy volunteers is 0.4-1.0 µM 8.

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MGO can be produced by the glycolysis 9, sorbitol pathway 9, lipid peroxidation 10,

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catabolism of threonine 11 and metabolism of ketone bodies 12.

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irreversibly with lysine and arginine residues of protein to form fluorescent and

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cross-linking AGEs, such as 1,3-di(Nε-lysino)-4-methyl-imidazolium 13, and

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non-fluorescent/non-cross-linking AGEs, including Nε-(1-carboxyethyl)lysine (CML)

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14

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formation though blocking the carbonyl or dicarbonyl groups and thus may prevent

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diabetes and its complications.

77 78

For example, the concentration of MGO in plasma of diabetic patients is

and argpyrimidine 15.

Free MGO can react

Several dietary flavonoids have been shown to inhibit AGE

The principal curcuminoid of the popular Indian spice turmeric, such as curcumin, demethoxycurcumin and bis-demethoxycurcumin, are rich in the root of the 3



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Curcuma longa, a member of the ginger family (Zingiberaceae) 16.

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curcuminoids, curcumin has been shown to have many biological functions, such as

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antioxidant 17, anti-inflammatory 18 and anticancer activities 19 and to act as a potential

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agent against various chronic diseases such as diabetes 20.

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has been shown to inhibit protein glycation in erythrocytes and the formation of AGE

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as well as cross-linking of collagen in diabetic animals 21.

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whether inhibition of AGE formation by curcumin is attributable to its MGO-trapping

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capacity.

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using HPLC and determined the formation of curcumin-MGO adducts using

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HPLC/ESI-MS-MS under physiological conditions (pH 7.4, 37°C).

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employed two curcumin analogues, dimethoxycurcumin (DIMC) and ferulic acid, to

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investigate the possible mechanism of MGO-trapping by curcumin using HPLC

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MS-MS.

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cells as the cell model to determine the effects of curcumin on MGO-trapping

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capacity.

Among these

Importantly, curcumin

However, little is known

In the present study, we investigated the trapping capacity of curcumin

We further

In addition, we used human umbilical vein endothelial cells (HUVECs)

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Materials and Methods

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Materials

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Curcumin (with purity≧ 98%, as claimed by suppler and determined in our

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laboratory using HPLC) was purchased from Tianjin Zhongxin Pharmaceutical

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Research and Design Center (China).

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(2-MQ), 5-methylquinoxaline (5-MQ), 1, 2-diaminobenzene (DB) and ferulic acid

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were purchased from Sigma (St. Louis, MO).

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acentonitrile were purchased from Panreac (Barcelona, Spain) and Li Chrosolv

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(Whitehouse Station, USA).

MGO (1.45 mM), 2-methylquinoxaline

HPLC-grade acetic acid and

DIMC and ferulic acid (with purity≧ 98%, as claimed 4


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by suppler) was obtained from Laila Impex (Vijayawada, India).

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purchased from Biodesign International (Saco, USA)

CML antibody was

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Cell cultures and cell proliferation

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Human umbilical vein endothelial cells (HUVECs) were obtained from Food

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Industry Research and Development Institute (FIRDI, Taiwan) and cultured in M199

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medium (Invitrogen) containing 20% (v:v) FBS, 1.5 g/L NaHCO3, 2 mM L-glutamine,

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30 µg/mL ECGS, penicillin (100 kU/L) and streptomycin (100 kU/L) in a humidified

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incubator under 5% CO2 and 95% air at 37°C.

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using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, as

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described previously 22.

The cell proliferation was measured

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Determination of the MGO-trapping capacity of curcumin and curcumin analogues

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by HPLC The chemical structure of curcumin, DIMC, and ferulic acid is shown in Figure 1.

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A mixture of MGO (1.45 mM) with curcumin (0.48 mM, 0.73 mM, 1.45 mM), DIMC

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(0.48 mM) or ferulic acid (0.48 mM) in phosphate buffer solution (pH 7.4) at 37°C

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was shaken at 40 rpm for 0-72 h.

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carbonyl was allowed to react with DB (30 mM) for 1 h to form stable 2-MQ for

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reliable quantification 23.

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was monitored using HPLC with 5-MQ as internal standard.

127

MGO reduction was calculated using the equation: % reduction = [(amount of MGO

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in control – amount of MGO in test compound)/amount of MGO in control] × 100%

129

24

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chromatography pump and a diode array detector (Hitachi, Japan).

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column Phenomenex® Luna C18 column (4.6 mm ID × 250 mm, 5 µm particle size)

.

Because MGO coeluted with mobile phase, this

The amount of 2-MQ derived from residual MGO and DB The percentage of

The level of MQ was analyzed using HPLC associated with a Hitachi L-7100

5


A reversed-phase


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was used.

HPLC was performed under binary gradient elution, which contained

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mobile phases A (distill water containing 0.2% acetic acid) and B (100% acetonitrile).

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All solvents were filtered through 0.2 µm Nylaflo membrane filter.

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was maintained at 1 mL/min.

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mobile phase B for 9 min and then with 95% mobile phase B for 1 min.

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injection volume was 20 µL for each sample solution, and the wavelength of UV

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detector was set at 313 nm for MQ.

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obtained using MGO as standard.

The flow rate

A linear gradient elution was carried out with 8-95% The

A linear equation y = 0.075x + 0.0051 was

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The formation of MGO adducts of curcumin using HPLC-MS-MS analysis. A mixture of curcumin (0.48 mM) with MGO (1.45 mM) in phosphate buffer

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solution (pH 7.4) was shaken at 40 rpm for 24 h at 37°C, followed by immediate

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analysis using HPLC/ESI-MS-MS.

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spectrometric analysis of curcumin and its related degraded compounds was

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performed as described previously 25, 26 with minor modification.

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curcumin-MGO adducts was conducted on a Phenomenex® Luna C18 column (2.00

148

mm ID× 150 mm, 3.0 µm particle size) using an HPLC system that consisted of a

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Finnigan Surveyor module separation system and a photodiode-array (PDA) detector

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(Thermo Electron Co., MA, USA).

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gradient elution using two solvents: solvent A (water containing 0.1% formic acid)

152

and solvent B (acetonitrile containing 0.1% formic acid).

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elution process was at 0.2 mL/min.

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30-95% B for 30 min and then with 95% B isocratic elution for 5 min.

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absorption spectra of eluted compounds were scanned within 190 to 600 nm using the

156

in-line PDA detector monitored at 230, 280 and 425 nm, respectively.

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compounds having been eluted and separated were further identified with a Finnigan

The HPLC/electrospray ionization (ESI) mass

In brief,

The elution solvent system was performed by

The flow rate during the

A linear gradient elution was carried out with

6


The

The

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LCQ Advantage MAX ion trap mass spectrometer.

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with a negative ionization mode.

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spray needle voltage, 3.5 kV; capillary voltage, −16 V; tube lens offset, −55 V; ion

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transfer capillary temperature, 320°C; nitrogen sheath gas, 45 and auxiliary gas, 5

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(arbitrary units).

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microscans and a maximum ion injection time of 200 ms.

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sample was directly injected into the column using a Rheodyne (model 7725i)

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injection valve.

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was used as the damping and collision gas, the collision energy was 35 V.

167 168 169

Determination of intracellular MGO levels

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The system was operated in ESI

The typical operating parameters were as follows:

Mass spectra were acquired in m/z range of 150-600, with five A volume of 20 µL

In addition, the ESI-MS/MS conditions were as follows: helium

Intracellular MGO levels were analyzed using HPLC, as described previously 27.

171

In brief, the cells were washed twice and scraped into PBS.

After sonication, the

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homogenate was used for MGO level analysis and protein determination. MGO was

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derivatized with DB to form 2-MQ, for which the homogenate was incubated in the

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dark with perchloric acid (0.45 N final concentration) and DB (10 mM final

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concentration) for 24 h at room temperature.

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standard (5-MQ) were measured by an HPLC system, as described above.

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intracellular MGO concentrations were calculated by a standard curve of MGO (0 to

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10 µM).

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(LOQ = 0.73 µM) were obtained based on the equations: LOD = 3.3 × δ/S and LOQ =

180

3.3 × LOD, where δ is the standard deviation of intercept and S is the slope from the

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linear equation of the standard curve: y = 0.075x + 0.0051 (R2 = 0.9999).

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Intracellular MGO levels were expressed as: nmol MGO/mg protein.

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levels were determined by the Coomassie blue (Bradford, Richmond, CA).

The levels of 2-MQ and the internal The

Values of limit of detection (LOD = 0.22 µM) and limit of quantification

184 7


The protein


185 186

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Determination of the protein levels of CML in HUVECs using western blotting Protein expression of CML was measured by western blotting.

In cell culture

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experiments, the medium was removed and cells were rinsed with PBS twice.

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the addition of 0.5 ml of cold RIPA buffer and protease inhibitors, cells were scraped

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and mildly votexed at 0°C for 20 min, and the cell lysates were then subjected to

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centrifugation (10,000 g for 30 min at 4°C).

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supernatant was resolved on SDS-PAGE and transferred onto a PVDF membrane.

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After blocking with TBS buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.4)

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containing 5% nonfat milk, the membrane was incubated with monoclonal antibody

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against human CML, followed by incubation with horseradish peroxidase-conjugated

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anti-goat IgG, and then visualized using an ECL chemiluminescent detection kit

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(Amersham, Sweden). The relative density of the immunoreactive bands was

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quantified by densitometry (Gel Pro Analyzer TM, version 3.0, Media Cybernetics,

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USA).

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Statistical Analysis

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After

An amount of protein (50 µg) from the

All experiments were repeated at least three times.

Values are expressed as

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means ± SD and analyzed using one way ANOVA followed by LSD test for

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comparisons of group means.

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for Windows, version 10 (SPSS, Inc.); a P value < 0.05 is considered statistically

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significant.

206 207 208 209 210 211 212

All statistical analyses were performed using SPSS

Results and Discussion The MGO-trapping capacity of curcumin Recently, some trapping agents of reactive dicarbonyl species from dietary sources, such as tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) 3, apple 8


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polyphenols phloretin and phloridzin 28, cinnamon proanthocyanidins 29,

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phlorotanninsfrom brown algae (Fucus vesiculosus) 30 and stilbene glucoside from

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polygonum multiflorum thunb 31 have been shown to possess MGO-trapping capacity.

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In this study, we found that curcumin significantly and time-dependently trapped

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MGO under physiological conditions up to 24 h (51% reduction, P < 0.05), and the

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effect remained unchanged up to 72 h of incubation (Fig. 2A).

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how many molecules of MGO could be trapped by one molecule of curcumin by

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using different ratios of curcumin to MGO (1:1, 1:2, 1:3).

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molecule of curcumin trapped approximately one molecule of MGO at 1:2 and 1:3

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ratios at 24 h of incubation (Fig. 2B)

We then determined

Results reveal that one

223 224 225

Curcumin-MGO adduct formation We further identified the curcumin-MGO adducts using HPLC/ESI-MS-MS

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after incubation of curcumin with MGO at 1:3 ratio for 24 h.

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three major peaks were observed, with retention times of 11.42, 17.65, 20.05 min,

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respectively.

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retention time of pure curcumin (Fig. 3B) and the molecular ion m/z 367 ([M-H]-)

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(Table 1).

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curcumin was m/z 217 and m/z 175, which are consistent with those of a previous

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study 26.

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the fragment ion m/z 217 ([M-150-H]-) via a β-hydrogen shift to the double bond of

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one of the diketones of deprotonated curcumin 32.

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fragment ion m/z 175 ([M-192-H]-) by ring-closure reaction from deprotonated

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curcumin 26.

237 238

As shown in Fig. 3A,

Data suggest that the peak 3 is curcumin, as evidenced by the same

In addition, the MS/MS spectrum of the molecular ion m/z 367 of

These results suggest that curcumin may lose a neutral moiety to generate

Curcumin may also generate the

Several lines of evidence support that the peak 1 is a curcumin-like compound. First, the molecular ion of peak 1 is m/z 439 ([M-H]-) which may lose one molecule of 9



239

H2O to generate the fragment ion m/z 421 ([M-18-H]-) and subsequently lose a neutral

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moiety to form fragment ion m/z 271 ([M-18-150-H]-) (Table 1), suggesting that peak

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1 undergoes the same degradation as does curcumin.

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absorbance for peak 1 were 429, 275 and 236 nm, which are similar to those of

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curcumin (Table 1).

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which equals the molecular weight of MGO (72 dalton) plus that of curcumin (368

245

dalton) (Table 1).

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adduct.

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because the molecular ion of peak 2 is m/z 549 ([M-H]-) which should lose 180 dalton

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to generate the fragment ion m/z 369 ([M-180-H]-) (Table 1).

249

molecular weight of di-MGO (72×2 =144 dalton) or tri-MGO (72×3 =216 dalton) is

250

different from 180 dalton.

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compound.

Second, the UV maximal

Moreover, the molecular weight of peak 1 was 440 dalton,

Thus, it is reasonable to speculate that peak 1 is a curcumin-MGO

However, peak 2 is unlikely to be di-MGO or tri-MGO adducts of curcumin

In addition, the

Further study is needed to identify peak 2, an unknown

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Curcumin traps MGO by its diketone group, but not by its phenol groups We then used two curcumin analogues, dimethoxycurcumin (DIMC) and ferulic

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acid to determine whether the MGO-trapping site of curcumin is its diketone group or

256

phenol groups.

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one methoxy group, and one hydroxyl groups in each of the two benzene moieties in

258

curcumin (Fig. 1).

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carboxylic group rather than ketone group (Fig. 1).

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0.48 mM ferulic acid with 1.45 mM MGO (i.e., 1:3 ratio) at 37°C for 24 h, we found

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that DIMC reduced the level of MGO by 31% (Fig. 4A), which is equivalent to one

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DIMC molecule trapping one MGO molecule (i.e., 1.45 mM MGO× 31%/ 0.48 mM

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DIMC) (Fig. 4B).

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DIMC are equivalent to those of curcumin (Figs. 4A and 4B).

The structure of DIMC contains two methoxy groups, rather than

The structure of ferulic acid, a half structure of curcumin, has a By reacting 0.48 mM DIMC or

Notably, the reduced MGO level and the MGO-trapping ratio of

10


In contrast, ferulic

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acid did not trap MGO (~0.7%) even though there is a hydroxyl group (C3) in its

266

benzene moiety, which could render C4 a high-electron density carbon under alkaline

267

conditions.

268

and C7) with meta-substitution on the A ring and two unsubstituted carbons (C6 and

269

C8), which can react with MGO owing to the high-electron density upon dissociation

270

of a hydrogen atom from two hydroxyl groups under alkaline conditions 3, 28.

271

EGCG, ferulic acid has only one hydroxyl group in the benzene moiety but the

272

electron density of C4 carbon of ferulic acid may not be strong enough to trap MGO.

273

Thus, the phenol structure of curcumin, like that of ferulic acid, fails to trap MGO at

274

the un-substituted carbons (C4) of the benzene rings.

275

Previous reports have revealed that EGCG has two hydroxyl groups (C5

Another functional group of curcumin is β-diketone group.

Unlike

The two hydrogen

276

atoms on the carbon (C10) between the two keto carbons are quite acidic and can be

277

converted completely to enolate anions under alkaline conditions 33.

278

negative charge on oxygen atom is shared by the carbon atoms between the two

279

β-diketone through resonance, making this carbon atom (C10) a nucleophile.

280

addition, the pKa of hydrogen (~9) on the carbon between the two keto carbons is

281

lower than that (~10) of hydroxyl group of phenol 33, therefore, this carbon between

282

the β-diketone is more prone to losing an H+ ion.

283

the evidence from the literature, we reasonably speculate that the nucleophilicity of

284

carbon atom (C10) between the two keto carbons facilitates the addition of MGO at

285

this position (Fig. 5) to form a mono-MGO adduct of curcumin, by the reaction of

286

curcumin with MGO at 1:3 ratio.

Moreover, the

In

Based on these observations and

287 288 289 290

Curcumin decreases intracellular MGO levels in HUVECs incubated with MGO Curcumin at low concentrations (0-2.5 µM) did not affect cell proliferation at 12 and 24 h of incubation (Fig. 6).

In contrast, curcumin at higher concentrations (5-20 11



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µM) significantly inhibited cell proliferation; for instance, curcumin at 20 µM

292

inhibited proliferation by 55% (P = 0.001) and 57% (P = 0.001) at 12 h and 24 h of

293

incubation, respectively.

294

0.25-2.5 µM curcumin for the following studies.

295

physiological conditions, we further determined the effects of curcumin on

296

MGO-trapping capacity in HUVECs cells.

297

with exogenously added MGO (30 µM) for up to 5 days significantly increased

298

intracellular MGO levels and that this effect was the strongest on the third day of

299

incubation (by 3-fold, P < 0.05), after which the effect weakened slightly at 4-5 days

300

of incubation (Fig. 7A).

301

(0.25-2.5 µM) for 24 h significantly reduced the levels of intracellular MGO induced

302

by exogenous MGO on the third day of incubation (Fig. 7B).

Therefore, we chose a preincubation time of 24 h and Since curcumin trap MGO under

We found that incubation of HUVECs

In addition, pre-treatment of HUVECs with curcumin

Elevated levels of MGO cause carbonyl stress and irreversibly modify lysine

303 304

and arginine residues of protein to form AGEs in vivo 34.

305

MGO-derivatized CML is the most abundant AGEs in proteins 35.

306

exogenous MGO (30 µM) significantly increased protein expression of CML in

307

HUVECs during 5 days of incubation and that the effect was the strongest on the

308

fourth and fifth days of incubation (by 2.1 and 2.3-fold, respectively, P < 0.05) (Fig.

309

8A).

310

the exogenous MGO-induced CML formation in a concentration-dependent manner

311

on the fourth day of incubation (Fig. 8B).

312

trapped MGO in a cell-free system but also decreased the exogenous MGO-induced

313

levels of intracellular MGO and protein expression of CML in HUVECs, indicating

314

that curcumin can inhibit the formation of AGEs through its MGO-trapping capacity.

315 316

It has been shown that We found that

Pre-treatment of HUVECs with curcumin (0.25-2.5 µM) for 24 h ameliorated

Thus, curcumin not only effectively

In conclusion, we have shown that curcumin is an efficient MGO-trapping agent in the cell free system as well as in human umbilical vein endothelial cells 12


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(HUVECs), leading to decreased CML expression.

In addition, we demonstrate that

318

the major active site of curcumin is at the carbon atom (C10) between the two keto

319

carbons, which can trap one molecule of MGO to form a curcumin-MGO adduct.

320

These results support the notion that AGE inhibitors from natural foodstuffs may be

321

potential therapeutic agents for delaying and preventing diabetic complication.

322 323

Acknowledgment This work was supported in part by the Ministry of Education, Taiwan, R.O.C.

324 325

under the ATU plan.

326 327

Reference

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Group. N Engl J Med 1993, 329, (14), 977-86. Brownlee, M.; Cerami, A.; Vlassara, H., Advanced glycosylation end products in tissue and the biochemical basis of diabetic complications. N Engl J Med

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1988, 318, (20), 1315-21. 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

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species. Chem Res Toxicol 2007, 20, (12), 1862-70. Wu, C. H.; Huang, S. M.; Lin, J. A.; Yen, G. C., Inhibition of advanced

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glycation endproduct formation by foodstuffs. Food Funct 2011, 2, (5), 224-34. Shibamoto, T., Analytical methods for trace levels of reactive carbonyl compounds formed in lipid peroxidation systems. J Pharm Biomed Anal 2006, 41, (1), 12-25.

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Group, T. D. C. a. C. T. R., The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. The Diabetes Control and Complications Trial Research

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Aldini, G.; Dalle-Donne, I.; Facino, R. M.; Milzani, A.; Carini, M., Intervention strategies to inhibit protein carbonylation by lipoxidation-derived reactive carbonyls. Med Res Rev 2007, 27, (6), 817-68. Beisswenger, P. J.; Howell, S. K.; Nelson, R. G.; Mauer, M.; Szwergold, B. S., Alpha-oxoaldehyde metabolism and diabetic complications. Biochem Soc Trans 2003, 31, (Pt 6), 1358-63. 13



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Khuhawar, M.; Kandhro, A.; Khand, F., Liquid chromatographic

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determination of glyoxal and methylglyoxal from serum of diabetic patients

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using meso-stilbenediamine as derivatizing reagent. Analytical letters 2006, 39, (10), 2205-2215. Kalapos, M. P., Methylglyoxal in living organisms: chemistry, biochemistry,

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toxicology and biological implications. Toxicol Lett 1999, 110, (3), 145-75. Turk, Z., Glycotoxines, carbonyl stress and relevance to diabetes and its

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complications. Physiol Res 2010, 59, (2), 147-56. Lyles, G. A.; Chalmers, J., The metabolism of aminoacetone to methylglyoxal by semicarbazide-sensitive amine oxidase in human umbilical artery. Biochem Pharmacol 1992, 43, (7), 1409-14. Casazza, J. P.; Felver, M. E.; Veech, R. L., The metabolism of acetone in rat. J Biol Chem 1984, 259, (1), 231-6. Frye, E. B.; Degenhardt, T. P.; Thorpe, S. R.; Baynes, J. W., Role of the Maillard reaction in aging of tissue proteins. Advanced glycation end product-dependent increase in imidazolium cross-links in human lens proteins. J Biol Chem 1998, 273, (30), 18714-9. Ahmed, M. U.; Brinkmann Frye, E.; Degenhardt, T. P.; Thorpe, S. R.; Baynes, J. W., N-epsilon-(carboxyethyl)lysine, a product of the chemical modification

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of proteins by methylglyoxal, increases with age in human lens proteins.

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Biochem J 1997, 324 ( Pt 2), 565-70. Shipanova, I. N.; Glomb, M. A.; Nagaraj, R. H., Protein modification by

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methylglyoxal: chemical nature and synthetic mechanism of a major

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fluorescent adduct. Arch Biochem Biophys 1997, 344, (1), 29-36. Aggarwal, B. B.; Sundaram, C.; Malani, N.; Ichikawa, H., Curcumin: the

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Indian solid gold. Adv Exp Med Biol 2007, 595, 1-75. Reddy, A. C.; Lokesh, B. R., Effect of dietary turmeric (Curcuma longa) on

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iron-induced lipid peroxidation in the rat liver. Food Chem Toxicol 1994, 32, (3), 279-83. Zhang, F.; Altorki, N. K.; Mestre, J. R.; Subbaramaiah, K.; Dannenberg, A. J., Curcumin inhibits cyclooxygenase-2 transcription in bile acid- and phorbol ester-treated human gastrointestinal epithelial cells. Carcinogenesis 1999, 20, (3), 445-51. Duvoix, A.; Morceau, F.; Delhalle, S.; Schmitz, M.; Schnekenburger, M.; Galteau, M. M.; Dicato, M.; Diederich, M., Induction of apoptosis by curcumin: mediation by glutathione S-transferase P1-1 inhibition. Biochem Pharmacol 2003, 66, (8), 1475-83. Suryanarayana, P.; Saraswat, M.; Mrudula, T.; Krishna, T. P.; Krishnaswamy, 14


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diabetic cataract in rats. Invest Ophthalmol Vis Sci 2005, 46, (6), 2092-9. Jain, S. K.; Rains, J.; Jones, K., Effect of curcumin on protein glycosylation, lipid peroxidation, and oxygen radical generation in human red blood cells

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from Polygonum multiflorum Thunb.: a novel natural inhibitor of advanced

427

glycation end product formation by trapping of methylglyoxal. J Agric Food

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32.

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33.

Marye A. Fox, J. k. W., Organic chemistry. 3 rd ed.; Jones & Bartlett Learning:

34.

2004. Desai, K. M.; Wu, L., Free radical generation by methylglyoxal in tissues.

35.

Drug Metabol Drug Interact 2008, 23, (1-2), 151-73. Huang, S. M.; Hsu, C. L.; Chuang, H. C.; Shih, P. H.; Wu, C. H.; Yen, G. C., Inhibitory effect of vanillic acid on methylglyoxal-mediated glycation in apoptotic Neuro-2A cells. Neurotoxicology 2008, 29, (6), 1016-22.

441 442 443

16


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444

Table 1.

445

Retention time, UV-Vis and Mass spectral characteristics for curcumin and the related degradation compounds1 Compound

Rt (min)2

UV λmax (nm)

MW3

1

Curcumin-MGO

11.42

425, 275, 236

2

Unknown

17.65

3

Curcumin

20.05

Peak

(-) ESI-MS (m/z)4

(-) ESI-MS2 (m/z, Rel %)

440

439

421(100), 271(57)

455, 170, 246 sh, 299

550

549

429, 266, 237

368

367

491(100), 327(53), 369(38) 217(100), 175(48)

446 447

1.

After incubation of curcumin (0.48 mM) with MGO (1.45 mM) in phosphate buffer solution (pH 7.4) at 37°C for 24 h, the samples were immediately analyzed using HPLC/ESI-MS-MS.

448 449

2.

RT: retention time

450

3.

MW: molecular weight

451

4.

ESI-MS: electrospray ionization mass spectrometry

452 453 454

17



Page 18 of 28

455

Legends of figures

456

Figure 1. The structure of curcumin, dimethoxycurcumin (DIMC) and ferulic acid.

457 458

Figure 2. Methylglyoxal (MGO)-trapping capacity of curcumin under physiological

459

conditions (pH 7.4, 37°C).

460

mM) for up to 72 h of incubation (A). Effect of curcumin at different ratios of

461

curcumin to MGO (1:1, 1:2 and 1:3) on MGO-trapping capacity (B).

462

(0.48 mM, 0.73 mM,1.45 mM) was incubated with MGO (1.45 mM) for 1-24 h.

463

Values are means ± SD, n = 3; means without a common letter differ, P < 0.05.

Curcumin (1.45 mM) was incubated with MGO (1.45

Curcumin

464 465

Figure 3. HPLC/UV chromatogram of the product of curcumin incubated with

466

MGO at 1:3 ratio for 24 h (A) and pure curcumin (B).

467

compounds correspond to peaks 1-3 are marked.

The curcumin-related

468 469

Figure 4. Effect of curcumin analogues on Methylglyoxal (MGO)-trapping capacity

470

under physiological conditions (pH 7.4, 37°C).

471

capacity of curcumin, DIMC and ferulic acid under physiological conditions (pH 7.4,

472

37°C).

473

37°C for 24 h of incubation.

474

common letter differ, P < 0.05.

(A) Percentage MGO-trapping

(B) Molecular ration of MGO-trapping capacity of curcumin and DIMC at Values are means ± SD, n = 3; means without a

475 476

Figure 5. Proposed formation pathway of curcumin-MGO adducts under

477

physiological condition (pH 7.4, 37°C).

478 479

Figure 6. Effect of curcumin (0-20 µM) on cell proliferation in human umbilical 18


Page 19 of 28


480

vein endothelial cells (HUVECs) incubated for 12 and 24 h. Values are means ± SD,

481

n = 3; means without a common letter differ significantly, P < 0.05.

482 483

Figure 7. Intracellular methylglyoxal (MGO) in HUVECs incubated with MGO (30

484

µM) at 37°C for 0-5 day. (B) The level of intracellular MGO in HUVECs pretreated

485

with curcumin (0.25-2.5 µM) for 24 h followed by incubation with MGO (30 µM)

486

for 3 day. Values are means ± SD, n = 3; means without a common letter differ

487

significantly, P < 0.05.

488 489

Figure 8. Effect of Methylglyoxal (MGO) (30 µM) on the protein level of

490

Nε-(1-carboxyethyl)lysine (CML) in human umbilical vein endothelial cells

491

(HUVECs) during incubation for 5 day (A). Effect of curcumin pretreatment (24 h)

492

followed by incubation with MGO (30 µM) for 4 day on protein level CML in

493

HUVECs (B). Values are means ± SD, n = 3; means without a common letter differ

494

significantly, P < 0.05.

495 496 497 498 499 500 501 502 503 504 505 19



506

Figure 1

507 O

508

7

1

H3CO

O 9 10

8

509

13

11

15

3

12 17

5

HO

16 OCH3

14

6

2

19

4

18

Curcumin

510

OH

511 O

512

7

1

H3CO

O 9 10

8

513

3

H3CO

13

11

15 14

6

2

16 OCH3

12 17

5

19 18

4

OCH3

Dimethoxycurcumin

514 515

O

516 H3CO

7

1

OH

8

517 HO

518

9

6

2

5

3 4

Ferulic acid

519 520 521 522 523 524 525 526 527 528 529 530 531 20


Page 20 of 28

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532

Figure 2

533

(A)

534 535 536 537 538 539

541 542 543 544 545 546 547

(B) 1.4

MGO reduction (mol)/Curcumin (mol)

540

Cur:MGO-1:1 Cur:MGO-1:2 Cur:MGO-1:3

1.2 1.0 0.8 0.6 0.4 0.2 0.0 1

12

6

548 549 550 551 552 553 554 555 556 557 21


24

(h)


558

Figure 3

559

(A)

560 561

(B)

562 563

22


Page 22 of 28

Page 23 of 28


564 565

Figure 4

566

(A)

567 568 569 570 571 572 573

(B)

574 575

577 578 579

MGO reduction (mol)/Curcumin (mol)

576

1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 C urcu m in

D IM C

23



580

Page 24 of 28

Figure 5. O

581 582

H3CO 2

583

3

O 13

7

1 6

9

11

8 HO

584

16 OCH3

12 17

5 4

15 14

10

19 18

Curcumin

OH

585 586 587 O

O

588 H3CO

OCH3

589 H

590

H

HO

OH

OH-

591 592 593 594

O-

O

H3CO

OCH3 H H

595 596

O

HO O

597

OH

MGO

598 599 O

600 601

O

H3CO

OCH3

602 OH

HO

603 O

604 605

Curcumin-MGO adduct

606 607 608 609 610 611 24


OH

Page 25 of 28


612

Figure 6

613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 25



645

Figure7

646

(A)

647 648 649 650 651 652 653 654 655 656 657 658

(B)

659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 26


Page 26 of 28

Page 27 of 28


678 679

Figure 8 (A)

680 681 682 683 684 685 686 687 688 689 690 691 (B)

27



692

Page 28 of 28

TOC Graphic

693 O H3CO 2

O 13

7

1 6

9

11

8 3 HO

16 OCH3

12 17

5 4

15 14

10

19 18

Curcumin

O

OH

O

H3CO

OCH3 H

H

HO

OH

OH-

O-

O

H3CO

OCH3 H H O

HO O

O

OH

MGO

O

H3CO

OCH3

OH

HO O

Curcumin-MGO adduct

28


OH

Trapping of Methylglyoxal by Curcumin in Cell-Free Systems and in

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