Food Factors in Health Promotion and Disease Prevention - American

Chapter 23

Liquid Chromatography-Mass Spectrometry Method for Fructosylarginine, an Antioxidant in the Aged Garlic Extract 1,2

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Downloaded by CORNELL UNIV on September 5, 2016 | http://pubs.acs.org Publication Date: June 19, 2003 | doi: 10.1021/bk-2003-0851.ch023

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Kenjiro Ryu , Nagatoshi Ide , Makoto Ichikawa , Kozue Ogasawara , and Robert T . Rosen 1

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Center for Advanced Food Technology, Cook College, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901-8020 Healthcare Research Institute, Wakunaga Pharmaceutical Company, Ltd., 1624, Shimokotachi, Koda-cho, Takata-gun, Hiroshima 739-1195, Japan

A liquid chromatography-mass spectrometry (LC/MS) method for fructosylarginine (Fru-Arg) has been developed. Fru-Arg is an antioxidant isolated from aged garlic extract. Fru-Arg is less stable in basic conditions than in acidic or neutral conditions. The half-life of Fru-Arg was 2.6 and 20.5 hours at p H 12.8 and 11.0 respectively. No degradation was observed in a phosphate buffer of pH 4.0 or less within 24 hours. LC/MS method was developed for the direct determination of Fru-Arg in aged garlic extract using an amino column. The results were obtained by the LC/MS method without pre-treatment or extraction of the sample. This new method was demonstrated to be quite useful because of its high sensitivity and speed of analysis. A kinetic study was performed to study the decomposition of Fru-Arg at various pH.

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© 2003 American Chemical Society Shahidi et al.; Food Factors in Health Promotion and Disease Prevention ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


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Maillard reaction products in food are very important not only as the factors for flavor and color but also beneficial constituents for our health. The Maillard reaction is the reaction between amino (e.g. amino acids, peptides and proteins) and carbonyl compounds (e.g. sugars, acids and aldehydes). Since the discovery of the Maillard reaction, much research has been directed to reveal the whole sequence of this complicated reaction (1,2). Some researchers have also been attempting to understand the biological effects of these compounds including toxicity, digestibility and beneficial effects (1,3), It has been reported some Maillard reaction products have antimutagenic, antioxidant, antibiotic and anti-allergenie effects (2). Fructosylarginine (N-a-( 1 -deoxy-D-fructos-1 -yl)-L-arginine, Fru-Arg, chemical structure shown in Figure 1), is an Amadori compound which is the key intermediate in the Maillard reaction (4). Fru-Arg was previously isolated from aged garlic extract (AGE) in the course of searching for antioxidant constituents (5). Fru-Arg has a strong hydrogen peroxide scavenging activity and prevention effect against oxidized LDL-induced damage on endothelial cells. (6). It has been shown that Fru-Arg in A G E is generated by the Maillard reaction between arginine and glucose and increases during the aging process (5). Fru-Arg is also reported to be contained in red ginseng and to suppress the noradrenalin-induced hypertension in in vivo study (7,8). In this study, to examine the chemical character of Fru-Arg, we studied its kinetic proparties at various pH. This kinetic study showed this compound was labile under basic conditions. Also, a liquid chromatography-mass spectrometry (LC/MS) method for Fru-Arg was developed. L C / M S is very usefiil analytical method especially in the biomedical and biochemical area because of its sensitivity and selectivity (9). Vinale et al reported the quantification method for the Amadori compound, fructosyllysine (70), however this is the first report concerning a L C / M S method for Fru-Arg.

Figure L Chemical structure of fructosylarginine.

Shahidi et al.; Food Factors in Health Promotion and Disease Prevention ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Materials

Acetonitrile and water solvents were H P L C grade and were obtained from Fisher (Pittsburgh, PA). Acetic acid (glacial), ammonium acetate, ammonium hydroxide and ethanol were purchased from Fisher (Pittsburgh, PA). Arginine, dipotassium phosphate, disodium phosphate, Dowex 50Wx8, glucose, glucoseC monopotassium phosphate, monosodium phosphate, 2,3,5-triphenyl-2Htetrazolium chloride and trisodium phosphate were purchased from SigmaAldrich (Milwaukee, WI). Aged garlic extracts were supplied by Wakunaga Pharmaceutical Co., Ltd. (Osaka, Japan). The detail description about the aged garlic extract has been published in the U.S. Pharmacopoeia as garlic fluidextract made by soaking sliced garlic in aqueous alcohol for a length of time sufficient to extract the constituents (11). 1 3


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Methods

Synthesis and Purification Fru-Arg was synthesized by the modified method previously reported (7). Arginine 8.7 g (50 mmol) and glucose 18 g (100 mmol) were added into glacial acetic acid 20 mL. The mixture were stirred and heated at 50 °C for 3 hours. After the reaction, the mixture was subjected to ion-exchange column chromatography Dowex50Wx8 in N H / form (Sigma-Aldrich, Milwaukee, WI). After flushing with 1 L of deionized water, crude Fru-Arg was eluted with 1 L of 0.2 M ammonium hydroxide. Crude Fru-Arg was refined using column chromatography with cellulose microcrystalline (Merck, Darmstadt, Germany) followed by Cosmosil 140C18-OPN (Nacalai Tesque, Tokyo, Japan). The purity of the refined Fru-Arg was more than 95% as determined by H P L C using U V detection at 195 nm. Confirmation of purity was obtained using *H and C N M R . Fru-Arg- C , the internal standard, was synthesized in the same way as described above using glucose- C (> 99 atom % C ) . l j

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Kinetic Study 0.2 M phosphate buffers in various p H were prepared by mixing 0.2 M monosodium phosphate, disodium phosphate and trisodium phosphate solution using a p H meter. The p H values of the phosphate buffer used were 2.1, 4.0,


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6.0, 7.0, 7.4, 8.0, 9.0, 11.0, and 12.8. Fru-Arg was dissolved in 0.2 M phosphate buffer at p H 7.4 to make a 10.1 mg/mL solution. A 10 m L of aliquot of 0.2 M phosphate buffer was pre-incubated for 10 min at 37 °C. The experiments were performed at 37 °C and initiated by adding 100 pL of the Fru-Arg solution. A portion of 100 pL was taken from the reaction solution after 0, 1, 2, 4, 6, 8, 12, and 24 hours. The solution was added to 400 pL of the potassium-phosphate buffer ( K H P 0 17.7 g + K H P 0 12.2 g/L in water, which was diluted and used as mobile phase in following H P L C analysis) which was ice-colded. The sample solutions were stored in the deep freezer (-85 °C) until analyzed. The p H value of these buffer solutions was checked before and after the reaction, and remained unchanged up to 24 hour. The H P L C analysis was performed with the same conditions as below. Detection was at 195 nm and there were no interfering peaks or components.


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HPLC Analysis H P L C method was previously reported (5). Two m L of A G E was applied into an ion exchange column containing 4 m L of Dowex 50Wx8 (NH4 " form) resin. After washing with 40 m L of water, the sample solution was eluted with 0.2 M ammonium hydroxide (20 mL). The effluent was appropriately diluted with 0.2 M ammonium hydroxide, i f necessary. The H P L C system was a LC10VP (Shimadzu, Kyoto, Japan) with reaction coil heater: C R B - 6 A (Shimadzu, Kyoto, Japan). H P L C conditions were as follows, column: Shodex Asahipak NH2P-50 4E (Showa Denko K . K . , Tokyo, Japan); mobile phase: acetonitrile-10 m M potassium-phosphate buffer (pH 6.7) (31:19, v/v), 1.0 mL/min; reaction solution: 2,3,5-triphenyl-2H-tetrazolium chloride 2.0 g/L in 100 m M sodium hydroxide-ethanol (1:1, v/v), 0.2 mL/min; reaction coil: 95 °C, 0.5 mm i.d. χ 5 m; detection: absorbance at 480 nm; injection volume: 10 pL; column temperature: ambient. 4

LC/MS Analysis One hundred pL of A G E was added into 400 pL of internal standard solution (Fru-Arg- C approx. 100 pg/mL in 70 % ethanol). After shaking for 30 sec, the mixture was centrifiiged at 14,000 rpm for 15 min. The supernatant was directly injected into the L C / M S . L C / M S conditions were as follows. The H P L C system was a Varian 9012 (Varian, Sugar Land, T X ) and the mass spectrometer a V G Platform II (Micromass, Beverly, M A ) . Ionization mode: ESI positive (selected ion monitoring at m/z 337 for Fru-Arg and 343 for Re labeled internal standard), source temperature: 135 °C, cone voltage: 25 V , 13

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column: Shodex Asahipak NH2P-50 2D (Showa Denko K . K . , Tokyo, Japan); mobile phase: acetonitrile-50 m M ammonium acetate (75:25), flow rate: 0.2 mL/min; injection volume: 5 pL; column temperature: ambient. The column was rinsed with acetonitrile-50 m M ammonium acetate (40:60) after each analysis. The confirmation of identify was carried out at 50 V cone voltage. The m/z 112, 158 and 175 fragment ions were observed at a ratio to the protonated molecular ion (m/z 337) 56 %, 17 % and 29 % respectively.


Results and Discussion

Kinetic Study The relationships between the time and the remaining Fru-Arg at the various p H values were plotted on the logarithmic graph. Each plot was linear. Figure 2 shows the results at p H 11.0 and 12.8. No degradation was observed at p H 4.0 or less. Half-life of Fru-Arg was more than 24 hours at p H 9.0 or less. These results indicated that the degradation reaction of Fru-Arg within the range of p H 6.0 to 12.8 followed pseudo-first order kinetics. Each straight slope drawn in figure 2 allows to calculate the apparent first-order rate constants, k bs> at various pH. The relationship between ko and p H values is plotted in Figure 3. Obviously Fru-Arg is more labile in basic conditions than in neutral and acidic conditions. A s a degradation product, arginine was detected by H P L C (data not shown). This indicated that beta-elimination occurred in the decomposition process. In basic condition, Amadori compounds preferentially undergo 2,3-enolizations rather than 1,2-enolizations (4). Thus, Fru-Arg might decompose into arginine and 1-deoxyglucosone via 2,3-enolization. 0

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L C / M S Method The L C conditions were confirmed based on a H P L C method reported previously (5). B y replacing the modifier in mobile phase into with a volatile salt, the method became amenable for use in a mass spectrometer. Chromatograms of the aged garlic extract and Fru-Arg standard are shown in Figure 4. Due to the high selectivity of the mass spectrometer, any special preparation or purification of injected sample solutions was not necessary. Internal isotopically labeled standardization allowed not only the linear



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Figure 2. First-order plots for the degradation of Fru-Arg in pH 11.0 and 12.8. Each data represents the mean of triplicate studies with standard deviation.

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Food Factors in Health Promotion and Disease Prevention - American

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