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Supplement: Recent Advances on the Nutritional Effects Associated with the Use of Garlic as a Supplement

N-(1-Deoxy-D-fructos-1-yl)-L-Arginine, an Antioxidant Compound Identified in Aged Garlic Extract¹

Institute for OTC Research, Wakunaga Pharmaceutical Company, Hiroshima 739-1195, Japan

²To whom correspondence should be addressed. E-mail: ryu-k{at}wakunaga.co.jp.

ABSTRACT

Aged garlic extract (AGE) has been shown to have antioxidant activity. The organosulfur compounds, S-allyl-L-cysteine and S-allylmercapto-L-cysteine, are responsible, at least in part, for the antioxidant activity of AGE. To identify major active components, we fractionated AGE, using hydrogen peroxide scavenging activity as an antioxidative index. Strong activity in the amino acid fraction was found and the major active compound was identified as N-(1-deoxy-D-fructos-1-yl)-L-arginine (Fru-Arg). Antioxidant activity of Fru-Arg was comparable to that of ascorbic acid, scavenging hydrogen peroxide completely at 50 µmol/L and 37% at 10 µmol/L. Quantitative analysis using the established HPLC system revealed that AGE contained 2.1–2.4 mmol/L of Fru-Arg, but none was detected in either raw or heated garlic juice. Furthermore, it was shown that a minimum of 4 mo aging incubation was required for Fru-Arg to be generated. These findings indicate that the aging process is critical for the production of the antioxidant compound, Fru-Arg. These results may explain some of the variation in benefits among different commercially available garlic preparations.

KEY WORDS: • aged garlic extract • N-(1-deoxy-D-fructos-1-yl)-L-arginine • fructose-arginine • garlic • Maillard reaction • antioxidant • HPLC

Garlic has been reported to have antioxidant activities that may contribute to the observed benefits reported for its various preparations (Kourounakis and Rekka 1991 ). It has been suggested that ascorbic acid (vitamin C), a strong antioxidant, may reduce atherosclerosis (Duell 1996 ) and some kinds of cancer (van Poppel and van den Berg 1997 ). These findings suggest that antioxidant activity may be important for maintaining health as well as providing a therapy for ailments. There are some reports of antioxidant agents in garlic, including several sulfur-containing compounds such as alliin, diallylsulfides and allicin (Kourounakis and Rekka 1991 , Horie et al. 1992 , Prasad et al. 1995 ). The activities of these compounds may account in part for some of the effects of garlic. Recently, a garlic fluid extract, aged garlic extract (AGE),³ was shown to have antioxidant activities, including scavenging of active oxygen species (Imai et al. 1994 ) and inhibition of lipid peroxidation (Ohnishi and Kojinra 1997 ). S-allyl-L-cysteine and S-allylmercapto-L-cysteine were proposed to be responsible in part for the antioxidative activity of AGE (Ide et al. 1996 ). However, other compounds may also be involved.

The amino-carbonyl reaction, namely, the Maillard reaction, is a nonenzymatic reaction between reducing sugars and amino acids. Various kinds of compounds are generated through this reaction, some of which are known to possess antioxidant activity (Friedman 1996 ). AGE probably contains Maillard reaction products because garlic contains considerable amounts of amino acids and reducing sugars (Ohsumi and Hayashi 1994 , Ueda et al. 1991 ), and the garlic is extracted/aged in aqueous ethanol for several months. We focused, therefore, on Maillard reaction products and characterized the antioxidant activity of the amino acid fraction of AGE by examining hydrogen peroxide scavenging activity. Finally, we isolated and identified a Maillard reaction product, N-(1-deoxy-D-fructos-1-yl)-L-arginine (Fru-Arg), as a major antioxidant constituent. Here, we describe the antioxidant activity of Fru-Arg, establish a HPLC system for quantitative analysis and suggest a possible mechanism for its generation.

MATERIALS AND METHODS

Chemicals.

Ascorbic acid (Wako Pure Chemical, Osaka, Japan), horseradish peroxidase (Boehringer Mannheim, Mannheim, Germany), hydrogen peroxide (Wako Pure Chemical), 2,2'-azino-di-[3-ethylbenzthiazoline sulfonate (6)] (ABTS) (Boehringer Mannheim) and 2,3,5-triphenyl-2H-tetrazolium chloride (Kishida Chemicals, Osaka, Japan) were purchased commercially. Ninhydrin reagent was prepared by dissolving 0.2 g of ninhydrin in 10 mL of water. Naphthoresorcinol-phosphoric acid reagent was prepared by dissolving 0.05 g of naphthoresorcinol in 25 mL of ethanol followed by the addition of 2.5 mL of phosphoric acid.

Sample preparation.

Aged garlic extract (AGE) was manufactured by Wakunaga Pharmaceutical as follows: briefly, garlic cloves (Allium sativum L.) were sliced and soaked in a water/ethanol mixture, naturally extracted/aged for > 10 mo at room temperature and then concentrated. AGE used in this study contained 28% solid extracts. Raw (RGJ) and heated garlic juice (HGJ) were prepared as follows: raw garlic cloves were homogenized with an equivalent volume of water in a Waring blender. The homogenate was filtered through gauze and the filtrate was obtained (RGJ). HGJ was prepared in the same way as RGJ, except that garlic cloves were heated in an equivalent volume of water at 85°C for 30 or 360 min before homogenization.

Isolation of Fru-Arg.

The process for isolating Fru-Arg is summarized in Figure 1 . AGE (5 L) was partitioned between ethyl acetate and water. The water layer was fractionated by reversed-phase column chromatography using a MCI gel CHP20P (Mitsubishi Chemical, Tokyo, Japan). The water effluent was subjected to ion-exchange column chromatography using a Dowex50Wx8 H⁺ form resin (Dow Chemical, Midland, MI). The fraction eluted with 2 mol/L aqueous ammonium was concentrated to obtain the amino acid fraction. The fraction was subjected to ion-exchange column chromatography using an Amberlite IRA-405 AcO^- form resin (Rahm & Haas, Philadelphia, PA). The resultant fraction containing neutral and basic amino acids, eluted with water, was further fractionated by ion-exchange column chromatography on a Dowex50Wx8 NH₄+ form. The fraction eluted by 2 mol/L aqueous ammonium was concentrated and then fractionated by column chromatography on cellulose microcrystalline (Merck, Darmstadt, Germany) with n-butanol/acetone/diethylamine/water (10:10:2:5) as an eluent. Then, reversed-phase column chromatography on a Cosmosil 75C18 (Nacalai Tesque, Kyoto, Japan) was performed. Finally, Fru-Arg was isolated by ion-exchange column chromatography on a Dowex50Wx8 NH₄+ form with 0.2 mol/L aqueous ammonium. The fractionations described above were directed by TLC analysis with ninhydrin reagent and naphthoresorcinol-phosphoric acid reagent as indicators for amino acid residue and reducing sugar moiety, respectively. Fru-Arg was a yellowish powder and 700 mg was obtained from 5 L of AGE (0.014% wt/v solid extract).

View larger version (52K): [in this window] [in a new window]   Figure 1. Isolation scheme of Compound A from aged garlic extract (AGE); 700 mg of Compound A was obtained from 5 L of AGE (yield 0.014%).

Fru-Arg was synthesized by the method previously reported (Matsuura et al. 1994 ). Arginine (0.88 g) and glucose (1.8 g) were dissolved in 10 mL of glacial acetic acid for 2 h at 80°C with stirring. After the mixture was concentrated to dryness, it was subjected to ion-exchange column chromatography (Dowex50Wx8, NH₄+ form). After a water wash, Fru-Arg was eluted from the column with 0.2 mol/L aqueous ammonium. By concentrating the ammonium effluent, crude Fru-Arg (0.9 g) was obtained. The crude Fru-Arg was further purified by a series of chromatographic techniques.

Hydrogen peroxide scavenging assay.

The efficacy of scavenging hydrogen peroxide was measured as previously described (Okamoto et al. 1992 ). Samples were dissolved in 1.8 mL of 0.1 mol/L phosphate buffer (pH 6.0) in appropriate concentrations; 0.15 mL of 0.03 mmol/L hydrogen peroxide, 0.6 mL of horseradish peroxidase (6 U) and 0.6 mL of 0.1% ABTS were added to 1.8 mL of the sample solutions and incubated at 37°C for 15 min. The reaction mixtures were then incubated at room temperature for 15 min and the absorbance at 414 nm was measured.

HPLC analysis for determination of Fru-Arg content.

AGE, RGJ or HGJ (2 mL) was applied into an ion-exchange column containing 4 mL of Dowex50Wx8 NH₄+ form resin. After being washed with 40 mL of water, the sample solution was eluted by 0.2 mol/L aqueous ammonium (20 mL). The effluent was appropriately diluted with 0.2 mol/L aqueous ammonium, if necessary. HPLC analysis was performed according to the previously reported method (Reutter and Eichner 1989 ). We used a LC6A HPLC system (Shimadzu, Kyoto, Japan) with a reaction coil heater, BF400 (Yamato, Tokyo, Japan). HPLC conditions were as follows: column, Shodex Asahipak NH2P-50 4E (Showa Denko, Tokyo, Japan); mobile phase, acetonitrile-phospate buffer (6:4), [phosphate buffer, pH 7.0 (K₂HPO₄, 0.92 g + KH₂PO₄, 0.64 g)], 1.0 mL/min; reaction solution, 2,3,5-triphenyl-2H-tetrazolium chloride 2.0 g/(0.05 mol/L NaOH in 50% EtOH 1 L), 0.2 mL/min; reaction coil, 75°C, 0.5 mm i.d. x 4 m; detection, absorbance at 480 nm (W lamp); injection volume, 10 µL; column temperature, room temperature.

Other analyses.

TLC analyses were performed with Silica gel 60 F₂₅₄ (Merck) developed by n-BuOH/acetic acid/water (2:1:1), and spots were visualized by spraying ninhydrin reagent or naphthoresorcinol-phosphoric acid reagent. The arginine contents were determined by amino acid analyses using a standard method amino acid analyzer L-8500 (Hitachi, Tokyo, Japan). The solid extract contents were measured by FD-230 (80°C, 94 min; Kett Electric Laboratory, Tokyo, Japan). pH values were measured by pH meter F-22 (Horiba, Kyoto, Japan) at 25°C. The glucose contents were determined by a commercial kit according to the manufacture’s instructions (# 139 106; Boehringer Mannheim).

Spectroscopy.

Spectra were recorded by the following instruments: ¹H and ¹³C nuclear magnetic resonance (NMR). Spectra were recorded in D₂O on a JNM-ECP500 spectrometer (JEOL, Tokyo, Japan), operating at 500 and 126 MHz. A fast atom bombardment mass spectrum (FAB-MS) was obtained in the negative ion mode on a GC mate mass spectrometer (JEOL) (accelerating voltage, 1667 V; xenon atom beam, JEOL) with glycerol as matrix.

RESULTS

Isolation and identification of an antioxidative compound, Fru-Arg.

We fractionated AGE to characterize antioxidant constituents using hydrogen peroxide scavenging activity as an index. The isolation processes are summarized in Figure 1 . First, AGE was divided into water-soluble and water-insoluble fractions by solvent partition between water and ethyl acetate. Approximately 750 g of water-soluble extract and 0.3 g of water-insoluble extract were obtained from 5 L of AGE. Then, the water-soluble fraction was subdivided into five fractions using reversed-phase chromatography. The fraction containing high polar components was divided into sugar and amino acid fractions. The amino acid fraction was further fractionated using ion-exchange chromatography. Major activity was found in the basic amino acid fraction; finally, a single substance, designated Compound A, was isolated.

TLC analyses revealed that Compound A is positive for both ninhydrin and naphthoresorcinol-phosphoric acid reagents, indicating the presence of an amino residue as well as a sugar moiety. The NMR spectra (¹H and ¹³C) of Compound A suggested that the amino acid is arginine and the sugar is probably fructose. Indeed, the spectral data on ¹H NMR and ¹³C NMR were completely matched between Compound A and the authentic Fru-Arg, N-(1-deoxy-D-fructos-1-yl)-L-arginine (data not shown). The mobility on TLC did not differ between them. In addition, negative FAB-MS and two-dimensional NMR analyses confirmed the chemical structure of Compound A as Fru-Arg. The chemical structure and assignment of the respective chemical shifts in NMR are shown in Figure 2 and Table 1 . The fructose moiety was indicated to take the ß-pyranose form, determined from the small coupling constant of 5'-H (1.83 Hz) and the chemical shift value of 2'-C (100.37 ppm).

View larger version (8K): [in this window] [in a new window]   Figure 2. Chemical structure of Fru-Arg.

The hydrogen peroxide scavenging activity of Fru-Arg is shown in Table 2 . Fru-Arg showed activity comparable to that of ascorbic acid, scavenging hydrogen peroxide completely at 50 µmol/L and 37% at 10 µmol/L.

To examine whether Fru-Arg is a native constituent of garlic or an artificial product produced through the aging of garlic, we quantitated Fru-Arg, arginine and glucose in AGE, RGJ and HGJ. As shown in Table 3 , Fru-Arg was detected only in AGE (2.1–2.4 mmol/L; 0.69–0.81 g/L). The arginine contents were comparable in all of the preparations, but the glucose contents differed significantly among these samples. The glucose content in AGE was > 5 times that found in the other preparations. pH values were nearly the same in all preparations.

View larger version (21K): [in this window] [in a new window]   Figure 3. Generation of Fru-Arg during the aging process. Sliced garlic cloves were soaked/aged in aqueous ethanol for 1, 2, 4 and 10 mo. Each value represents the mean of triplicate studies. These data were corrected by adjusting the solid extract content to 28.2%; ND, not detected (<0.36 mmol/L).

DISCUSSION

Fru-Arg was identified as a major antioxidant compound in AGE. The hydrogen peroxide scavenging activity of Fru-Arg was comparable to that of ascorbic acid, suggesting that it could contribute to the pharmacologic effects of AGE through its antioxidant properties. Fru-Arg was detected in AGE, but not in RGJ or HGJ. These findings indicate that Fru-Arg is not a native constituent of garlic, nor is it generated simply by heating.

Fru-Arg is known as an Amadori compound, which is generated through a Maillard reaction as shown in Figure 4 (Ledl 1990 ). The Maillard reaction is a nonenzymatic reaction between amino acids and reducing sugars and is suggested to result in the browning of food. There are many reports about Maillard reaction products that show biologically beneficial effects such as antibiotic, antiallergenic, antimutagenic and antioxidant activities (Friedman 1996 ). Interestingly, Amadori compounds were also isolated and identified as biologically active constituents in Korean red ginseng, a popular medicinal plant (Kitao et al. 1995 , Matsuura et al. 1994 ). Recently, it was suggested that Maillard reaction products are responsible for the biological effects and quality of Chinese traditional medicines (Hirayama et al. 1996 ).

View larger version (20K): [in this window] [in a new window]   Figure 4. A possible pathway for generation of Amadori compounds including Fru-Arg (Ledl 1990 .

On the other hand, Fru-Arg was not detected in garlic juice heated at 85°C for 6 h, although Maillard reaction is a nonenzymatic reaction and easily occurs by heating amino acids and sugars. The lower glucose content in RGJ vs. AGE is a possible explanation (Table 3) . Indeed, we confirmed that Fru-Arg was generated by heating RGJ after addition of glucose (data not shown).

In conclusion, the aging process is critical for generation of the biologically active compound, Fru-Arg. The formation of this compound may account for some of the health benefits that have been attributed to AGE. Similarly, the presence of this compound may account for some of the variation in response among commercially available garlic preparations.

FOOTNOTES

¹ Presented at the conference "Recent Advances on the Nutritional Benefits Accompanying the Use of Garlic as a Supplement" held November 15–17, 1998 in Newport Beach, CA. The conference was supported by educational grants from Pennsylvania State University, Wakunaga of America, Ltd. and the National Cancer Institute. The proceedings of this conference are published as a supplement to The Journal of Nutrition. Guest editors: John Milner, The Pennsylvania State University, University Park, PA and Richard Rivlin, Weill Medical College of Cornell University and Memorial Sloan-Kettering Cancer Center, New York, NY.

³ Abbreviations: ABTS, 2,2'-azino-di-[3-ethylbenzthiazoline sulfonate (6)]; AGE, aged garlic extract; FAB-MS, fast atomic bombardment mass spectrometry; Fru-Arg, N-(1-deoxy-D-fructos-1-yl)-L-arginine; GC, gas chromatography; HGJ, heated garlic juice; NMR, nuclear magnetic resonance; RGJ, raw garlic juice.

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