Aged garlic extract restores nitric oxide bioavailability in cultured human endothelial cells even under conditions of homocysteine elevation.

ETHNOPHARMACOLOGICAL RELEVANCE: Supplementation with aged garlic extract (AGE) has been shown to restore impaired endothelium-dependent vasodilator response in subjects with acutely elevated plasma homocysteine (Hcy) levels after an oral methionine load and in patients with chronic coronary artery disease. Moreover, AGE has been shown to inhibit the progression of coronary calcifications in patients with coronary artery disease. The molecular mechanisms, by which AGE preserves endothelial function is unknown. Our objective was to explore whether AGE preserves endothelial nitric oxide (NO) output even under conditions of elevated Hcy levels by preventing oxidative inactivation of the NO synthase cofactor tetrahydrobiopterin. MATERIAL AND METHODS: Endothelial (EA.hy 926) cells were incubated with hypoxanthine, aminopterin, thymidine and methionine (HAT/MET) to increase cellular Hcy levels, and with and without AGE. Agonist stimulated NO output was measured using the fluorescent probe DAF-2, and cellular thiol levels (Hcy, cysteine, reduced and oxidized glutathione) and cellular tetrahydrobiopterin levels were measured by high performance liquid chromatography. RESULTS: HAT/MET incubation resulted in significantly increased cellular Hcy levels, unaffected by coincubation with AGE. Elevated Hcy went along with significantly decreased NO output (to 34.4 ± 4.4% of control) and levels of tetrahydrobiopterin (from 4.67 ± 2.17 to 2.17 ± 0.97 pmol/mg). Incubation with AGE (5mg/mL) in HAT/MET-treated cells prevented the declines in NO output and tetrahydrobiopterin levels. AGE increased cellular levels of cysteine and total glutathione, and prevented glutathione and tetrahydrobiopterin oxidation induced by elevated Hcy. CONCLUSION: Incubation with AGE preserved normal NO output from endothelial cells even under conditions of elevated Hcy levels by increasing cellular thiol antioxidant and prevention of tetrahydrobiopterin oxidation. This suggests that AGE might be useful in the prevention of endothelial dysfunction.

Aged garlic extract inhibits homocysteine-induced scavenger receptor CD36 expression and oxidized low-density lipoprotein cholesterol uptake in human macrophages in vitro.

AIM OF THE STUDY: Expression of CD36 scavenger receptors on macrophages is involved in oxidized low-density lipoprotein (OxLDL) uptake and foam cell formation during atherosclerotic lesion development. It has been shown previously in vitro and in vivo that the atherosclerotic risk factor homocysteine (Hcy) stimulates macrophage CD36 expression and OxLDL uptake. We now examined the effects of aged garlic extract (AGE), a garlic preparation enriched in water-soluble organic sulfur-containing compounds, on Hcy-induced CD36 expression and foam cell formation in human monocytes/macrophages. RESULTS: Incubation with Hcy (200 µM for 72h) in THP-1-derived macrophages and primary human macrophages caused a 37.8±5.2% and 60.7±4.2% increase in CD36 expression compared to control, respectively. Coincubation with AGE (5mg/ml) significantly suppressed CD36 expression in THP-1 derived macrophages by 48.6±9.0% compared to Hcy-incubated cells only. AGE (1-5mg/ml) dose dependently inhibited Hcy-induced CD36 expression in primary human macrophages, and decreased binding of nuclear proteins to a PPARγ response element. Preincubation with AGE significantly inhibited DiI-labeled OxLDL uptake. CONCLUSIONS: These data indicate that AGE inhibits CD36 expression and OxLDL uptake in human macrophages by modulating the PPARγ pathway, and suggest that the extract could be useful for the prevention of atherosclerotic lesions.

Aged garlic extract inhibits CD36 expression in human macrophages via modulation of the PPARgamma pathway.

Expression of CD36 scavenger receptors on macrophages is involved in oxidized low-density lipoprotein uptake and foam cell formation during atherosclerotic lesion development. We examined the effects of aged garlic extract (AGE), a garlic preparation enriched in water-soluble cysteinyl moieties that increases cellular total thiols and glutathione concentrations, on CD36 expression in human monocytes/macrophages (THP-1 cells and primary human monocytes). Compared to control, AGE (1-5 mg/mL) dose-dependently and significantly suppressed CD36 expression up to by 61.8 +/- 7.4% in THP-1-derived macrophages and up to 50.5 +/- 7.1% in primary human macrophages, respectively. Furthermore, AGE prevented induction of CD36 expression by the peroxisome proliferator activator receptor (PPAR) gamma agonist troglitazone, and decreased binding of nuclear proteins to a PPARgamma response element. AGE showed a stronger inhibitory effect on CD36 expression in THP-1 cells during simultaneous incubation with phorbol 12-myristate 13-acetate (PMA) compared to cells that had been pre-incubated with PMA. Furthermore, AGE decreased CD11b expression in a dose-dependent manner. These data indicate that AGE inhibits CD36 expression by modulating the PPARgamma pathway in human macrophages and monocytes differentiation into macrophages, and suggests that the extract could be useful for the prevention of atherosclerotic lesions.

Changes in organosulfur compounds in garlic cloves during storage.

We determined the changes in the contents of three gamma-glutamyl peptides and four sulfoxides in garlic cloves during storage at -3, 4, and 23 degrees C for 150 days using a validated high-performance liquid chromatography method that we reported recently. When garlic was stored at 4 degrees C for 150 days, marked conversion of the gamma-glutamyl peptides, gamma-L-glutamyl-S-allyl-L-cysteine and gamma-L-glutamyl-S-(trans-1-propenyl)-L-cysteine (GSPC), to sulfoxides, alliin and isoalliin, was observed. Interestingly, however, when garlic was stored at 23 degrees C, a decrease in GSPC and a marked increase in cycloalliin, rather than isoalliin, occurred. To elucidate in detail the mechanism involved, the conversion of isoalliin to cycloalliin in both buffer solutions (pH 4.6, 5.5, and 6.5) and garlic cloves at 25 and 35 degrees C was examined. Decreases in the concentration of isoalliin in both the solutions and the garlic cloves during storage followed first-order kinetics and coincided with the conversion of cycloalliin. Our data indicated that isoalliin produced enzymatically from GSPC is chemically converted to cycloalliin and that the cycloalliin content of garlic cloves increases during storage at higher temperature. These data may be useful for controlling the quality and biological activities of garlic and its preparations.

Determination of seven organosulfur compounds in garlic by high-performance liquid chromatography.

The properties of garlic (Allium sativum L.) are attributed to organosulfur compounds. Although these compounds change during cultivation and storage, there is no report of their simultaneous analysis. Here, a newly developed analytical method with a rapid and simple sample preparation to determine four sulfoxides and three gamma-glutamyl peptides in garlic is reported. All garlic samples were simply extracted with 90% methanol solution containing 0.01 N hydrochloric acid and prepared for analysis. Alliin, isoalliin, methiin, cycloalliin, and gamma-l-glutamyl-S-methyl-l-cysteine were determined by normal-phase HPLC using an aminopropyl-bonded column. gamma-l-Glutamyl-S-(2-propenyl)-l-cysteine and gamma-l-glutamyl-S-(trans-1-propenyl)-l-cysteine were separated on an octadecylsilane column. The overall recoveries were 97.1-102.3%, and the relative standard deviation values of intra- and interday precision were lower than 2.6 and 4.6%, respectively. This newly developed method offers some advantages over the currently accepted techniques including specificity, speed, and ease of use and would be useful for chemical and biological studies of garlic and its preparations.

Tetrahydro-beta-carboline derivatives in aged garlic extract show antioxidant properties.

This study used the hydroden peroxide scavenging assay to investigate antioxidant chemical constituents derived and separated from aged garlic extract, a unique garlic extract produced by soaking sliced garlic in an aqueous ethanol solution for >10 mo. Four types of 1, 2, 3, 4-tetrahydro-beta-carboline derivatives (THbetaCs); 1-methyl-1, 2, 3, 4-tetrahydro-beta-carboline-3-carboxylic acid, and 1-methyl-1, 2, 3, 4-tetrahydro-beta-carboline-1, 3-dicarboxylic acid (MTCdiC), from both diastereoisomers, were isolated and identified by use of liquid chromatography-mass spectrometry. All these compounds indicate strong hydrogen peroxide scavenging activities and inhibit 2, 2'-azobis(2-amidinopropane) hydrochloride-induced lipid peroxidation. Particularly, (1S, 3S)-MTCdiC had the most potent hydrogen peroxide scavenging activity, more than ascorbic acid. The (1R, 3S)- and (1S, 3S)-MTCdiC at 50-100 micromol/L and 10-100 micromol/L inhibited LPS-induced nitrite production. Interestingly, THbetaCs were not detected in raw garlic and other processed garlic preparations, but they were generated and increased during the natural aging garlic extraction process. These data suggest that THbetaCs, which are formed during the natural aging process, are potent antioxidants in aged garlic extract and thus may be useful for the prevention of diseases associated with oxidative damage.

Aged garlic extract inhibits homocysteine-induced CD36 expression and foam cell formation in human macrophages.

Elevated plasma homocysteine (Hcy) levels have been recognized as an independent risk factor for atherosclerotic vascular disease. During formation of early atherosclerotic lesions, expression of CD36, a class B scavenger receptor on macrophages, is crucially involved in the uptake of oxidized low-density lipoprotein (OxLDL) and foam-cell formation. We therefore determined the effects of Hcy on CD36 expression and foam cell formation in human monocytes/macrophages (THP-1) using flow cytometry, and the effects of aged garlic extract (AGE) on this process. Incubation of THP-1 cells with Hcy (200 micromol/L) for 72 h in the presence of phorbol 12-myristate 13-acetate (PMA) (10 nmol/L) caused a 37.8+/-5.2% increase in CD36 expression compared with PMA-stimulated cells without Hcy (P<0.01). Coincubation with AGE (5 g/L) significantly suppressed CD36 expression by 61.8+/-13.9%, compared with control conditions, and by 48.6+/-9.0% compared with Hcy-incubated cells (P<0.01). THP-1 cells in the presence of PMA (10 nmol/L) were incubated with Hcy or AGE for 72 h followed by incubation with 1,1'-dioctadecyl-3,3,3'3'-tetra-methylindocyanide percholorate (DiI)-labeled OxLDL for 3 h, and fluorescence intensity was measured by flow cytometry. AGE also inhibited DiI-labeled OxLDL uptake into PMA-stimulated THP-1 cells by 85.6+/-2.8% (P<0.01), but Hcy had no effects on it. Our data indicate that AGE inhibits CD36 expression and OxLDL uptake in macrophages and suggest that the extract could modulate the formation of early atherosclerotic lesions.

Aged garlic extract inhibits peroxynitrite-induced hemolysis.

Nitric oxide (NO), which is synthesized by constitutive NO synthase (cNOS), plays important roles in physiological functions of the cardiovascular system. However, NO, which is synthesized by inducible NOS, is detrimental when it reacts with superoxide to form peroxynitrite. Peroxynitrite is recognized as a powerful oxidant, and results in vascular or tissue damage. We have previously reported that aged garlic extract (AGE) enhances NO production through cNOS stimulation. In the present study, we determined the effect of AGE, its fractions or constituents on peroxynitrite-induced hemolysis using rat erythrocytes. Incubation of rat erythrocytes with peroxynitrite (300 microM) for 30 min at 37 degrees C caused 4-fold hemolysis. AGE (0.14-0.57 %w/v) added to an erythrocyte suspension was found to reduce peroxynitrite-induced hemolysis in a concentration-dependent manner. Of the AGE fractions, a polar fraction and a low-molecular-weight fraction both suppressed the hemolysis to the same degree as that seen with AGE. S-allylcysteine, one of the major compounds in AGE, also reduced hemolysis at 1-10 mM dose-dependently. These data indicate that AGE and its compounds protect erythrocytes from membrane damage induced by peroxynitrite, suggesting that AGE could be useful for prevention of cardiovascular diseases associated with oxidative stress or dysfunction of NO production.

Identification of six phenylpropanoids from garlic skin as major antioxidants.

The extract of garlic skins (peels) showed strong antioxidant activity, and some responsible constituents were isolated and identified. Garlic (Allium sativum L.) has been used as an herbal medicine, but there is no report on the health benefits of the skin or peel. In this study, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of garlic skin extract was evaluated. Using chromatographic techniques, the active constituents were isolated and subsequently identified. Analyses by high-performance liquid chromatography coupled with a photodiode array detector (HPLC-PDA) suggested that these compounds were phenylpropanoids, which had a characteristic absorbance at 300-320 nm. Liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance analyses allowed the chemical structures of the isolated constituents to be postulated. The proposed compounds were subsequently synthesized and compared with the constituents in the extract using HPLC-PDA and LC-MS. N-trans-Coumaroyloctopamine, N-trans-feruloyloctopamine, guaiacylglycerol-beta-ferulic acid ether, and guaiacylglycerol-beta-caffeic acid ether were identified as were trans-coumaric acid and trans-ferulic acid. Also, the antioxidant activities of these compounds were determined.

Antioxidant effects of tetrahydro-beta-carboline derivatives identified in aged garlic extract.

1,2,3,4-Tetrahydro-beta-carboline derivatives (THbetaCs) are formed through Pictet-Spengler chemical condensation between tryptophan and aldehydes during food production, storage and processing. In the present study, in order to identify the antioxidants in aged garlic extract (AGE), we fractionated it and identified four THbetaCs; 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acids (MTCC) and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-1,3-dicarboxylic acid (MTCdiC) in both diastereoisomers using liquid chromatography mass spectrometry (LC-MS). Interestingly, these compounds were not detected in raw garlic, but the contents increased during the natural aging process of garlic. In in vitro assay systems, all of these compounds have shown strong hydrogen peroxide scavenging activities. (1S, 3S)-MTCdiC was found to be stronger than the common antioxidant, ascorbic acid. MTCC and MTCdiC inhibited AAPH-induced lipid peroxidation. Both MTCdiCs also inhibited LPS-induced nitrite production from murine macrophages at 10-100 microM. Our data suggest that these compounds are potent antioxidants in AGE, and thus may be useful for prevention of disorders associated with oxidative stress.