Effects of S-1-propenylcysteine, a sulfur compound in aged garlic extract, on blood pressure and peripheral circulation in spontaneously hypertensive rats.

OBJECTIVES: This study was designed to investigate the antihypertensive effect of S-1-propenylcysteine, a characteristic sulfur compound in aged garlic extract, using a hypertensive rat model. METHODS: The blood pressure and tail blood flow of both spontaneously hypertensive rats and control Wistar Kyoto rats were measured by the tail-cuff method and the noncontact laser Doppler method, respectively, at various times after single oral administration of a test compound for 24 h. KEY FINDINGS: Treatment with S-1-propenylcysteine (6.5 mg/kg BW) significantly decreased the systolic blood pressure of spontaneously hypertensive rat approximately 10% at 3 h after administration, and thereafter, the systolic blood pressure gradually returned to the baseline level in 24 h. The effect of S-1-propenylcysteine was dose-dependent and was maximal at the dose of 6.5 mg/kg BW at 3 h. However, the other compounds such as S-allylcysteine and S-allylmercaptocysteine in aged garlic extract were ineffective. In addition, S-1-propenylcysteine had no effect on systolic blood pressure of control Wistar Kyoto rats. Furthermore, S-1-propenylcysteine significantly increased the blood flow at 3 h after administration at the dose of 6.5 mg/kg BW. CONCLUSIONS: S-1-propenylcysteine is a key constituent of aged garlic extract responsible for its antihypertensive effect, and the effect of S-1-propenylcysteine involves the improvement in peripheral circulation.

Aged garlic extract suppresses inflammation in apolipoprotein E-knockout mice.

SCOPE: Chronic inflammation plays a major role in the formation and progression of atherosclerotic plaques. To clarify the mode of action of aged garlic extract (AGE) to retard atherosclerosis, we investigated whether AGE suppresses the inflammation in apolipoprotein E-knockout (ApoE-KO) mice. METHODS AND RESULTS: ApoE-KO mice were fed standard diet with or without 3% AGE for 12 wk. AGE feeding inhibited the progression of atherosclerotic lesion by 27% and reduced the level of C-reactive protein (CRP) and thromboxane B2 (TXB2 ), a marker of platelet activation, in serum by 39 and 33%, respectively, compared to ApoE-KO mice without AGE treatment. AGE treatment also decreased the level of tumor necrosis factor alpha (TNF-α), a major stimulus inducing CRP production, in the liver by 35%. AGE decreased the level of interleukin-1 receptor-associated kinase 4 (IRAK4) by 60% and almost doubled the level of phospho-AMP-activated protein kinase (p-AMPK) in the liver. CONCLUSION: The anti-atherosclerotic effect of AGE involves the suppression of inflammation by reducing the serum level of CRP and TXB2 , and the protein level of TNF-α and IRAK4, and increasing AMPK activity in liver.

Aged garlic extract exerts endothelium-dependent vasorelaxant effect on rat aorta by increasing nitric oxide production.

BACKGROUND: Clinical trials have shown that aged garlic extract (AGE) is effective in reducing blood pressure of hypertensive patients. However, the mechanisms involved remain to be elucidated. PURPOSE: The aim of the present study was to investigate the vasorelaxant effect of AGE on the aorta and its mechanism of action in order to clarify the blood pressure-lowering action of AGE. METHODS: The vasorelaxant effect was evaluated in isolated rat aortic rings. After aortic rings were contracted by 3 × 10-6M norepinephrine (NE) for 30min, AGE and other test drugs were added to the aortic rings. All results were expressed as percentages of the maximal NE-induced contraction. RESULTS: AGE induced the concentration-dependent vasorelaxation of isolated rat aortic rings that had been precontracted with norepinephrine. The effect of AGE was severely impaired in aortic rings lacking endothelium. In addition, the effect of AGE was inhibited by a nitric oxide synthase (NOS) inhibitor and a nitric oxide (NO) scavenger. Moreover, AGE treatment of aorta significantly increased the NO production. When various constituents of AGE were tested, the vasorelaxation of aorta was observed only in the presence of L-arginine, a substrate of NOS. CONCLUSION: AGE causes endothelium-dependent vasorelaxation of aorta via stimulation of NO production and that L-arginine in AGE serves as a key agent for NOS-mediated NO production.

Aged garlic extract suppresses the increase of plasma glycated albumin level and enhances the AMP-activated protein kinase in adipose tissue in TSOD mice.

SCOPE: In this study, we investigated the effect of aged garlic extract (AGE) on the high level of blood glucose in Tsumura Suzuki Obese-Diabetes (TSOD) mice. METHODS AND RESULTS: TSOD mice were fed standard diet with or without 2% AGE for 19 weeks. AGE treatment lowered the blood glucose level and significantly reduced the plasma level of glycated albumin in TSOD mice as compared with those without AGE treatment. In addition, AGE treatment increased the level of phosphorylated AMP-activated protein kinase (AMPK) in the adipose tissue, liver and muscle that played an important role in the maintenance of insulin sensitivity. Moreover, AGE treatment also suppressed the mRNA expression of fatty acid synthase, a known factor regulated by AMPK, and monocyte chemoattractant protein 1, one of the representative inflammatory chemokines, in the adipose tissue but not in the liver. CONCLUSION: AGE treatment suppresses the increase of plasma glycated albumin level in TSOD mice and this effect is accompanied by the activation of AMPK in adipose tissue, and suggests that AGE may play a potential role in the prevention and treatment of type 2 diabetes.

Aged garlic extract suppresses platelet aggregation by changing the functional property of platelets.

Aged garlic extract (AGE), a garlic preparation rich in water-soluble cysteinyl moieties, has been reported to have multiple beneficial effects on cardiovascular disease including inhibition of platelet aggregation. However, the mode of AGE action on platelets has not been clear. In this study, we examined the effect of AGE on the functional property of platelet by administering AGE to rats and evaluating the platelet aggregation in response to collagen in vitro. We found that AGE treatment significantly reduced the ability of platelet to aggregate and this effect of AGE was manifested on the 14 day, but not 7 day of treatment. In addition, AGE treatment produced platelets that responded to collagen by significantly increasing the amount of both the extracellular ATP and the extra- and intracellular TXB2. AGE treatment also dose-dependently suppressed the phosphorylation of collagen-induced ERK, p38 and JNK. However, AGE administration did not affect the bleeding time. These findings suggest that AGE treatment results in suppression of platelet aggregation by changing the functional property of platelets to respond to collagen.

Aged Garlic Extract Suppresses the Development of Atherosclerosis in Apolipoprotein E-Knockout Mice.

BACKGROUND: Aged garlic extract (AGE) has been shown to retard the progression of coronary calcification in patients with coronary artery disease. OBJECTIVE: To clarify the mechanism of AGE's action to retard atherosclerosis, we investigated whether AGE suppresses the formation and progression of atherosclerosis in Apolipoprotein E (Apoe)-knockout (ApoE-KO) mice. METHODS: Male C57BL/6J mice (control mice, 5 wk old) were fed a standard diet, whereas male ApoE-KO mice (5 wk old) were fed a standard diet with or without 3% AGE for 12 or 24 wk. After the treatment, blood samples, aortas, and spleens were collected from all mice. Concentrations of total cholesterol (TC), HDL cholesterol, and triglycerides (TGs) in serum were measured. The area of atherosclerotic lesion in the aorta was examined by Oil Red O staining. The relative abundances of monocytes plus macrophages (CD11b(+) cells) and interferon-γ-producing CD4(+) T cells in spleen were assessed by flow cytometric analysis. RESULTS: The atherosclerotic lesion areas in the aortas of ApoE-KO mice were 87 and 114 times as great (P < 0.01) as those in control mice at 12 and 24 wk, respectively. AGE feeding significantly inhibited the progression of atherosclerotic lesion area in ApoE-KO mice by 22% (P < 0.05) at 12 wk. In addition, serum concentrations of TC and TGs in ApoE-KO mice were significantly higher than those in control mice at 12 and 24 wk. Treatment with AGE significantly suppressed the increases in serum concentrations of TC and TGs in ApoE-KO mice by 21% (P < 0.05) and 19% (P < 0.05) at 24 wk, respectively, and reduced the relative abundance of CD11b(+) cells in ApoE-KO mice by 24% (P < 0.05) at 12 wk. CONCLUSION: These data suggest that the antiatherosclerotic activity of AGE is at least partly due to the suppression of inflammation and lipid deposition in the vessels during the early stage of atherosclerotic development in ApoE-KO mice.

Aged garlic extract enhances heme oxygenase-1 and glutamate-cysteine ligase modifier subunit expression via the nuclear factor erythroid 2-related factor 2-antioxidant response element signaling pathway in human endothelial cells.

The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway defends cells against oxidative stress and regulates the cellular redox balance. Activation of this pathway induces a variety of antioxidant enzymes, resulting in the protection of our bodies against oxidative damage. It has been reported that aged garlic extract (AGE), a garlic preparation that is rich in water-soluble cysteinyl moieties, reduces oxidative stress and helps to ameliorate of cardiovascular, renal and hepatic diseases. We hypothesized that AGE enhances the expression of antioxidant enzymes via the Nrf2-ARE pathway in human umbilical vein endothelial cells in culture. Gene expression of antioxidant enzymes was measured using real-time polymerase chain reaction. Nuclear accumulation of Nrf2 and antioxidant enzymes expression were evaluated using western blotting analyses. We found that AGE promoted the accumulation of Nrf2 into the nucleus in a time- and dose-dependent manner and increased the gene expression and polypeptide level of heme oxygenase-1 (HO-1) and glutamate-cysteine ligase modifier subunit (GCLM). Moreover, the effect of AGE in elevating the gene expression of HO-1 and GCLM was found to be mediated via Nrf2 activation in human umbilical vein endothelial cells. Taken together, these observations suggest that AGE induces the expression of HO-1 and GCLM, which are antioxidant enzymes, via activation of the Nrf2-ARE signaling pathway.

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 scavenges superoxide radicals.

There is increasing evidence to suggest that many degenerative or pathological processes, such as aging, cancer, and coronary heart disease, are related to reactive oxygen species and radical-mediated reactions. We examined the effectiveness of aged garlic extract (AGE), a garlic preparation rich in water-soluble cysteinyl moieties, and its component for scavenging of superoxide by using the hypoxanthine-xanthine oxidase and human neutrophils. In the hypoxanthine-xanthine oxidase system, electron spin resonance showed that aged garlic extract scavenged superoxide radicals in a dose-dependent manner up to 54%. The EC(50) value of aged garlic extract for the superoxide radical scavenging effect was 0.80 mg/ml. N-α-(1-deoxy-D-fructos-1-yl)-L-arginine (25.9%) and (1S, 3S)-1-methyl-1,2,3,4-tetrahydro-ß-carboline-1,3-dicarboxylic acid (20.8%), water-soluble moieties of AGE, also exerted superoxide scavenging effects. Phorbol 12-myristate 13-acetate-activated human neutrophils produced superoxide radical of 56.6 ± 9.27 nmol/min/10(7) cells. Aged garlic extract (3 mg/ml) significantly inhibited superoxide production in comparison to the control. These data suggest that aged garlic extract may be useful for preventing diseases associated with reactive oxygen species.

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.

Garlic as an anti-fatigue agent.

More than three thousand publications in the past have confirmed the efficacy of garlic for the prevention and treatment of a variety of diseases, acknowledging and validating its traditional uses. Garlic is also used for the treatment of fatigue, although the mechanism involved remain unclear. The anti-fatigue function of garlic may be closely related to its many favorable biological and pharmacological effects. In animal studies, garlic has been shown to promote exercise endurance. Differences in the methods of processing garlic result in differences in the intensity of its anti-fatigue effect, and the most favorable form of processing has been shown to be extraction of raw garlic followed by its natural aging for a long period in a water-ethanol mixture. In human studies, it has been confirmed that garlic produces symptomatic improvement in persons with physical fatigue, systemic fatigue due to cold, or lassitude of indefinite cause, suggesting that garlic can resolve fatigue through a variety of actions. Recently, primarily in Japan, attempts have been made to measure the intensity of fatigue objectively and quantitatively using biomarkers. Currently available data strongly suggest that garlic may be a promising anti-fatigue agent, and that further studies to elucidate its application are warranted.

Aged garlic extract ameliorates physical fatigue.

Aged garlic extract (AGE) has recently received attention as a potent anti-fatigue agent. The principal aim of this study was to elucidate the mechanism responsible for the ameliorating effect of AGE on physical fatigue in rats caused by repeated endurance exercise on a mechanical treadmill apparatus. Rats were subjected to endurance exercise 5 times per week for 4 weeks. AGE at a dosage of 2.86 g/kg was administrated to rats 30 min before every exercise. Succinate dehydrogenase (SDH) activity in the gastrocnemius and soleus muscles and superoxide dismutase (SOD) activity, nitric oxide (NO) metabolites, and lactic acid concentration in plasma were evaluated as biomarkers of physical fatigue. SDH activity was increased 2-4-fold by repeated endurance exercise in comparison with unexercised (intact) rats, and AGE further up-regulated this activity by 40%. SOD activity was increased 5-fold, whereas AGE maintained it at a level equivalent to that in intact rats. Levels of NO metabolites were slightly decreased, whereas AGE enhanced them 2-fold. Lactic acid concentration was not changed in any of the groups. These results indicate that AGE may facilitate the turnover of aerobic glucose metabolism, attenuate oxidative stress, and promote oxygen supply based on vasodilation, suggesting that AGE ameliorates the various impairments associated with physical fatigue.

Aged garlic extract maintains cardiovascular homeostasis in mice and rats.

Nitric oxide (NO) plays an important role in controlling the physiological functions of the cardiovascular system. However, toxic peroxynitrite is produced by the reaction of NO with superoxide. We investigated the effect of aged garlic extract (AGE) on NO production, and on oxidative stress induced by peroxynitrite. A single dose of AGE temporarily increased NO production by 30-40% between 15 and 60 min after administration to mice. The time course of the fluctuation in NO levels in the AGE-treated group clearly differed from that in a group treated with an inducible NO synthase (iNOS) inducer. A selective constitutive NOS (cNOS) inhibitor overcame the effect of AGE. These results indicate that AGE increases NO production by activating cNOS, but not iNOS. In another experiment, the addition of AGE to a rat erythrocyte suspension reduced the rate of peroxynitrite-induced hemolysis in a concentration-dependent manner, suggesting that AGE protects erythrocytes from membrane damage induced by peroxinitrite. Because an increase in NO derived from cNOS and protection against peroxynitrite are important factors in the prevention of cardiovascular disease, our data strongly suggest that AGE could be useful in preventing cardiovascular diseases associated with oxidative stress or dysfunctions of NO production.

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.

Aged garlic extract enhances production of nitric oxide.

Nitric oxide (NO) controls several physiological functions of the cardiovascular system. Three kinds of NO synthases (NOSs), neuronal constitutive NOS (ncNOS), inducible NOS (iNOS) and endothelial constitutive NOS (ecNOS), were responsible for NO biosynthesis. This study investigated the effect of aged garlic extract (AGE) on NO production by measuring the NO metabolites nitrite and nitrate in the plasma of mice. AGE (2.86 g/kg, p.o.) temporarily increased NO production by 30-40% from 15 to 60 min after administration. The time course of the fluctuation in NO levels in the AGE-treated group was clearly different to that in a group of mice treated with lipopolysaccharides, a typical iNOS inducer. Arginine (63 mg/kg, p.o.) at the equivalent dose of AGE did not increase NO production. However diphenyleneiodonium chloride (1 mg/kg, i.p.), a selective cNOS inhibitor, administered prior to AGE, overcame the effect of AGE. These results indicate that AGE increased NO production by activating cNOS, but not iNOS. The arginine contained in AGE was not responsible for the effect. AGE may be a useful tool for the prevention of cardiovascular disease.