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Healthcare Research Institute, Wakunaga Pharmaceutical Co. Ltd, Hiroshima, 739-1195, Japan
3 To whom correspondence should be addressed. E-mail: morihara_n{at}wakunaga.co.jp.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • garlic • nitric oxide • peroxynitrite • cardiovascular diseases
Since ancient times, people of many different cultures have considered garlic (Allium sativum) to be a valuable healing agent. Garlic has been used as a cure for various ailments, including heart disease, cancer, and infection (1). However, chronic administration of raw garlic causes diverse toxic effects, such as anemia, weight loss, and growth reduction (2). Aged garlic extract (AGE),4 extracted for >10 mo, is less irritating and does not induce the toxic changes mentioned above (2,3). Furthermore, a large number of pharmacological studies found that AGE and its components possess antioxidative (4,5), antiaging (6), immunomodulatory (7), cardiovascular (8–11), and hepatoprotective (12,13) properties.
Nitric oxide (NO) is synthesized from L-arginine by NO synthases (NOS) in many of the cells of the cardiovascular system, including endothelial cells, macrophages, smooth muscle cells, platelets, and fibroblasts (14). Three kinds of NOS, i.e., neuronal NOS, inducible NOS (iNOS), and endothelial NOS, were reported to be responsible for NO biosynthesis in these cells (15). The small quantity of NO produced by constitutive NOS (cNOS; neuronal NOS and endothelial NOS) is an important cellular messenger with a major role in controlling physiological functions in the cardiovascular system (16,17). However, when excess NO is produced through upregulation of iNOS, toxic peroxynitrite is produced by a reaction with superoxide (18,19). Peroxynitrite is a potent oxidant that was shown to oxidize lipids and LDL and to promote platelet aggregation, thus aggravating the atherogenic process (20). Thus, NO possesses the character of a double-edged sword.
In this study, we examined the effect of AGE on NO production, and on oxidative stress induced by peroxynitrite.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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286 g/L solid material and 6.3 g/L arginine. Chemicals. We purchased (±)-(E)-4-Ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR3), NG-monomethyl-L-arginine acetate (L-NMMA), and peroxynitrite from Dojindo Laboratories. Arginine, zinc sulfate, phosphoric acid, sulfanilamide, N-1-naphthylethylenediamine, sodium nitrite, potassium nitrate, sodium pyruvate, sodium chloride, disodium hydrogenphosphate and sodium dihydrogenphosphate were obtained from Wako Pure Chemical Industries. Diphenyleneiodonium chloride (DPI) was purchased from Research Biochemical International. Lipopolysaccharide (LPS; W E. coli 055:B5) was obtained from DIFCO Laboratories. FAD, NADPH, nitrate reductase (EC 1.6.6.2), and lactate dehydrogenase (EC 1.1.1.27) were purchased from Boehringer Mannheim. LPS, L-NMMA, and DPI were dissolved in sterile saline. NOR3 was suspended in sterile saline. Other reagents were dissolved in distilled water.
Experiment 1: Effect of AGE on NO production
Male ddY mice (5 wk old) were purchased from Japan SLC and housed, 4–6/plastic cage, under a 12-h light:dark cycle for 1 wk before use in the experiment. They had free access to a commercial diet (CE-2, Clea) and water. They were killed by bleeding after collection of blood under anesthesia. The in vivo experiments were approved by the Wakunaga Pharmaceutical Company Institutional Animal Care and Use Committee.
AGE (2.86 g/kg body weight, orally), NOR3 (10 mg/kg, orally), or L-NMMA (40 mg/kg, i.p.) was administered to the mice. Blood samples were collected 15 min after administration in the AGE and NOR3 groups and 1 h after administration in the L-NMMA group.
Separately, AGE (2.86 g/kg, orally) or LPS (30 mg/kg, i.p.) was administered to the mice, and blood samples were collected to measure changes in NO metabolites over time.
To clarify the mechanisms involved in the fluctuation of NO metabolites after AGE administration, mice were given AGE (2.86 g/kg, orally) or a dose of arginine equivalent to the arginine content of the AGE (63 mg/kg, orally). DPI (1 mg/kg, i.p.) was administered 2 h before the AGE, based on its duration of effect. Blood samples were collected to measure changes in NO metabolites over time.
Blood samples were taken from the right ventricle of the anesthetized mice with a heparinized syringe. Plasma obtained by centrifuging (1200 x g; 15 min) the heparinized blood was used for the measurement of NO metabolites. The stable NO metabolites, nitrite (NO2–) and nitrate (NO3–), were used as an index of NO production (14) and were determined using the method of Schmidt et al. (21). Briefly, the plasma was diluted 4-fold with distilled water. NADPH, FAD, and nitrate reductase were added to final concentrations of 50 µmol/L, 5 µmol/L, and 200 U/L, respectively. The samples were incubated for 20 min at 37°C, then lactate dehydrogenase and sodium pyruvate were added to final concentrations of 10 mg/L and 10 mmol/L, respectively. The samples were further incubated for 5 min at 37°C to oxidize the NADPH, and nitrite was measured using the Griess reaction. The samples were deproteinized by adding zinc sulfate to a final concentration of 93 mmol/L. After centrifugation at 1000 x g for 15 min at room temperature, the supernatant was mixed with the same quantity of Griess reagent (5.8 mmol/L sulfanilamide, 25 g/L phosphoric acid, and 5.3 mmol/L N-1-naphthylethylenediamine). After 10 min of color development at room temperature, the absorbance was measured at a wavelength of 540 nm. Each plasma sample was assayed in duplicate. Control values were obtained by treating the samples as described but using 25 g/L phosphoric acid instead of complete Griess reagent. Calibration curves were constructed using sodium nitrite and potassium nitrate in distilled water. The values obtained by this procedure reflect the total nitrite plus nitrate levels in the samples.
Experiment 2: Effect of AGE on peroxynitrite-induced oxidative stress
Male Wistar rats (5 wk old) were purchased from Japan SLC and housed under a 12-h light:dark cycle for about 1 wk before use in the experiment. They had free access to a commercial diet (CE-2, Clea) and water. Blood was taken from the abdominal aortae of the anesthetized rats using a heparinized syringe. The erythrocytes were separated from the plasma and buffy coat by centrifugation at 1000 x g for 10 min at 4°C, then washed 3 times with 10 volumes of PBS. The washed erythrocytes were suspended in PBS, and the hematocrit was measured using a Particle Counter PC-608 (ERMA). The hematocrits of the erythrocyte suspensions used in the experiments were adjusted to 10% using PBS. The erythrocyte suspensions were preincubated with various concentrations of AGE (1.4–5.7 g/L) at 37°C for 5 min. Peroxynitrite (300 µmol/L) was then added to each erythrocyte suspension to induce peroxidation, followed by further incubation for 30 min at 37°C. Each erythrocyte suspension was then separated by centrifugation at 1000 x g for 5 min at 4°C and the absorbance of the supernatant was measured at 540 nm. By comparing these values with the absorbance of distilled water (considered equivalent to 100% hemolysis), the percentage hemolysis obtained with peroxynitrite was calculated.
Statistical analysis. The data are expressed as means ± SEM. Significant differences between means were determined using one-way ANOVA followed by Duncan's or Scheffé's multiple-comparison test. All statistical analyses were performed using STATISTICATM (StatSoft Japan).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Fluctuations in NO production were investigated after administration of AGE, NOR3, and L-NMMA. Both AGE and NOR3 (a NO donor) increased NO levels in the plasma compared with the control (P < 0.05 and P < 0.01, respectively). On the other hand, the NOS inhibitor L-NMMA reduced NO levels in the plasma compared with the control (P < 0.01, Table 1).
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The addition of peroxynitrite to rat erythrocyte suspensions produced 4 times more hemolysis than in control erythrocyte suspensions without peroxynitrite (Table 2). AGE (1.4–5.7 g/L) significantly inhibited this increase in hemolysis in a dose-dependent manner (P < 0.05 or P < 0.01). However, AGE (1.4–5.7 g/L) did not affect the degree of hemolysis that occurred in the absence of peroxynitrite.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We investigated NO production in the body by measuring stable NO metabolites in the blood. We confirmed that levels of NO metabolites change in the presence of a NO donor or NOS inhibitor. Both AGE and an NO donor significantly increased NO levels (Table 1). AGE rapidly increased NO production by 30–40% after administration, but NO production had returned to the basal level at 120 min after administration (Fig. 1). This rapid response may be explained by the fact that S-allylcysteine, one of the major compounds in AGE, is rapidly and easily absorbed from the gastrointestinal tract (within 15 min of administration of AGE) in animals (31).
To clarify the mechanism by which AGE increases NO production, we investigated the effect of DPI (a selective cNOS inhibitor) on NO production. DPI significantly decreased the NO level in mouse plasma at 60 and 120 min after treatment compared with the basal value (Fig. 3). We determined the time of administration of DPI based on the absorption of AGE and the activity profile of DPI. NO production was inhibited when DPI was administered 120 min before AGE, in contrast to the increase seen when AGE was administered alone (Fig. 4). Because our AGE contained 6.3 g/L arginine (a substrate of NOS), we investigated the effect of an equivalent dose of arginine. However, treatment with arginine alone did not increase NO production (Fig. 4). These results suggest that the enhancement of NO production that occurred after administration of AGE does not depend on the arginine content of the AGE, and could therefore be caused by an increase in cNOS activity. LPS (an iNOS inducer) began to increase NO production at 120 min after administration. Levels had increased by 5.2-fold at 360 min and continued to rise for up to 720 min after administration (Fig. 2). Nevertheless the pattern of NO production in response to AGE was obviously different from that seen with LPS (Figs 1 and 2). Thus, the results obtained after administering LPS further substantiated the theory that NO production after the administration of AGE might result from cNOS activity. Although cNOS normally exists as an inactive homodimer, its expression and activation depend on the intracellular Ca2+ concentration. We therefore hypothesized that AGE may activate cNOS by accelerating Ca2+ influx.
This study also demonstrated that AGE significantly suppresses peroxynitrite-induced hemolysis in a dose-dependent manner (Table 2). Kondo et al. (30) showed that the antioxidant actions of albumin, glutathione, and N-acetylcysteine suppress peroxynitrite-induced hemolysis. It is thought that the antioxidant activity of albumin in the vascular compartment results from the scavenging of reactive oxygen and nitrogen species that are generated by basal aerobic metabolism (32,33). The main constituent responsible for the antioxidant properties of albumin is the thiol group within the cysteine moiety (34). In addition, the thiol residue of albumin reacts preferentially with peroxynitrite leading to thiol oxidation (35). Because glutathione and N-acetylcysteine also contain thiol groups, we consider that their effects are likely to be similar to that of albumin. AGE contains not only sulfur-containing compounds derived from garlic, but also other compounds such as Maillard reaction products formed during its natural aging process. Compared with the common antioxidants mentioned above, garlic compounds do not possess a free thiol group, but contain an S-allyl moiety or an allyl sulfoxide moiety. These may be important for the prevention of peroxynitrite-induced hemolysis. Other potential mechanisms of action may involve scavenging of peroxynitrite or erythrocyte membrane stabilization (36,37).
NO has different effects in cardiovascular disease depending on whether it is produced by cNOS or iNOS. Although the small quantity produced via cNOS in atherosclerotic lesions has a protective effect, peroxynitrite arising from the larger quantities of NO produced by iNOS contributes to vascular injury (36,38). In this report, we demonstrated that AGE increases NO levels through stimulation of cNOS, but not iNOS and protects against peroxynitrite-induced damage. These findings suggest that AGE may be extremely useful in the prevention of cardiovascular disease. It was reported that fresh garlic powder increases cNOS activity, but a quantity of arginine equivalent to that found in fresh garlic powder does not affect cNOS activity (39,40). Garlic contains many other amino acids, and Das et al. (40) speculated that any of these might be responsible for the increase in NOS activity in response to fresh garlic powder. Furthermore, wild garlic (Allium ursinum), another species of garlic, was found to block NOS inhibitor–induced hypertension by antagonizing the inhibitory effect on NO production in vivo (41). Because cardiovascular disease is generally chronic in nature, long-term intake of garlic may contribute to its prevention. However, it is not practical to use raw garlic because this has a variety of unwanted side effects such as anemia, weight loss, and growth reduction (2). AGE, which is extracted over a period of 10 mo, is less irritating and less toxic, so that intake can be tolerated for extended periods of time without the side effects of raw garlic (2,3). For this reason AGE could be useful for the long-term prevention of cardiovascular disease.
In conclusion, the study indicates that AGE increases NO levels by stimulating cNOS, but not iNOS, and that this increase is not due to the arginine content of AGE. Moreover, AGE protects erythrocytes from peroxynitrite-induced membrane damage. These findings suggest that chronic intake of AGE could be useful for the prevention of cardiovascular diseases resulting from oxidative stress associated with high iNOS activity or dysfunctions of NO production.
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FOOTNOTES |
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2 Author disclosure: No relationships to disclose.
4 Abbreviations used: AGE, aged garlic extract; cNOS, constitutive NOS; DPI, diphenyleneiodonium chloride; iNOS, inducible NOS; L-NMMA, NG-monomethyl-L-arginine acetate; LPS, lipopolysaccharide; NO, nitric oxide; NOR3, (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide, NOS, NO synthases.
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