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School of Biomolecular Sciences, Liverpool John Moores University, Liverpool L3 3AF, England, UK
4 To whom correspondence should be addressed: Email: k.rahman{at}livjm.ac.uk.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • aged garlic extract • platelets • inhibition • calcium • ADP
Garlic (Allium sativum) has been used for centuries both as a flavoring agent and as a therapeutic agent for the treatment of a variety of human diseases and disorders (1). Properties of garlic that have attracted much attention are its role as an antioxidant and its ability to reduce cardiovascular disease and cancer. Because of the intense interest in garlic, various commercial preparations have become available over the years. These include garlic powder tablets, oil of steam-distilled garlic, oil of oil-macerated garlic, and ether-extracted oil of garlic. Another such preparation is aged garlic extract (AGE)5, which is prepared by soaking sliced raw garlic in 15–20% aqueous ethanol for up to 20 mo at room temperature. This extract is then filtered and concentrated under reduced pressure at low temperatures and is marketed in both dry and liquid forms. This process causes considerable loss of allicin and increases the concentration of newer compounds, many of which are sulfur-based and water-soluble. The major sulfur compound, S-allylcysteine (SAC), is used to standardize AGE, and some of its other constituents are shown in Table 1.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Triton-X-100 was obtained from BDH laboratory supplies.
AGE and its constituents, alliin, cycloalliin, S-allyl-L-cysteine, S-methyl-L-cysteine (SMC), S-ethylcysteine (SEC), S-1-proponyl-L-cysteine (SPC), S-allylmercapto-L-cysteine (SAMC), fructosyl-arginine, and ß-chlorogenin (BC), were donated by Wakunaga of America.
Preparation of human platelet-rich plasma. This study was approved by the ethics committee of Liverpool John Moores University and informed consent was obtained from all volunteers prior to blood donation. Blood samples were collected from apparently healthy volunteers who had not taken medication that could affect platelet function for at least 2 wk prior to the study. Blood was mixed with 3.8% (wt:v) trisodium citrate as the anticoagulant in the ratio 9:1 (v:v). Platelet-rich plasma (PRP) was prepared by centrifugation of whole citrated blood for 10 min at 150 x g. Platelet-poor plasma (PPP) was prepared by further centrifuging the blood for 20 min at 1500 x g. Platelet count for the PRP was adjusted if necessary to 2.5 ± 0.5 x 105 cells/mL by diluting with PRP. All experiments were conducted within 3 h of blood donation.
Platelet aggregation. Platelet aggregation was performed in a PAP-4D Platelet Aggregation Profiler (Bio/Data Corporation) as described by Rahman and Billington (9). Briefly, platelet aggregation was carried out in 0.2 mL of PRP and was initiated by adding 0.02 mL of ADP (8µmol/L) or A23186 (5µmol/L). In experiments investigating the effects of AGE and its constituents on calcium mobilization, 1 mmol/L of CaCl2 was added to the incubation medium prior to initiating platelet aggregation. Due to the metal-chelating properties of AGE and its dark color, a diethyl-ether extract was prepared by diluting 1 part of AGE with 2 parts of diethyl ether; this was left to stand at room temperature for 5 min, after which the diethyl-ether extract was removed and dried under oxygen-free nitrogen gas. The residue was resuspended in phosphate-buffered saline, pH 7.2 to its original volume (5). AGE and its diethyl-ether extract were tested up to a final concentration of 25% (v:v) while the constituents of AGE were tested up to a final concentration of 1 mmol/L. In some experiments the effect of prostaglandin E1 (PGE1) on ADP induced platelet aggregation was also investigated. For these experiments PGE1 was dissolved in DMSO, and further dilutions were performed up to a final concentration of 250µg/mL in phosphate buffered saline (PBS). The reaction was allowed to run for 5 min and aggregation was calculated as percentage aggregation after the addition of agonists and was compared with that of the control.
Disaggregation studies.
This was essentially carried out as reported by Samal and Loiko (12). AGE, its constituents, or its diethyl-ether extract, was added to PRP and aggregation was induced by the addition of ADP. These experiments were only performed if the maximum aggregation was 
60%. AGE and diethyl-ether extract were investigated up to a concentration of 25% (v:v) and the constituents mixture of AGE was investigated up to a concentration of 1 mmol/L.
Calcium measurement studies. Fura-2/AM at a final concentration of 2 µmol/L was added to PRP, and the mixture was incubated for 45 min at 37°C. After washing, Fura-2–loaded platelets were resuspended in HEPES-Tyrode's buffer, pH 7.4 at a concentration of 2.5 x 105 cells/mL. Washed platelets were incubated with either AGE or its diethyl-ether extract at a final concentration of 25% (v:v) for 10 min at 37°C, respectively. Finally, the platelets were stimulated by the addition of ADP at a final concentration of 8 µmol/L or by the Ca2+ ionophore A23187 at a final concentration of 5 µmol/L. Fura-2 fluorescence was measured using a VARIAN Cary Eclipse Fluorescence Spectrophotometer (Australia) with an excitation wavelength alternating every 0.5 s from 340 to 380 nm; the emission wavelength was set at 510 nm. The [Ca2+]i values were determined from the ratio of fura-2 fluorescence intensity at 340 and 380 nm, using the ratio-scan function and the software version 1.1(132) for the Cary Eclipse machine.
Statistical analysis. Differences in the presence and absence of AGE and other compounds were analyzed by Students' paired t test, using the Minitab statistical software package. P < 0.05 was considered significant. Values are means ± SEM.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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75% platelet aggregation was observed. In the presence of AGE, inhibition of platelet aggregation occurred in a concentration-dependent manner and achieved significance between 1.56–25% (v:v), (Fig. 1A). A diethyl-ether extract of AGE up to 25% (v:v) had no effect on the ability of platelets to aggregate (results not shown). Individual components of AGE (see Table 1) were also tested for their effects on ADP-induced platelet aggregation. All constituents displayed a biphasic pattern of inhibition that was only significant for SEC at 0.78 µmol/L, SMC at 3.125, 6.25 µmol/L, SPC at 0.78, 3.125, 6.25 µmol/L, and BC at 0.78, 3.125 and 25 µmol/L, respectively (results not shown). However, when these constituents were combined as a mixture, a biphasic pattern was again observed, although no significant inhibition of platelet aggregation was apparent (Fig. 1B). The amino acids, L-arginine, L-cysteine, and L-methionine are also present in AGE. L-Methionine showed significant inhibition at concentrations of 0.00078, 0.00625, and 0.1 mmol/L, while L-arginine and L-cysteine displayed significant inhibition of platelet aggregation only at a concentration of 9 mmol/L (Fig. 1C). The amino acids tested as a mixture up to a concentration of 1 mmol/L had no effect on platelet aggregation (results not shown). However, when the mixture of the 9 constituents (Table 1) was tested in the presence of the three amino acids, inhibition of platelet aggregation was only significant at 1 mmol/L (Fig. 1D).
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85% platelet aggregation was observed (Fig. 3A). The use of A23187 also resulted in a peak in intracellular calcium at 0.5 s (Fig. 3B). When these experiments were repeated in the presence of AGE, significant inhibition of platelet aggregation was observed at a concentration of 0.78, 6.25–25% (v:v) (Fig. 3A). Similarly, when AGE was used at a concentration of 25% (v:v), the rise in intracellular calcium ions was abolished (Fig. 3B). ADP at a concentration of 8µmol/L also resulted in an increase in intracellular calcium ion concentration similar to that seen with A23187 (Fig. 3C). The presence of AGE at a concentration of 25 (v:v) led to the abolishment of the peak in intracellular calcium-ion concentration (Fig. 3C). In contrast, a diethyl-ether extract of AGE failed to suppress calcium mobilization when platelets were stimulated with either ADP or A23187 (results not shown).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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AGE is a complex mixture with relatively high concentrations of water-soluble compounds and low concentrations of oil-soluble compounds, and it is standardized by S-allylcysteine, its major organosulfur constituent. It also contains the amino acids L-arginine, L-cysteine, L-methionine, and other major constituents shown in Table 1. In this study 5% aggregation was achieved when platelet aggregation was initiated with ADP at a concentration of 8 µmol/L. AGE significantly inhibited platelet aggregation between 1.56–25% (v:v) (Fig. 1A). Because a diethyl-ether extract of AGE failed to show any inhibition of ADP-induced platelet aggregation (results not shown), it is highly likely that the water-soluble compounds present in AGE are the ones responsible for its antiplatelet aggregation reaction. However, there are other components present in the diethyl-ether extract of AGE that have antioxidative properties (5). The individual constituents of AGE shown in Table 1 were tested for their antiaggregatory affects on platelets. A biphasic pattern of inhibition of platelet aggregation was observed, but only for SEC at 0.78 µmol/L, SMC at 3.125 and 6.25 µmol/L, SPC at 0.78, 3.125, and 6.25 µmol/L, and BC at 0.78, 3.125, and 25 µmol/L (results not shown). The mixture of all these constituents also failed to show any significant inhibition of platelet aggregation, although a biphasic pattern of inhibition was again apparent (Fig. 1B). The amino acids were also tested for their possible antiaggregatory effects on platelet aggregation. L-Methionine significantly inhibited platelet aggregation at concentrations of 0.00078, 0.00625, and 0.1 mmol/L, whereas L-arginine and L-cysteine displayed significant inhibition at a concentration of 9 mmol/L (Fig. 1C). However, the mixture of amino acids up to a concentration of 1 mmol/L had no effect on platelet aggregation (results not shown), and neither did the mixture of amino acids plus other constituents (Fig.1D). This strongly implies that for AGE to be effective, all the constituents (some of which were not tested in this study) must be present in a synergistic manner. In support of this, it has been shown previously that AGE is more potent than its constituents in its ability to lower cholesterol (14,15). Another possible explanation is that the constituents of AGE must be present at certain concentrations, or that, once these constituents are absorbed in the gut, they are transformed into more potent molecules.
Prostaglandin is a well-established inhibitor of platelet aggregation and is used in the treatment of atherosclerosis (13,16). It has also been reported that when PGE1 is added to aggregated platelets, it causes them to disaggregate (13). Platelet activity is, in part, regulated by cyclic adenosine monophosphate (cAMP). Endothelial cells contain prostacyclin synthetase, which produces prostaglandin I2 from endoperoxides; whereas platelets contain thromboxane synthetase, which produces TXA2 (17). The formation of TXA2 in platelets induces platelet aggregation, which is accompanied by the platelet-release reaction whereby serotonin and other granule components are expelled from platelet stores. TXA2 also induces a rise in the concentration of ionized calcium in the platelet cytosol and a decrease in platelet cAMP formation by inhibiting adenylyl cyclase. Prostaglandin I2 binds to specific receptors on the surface of the platelets and stimulates adenylyl cyclase. The resulting increase in platelet cAMP leads to calcium reuptake by the dense tubular system and thereby inhibits platelet activation, platelet granulation secretion, and thus platelet aggregation. In confirmation, PGE1, a known stimulator of platelet cAMP, caused significant inhibition of platelet aggregation when these were preincubated with PGE1 up to a concentration of 250 µg/mL and stimulated with ADP (18) (Fig. 2A). Because PGE1 is reported to cause disaggregation of stimulated platelets, AGE, its constituents, and a diethyl-ether extract of AGE were tested to see if they would behave in a similar manner. AGE caused disaggregation of platelets at concentrations of 12.5 and 25% (v:v), whereas the constituents of AGE, tested as a mixture, caused the disaggregation at 0.25 and 1 mmol/L, respectively (Fig. 2B,D). In contrast, the diethyl-ether extract of AGE failed to show any disaggregation of platelets (Fig. 2C), which confirms earlier data that the diethyl-ether extract has no effect on platelet aggregation induced by ADP (results not shown). Aged garlic extract is reported to enhance nitric oxide production (19) and the arginine present in AGE is not responsible for this effect. It is likely that the platelet-derived nitric oxide contributes to the process of platelet disaggregation. The inhibition of phosphoinositide 3-kinase (PI3-kinase) has been shown to cause platelet disaggegation, and the incubation of platelets with PI3-kinase inhibitors leads to a dose-dependent increase in platelet nitric oxide and cyclic adenosine monophosphate (cGMP) levels (20). AGE could also be reducing thromboxane formation because fresh garlic extract has been shown to do this (21), or it is binding to thromboxane A2 receptors in a similar manner to that observed with the inhibitory effects of flavonoids on platelet function (22). It has also been reported that N-ethylmaleimide causes the disaggregation of both ADP- and thrombin-induced platelet aggregation, and that this disaggregation is a result of the removal of calcium ions from the platelet cytosol (12). Therefore, the effects of AGE on calcium mobilization were investigated in both A23187 and ADP-stimulated platelets.
Although variable results were obtained in the calcium-mobilization experiments, the underlying trends were similar. In the presence of A23187, 85% aggregation of platelets was observed. When these experiments were repeated in the presence of AGE, significant inhibition of platelet aggregation occurred at concentrations of 0.78 and 6.25–25% (v:v) (Fig. 3A). This implies that the antiaggregatory effect of AGE may be related to the intraplatelet mobilization of calcium ions. Additional support of this can be seen in Fig. 3B: when platelets are stimulated with A23187, a peak in intracellular calcium ion concentration is observed at 0.5 s, and this peak is also abolished in the presence of a 25% (v:v) concentration of AGE. ADP-stimulated platelets also show a peak in intracellular calcium concentration at 0.5 s, and again, this peak was abolished in the presence of a 25% (v:v) concentration of AGE (Fig. 3C). An interesting observation was that, in both A23187 and ADP-stimulated platelets in the presence of AGE, the initial concentration of calcium ions was significantly less than when the experiments were performed in the absence of AGE (Fig. 3B,C). This could be due to the metal chelation properties of AGE, as reported earlier (5). In support of this, garlic extract has been shown to strongly inhibit calcium binding and arteriosclerotic nanoplaque formation (23). Interestingly, a diethyl-ether extract of AGE up to a concentration of 25% (v:v) had no effect on intraplatelet calcium mobilization (results not shown). The garlic component, diallyl trisulfide, is reported to inhibit platelet aggregation and calcium mobilization in a concentration-dependent manner without increasing intracellular cAMP and cGMP levels. Diallyl trisulfide also had no effect on thromboxane A2 production and no effect on inositol-1,4,5-triphosphate formation. Hence, it is possible that AGE acts in a similar fashion and suppresses calcium mobilization at a step distal to the formation of inositol-1,4,5-triphosphate (24). Another speculation is that AGE may inhibit phospholipase A2, thus reducing levels of lysophosphatidic acid, which causes platelet aggregation and increases intracellular calcium ions (25). AGE could also exert its affects through intracellular signals that inhibit H2O2-induced platelet aggregation and the accompanying increase in intracellular calcium ions (26). Finally, it is possible that AGE can bind to GPIIb/IIIa receptors on the platelet membrane and change its conformation so that platelets have a reduced affinity to bind fibrinogen.
To conclude, the results reported in this study indicate that AGE acts in a synergistic manner in its inhibitory effect on platelet aggregation. The mechanisms involved appear to be multiple in nature and may involve membrane fluidity changes, inhibition of phospholipase C, inhibition of calcium mobilization, increase in nitric oxide and cAMP production, and inhibition of TXA2, all of which will lead to an inhibition of platelet aggregation. The results reported here need to be validated further, and the mechanisms that inhibit platelet aggregation by AGE in vivo need to be established.
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FOOTNOTES |
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2 Author disclosure: No relationships to disclose.
3 This study was supported by a grant from Wakunaga of America.
5 Abbreviations used: AGE, aged garlic extract; ADP, adenosine diphosphate; ALN, alliin; BC, ß-chlorogenin; CA, cycloalliin; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanine monophosphate; DMSO, dimethyl sulphoxide; FRA, fructosyl-arginine; HEPES, N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid]; SAC, S-allyl-L-cysteine; SAMC, S-allylmercapto-L-cysteine; SEC, S-ethylcysteine; SMC, S-methyl-L-cysteine; SPC, S-1-proponyl-L-cysteine; PGE1, prostaglandin E1; PI3-kinase, phosphoinositide 3-kinase; PPP, platelet-poor plasma; PRP, platelet-rich plasma; TXA2, thromboxane A2.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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