DL-homocysteine (Hcy), 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), bradykinin, 1,1-diphenyl-2-picrylhydrazyl (DPPH), dimethyl sulfoxide, propranolol, n-butanol and other chemicals were high-grade products purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). N^G-nitro-_L-arginine (_L-NNA) and glibenclamide (Glib) were obtained from MP Biomedicals, LLC (Santa Ana, CA, USA). KH solution was composed of 70.2 mM NaCl, 4.2 mM KCl, 2.8 mM CaCl₂, 2.7 mM MgSO₄, 21.0 mM NaHCO₃, 0.2 mM KH₂PO₄ and 9.8 mM glucose, and the pH was adjusted to 7.4.

A total of 600 g fresh ginger rhizome was soaked in 2.5 l ethanol. The extracts were refluxed at 78°C for 2 h; this was repeated three times. Subsequently, the filtrate was concentrated in a rotary evaporator. The weight of extracts was ~34.2 g (yield, 5.7%). The residue was then suspended in 50 ml water and extracted with 50 ml chloroform twice, after which the chloroform partition was evaporated to obtain 8.4 g residue (GCE). The aqueous phase was partitioned with n-butanol. The n-butanol partition was evaporated to obtain 6.3 g residue (ginger n-butanol extract, GNE). The water extract underwent reverse osmosis to obtain 19.5 g residue (ginger water extract, GWE); this process is summarized in Fig. 1. The stock solution of ginger extraction was prepared by dimethyl sulfoxide to dilute for further experiments.

Porcine hearts were freshly obtained from the local abattoir, immersed in cold 0.9% NaCl at 4°C and were transported to the research laboratory. Excess connective tissue was removed and the arteries were cut into 5-mm rings. Endothelium-intact and -denuded porcine coronary artery rings were prepared, and the rings were then mounted with two stainless steel hooks in 10 ml KH solution-filled organ baths. KH solution was kept in oxygenated conditions (95% O₂ and 5% CO₂) at 37°C and was replaced every 15 min to maintain continuous equilibration. The rings were perfused with 30 mM KCl in the organ bath until tonic phase contraction was achieved, as previously described (21) before pretreatment with 100 µM L-NNA, 10 µM ODQ, 10 µg/ml indomethacin, 20 µM propranolol, 1 µM Glib, 100 µM Hcy, 30 mM bradykinin and 77.5 mM H₂O₂, respectively, for indicated period of time.

The porcine coronary arteries were harvested, cut into numerous 5-mm rings, and were maintained in 5 ml organ baths containing 95% O₂ and 5% CO₂ at 37°C. Ginger extracts were individually added to the 5-mm rings for 30 min and relaxation was observed. Alterations in tension were recorded using a Grass Force displacement transducer (model FT03; Grass; Natus Medical Incorporated, Pleasanton, CA, USA).

The DPPH radical scavenging assay was performed according to the method described by Sakanashi et al (21). Briefly, in each well of a 96-well plate, 50 µl sample extract was added to 150 µl 0.25 mM DPPH methanolic solution. After mixing thoroughly, the reactants were incubated in the dark for 30 min at room temperature. The control was prepared by mixing 50 µl methanol with 150 µl DPPH. The absorbance was detected at 517 nm using a spectrophotometer. Samples were measured in triplicate.

The levels of superoxide anion produced by endothelial cells of the porcine arteries were detected using the lucigenin-enhanced chemiluminescence method, as previously described by Sun et al (22). Briefly, the samples of GCE, GNE and GWE were mixed with 5 µM lucigenin for 6 min. Time-based reading was recorded in a 5 min period using a luminometer. The area of each vessel segment was measured using a caliper and was used to normalize the data for each sample.

Following treatment with or without GCE, GWE and GNE, porcine coronary artery endothelial cells were collected as previously described (23) and mixed with protein lysis buffer [50 mM Tris-HCl (pH 7.4), 1 mM NaF, 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethane-sulfonyl fluoride, 1% NP-40 and 10 µg/ml leupeptin] on ice. The samples were homogenized for 20 sec, incubated for 20 min on ice and centrifuged at 15,000 × g for 30 min at room temperature. The supernatants were then transferred into new tubes for protein quantification, as previously described (24,25).

A total of 50 µg protein was loaded and separated by 10% SDS-PAGE. The samples in the gels were then transferred onto polyvinylidene difluoride membranes. The membranes were incubated with 0.1% PBS-Tween containing 5% non-fat milk for 30 min at room temperature, and were then hybridized with cyclooxygenase-2 (COX-2; cat. no. GTX100656; 1:1,000 dilution; GenTex, Hsinchu, Taiwan), inducible nitric oxide synthase (iNOS; cat. no. GTX130246; 1:1,000 dilution; GenTex), endothelial nitric oxide synthase (eNOS; cat. no. 3GTX129843; 1:1,000 dilution; GenTex) and β-actin (cat. no. GTX109639; 1:5,000 dilution; GenTex) primary antibodies. Subsequently, membranes were incubated with horseradish peroxidase-conjugated rabbit IgG antibody (cat. no. GTX213110-01; 1:10,000 dilution; GenTex) at room temperature for 1 h and were then visualized using Immobilon Western HRP substrate kit (EMD Millipore, Billerica, MA, USA). Densitometric analysis of each band was performed utilizing National Institutes of Health (NIH) ImageJ 1.47 software (NIH, Bethesda, MD, USA).

Data are presented as the means ± standard deviation from at least three separate experiments. Statistical data were analyzed using one-way ANOVA with post hoc Dunnett's test for comparing groups to the control by SPSS version 13.0 for Windows (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significantly difference.

The three varieties of ginger extract (GCE, GNE and GWE) were prepared according to the diagram presented in Fig. 1. These three ginger extracts were used in the present study to explore their effects on the vasorelaxation of porcine coronary artery rings.

Porcine coronary arteries were suspended in an organ bath. Various amounts of GCE (1, 3, 10, 30 and 100 µg/ml) were added to the porcine coronary arteries; water was used as a vehicle control. A dose-dependent increase in relaxation was observed in response to GCE (Fig. 2).

The results of the present study indicated that GCE induced endothelium-dependent vasorelaxation. Endothelium-intact and -denuded porcine coronary artery rings were incubated with various amounts of GCE (1, 3, 10, 30 and 100 µg/ml). GCE was able to reduce KCl-induced contraction and increase vasorelaxation from 27 to 99% in the endothelium-intact porcine coronary artery rings, whereas GCE exerted mild effects on the vasorelaxation of denuded porcine coronary artery rings (from 15 to 93%) (Fig. 3). Based on these data, it was suggested that endothelium-dependent relaxation was increased in porcine coronary arteries following GCE exposure.

Numerous in vitro and in vivo studies have reported that endothelium-dependent relaxation and vasodilatation persist in the presence of NOS inhibitors, including L-arginine analogues, such as _L-NNA (23,26). Porcine coronary artery rings were pretreated in the absence (control) or presence of 100 µM _L-NNA for 20 min, and were then incubated with 30 mM KCl to induce contraction until the tonic phase (27). Relaxation was examined in the presence of various concentrations of GCE (1, 3, 10, 30 and 100 µg/ml) in an organ bath. GCE induced relaxation of porcine coronary artery rings from 13 to 86% without L-NNA pretreatment. Conversely, GCE (100 µg/ml) induced relaxation of porcine coronary artery rings to 66%, in the presence of _L-NNA (Fig. 4). These results revealed that GCE-induced relaxation of porcine coronary arteries may be mediated by the NOS signaling pathway.

The present study determined the effects of GCE on sGC-induced relaxation. Porcine coronary artery rings were pretreated in the absence (control) or presence of 10 µM ODQ for 20 min, and were then incubated with 30 mM KCl to induce contraction until the tonic phase. Relaxation was examined in the presence of various concentrations of GCE (1, 3, 10, 30 and 100 µg/ml) in an organ bath. GCE induced an increase in relaxation from 8 to 87% in porcine coronary artery rings following pretreatment without 10 µM ODQ. Conversely, relaxation of porcine coronary artery rings was significantly reduced following pretreatment with 10 µM ODQ and treatment with GCE at 30 and 100 µg/ml (Fig. 5). These results indicated that NO is a vital factor in GCE-induced relaxation of porcine coronary arteries.

The present study further examined the effects of GCE on relaxation following treatment with indomethacin, which is an inhibitor of COX. Porcine coronary artery rings were pretreated in the absence (control) or presence of 1 µg/ml indomethacin for 20 min, and were then incubated with 30 mM KCl to induce contraction until the tonic phase. Relaxation was examined in the presence of various concentrations of GCE (1, 3, 10, 30 and 100 µg/ml) in an organ bath. GCE induced an increase in relaxation from 15 to 100% in porcine coronary artery rings without 1 µg/ml indomethacin treatment. Conversely, following pretreatment with 1 µg/ml indomethacin and treatment with low concentrations of GCE (3–30 µg/ml), relaxation of porcine coronary artery rings was significantly attenuated (Fig. 6). These results suggested that GCE attenuated relaxation induced by arachidonic acid. GCE-induced relaxation of porcine coronary arteries may be through COX pathway.

β-blockers have been widely used in the treatment of numerous cardiovascular diseases, particularly hypertension and atherosclerosis (28). Some β1-adrenergic receptor blockers cause vasodilation by increasing NO (29). The present study examined the effects of GCE on relaxation induced by propranolol, which is a β-blocker. Porcine coronary artery rings were pretreated in the absence (control) or presence of 20 µM propranolol for 20 min, and were then incubated with 30 mM KCl to induce contraction until the tonic phase. Relaxation was examined in the presence of various amounts of GCE (1, 3, 10, 30 and 100 µg/ml) in an organ bath. GCE at 3–30 µg/ml induced an increase in relaxation from 11 to 91% in porcine coronary artery rings without pretreatment with 20 µM propranolol (Fig. 7). These results indicated that GCE exhibited no apparent effect on propranolol-induced relaxation.

K_ATP channels are activated and opened by declining intracellular ATP levels and elevated cAMP concentration, which leads to hyperpolarization of endothelial cells and the promotion of NO formation in vitro (30,31). It has been suggested that endothelial cell hyperpolarization may contribute to vascular relaxation. K_ATP channels are inhibited by sulfonylurea agents, including Glib (31,32). The present study examined the effects of Glib, a K_ATP channel blocker, on GCE-induced relaxation. Porcine coronary artery rings were pretreated in the absence (control) or presence of 1 µM Glib for 60 min, and were then incubated with 30 mM KCl to induce contraction until the tonic phase. Relaxation was examined in the presence of various amounts of GCE (1, 3, 10, 30 and 100 µg/ml) in an organ bath. GCE induced an increase in relaxation from 15 to 76% in the porcine coronary artery rings without 1 µM Glib pretreatment (Fig. 8). These results suggested that Glib had no effect on GCE-induced relaxation.

The present study investigated the effects of GCE on Hcy-induced endothelial cell damage. Porcine coronary artery rings were incubated with 30 µg/ml GCE for 15 min, and were then treated with 100 µM Hcy for 30 min. Porcine coronary artery rings were placed in an organ bath containing 30 mM KCl to induce contraction until the tonic phase. Relaxation was examined following the addition of 30 mM bradykinin into the organ bath. Hcy reduced relaxation, whereas GCE significantly prevented Hcy-induced endothelial dysfunction (Fig. 9). These results indicated that GCE may improve Hcy-induced endothelial cell damage.

The present study clarified the effects of GCE on H₂O₂-induced endothelial cell damage. Porcine coronary artery rings were incubated with 30 µg/ml GCE for 15 min, and were then placed in an organ bath containing 30 mM KCl to induce contraction until the tonic phase. Rings were treated with 77.5 mM H₂O₂ for 15 min and contraction was examined. H₂O₂ induced endothelial contraction, whereas GCE significantly prevented H₂O₂-induced endothelial dysfunction (Fig. 10). These data revealed that GCE may attenuate H₂O₂-induced endothelial cell injury.

Reactive oxygen species (ROS) are produced under oxidative stress and adverse cellular environments (33). Vitamins E and C, β-carotene, flavonoids and polyphenols have previously been demonstrated to possess free radical-scavenging abilities (34). In the present study, the antioxidant properties of ginger extracts were individually determined according to DPPH and lucigenin-enhanced chemiluminescence assays. DPPH absorbance decreased from 0.70 to 0.24, as GCE concentration increased from 62.5 to 1,000 µg/ml (Fig. 11A). The rate of inhibition was increased from 40 to 85% in a dose-dependent manner. DPPH absorbance decreased from 0.79 to 0.39, as GNE concentration increased from 62.5 to 1,000 µg/ml. The rate of inhibition was increased from 37 to 78% in a dose-dependent manner (Fig. 11B). DPPH absorbance decreased from 0.73 to 0.66, as GWE concentration increased from 62.5 to 1,000 µg/ml. The rate of inhibition was increased from 42 to 48% (Fig. 11C). These findings indicated that GCE possesses a stronger ability to reduce free radical levels. To determine whether ginger extracts possess H₂O₂-scavenging abilities, a lucigenin-enhanced chemiluminescence assay was conducted. Various concentrations of GCE, GNE and GWE were used to evaluate their ability to remove H₂O₂. The H₂O₂-scavenging ability was increased from 10 to 52% in response to GCE (Fig. 12A). The H₂O₂-scavenging ability was increased from 68 to 94% in response to GNE (Fig. 12B) and from 63 to 90% in response to GWE (Fig. 12C). These findings indicated that GCE may possess a stronger antioxidant ability to scavenge free radicals.

The present study investigated the effects of ginger extracts on Hcy-induced endothelial cell damage by analyzing the protein expression levels of endothelial NOS (eNOS), iNOS and COX-2. Hcy increased eNOS, iNOS and COX-2 expression. In the absence of Hcy, GWE induced eNOS, maintained iNOS and reduced COX-2 expression (Fig. 13A). Conversely, low concentration (10 µg/ml) of GWE slightly reduced eNOS, slightly induced iNOS and reduced COX-2 expression in the presence of Hcy. A high concentration (30 µg/ml) of GWE markedly reduced the expression levels of eNOS, iNOS and COX-2 in the presence of Hcy. GCE markedly reduced eNOS, iNOS and COX-2 expression in the presence of Hcy, whereas GNE markedly induced eNOS, iNOS and COX-2 expression in the presence of Hcy. These findings indicated that GCE exerts stronger vasoprotective effects. In addition, eNOS expression was quantified from western blot analysis (Fig. 13B). These data suggested that GCE, not GWE or GNE, possesses a strong vasoprotective effect.