Apolipoprotein E (ApoE) and low-density lipoproteins receptor (LDLR) deficient mice (Apoe^−/− and Ldlr^−/− double knockout mice, 129xC57BL/6J background) were obtained from Jackson Laboratory (Bar Harbor, ME, USA) and bred in Kobe Gakuin University. All experiments were conducted in accordance with the ethical principles of animal care of Kobe Gakuin University and the guiding principles for the care and use of animals in the field of physiological sciences published by the Physiological Society of Japan [24] . Euthanasia was performed by CO₂ inhalation or cervical dislocation after experiments. Myricetin, purity more than 97% was purchased from Tokyo Chemical Industry Co., Ltd., Tokyo, Japan and glucosyl hesperidin (G-hesperidin; purity of 78.8%) was kindly provided by Toyo Sugar Refining Co., Ltd, Tokyo, Japan. Control animals (N = 8) were fed high fat diet (F2HFD1), obtained from Oriental Yeast Co., LTD., Tokyo, Japan, to induce atherosclerosis. Myricetin or G-hesperidin were mixed with the high fat diet at concentrations of 0.06% and 0.12% and 0.3% and 0.6%, respectively. The animals (N = 8 in each group) were given myricetin diet or G-hesperidin diet and water ad libitum. Each animal was kept in an individual cage and water and food were changed at 9:00 am every day. Consumption was assessed by weighing the diet and water on each occasion. The daily average doses of myricetin and G-hesperidin, calculated from the food intake in each group were adjusted to approximately 100 and 200 mg/kg animal body weight/day [25] [26] and 250 mg/kg and 500 mg/kg animal body weight/day [16] , respectively (Table 1).

Closed cranial windows were created as described by Morii et al. [27] and thrombotic tendency was measured as reported previously [16] [28] .

In brief, animals were ventilated artificially after anesthetizing and Evans blue was administered through a femoral vein. Closed cranial windows were formed with 2 mm diameter in the right parietal bone and artificial cerebrospinal fluid was infused continuously within this window. The animals were placed in a stereotaxic frame on the stage of an optical microscope and the cerebral vessels on the surface of the brain were monitored and recorded on videotape with a CCD camera (Pulnix, Takenaka System, Kyoto). A He-Ne laser beam (15 μm in diameter) was focused on the center of selected blood vessels through the optical path of the microscope and thrombi were formed in venules (25 - 40 μm) by repeated irradiation for 10 s at 20 s intervals at a power of 13 mW. The number of laser pulses required to generate an occlusive thrombus was used as an index of thrombotic tendency.

Arteriosclerosis was assessed by estimating the development of atherosclerotic lesions as a percent of the entire surface area of the aorta [29] [30] . Briefly, hearts were exposed by abdominal incision and 50 mL of phosphate buffered saline (pH 7.4) infused through butterfly needle, followed by 10% neutral buffered formalin solution by reflex through a femoral artery. The aortic arch was carefully dissected from connective tissue and minor branching blood vessels. The extracted vessels were kept in 10% neutral buffered formalin solution until processed. The vessels were then incised along the longitudinal plane, inverted on a black rubber sheet and held in place using stainless steel pins. The tissue was washed with distilled water for 30 s, treated with 60% isopropyl alcohol for 1 min, and stained with Oil Red O stain solution at 37˚C for 15 min. Finally, the vessel was washed with 60% isopropyl alcohol and distilled water.

The Oil Red O stained specimens were photographed using a digital camera (Olympus Tokyo Japan). Images were transferred to a PC and analyzed using NIH Image. The entire area of the dissected blood vessels and the area of atherosclerotic lesions identified by Oil Red O staining were calculated. The percentages of Oil Red O staining area to entire area were used as an index of atherosclerotic development.

The results illustrate the number of animals (N) and the mean and standard error (SEM) of each experiment. Statistical evaluation was performed by one-way analysis of variance (ANOVA), by the post hoc test of Dunnett and Mann-Whitney test using commercially available statistical packages (Prism 5.0; GraphPad Software, Inc., San Diego, CA, USA). A value of p < 0.05 was considered to be statistically significant.

The doses of myricetin (100 and 200 mg/kg/day) and G-hesperidin (250 and 500 mg/kg/day) were adjusted every day for 8 weeks, respectively by weighing the consumption of high fat diet (F2HFD1) mixed with myricetin or G-hesperidin. Changes in body weight over time after administration of myricetin and G-hesperidin from 8 weeks of age up to 16 weeks of age are shown in Figure 2. The body weights increased with age, and there were no statistically significant differences among these groups.

Thrombogenesis in cerebral vessels for each group of animals, as assessed by the He-Ne laser technique, is summarized in Figure 3. The number of laser pulses

required to generate occlusive thrombi in pial vessels in control and myricetin-fed mice at 100 and 200 mg/kg/day were 3.9 ± 0.2 (N = 5), 5.8 ± 0.5 (N = 4), 6.4 ± 0.4 (N = 5), respectively and G-hesperidin-fed animals at 250 and 500 mg/kg/day were 6.0 ± 0.6 (N = 4) and 7.5 ± 0.2 (N = 8), respectively. Thrombus generation was significantly delayed at both concentrations of myricetin and G-hesperidin as shown in Figure 3. (p < 0.01 and p < 0.001). These results were consistent with our former studies demonstrating that flavonoids, including hesperidin, naringin and nobiletin, inhibited thrombus generation, possibly by scavenging free radicals and resulting in increased nitric oxide (NO) which inhibits platelet aggregation [16] [17] . Our new data provided further evidence that mechanisms underlying the effects of the flavonoids were related to strong antioxidative properties.

Mechanisms of He-Ne laser induced thrombus generation in small blood vessels may depend on free radical and/or active oxygen produced in response to laser irradiation [31] , and such reactive oxygen species (ROS) may be involved in vascular dysfunction. ROS reduce levels of bioactive NO by conversion into toxic peroxynitrite (ONOO⁻) leading to a deterioration in protective endothelial function [32] [33] . Povlishock demonstrated that the endothelial dysfunction caused by He-Ne laser irradiation induced platelet aggregation without cell-surface denudation [34] . Furthermore, Wachowicz concluded that ROS are produced during platelet aggregation and play an important role in platelet function [35] . Other studies have shown, however, that flavonoids blocked platelet aggregation by inhibiting thromboxane receptors [36] or thromboxane A₂ [37] .

In alternative studies, Rong et al. reported that hesperidin pretreatment protected neonatal rats from brain injury by reducing free radicals [38] , and Mastantuono et al. also reported that hesperidin, diosmin, and apigenin had dose-related protective effects on microvascular damage induced by hypoperfusion and reperfusion in the rat pial circulation, possibly related to a decrease in ROS production [39] .

Our previous studies indicated that the antioxidant activity of G-hesperidin inhibited He-Ne laser induced thrombosis by scavenging ROS [16] , and overall the present studies are consistent with the likelihood that the adverse effects of ROS produced by laser irradiation and platelet aggregation were moderated by the flavonoids

The entire area of the dissected blood vessels and the area of atherosclerotic lesions identified by Oil Red O staining in control, myricetin at 200 mg/kg/day and G-hesperidin at 500 mg/kg/day are shown in Figure 4. The lipid-stained areas in control were dominant and clear, as compared to myricetin and G-hesperidin treated aorta. The severity of atherogenesis, as expressed as percentages of lipid-stained areas to entire area of the aorta is shown in Figure 5. The percentages in control, myricetin and G-hesperidin treated mice were 33.4 ± 3.1% (N = 8), 14.2 ± 2.3% (N = 8; p < 0.001 vs. control) and 22.0 ± 1.4% (N = 8; p < 0.001 vs. control), respectively. The data clearly demonstrated that the control

Apoe^−/− and Ldlr^−/− double knockout mice fed high fat diet for eight weeks accumulated lipid over approximately about one third of the entire aorta, and that the flavonoids significantly prevented this deposit of lipid.

Therapeutic flavonoids including quercetin, kaempferol, myricetin, apigenin, and luteolin are well known to reduce the incidence of coronary disease in elderly humans [11] . Atherosclerosis is widely considered to be a chronic inflammatory disease, and excess ROS, generated by oxidative stress, has emerged as a critical common mechanism involved in vascular injury in both the development and progression of atherosclerosis [20] . It is generally believed that the potent antioxidant activity of polyphenols and their inhibitory effects on LDL oxidation support antiatherogenic properties [4] [10] . In addition, Bertin et al. reported that myricetin appeared to be the most active of selected plant-derived polyhydroxyl compounds, with prominent capacities against oxidized LDL and ROS production [40] . Our finding that the inhibition of both thrombotic tendency and atherogenesis by myricetin and G-hesperidin, were in keeping with their strong antioxidant potential, although other factors also might be involved. In this context, Sun et al, recently suggested that hesperidin moderated atherosclerosis not only by anti-oxidative and anti-inflammatory action but also by other pleiotropic effects, including improvement of insulin resistance, amelioration of lipid profiles and inhibition of macrophage foam cell formation [41] . Our results provide convincing direct evidence that myricetin and G-hesperidin inhibited thrombogenesis and atherogenesis. Precise mechanisms underlying this important inhibitory action remain to be fully characterized.