Protective Effects of Nobiletin on Hypertension and Cerebral Thrombosis in Stroke-Prone Spontaneously Hypertensive Rats ( SHRSP ) *

Some citrus flavonoids have been reported to possess antioxidant activities that moderate endothelial dysfunction and show protective effects on cardiovascular disease. We have investigated the protective effects of nobiletin (5,6,7,8,3’,4’-hexamethoxy flavone) derived from the peel of Citrus depressa Hayata (Shiikuwasha), a citrus fruit produced in Okinawa prefecture in Japan on hypertension and thrombogenicity in cerebral vessels of stroke-prone spontaneously hypertensive rats (SHRSP). Nobiletin was added to the diet of male SHRSP (7 weeks old) for 4 weeks. The age-related increase in systolic blood pressure usually observed in SHRSP was significantly suppressed in the treated animals. Thrombogenesis in pial blood vessels, determined using a He-Ne laser technique, and antioxidant activity, assessed by measuring urinary 8-hydroxy-2’-deoxyguanosine (8-OHdG), were significantly reduced after treatment. Urinary nitric oxide (NO) metabolites and acetylcholine-induced endothelial relaxation were increased after dietary intervention. These results strongly suggested that antihypertensive and antithrombotic effects of nobiletin may be related to an increase in bioavailable NO, possibly mediated by the scavenging of reactive oxygen species (ROS).

We have previously studied the antihypertensive and antithrombotic effects of hesperidin and naringin, and demonstrated that these citrus flavanone glycosides moderated hypertension and thrombogenesis in cerebral vessels of stroke-prone spontaneously hypertensive rats (SHRSP) [18].Our results indicated that mechanisms underlying the effects of the flavonoids were related to strong antioxidative properties.Many studies have indicated that SHRSP are exposed to high oxidative stress and ROS contribute to the maintenance of hypertension [19][20][21].Increased ROS are involved in mechanisms of vascular dysfunction and reduce the amount of bioactive nitric oxide (NO) by chemical inactivation to form toxic peroxynitrite (ONOO − ) and deteriorate hypertension and endothelial dysfunction [22][23][24].In addition, oxidized low-density lipoproteins (ox-LDL) appear to dysregulate the homeostasis between blood and vascular cells, alterate endothelial function, and promote inflammatory, thrombotic and atherogenetic processes [24][25][26].Nobiletin has been reported to inhibit ROS production and unregulate uptake of ox-LDL via scavenger receptors in macrophages and monocytes [15,27].Nobiletin also at-tenuates very-low-density lipoprotein (VLDL) overproduction, dyslipidemia, and atherosclerosis in mice [28].
The potential cardiovascular effects of polymethoxylated flavonoids were investigated by Robbins et al. [29,30] who demonstrated that nobiletin inhibited ADPinduced platelet aggregation and thrombosis.In addition, Sempiska et al. [31] showed that nobiletin and other flavonoids suppressed platelet adhesion to glasswool.
Shiikuwasha fruit (Citrus depressa Hayata) has been reported to contain higher concentrations of nobiletin and tangeretin than in other citrus fruits [1], and recent studies of extracts of Shiikuwasha have focused on various beneficial effects.Akachi et al. reported that Shiikuwasha juice suppressed liver injury by D-galactosamine [32] and obesity mediated by a high fat diet [33].
In the present communication, we have investigated the beneficial cardiovascular activities of nobiletin purified from the peel of Shiikuwasha fruits.We assessed blood pressures and thrombogenesis in cerebral vessels of SHRSP after ingestion of nobiletin added to the daily diet.We demonstrated that nobiletin moderated the usual age-related increase in blood pressure, and inhibited He-Ne laser induced thrombogenesis in cerebral vessels.Furthermore, dietary intervention significantly reduced antioxidant activity in SHRSP, and increased urinary excretion of NO metabolites and acetylcholine-induced endothelial relaxation.These results suggest that antihypertensive and antithrombogenic effects of nobiletin were possibly mediated by suppression of ROS and increased levels of bioavailable NO.

Animals and Diets
Male stroke prone spontaneously hypertensive rats (SHRSP), 6 weeks old, were obtained from Japan SLC (Hamamatsu, Japan).The animals were used after acclimatization in the laboratory for one week.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 by the Physiological Society of Japan [34].The rats were euthanized with an overdose of sodium pentobarbital following the experiments.
The powder diet was purchased from Funabashi Farm (Funabashi SP diet, Chiba, Japan).Purified nobiletin (purity 96.6%) from the peel of Okinawan Shiikuwasha was provided by ARKRAY Inc. (Karada Support Institute, Kyoto, Japan).Nobiletin was mixed with the normal powdered diet for SHRSP (Funabashi SP diet, Funabashi, Japan) at the indicated concentrations.The nobiletin treated animals were classified into three groups 1) a control group, which was given the standard Funabashi SP diet; 2) nobiletin groups which were given either 20 mg/kg/day nobiletin (c1) or 40 mg/kg/day nobiletin (c2) for 4 weeks from 7 to 11 weeks of age.All animals were allowed water ad libitum.Food and water intake and body weights were measured every day.
Blood pressures were measured once a week using a tail-cuff plethysmograph (LE5001, Pan.Lab, Barcelona, Spain).

Measurement of Thrombogenesis
Closed cranial windows were created as described by Morii et al. [35] and thrombotic tendency was measured as previously reported [36,37].In outline, animals were anesthetized with sodium pentobarbital (60 mg/kg) and artificially ventilated with a mixture of oxygen in air.Femoral veins were exposed and blood vessels were cannulated with polyethylene tubing (PE50, Becton Dickinson and Company, Sparks, MD, USA).Evans blue was administered through the femoral vein.A craniotomy was performed using a hand drill to form a cranial window, 5 mm in diameter, in the center of the right pariet al bone.A coverslip was placed on the window and fixed with dental resin.Cerebrospinal fluid was continuously infused within the cranial window and the intracranial pressure was adjusted to 3 -5 mm Hg to avoid brain herniation.The animals were stablized in a stereotaxic frame and were placed on the stage of an Olympus BH2 microscope equipped with a long working distance objective.The cerebral vessels were monitored using a charge-coupled device (CCD) camera (Pulnix, Takenaka System, Kyoto) and recorded on videotape.A He-Ne laser beam (15 µm in diameter) was focused on the center of the selected blood vessels through the optical path of the microscope and thrombi were formed by repeated irradiation for 10 sec at 20 sec intervals at a power 13 mW.The number of laser pulses needed to induce the formation of an occlusive thrombus was used as an index of thrombotic tendency.

Determination of Urinary 8-OHdG and NO
Metabolites (NO 2 /NO 3 ) Assays were performed before and 4 weeks after each diet.The animals were kept in metabolic cages and urine samples were collected for 24 h.Samples were stored at −80˚C until assayed.8-hydroxy-2'-deoxyguanosine (8-OHdG) was determined using a competitive enzymelinked immunosorbent assay (ELISA; 8-OHdG check, Japan Institute for the Control of Ageing, Shizuoka, Japan).Urinary NO metabolites (NO 2 /NO 3 ) concentrations were determined using Griess reagent (Assay kit-C; Dojindo Laboratory, Kumamoto, Japan).Rings (3 mm wide) were cut and three segments from each animal were mounted on stainless steel hooks and suspended in a water-jacketed 5 ml tissue bath filled with KHS maintained at 37˚C and aerated with 95% O 2 and 5% CO 2 (Easy Magnus System U-5A (4-channels), Iwashiya Kishimoto Medical Instruments, Kyoto, Japan).Aortic rings were stretched to the equivalent of 1.5 g tension for 90 min before commencement of the experiment.After stretching, the aortic rings were equilibrated and contracted with phenylephrine (10 −5 mol/L).Relaxation was measured in the presence or absence of endothelium, by adding acetylcholine (10 −5 mol/L).Relaxation was calculated as a percentage of precontractile vascular tone.The tissue responses were recorded using isometric transducers (Easy Magnus System U-5A, Iwashiya Kishimoto Medical Instruments, Kyoto, Japan) and recorders (SEKONIC SS-250F Recorder, Tokyo, Japan).

Statistical Analyses
Statistical analyses were performed with a commercially available statistical package (Prism 5.0; GraphPad Software, Inc., San Diego, CA, USA).Results are expressed as the number of animals (N) and the mean and standard error (SE) of each experiment.When more than 2 groups were evaluated, a One-way analysis of variance (oneway ANOVA) was followed by post hoc tests of Dunnett.For comparisons of two groups, Student's t-test was used.A value of p < 0.05 was considered to be statistically significant.

Body Weight, Systolic Blood Pressure
Nobiletin were given for 4 weeks from 7 weeks old to 11 weeks old, respectively.Body weight increased over time up to 10 weeks of age as shown in Figure 1, and was increased significantly between 7 and 9 weeks old at 40 mg/kg/day in the nobiletin treated rats.Systolic blood pressures increased with age in control animals, and nobiletin suppressed this increase significantly (Figure 2).

Effects of Nobiletin on Cerebral Thrombosis
Thrombogenesis in cerebral microvessls for each group of animals, as assessed using the He-Ne laser technique, is illustrated in Figure 3.The number of laser pulses   required to generate occlusive thrombi in pial venules in control, nobiletin at 20, nobiletin at 40 mg/kg/day were 6.2 ± 0.4 (n = 6), 8.7 ± 0.8 (n = 6), 11.9 ± 0.5 (n = 5), respectively.The differences between the nobiletin groups at 20 mg/kg/day (p < 0.05) and 40 mg/kg/day (p < 0.01) were significantly higher than control.

Effects of Nobiletin on Oxidative Stress
The amounts of 8-OHdG in urine collected for 24 hr before and after dietary intervention, adjusted for body weight are shown in Figure 4.There were no significant differences in the amounts of urinary 8-OHdG in the control group before and after the experimental period.
The amounts of urinary 8-OHdG were significantly decreased after the experimental diet in the nobiletin group (p < 0.05).

Discussion
We have demonstrated significant cardiovascular effects of nobiletin.The polymethoxylated flavone moderated the usual age-related increase in blood pressure and thrombogenicity in cerebral vessels in stroke prone spontaneously hypertensive rats (SHRSP) after ingestion for 4 weeks.Nobiletin has been reported to exhibit anti-inflammatory, anticarcinogenic and antitumor and antioxidant activities [3,4,13,14].Our findings indicate that the antioxidant activities of these compounds prevented conversion of nitric oxide (NO) into peroxynitrite (ONOO − ) and increased the levels bioavailable NO, mediating an increase in vasorelaxation and leading to a decrease in blood pressure.In addition, we demonstrated that urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels were significantly decreased after ingestion of nobiletin for 4 weeks.In this context, El Haouari et al. reported that reactive oxygen species (ROS) generated in hypertensive animal models stimulated platelets [38], and others have shown that chronic exposure to oxidative stress in spontaneously hypertensive rat (SHR) and SHRSP might contribute to hypertension [19][20][21]22,39].In our studies, nobiletin mixed with normal diet and given for 4 weeks to SHRSP appeared to moderate oxidative stress leading to increases in the urinary excretion of NO metabolites and suppression of enhanced blood pressures and thrombogenesis in cerebral blood vessels.Recent studies have suggested that ROS produced in receptor-mediated platelet activation participate in the regulation of platelet function [40][41][42].It is conceivable, therefore, that the antioxidant activities of nobiletin inhibited platelets activities.These effects of nobiletin might have contributed to inhibition of thrombus formation in the current inves tigations.On the other hand, Guerrero et al. suggested that the mechanisms underlying inhibition of platelet function mediated by some flavonoids such as apigenin, genistein, luteolin, quercetin were related to inhibition of the thromboxane A 2 signalling pathways and antagonizing thromboxane A 2 receptors [43].Further studies are required, therefore, to examine the possible role of interactions between polymethoxylated flavones and platelet thromboxane A 2 receptors in the inhibition of platelet function.Our data on the antithrombotic effects of nobiletin were in keeping with the older studies of Robbins and Sempiska et al.Robbins reported that nobiletin inhibited ADP-induced pulmonary thrombosis in rats in vivo and ADP-induced platelet aggregation in vitro [29,30].Sempiska et al., reported that nobiletin and other flavonoids suppressed adhesion of platelets to glasswool [31].In addition, Hirata et al. suggested that nobiletin could act as an anticoagulant by inhibiting tissue factor [44].Our findings that nobiletin inhibited thrombogenesis in cerebral blood vessels in SHRSP are consistent with these reports.
In conclusion, our results suggested that antioxidant activities of nobiletin contributed to antihypertensive and antithrombotic properties.Precise mechanisms underlying these effects remain to be clarified, however.
Nevertheless, the findings offer the challenging possibility that pharmacological administration of nobiletin could have beneficial effects for the prevention of cardiovascular diseases.

Figure 1 .
Figure 1.The effect of nobiletin on body weight.SHRSP were purchased from a local supplier at 6 weeks old and acclimatized for a week.Nobiletin was fed from 7 weeks old for 4 weeks.Body weights were measured weekly.Statistical analyses were performed by one-way analysis of variance (ANOVA) and by post hoc test of Dunnett.* : p < 0.05.

Figure 2 .
Figure 2. The effect of nobiletin on systolic blood pressure.SHRSP were purchased from a local supplier at 6 weeks old and acclimatized for a week.Nobiletin was fed from 7 weeks old for 4 weeks.Blood pressure was measured weekly.Statistical analyses were performed by one-way analysis of variance (ANOVA) and by post hoc test of Dunnett.* : p < 0.05, ** : <0.01 vs Control.

Figure 3 .
Figure 3.The effect nobiletin on cerebral thombogenesis in SHRSP.Cerebral thrombogenesis was assessed using a He-Ne laser technique after ingestion of nobiletin for 4 weeks as described in Materials and Methods.Statistical analyses were performed by one-way analysis of variance (ANOVA) and by post hoc test of Dunnett.* : p < 0.05, ** : < 0.01 vs Control.

Figure 4 .
Figure 4.The antioxidant effect of nobiletin.Oxidative stress was assessed by measurements of 8-OHdG in 24 hr urine samples collected before and after ingestion of nobiletin for 4 weeks.Statistical analysis was performed by unpaired t-test.* : p < 0.05.

Figure 5 .
Figure 5.The effect of nobiletin on nitric oxide (NO) metabolites (NO 2 /NO 3 ).NO 2 /NO 3 was measured, using Griess reagent, in 24 hr urine samples collected before and after ingestion of nobiletin for 4 weeks.Statistical analysis was performed by unpaired t-test.NO metabolites were significantly decreased in control and increased 4 weeks after ingestion of nobiletin.* : p < 0.05.