A Novel Role of Eruca sativa Mill. (Rocket) Extract: Antiplatelet (NF-κB Inhibition) and Antithrombotic Activities

Background: Epidemiological studies have shown the prevention of cardiovascular diseases through the regular consumption of vegetables. Eruca sativa Mill., commonly known as rocket, is a leafy vegetable that has anti-inflammatory activity. However, its antiplatelet and antithrombotic activities have not been described. Methods: Eruca sativa Mill. aqueous extract (0.1 to 1 mg/mL), was evaluated on human platelets: (i) P-selectin expression by flow cytometry; (ii) platelet aggregation induced by ADP, collagen and arachidonic acid; (iii) IL-1β, TGF-β1, CCL5 and thromboxane B2 release; and (iv) activation of NF-κB and PKA by western blot. Furthermore, (v) antithrombotic activity (200 mg/kg) and (vi) bleeding time in murine models were evaluated. Results: Eruca sativa Mill. aqueous extract (0.1 to 1 mg/mL) inhibited P-selectin expression and platelet aggregation induced by ADP. The release of platelet inflammatory mediators (IL-1β, TGF-β1, CCL5 and thromboxane B2) induced by ADP was inhibited by Eruca sativa Mill. aqueous extract. Furthermore, Eruca sativa Mill. aqueous extract inhibited NF-κB activation. Finally, in murine models, Eruca sativa Mill. aqueous extract showed significant antithrombotic activity and a slight effect on bleeding time. Conclusion: Eruca sativa Mill. presents antiplatelet and antithrombotic activity.


Processing Material
Eruca sativa Mill. cv. Sauvage leaves were harvested from a crop obtained from a commercial hydroponic company in the Region of Maule, Chile, 25 days after sowing. The float Speedling system was used to grow this crop [14,15].

Preparation of Extract
Extract from Eruca sativa Mill. was obtained according to Fuentes et al. [16]. In brief, the samples were comminuted in a blender and mixed with water:methanol, 7:3 v/v, then sonicated and centrifuged for 10 min at 700× g. Then, the supernatant was lyophilized at −45 °C (freeze dried) and stored at −80 °C until use.

Preparation of Human Platelet Suspensions
After receiving written informed consent, venous blood samples were taken from six young healthy volunteers. The protocol was authorized by the ethics committee of the Universidad de Talca in accordance with the Declaration of Helsinki (approved by the 18th World Medical Assembly in Helsinki, Finland, 1964). The samples were placed in 3.2% citrate tubes (9:1 v/v) by phlebotomy with a vacuum tube system (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA). Samples obtained from each volunteer were processed independently for each assay and centrifuged (DCS-16 Centrifugal Presvac RV) at 240× g for 10 min to obtain platelet-rich plasma (PRP). Subsequently, two-thirds of PRP were removed and centrifuged (10 min at 650× g). The pellet was then washed with HEPES-Tyrode's buffer containing PGE1 (120 nmol/L). Washed platelets were prepared in HEPES-Tyrode's buffer at a concentration of 200 × 10 9 platelets/L (Bayer Advia 60 Hematology System, Tarrytown, NY, USA). After blood samples were taken, platelets were kept at 4 °C during all of the isolation steps.

Flow Cytometry Analysis for P-Selectin
P-selectin expression on platelet surface was analyzed by flow cytometry [17]. Briefly, 480 µL of washed platelets were pre-incubated with 20 µL of vehicle (DMSO 0.2%) or Eruca sativa Mill. extract (0.1 to 1 mg/mL) for 3 min, followed by 6 minutes of stimulation at 37 °C with ADP 8 μmol/L. To determine platelet P-selectin expression, 50 µL of the sample were mixed with saturated concentrations of anti-CD62P-PE and anti-CD61-FITC and incubated for 25 min in the dark. Samples were then acquired and analyzed in an Accuri C6 flow cytometer (BD, Biosciences, San Diego, CA, USA). Platelet populations were gated on cell size using forward scatter (FSC) vs. side scatter (SSC) and CD61 positivity to distinguish them from electronic noise. The light scatter and fluorescence channels were set at logarithmic gain, and 5000 events per sample were analyzed. Fluorescence intensities of differentially-stained populations were expressed as the mean channel value using the BD Accuri C6 Software (BD Biosciences, San Diego, CA, USA). All measurements were performed from six separate platelet donors.

Measurement of Platelet Aggregation
Platelet aggregation was monitored by light transmission according to Born and Cross [18], using a lumi-aggregometer (Chrono-Log, Havertown, PA, USA). Briefly, 480 μL of PRP in the reaction vessel were pre-incubated with 20 μL of vehicle (DMSO 0.2%) or Eruca sativa Mill. extract (0.1 to 1 mg/mL). After 3 min of incubation, 20 μL of agonist (ADP 8 µmol/L, collagen 1.5 μg/mL or AA 1 mmol/L) were added to initiate platelet aggregation, which was measured for 6 min. The platelet aggregation (maximal amplitude (%)) was determined by AGGRO/LINK software (Chrono-Log, Havertown, PA, USA). The inhibition of the maximal platelet aggregation was expressed as a percentage with respect to control (DMSO 0.2%). The concentration required to inhibit platelet aggregation by 50% (IC50) was calculated from the dose-response curves. All measurements were performed from six separate platelet donors.

Measurement of cAMP Levels in Human Platelets
The effect of Eruca sativa Mill. extract (0.1 to 1 mg/mL) on cAMP platelet levels was evaluated in 480 µL of washed platelets (200 × 10 9 platelets/L) after a 5-min incubation period without stirring. The platelet reaction was stopped in ice-cold 15% trichloroacetic acid, and precipitated proteins were removed by centrifugation. Samples were stored at −70 °C until analysis. Before determination, samples were dissolved in 200 µL PBS at pH 6.2. The cAMP Parameter Assay Kit (R&D Systems, Minneapolis, MN, USA) was used. All measurements were performed from six separate platelet donors.

Western Blotting
Washed platelets (200 × 10 9 platelets/L) were pre-incubated with vehicle (DMSO 0.2%) or Eruca sativa Mill. extract (0.1 to 1 mg/mL) for 3 min and activated with ADP (8 µmol/L) for 6 min. Then, platelets were lysed with 0.2 mL of lysis buffer in ice for 30 min and heated for 10 min at 95 °C. Equal quantities of total protein (30 μg) were subjected to SDS-PAGE under reducing conditions and transferred to a nitrocellulose membrane. The proteins were detected with anti-phospho-PKA, anti-phospho-NF-κB p65 and anti-γ-tubulin antibodies. All measurements were performed from six separate platelet donors.

Murine Model of Thrombosis
This study was carried out under recommendations by the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Talca. All efforts were made to minimize suffering. Thrombosis in mice was performed by photochemical injury using modified methods described by Przyklenk and Whittaker [19]. Briefly, C57BL/6 mice (12-16 weeks old) were anesthetized with a combination of tribromoethanol (270 mg/kg) and xylazine (13 mg/kg). Thrombosis was induced by an injection of 50 mg/kg rose bengal through the tail vein followed by illumination of the exposed mesenteric artery with a 1.5-mW green light laser (532 nm). Blood flow was monitored for 60 min, and stable occlusion was defined as a blood flow of 0 mL/min for 3 min. Eruca sativa Mill. extract (200 mg/kg, n = 6), vehicle (DMSO 0.2% group, n = 6) and ASA (200 mg/kg, n = 6) were administered intraperitoneally 30 min before the experiment. After laser exposure, the injury image generated was recorded with a charge-coupled device camera (Lumenera Corporation, Ottawa, ON, Canada). The image was analyzed with ImageJ software (version 1.26t, NIH, USA). The region of interest (vessel occlusion) was defined as the target artery, which included the portion of the target artery that was larger than the maximum injured area. Using software tools, thrombus size was measured in the region of interest. Rectal temperatures were similar and within the physiological range between all experimental animals throughout the experimental period.

Bleeding Assay
C57BL/6 mice were anesthetized with a combination of tribromoethanol (270 mg/kg) and xylazine (13 mg/kg) and placed prone on a warming pad from which the tail protruded. The same amounts of Eruca sativa Mill. extract (200 mg/kg, n = 6, intraperitoneally), ASA (200 mg/kg, n = 6, intraperitoneally) or vehicle (DMSO 0.2%, n = 6, intraperitoneally) were given as described in the thrombosis model. An incision was made on the ventral surface of the mice tails about 2 mm from the tip [20]. The bleeding time was measured in seconds (s) until bleeding stopped.

Statistical Analysis
Data were analyzed using SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA) and expressed as the mean ± standard error of mean (SEM). Six or more independent experiments were performed for the different assays. Results were expressed as a percentage of inhibition or as a percentage of control (as 100%). The fifty-percent inhibitory concentration (IC50) of Eruca sativa Mill. extract was calculated from the dose-response curves. Differences between groups were analyzed by a one-way analysis of variance (ANOVA) using Tukey's post hoc test. p-values <0.05 were considered significant.

Effect of Eruca sativa Mill. Extract on Platelet Activation
The effect of Eruca sativa Mill. extract on P-selectin expression in human platelets after stimulation by ADP in PRP was measured by flow cytometry (Figure 1). In the presence of Eruca sativa Mill. extract (1 mg/mL), the P-selectin expression was inhibited from 58 ± 3 to 45 ± 3% (p < 0.05).

Effect of Eruca sativa Mill. Extract on Platelet Aggregation
The effects of Eruca sativa Mill. extract on platelet aggregation induced by ADP, collagen and AA are shown in Figure 2. Eruca sativa Mill. extract inhibited ADP-induced platelet aggregation with a 50% inhibitory concentration (IC50) of 0.71 mg/mL. In addition, Eruca sativa Mill. extract only showed a mild inhibitory effect (17 ± 4 and 16% ± 3%, p < 0.05) over collagen and AA-induced platelet aggregation at a concentration of 1 mg/mL.

Eruca sativa Mill. Extract and Intraplatelet Levels of cAMP
We investigated whether platelet inhibition by Eruca sativa Mill. extract was mediated by changes of intraplatelet cAMP levels. Eruca sativa Mill. extract (0.1 to 1 mg/mL) did not show any effect on intraplatelet levels of cAMP. Levels of cAMP in resting platelets were marked lower than those observed in PGE1 (0.02 mmol/L)-treated platelets (p < 0.001).

Effects of Eruca sativa Mill. Extract on PKA and NF-κB
PKA activation by cAMP phosphorylates multiple target proteins in numerous platelet inhibitory pathways. Here, the treatment of washed platelets with Eruca sativa Mill. extract (0.1 to 1 mg/mL) did not increase the phosphorylation of PKA (Figure 4).

Figure 4. Effect of Eruca sativa
Mill. extract on phospho-PKA in activated platelets by ADP (8 μmol/L). The activated group corresponds to prostaglandin E1 (PGE1) plus ADP. Data are presented as the mean ± SEM of n = 6 experiments. *** p < 0.001 indicates the difference between activated and basal groups as analyzed by one-way ANOVA and Tukey's post hoc test.

Effect of Eruca sativa Mill. Extract on Arterial Thrombus Formation and Bleeding Time
As shown in Figure 6, the mesenteric artery of untreated mice (control) was completely occluded by a stable bulky thrombus 30 min after laser injury. In contrast, one intraperitoneal bolus injection of Eruca sativa Mill. extract (200 mg/kg) delayed vessel occlusion to 60 min and reduced the maximum occlusion (occlusion for 100%) to 57% ± 2% (p < 0.01).
We measured Eruca sativa Mill. extract-induced C57BL/6 mouse blood loss after tail snip at the same concentration that was used for arterial thrombus formation in vivo (200 mg/kg, a single bolus intraperitoneally injection). In this study, the same antithrombotic concentration used of Eruca sativa Mill. extract did not cause significant bleeding measured by tail snip. Thus, the bleeding time by Eruca sativa Mill. extract of 187 ± 19 s (n = 6) was not statistically significantly higher than the control (171 ± 26 s, n = 6) (p > 0.05).

Figure 5. Effect of Eruca sativa
Mill. extract on phospho-NF-κB p65 in activated platelets by ADP (8 μmol/L). Washed platelets were collected, and subcellular extracts were analyzed for phospho-NF-κB p65, as described in the Experimental Section. Data are presented as the mean ± SEM of n = 6 experiments. ** p < 0.01 and *** p < 0.001 indicates differences between activated and Eruca sativa Mill. extract groups as analyzed by one-way ANOVA and Tukey's post hoc test.

Discussion
In this study, we demonstrated for the first time that Eruca sativa Mill. extract inhibits platelet aggregation and activation, reduces platelet release of atherosclerotic-related inflammatory mediators (thromboxane B2, CCL5, TGF-1β and IL-1β levels) and decreases in vivo thrombus formation. In addition, evidence is provided that Eruca sativa Mill. extract antiplatelet activities are associated with NF-κB inhibition.
Although antiplatelet drugs have a cardiovascular protective function, many also have side effects (including headaches, gastrointestinal symptoms, skin rash and bleeding) [22]. Therefore, there is an urgent need to identify more effective and safe antiplatelet and antithrombotic agents. In this way, herbs, medicinal plants, spices and vegetables are a potential source to help combat various diseases, including CVD. In recent years, Eruca sativa Mill. rocket leaves, a member of the Brassicaceae family, have been eaten (at different ontogenic stages) all over the world. The leaves are eaten raw or cooked, and Eruca sativa Mill. flowers are also consumed [23,24]. The seeds, roots, leaves and flowers of Eruca sativa Mill. contain different flavonoid profiles [1]. Kaempferol derivatives represent the major group of phenolics present in Eruca sativa Mill. leaves (77%-88% of total phenolics), followed by quercetin and isorhamnetin-3,4-diglucoside, representing 9% and 16.3% of the total phenolics, respectively [25,26]. The antiplatelet activity of Eruca sativa Mill., could be by the presence of kaempferol, quercetin and isorhamnetin [27][28][29].
In this study, Eruca sativa Mill. extract displayed in vitro and in vivo antiplatelet activities. Thus, Eruca sativa Mill. extract significantly inhibits platelet activation (less P-selectin expression) and platelet aggregation induced by ADP and showed only a low inhibition over collagen and AA.
Using a murine model of real-time thrombus formation [32], we demonstrated, for the first time, that Eruca sativa Mill. extract prevented thrombus growth in vivo. Moreover, antiplatelet drugs that are currently available inevitably increase bleeding risk at antithrombotic doses [33]. This study showed that Eruca sativa Mill. extract possesses antithrombotic efficacy without significant bleeding.

Conclusions
Rocket extract shows antiplatelet activity (inhibition of platelet activation, aggregation and release of inflammatory mediators), and the mechanism of action could be by NF-κB inhibition. However, further studies are needed to expand Eruca sativa Mill. extract properties in the setting of the NF-κB pathway.