http://informahealthcare.com/plt ISSN: 0953-7104 (print), 1369-1635 (electronic) Platelets, Early Online: 1–6 ! 2014 Informa UK Ltd. DOI: 10.3109/09537104.2014.898747
ORIGINAL ARTICLE
Strawberry extract presents antiplatelet activity by inhibition of inflammatory mediator of atherosclerosis (sP-selectin, sCD40L, RANTES, and IL-1b) and thrombus formation
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Marcelo Alarco´n1,2*, Eduardo Fuentes1,2*, Natalia Olate1, Simon Navarrete1, Gilda Carrasco2,3, & Iva´n Palomo1,2 1
Department of Clinical Biochemistry and Immunohematology, Faculty of Health Sciences, Interdisciplinary Excellence Research Program on Healthy Aging (PIEI-ES), Universidad de Talca, Talca, Chile, 2Centro de Estudios en Alimentos Procesados (CEAP), CONICYT-Regional, Gore Maule, Talca, Chile, and 3Department of Horticulture, Faculty of Agricultural Sciences, Universidad de Talca. Talca, Chile Abstract
Keywords
Cardiovascular disease prevention is of high priority in developed countries. Healthy eating habits including the regular intake of an antithrombotic diet (fruit and vegetables) may contribute to prevention. Platelet function is a critical factor in arterial thrombosis and the effect strawberries have is still unclear. Therefore, the aim of this study was to systematically examine the action of strawberries in preventing platelet activation and thrombus formation. Strawberry extract concentration-dependently (0.1–1 mg/ml) inhibited platelet aggregation induced by ADP and arachidonic acid. At the same concentrations as strawberry inhibits platelet aggregation, it significantly decreased sP-selectin, sCD40L, RANTES, and IL-1b levels. The strawberry may exert significant protective effects on thromboembolic-related disorders by inhibiting platelet aggregation. Also, this suggests that antithrombotic activity may have novel anti-inflammatory effects.
Antiplatelet, antithrombotic, IL-1b, P-selectin, RANTES, sCD40L, strawberry
Introduction Platelets are involved in the formation of thrombus that play an important role in heart attacks, strokes, and peripheral vascular disease [1]. Given that platelets play a central role in the atherosclerotic-related inflammatory response and the subsequent thrombotic event, a variety of antiplatelet agents have been developed [2, 3]. Antiplatelet drugs are members of a class of pharmaceuticals that decrease platelet aggregation and inhibit thrombus formation. Antiplatelet drugs can be classified into three groups: (i) prevent cardiovascular diseases (CVD) (primary prevention), (ii) treat an acute disease, and (iii) treat a chronic disease (secondary prevention) [4]. The most commonly used antiplatelet agent is aspirin, other oral agents may also be prescribed (ticlopidine, clopidogrel, dipyridamole, abciximab, and eptifibatide, among others) [5]. Each antiplatelet agent affects platelets in slightly different ways and may have specific side effects, such as internal bleeding and gastrointestinal adverse effects, among others [6, 7]. Moreover, recent analyses suggest that there is a concept of the cardio protective properties of the dietary habits within the Mediterranean regions [8]. In this context, together with regular physical activity and smoking cessation, both coronary heart disease and strokes could be avoided by healthy food choices that *These authors contributed equally to this work and are the first authors. Correspondence: G. Iva´n Palomo, Ph.D., Department of Clinical Biochemistry and Immunohematology, Faculty of Health Sciences, Universidad de Talca, Chile., Talca, Chile. E-mail: [email protected] Eduardo Fuentes, Ph.D. Student, Immunology and Haematology Laboratory, Faculty of Health Sciences, Universidad de Talca, Talca, Chile. E-mail: [email protected]
History Received 30 July 2013 Revised 17 February 2014 Accepted 24 February 2014 Published online 18 April 2014
are consistent with the Mediterranean Diet [9]. Within a Mediterranean Diet, a higher intake of fruit and vegetables may protect against CVD and support current dietary guidelines to increase fruit and vegetable intake [10, 11]. Preliminary studies have demonstrated that platelet anti-aggregant activity of fruit (red grapes, strawberries, kiwis, and pineapples) and vegetables (garlic, onions, green onions, melons, and tomatoes) [12–16]. There is evidence suggesting that dietary strawberry powder (containing several key nutrients) may be associated with reduced risk factors for CVD, stroke, and diabetes in obese volunteers [17–19]. Yet, the mechanisms by which the strawberry may interfere with platelet function remain to be fully elucidated. The present study was aimed at investigating the effect of aqueous extract from the strawberry on platelet inflammatory mediators of atherosclerosis (sP-selectin, sCD40L, RANTES, and IL-1b) and in vivo thrombus formation.
Materials and methods Reagents Sodium chloride (p.a.) was obtained from Arquimed (Santiago, Chile). Adenosine 50 -diphosphate (ADP), thrombin receptor activator peptide 6 (TRAP-6), arachidonic acid (AA), rose bengal, prostaglandin E1 (PGE1), and acetylsalicylic acid (ASA) were obtained from Sigma-Aldrich (St. Louis, MO). Collagen type I was obtained from Chrono-Log corp. (Havertown, PA). Processing material The variety of strawberry called Camarosa was selected for the study. Ripe fruits were obtained from the Centro Regional de Abastecimiento in Talca, Chile.
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Preparation of extract Total aqueous extract from the strawberries was obtained according to Fuentes et al. [20]. Briefly, the samples were comminuted in a blender, sonicated, and centrifuged for 10 minutes at 700 g. Then the supernatant was filtered and lyophilized at 45 C (freeze dried), and stored at 80 C until use.
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Preparation of human washed platelet suspensions The protocol was authorized by the ethics committee of Universidad de Talca in accordance with the Declaration of Helsinki (approved by the 18th World Medical Assembly in Helsinki, Finland, 1964). Written informed consent was obtained from six young healthy volunteers who agreed to participate in the study. Venous blood samples were taken according to the ‘‘British Committee for Standards in Hematology’’ [21]. 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) and needle of 21 G [21]. Samples from each volunteer were processed independently for each assay, and centrifuged (DCS-16 Centrifugal Presvac RV) at 240 g for 10 minutes to obtain platelet-rich plasma (PRP). Then two-thirds of PRP was removed and centrifuged (10 minutes at 650 g). The pellet was washed with HEPES-Tyrode’s buffer containing PGE1 (120 nmol/l). Washed platelets were prepared in HEPESTyrode’s buffer at a concentration 200 106 platelets/ml (Bayer Advia 60 Hematology System, Tarrytown, NY). Platelets were kept at 4 C during all the isolation steps after blood drawing. Platelet aggregation assay Platelet aggregation was monitored by light transmission according to Born and Cross [22], using a lumi-aggregometer. Briefly, 480 ml of washed platelets (200 109 platelets/l) were preincubated with 20 ml of saline, ASA (0.3 mmol/l), or strawberry extract (0.1–1 mg/ml) for 3 minutes. Following this, 20 ml of agonist (ADP 8 mmol/l, collagen 15 mg/ml, TRAP-6 30 mmol/l, or AA 1 mmol/l) was added and platelet aggregation was registered for 6 min. All measurements were performed in triplicate. Platelet aggregation results (maximal amplitude [%], slope, area under the curve (AUC) and lag time [s]) were determined by the AGGRO/ LINK (Chrono-Log, Havertown, PA). Strawberry extract inhibition of the maximal platelet aggregation was expressed as a percentage of the control (saline). To evaluate the stability, the strawberry extract was subjected to 100 C for 1 hour, kept at 4 C for 5 minutes, and then used in platelet aggregation in vitro studies.
Platelets, Early Online: 1–6
Whittaker [23]. Briefly, C57BL/6 mice (12–16 weeks old) were anesthetized with a combination of tribromoethanol (270 mg/kg) and xylazine (13 mg/kg). The mesentery was exposed by performing a central incision in the abdomen, permitting the visualization of thrombus development in mesenteric vessels. Thrombosis was induced by an injection of 50 mg/kg rose bengal through the tail vein followed by illumination of the exposed mesenteric area with a diode laser (1.5-mW over objective lens, 532 nm, diameter of 5 mm2). Blood flow was monitored for 60 minutes and stable occlusion was defined as a blood flow of 0 ml/minute for 3 minutes. Saline (control group, n ¼ 6), ASA (200 mg/kg, n ¼ 6), or strawberry extract (200 mg/kg, n ¼ 6) were administered intraperitoneally 30 minutes before the experiment. Rectal temperatures were similar and within the physiological range in all experimental animals throughout the experimental period. Thrombus size analysis After laser exposure, the injury image generated was recorded with a charge-coupled device camera (Lumenera Corporation, Ottawa, ON). The image was analyzed with ImageJ software (NIH, Bethesda, MD). The region of interest (ROI) was defined as the target artery that included a portion of the target artery that is larger than the maximum injured area. Using software tools, thrombus size was measured in the ROI. Statistical analysis Data were analyzed using SPSS version 17.0 (SPSS, Inc., Chicago, IL) and expressed as mean ± standard error of mean (SEM). Three or more independent experiments were performed for the different assays. Results were expressed as percent inhibition or as a percentage of control (as 100%). Differences between groups were analyzed by the Student paired or unpaired t-test and a one-way analysis of variance (ANOVA) using Tukey’s post-hoc test. p Values 50.05 were considered significant.
Results and discussion Effects of strawberry extract on platelet aggregation The effects of strawberry extract on ADP-, collagen-, TRAP-6, and AA- induced platelet aggregation are presented in the Table I. Strawberry extract inhibited platelet aggregation induced by ADP and AA, respectively, but to a different extent. Considering the different agonists tested in this study, the inhibition of platelet aggregation by the strawberry extract was in the following order: AA (65 ± 5%, p50.05)4ADP (55 ± 4%, p50.05) 4TRAP-6 Table I. Antiplatelet activity of aqueous extract from strawberry.
Platelet inflammatory mediators Soluble CD40 ligand (sCD40L), RANTES, and IL-1b were determined using human quantikine ELISA kits (R&D systems, Minneapolis, MN) and soluble P-selectin (sP-selectin) was determined by ELISA according to the manufacturer’s instructions (Invitrogen Corporation, Carlsbad, CA). Briefly, washed platelets were pretreated with saline, ASA (0.3 mmol/l), or strawberry extract (0.5–1 mg/ml) for 15 minutes at 37 C and then stimulated with thrombin (2 U/ml) for 45 minutes at 37 C. Supernatants were collected after centrifugation (2000 g, 10 minutes, 4 C) and stored at 70 C until use.
ADP
Collagen TRAP-6
AA
Maximum aggregation (%) Strawberry extract 48 ± 3* 88 ± 5 88 ± 4 36 ± 5* Negative control 86 ± 5 89 ± 4 91 ± 6 85 ± 5 Slope Strawberry extract 66 ± 4* 106 ± 6 118 ± 7 41 ± 5* Negative control 109 ± 12 111 ± 16 101 ± 9 116 ± 12 AUC Strawberry extract 220 ± 28* 334 ± 19 430 ± 10 134 ± 15* Negative control 383 ± 21 299 ± 25 474 ± 51 398 ± 62 Lag time (s) Strawberry extract 29 ± 1* 43 ± 2 25 ± 2 42 ± 2* Negative control 10 ± 1 46 ± 1 23 ± 1 33 ± 1
In vivo murine model of thrombosis Thrombosis in mice was performed by photochemical injury using a modification in the methods described by Przyklenk and
Values are presented as mean ± SEM (n ¼ 6). ADP 8 mmol/l, Collagen 1.5 mg/ml, TRAP-6 30 mmol/l, and AA 1 mmol/l. Extract at 1 mg/ml. *p50.05 vs. negative control (saline 0.9%). AUC: area under the curve.
DOI: 10.3109/09537104.2014.898747
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(10 ± 5%, p40.05) 4 collagen (2 ± 2%, p40.05) as compared with the control. Therefore, strawberry extract in a concentrationdependently (0.1, 0.5, and 1 mg/ml) inhibited human platelet aggregation stimulated by AA and ADP (Figure 1). Thus, the platelet aggregation induced by AA in the presence of strawberry extract at 0.5 and 1 mg/ml was inhibited by 23 ± 5 and 65 ± 5% (p50.05), respectively. While platelet aggregation stimulated by ADP was inhibited by 23 ± 5, 39 ± 6, and 55 ± 4% at 0.1, 0.5, and 1 mg/ml (p50.05). Furthermore strawberry extract, both with AA- and ADP-induced platelet aggregation inhibited slope, AUC and lag time as compared with the control (p50.05).
Figure 1. Strawberry extract concentration-dependently inhibited platelet aggregation. Effect of strawberry extract on ADP (8 mmol/l) and AA (1 mmol/l) induced platelet aggregation. Results were expressed as % inhibition (mean ± SEM, n ¼ 6).
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After being subjected to 100 C, strawberry extract retained its ability to inhibit platelet activation induced by shear force, similar results were reported by Naemura et al. [24]. Therefore, the thermal stability of strawberry extract, unlike the thermal stability of Solanum lycopersicum [25], is dependent on the agonist used. Effects of strawberry extract on platelet sP-selectin, sCD40L, RANTES, and IL-1b It has been described in the last decade that the secretion of platelet pro-inflammatory molecules (sCD40L, RANTES, sP-selectin, and IL-1b) may play a pathogenic role in both the long-term atherosclerotic process, and in the triggering and propagation of acute coronary syndromes [26, 27]. In this study, we have demonstrated, for the first time that strawberry extract inhibited platelet inflammatory mediators of atherosclerosis (sP-selectin, sCD40L, RANTES, and IL-1b). In non-stimulated platelets, P-selectin, sCD40L, RANTES, and IL-1b release were minimal, whereas it was markedly enhanced after stimulation with thrombin (2 U/ml). Pretreatment of washed platelet with the strawberry extract (0.5–1 mg/ml) significantly inhibited thrombin-induced P-selectin expression. Concretely, thrombin-induced sP-selectin release was inhibited by 23 ± 4 (p50.05) and 37 ± 4% (p50.01), in the presence of strawberry extract at 0.5 and 1 mg/ml, respectively (Figure 2A). Moreover, as platelets are considered the major source of sCD40L in blood and sCD40L play a pivotal role in inflammation and atherosclerosis [28], we examined the effect of strawberry extract on platelet sCD40L release. As observed in Figure 2(B),
Figure 2. Effects of strawberry extract on release of sP-selectin, sCD40L, RANTES, and IL-1b from platelets. Washed platelets were pretreated with saline, ASA (acetylsalicylic acid) 0.3 mmol/l or strawberry extract (0.5–1 mmol/l) for 15 minutes at 37 C and then stimulated by thrombin (2 U/ml) for determination of sP-selectin (A), sCD40L (B), RANTES (C), and IL-1b (D). The graph depicts the mean ± SEM of n ¼ 6 experiments. *p50.05 and **p50.01 as compared with the thrombin activated group, analyzed by Student’s t-test.
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Figure 3. Strawberry extract inhibited arterial thrombosis formation. (A) Representative images of thrombus formation after laser irradiation in the saline control group (n ¼ 6); ASA, acetylsalicylic acid (200 mg/kg, n ¼ 6) and strawberry extract (200 mg/kg, n ¼ 6) to 60 minutes. (B) Time course changes of thrombus growth rate as described in Materials and methods section. I, intima; M, media; A, adventitia.
we found that strawberry extract significantly reduced thrombininduced sCD40L platelet release by 24 ± 3 (p50.05) and 43 ± 3% (p50.01) at concentrations of 0.5 and 1 mg/ml, respectively. It has been suggested that alteration of the level of the RANTES affects the progress of atherogenesis through the inflammatory pathway in patients with cardiovascular risk factors [29]. Moreover, IL-1b has been identified as a major mediator of platelet-induced activation and endothelial dysfunction [30]. In this study, the strawberry extract inhibited two important platelet mediators of inflammation, both RANTES and IL-1b. Thus, the strawberry extract at 1 mg/ml attenuated the effect of thrombininduced RANTES and IL-1b release by 41 ± 4 (p50.05) and 37 ± 4% (p50.01), respectively (Figure 2C and D). In this way, regular consumption of strawberries for 6 weeks may provide protection against high carbohydrate/fat meal-induced increase of inflammatory mediators (e.g. IL-1b) [31].
Effects of strawberry extract on arterial thrombus formation in vivo All animals in this study showed similar physiological values for rectal temperature before and after of thrombosis model among groups (data not shown).
Despite the benefits associated with current antiplatelet therapy, significant clinical limitations are associated with the use of aspirin and P2Y12 ADP receptor antagonist [32]. In this study, the strawberry extract presented inhibitory activity on human platelets aggregation and each constituent may possess multiply targets, and they may exert pleiotropic and synergistic effects. Thus, this prevents the access of the arachidonic aid to and the interactions of ADP with P2Y12 and with P2Y1, resulting in an inhibition of platelet thrombus formation under arterial flow [33–35]. It is known that strawberry is rich in polyphenols. We hypothesized that the mechanisms of antiplatelet action could be via the activation of PPARs, an increase of cAMP levels and inhibition of phospholipases C, NADPH oxidase, platelet isoprostanes, NF-kB and thromboxane A2, among others [36–38]. The dramatic inhibitory effects of the strawberry extract on platelet aggregation, and P-selectin, sCD40L, RANTES, and IL1b release supported the assessment of strawberry extract effectiveness to inhibit in vitro thrombus formation [39]. Taking into consideration all these findings, we provided further in vivo evidence of such in vitro-related strawberry extract- antithrombotic effects. To examine the in vivo antithrombotic activity of the strawberry extract, we evaluated the effects of the strawberry
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DOI: 10.3109/09537104.2014.898747
extract on laser-injured thrombus formation in mice mesenteric artery in vivo. As shown in Figure 3, in untreated mice (control), the mesenteric artery was totally blocked by a stable bulky thrombus at 20 minutes. In contrast, further analysis revealed that the time taken to form the artery thrombosis (42500 mm2) was drastically prolonged in strawberry extracttreated mice compared with mice receiving the same volume of vehicle. Thus, one intraperitoneally bolus injection of strawberry extract (200 mg/kg) 30 minutes before laser injury prevented thrombus formation over 60 minutes after laser injury, only achieving a maximum of occlusion of 35 ± 2% (p50.001 vs. control) (Figure 3). Although, in this study we used a different strawberry variety than the one published by other studies, the same antithrombotic effects have been identified [24]. Although antiplatelet drugs have proven to be beneficial in patients with clinical evidence of CVD, the outcome still remains poor. Currently, ASA is the gold standard for secondary prevention of stroke of vascular origin; however, it is frequently associated with serious adverse effects (internal bleeding and gastrointestinal adverse effects, among others) [6] and its effectiveness in primary prevention is still a matter of debate [40]. In this study, using a murine model of real-time thrombus formation, we demonstrate that the strawberry extract inhibited arterial thrombus growth at the same concentration as that of aspirin, a widely used antiplatelet agent as shown in the Figure 3. According to the previous results, aqueous fraction from Solanum lycopersicum (70 mg/kg) had a 20% reduction from baseline ex vivo platelet aggregation response in acute human study [41]. Therefore, according to the same antiplatelet effect in vitro and in vivo by strawberry it is possible to estimate that the amount of strawberry extract necessary in humans for proven antiplatelet effects is about 70 mg/kg. However, additional studies are needed to confirm the strawberry extract dose to observe acute and chronic antiplatelet effects in humans.
Conclusion In this study, we have demonstrated that aqueous extract from strawberries displayed a range of antiplatelet activities targeting different platelet activation responses in vitro and inhibition of thrombus formation. Based on these effects, it is possible to consider the strawberry as a functional food that could provide health benefits beyond basic nutrition.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This work was funded by the CONICYT REGIONAL/GORE MAULE/CEAP/R09I2001, Interdisciplinary Excellence Research Program on Healthy Aging (PIEI-ES), and supported by grant no. 1130216 (I. P., M. G., R. M., M. A., and J. C.) from Fondecyt, Chile.
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ORIGINAL ARTICLE
Strawberry extract presents antiplatelet activity by inhibition of inflammatory mediator of atherosclerosis (sP-selectin, sCD40L, RANTES, and IL-1b) and thrombus formation
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Marcelo Alarco´n1,2*, Eduardo Fuentes1,2*, Natalia Olate1, Simon Navarrete1, Gilda Carrasco2,3, & Iva´n Palomo1,2 1
Department of Clinical Biochemistry and Immunohematology, Faculty of Health Sciences, Interdisciplinary Excellence Research Program on Healthy Aging (PIEI-ES), Universidad de Talca, Talca, Chile, 2Centro de Estudios en Alimentos Procesados (CEAP), CONICYT-Regional, Gore Maule, Talca, Chile, and 3Department of Horticulture, Faculty of Agricultural Sciences, Universidad de Talca. Talca, Chile Abstract
Keywords
Cardiovascular disease prevention is of high priority in developed countries. Healthy eating habits including the regular intake of an antithrombotic diet (fruit and vegetables) may contribute to prevention. Platelet function is a critical factor in arterial thrombosis and the effect strawberries have is still unclear. Therefore, the aim of this study was to systematically examine the action of strawberries in preventing platelet activation and thrombus formation. Strawberry extract concentration-dependently (0.1–1 mg/ml) inhibited platelet aggregation induced by ADP and arachidonic acid. At the same concentrations as strawberry inhibits platelet aggregation, it significantly decreased sP-selectin, sCD40L, RANTES, and IL-1b levels. The strawberry may exert significant protective effects on thromboembolic-related disorders by inhibiting platelet aggregation. Also, this suggests that antithrombotic activity may have novel anti-inflammatory effects.
Antiplatelet, antithrombotic, IL-1b, P-selectin, RANTES, sCD40L, strawberry
Introduction Platelets are involved in the formation of thrombus that play an important role in heart attacks, strokes, and peripheral vascular disease [1]. Given that platelets play a central role in the atherosclerotic-related inflammatory response and the subsequent thrombotic event, a variety of antiplatelet agents have been developed [2, 3]. Antiplatelet drugs are members of a class of pharmaceuticals that decrease platelet aggregation and inhibit thrombus formation. Antiplatelet drugs can be classified into three groups: (i) prevent cardiovascular diseases (CVD) (primary prevention), (ii) treat an acute disease, and (iii) treat a chronic disease (secondary prevention) [4]. The most commonly used antiplatelet agent is aspirin, other oral agents may also be prescribed (ticlopidine, clopidogrel, dipyridamole, abciximab, and eptifibatide, among others) [5]. Each antiplatelet agent affects platelets in slightly different ways and may have specific side effects, such as internal bleeding and gastrointestinal adverse effects, among others [6, 7]. Moreover, recent analyses suggest that there is a concept of the cardio protective properties of the dietary habits within the Mediterranean regions [8]. In this context, together with regular physical activity and smoking cessation, both coronary heart disease and strokes could be avoided by healthy food choices that *These authors contributed equally to this work and are the first authors. Correspondence: G. Iva´n Palomo, Ph.D., Department of Clinical Biochemistry and Immunohematology, Faculty of Health Sciences, Universidad de Talca, Chile., Talca, Chile. E-mail: [email protected] Eduardo Fuentes, Ph.D. Student, Immunology and Haematology Laboratory, Faculty of Health Sciences, Universidad de Talca, Talca, Chile. E-mail: [email protected]
History Received 30 July 2013 Revised 17 February 2014 Accepted 24 February 2014 Published online 18 April 2014
are consistent with the Mediterranean Diet [9]. Within a Mediterranean Diet, a higher intake of fruit and vegetables may protect against CVD and support current dietary guidelines to increase fruit and vegetable intake [10, 11]. Preliminary studies have demonstrated that platelet anti-aggregant activity of fruit (red grapes, strawberries, kiwis, and pineapples) and vegetables (garlic, onions, green onions, melons, and tomatoes) [12–16]. There is evidence suggesting that dietary strawberry powder (containing several key nutrients) may be associated with reduced risk factors for CVD, stroke, and diabetes in obese volunteers [17–19]. Yet, the mechanisms by which the strawberry may interfere with platelet function remain to be fully elucidated. The present study was aimed at investigating the effect of aqueous extract from the strawberry on platelet inflammatory mediators of atherosclerosis (sP-selectin, sCD40L, RANTES, and IL-1b) and in vivo thrombus formation.
Materials and methods Reagents Sodium chloride (p.a.) was obtained from Arquimed (Santiago, Chile). Adenosine 50 -diphosphate (ADP), thrombin receptor activator peptide 6 (TRAP-6), arachidonic acid (AA), rose bengal, prostaglandin E1 (PGE1), and acetylsalicylic acid (ASA) were obtained from Sigma-Aldrich (St. Louis, MO). Collagen type I was obtained from Chrono-Log corp. (Havertown, PA). Processing material The variety of strawberry called Camarosa was selected for the study. Ripe fruits were obtained from the Centro Regional de Abastecimiento in Talca, Chile.
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Preparation of extract Total aqueous extract from the strawberries was obtained according to Fuentes et al. [20]. Briefly, the samples were comminuted in a blender, sonicated, and centrifuged for 10 minutes at 700 g. Then the supernatant was filtered and lyophilized at 45 C (freeze dried), and stored at 80 C until use.
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Preparation of human washed platelet suspensions The protocol was authorized by the ethics committee of Universidad de Talca in accordance with the Declaration of Helsinki (approved by the 18th World Medical Assembly in Helsinki, Finland, 1964). Written informed consent was obtained from six young healthy volunteers who agreed to participate in the study. Venous blood samples were taken according to the ‘‘British Committee for Standards in Hematology’’ [21]. 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) and needle of 21 G [21]. Samples from each volunteer were processed independently for each assay, and centrifuged (DCS-16 Centrifugal Presvac RV) at 240 g for 10 minutes to obtain platelet-rich plasma (PRP). Then two-thirds of PRP was removed and centrifuged (10 minutes at 650 g). The pellet was washed with HEPES-Tyrode’s buffer containing PGE1 (120 nmol/l). Washed platelets were prepared in HEPESTyrode’s buffer at a concentration 200 106 platelets/ml (Bayer Advia 60 Hematology System, Tarrytown, NY). Platelets were kept at 4 C during all the isolation steps after blood drawing. Platelet aggregation assay Platelet aggregation was monitored by light transmission according to Born and Cross [22], using a lumi-aggregometer. Briefly, 480 ml of washed platelets (200 109 platelets/l) were preincubated with 20 ml of saline, ASA (0.3 mmol/l), or strawberry extract (0.1–1 mg/ml) for 3 minutes. Following this, 20 ml of agonist (ADP 8 mmol/l, collagen 15 mg/ml, TRAP-6 30 mmol/l, or AA 1 mmol/l) was added and platelet aggregation was registered for 6 min. All measurements were performed in triplicate. Platelet aggregation results (maximal amplitude [%], slope, area under the curve (AUC) and lag time [s]) were determined by the AGGRO/ LINK (Chrono-Log, Havertown, PA). Strawberry extract inhibition of the maximal platelet aggregation was expressed as a percentage of the control (saline). To evaluate the stability, the strawberry extract was subjected to 100 C for 1 hour, kept at 4 C for 5 minutes, and then used in platelet aggregation in vitro studies.
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Whittaker [23]. Briefly, C57BL/6 mice (12–16 weeks old) were anesthetized with a combination of tribromoethanol (270 mg/kg) and xylazine (13 mg/kg). The mesentery was exposed by performing a central incision in the abdomen, permitting the visualization of thrombus development in mesenteric vessels. Thrombosis was induced by an injection of 50 mg/kg rose bengal through the tail vein followed by illumination of the exposed mesenteric area with a diode laser (1.5-mW over objective lens, 532 nm, diameter of 5 mm2). Blood flow was monitored for 60 minutes and stable occlusion was defined as a blood flow of 0 ml/minute for 3 minutes. Saline (control group, n ¼ 6), ASA (200 mg/kg, n ¼ 6), or strawberry extract (200 mg/kg, n ¼ 6) were administered intraperitoneally 30 minutes before the experiment. Rectal temperatures were similar and within the physiological range in all experimental animals throughout the experimental period. Thrombus size analysis After laser exposure, the injury image generated was recorded with a charge-coupled device camera (Lumenera Corporation, Ottawa, ON). The image was analyzed with ImageJ software (NIH, Bethesda, MD). The region of interest (ROI) was defined as the target artery that included a portion of the target artery that is larger than the maximum injured area. Using software tools, thrombus size was measured in the ROI. Statistical analysis Data were analyzed using SPSS version 17.0 (SPSS, Inc., Chicago, IL) and expressed as mean ± standard error of mean (SEM). Three or more independent experiments were performed for the different assays. Results were expressed as percent inhibition or as a percentage of control (as 100%). Differences between groups were analyzed by the Student paired or unpaired t-test and a one-way analysis of variance (ANOVA) using Tukey’s post-hoc test. p Values 50.05 were considered significant.
Results and discussion Effects of strawberry extract on platelet aggregation The effects of strawberry extract on ADP-, collagen-, TRAP-6, and AA- induced platelet aggregation are presented in the Table I. Strawberry extract inhibited platelet aggregation induced by ADP and AA, respectively, but to a different extent. Considering the different agonists tested in this study, the inhibition of platelet aggregation by the strawberry extract was in the following order: AA (65 ± 5%, p50.05)4ADP (55 ± 4%, p50.05) 4TRAP-6 Table I. Antiplatelet activity of aqueous extract from strawberry.
Platelet inflammatory mediators Soluble CD40 ligand (sCD40L), RANTES, and IL-1b were determined using human quantikine ELISA kits (R&D systems, Minneapolis, MN) and soluble P-selectin (sP-selectin) was determined by ELISA according to the manufacturer’s instructions (Invitrogen Corporation, Carlsbad, CA). Briefly, washed platelets were pretreated with saline, ASA (0.3 mmol/l), or strawberry extract (0.5–1 mg/ml) for 15 minutes at 37 C and then stimulated with thrombin (2 U/ml) for 45 minutes at 37 C. Supernatants were collected after centrifugation (2000 g, 10 minutes, 4 C) and stored at 70 C until use.
ADP
Collagen TRAP-6
AA
Maximum aggregation (%) Strawberry extract 48 ± 3* 88 ± 5 88 ± 4 36 ± 5* Negative control 86 ± 5 89 ± 4 91 ± 6 85 ± 5 Slope Strawberry extract 66 ± 4* 106 ± 6 118 ± 7 41 ± 5* Negative control 109 ± 12 111 ± 16 101 ± 9 116 ± 12 AUC Strawberry extract 220 ± 28* 334 ± 19 430 ± 10 134 ± 15* Negative control 383 ± 21 299 ± 25 474 ± 51 398 ± 62 Lag time (s) Strawberry extract 29 ± 1* 43 ± 2 25 ± 2 42 ± 2* Negative control 10 ± 1 46 ± 1 23 ± 1 33 ± 1
In vivo murine model of thrombosis Thrombosis in mice was performed by photochemical injury using a modification in the methods described by Przyklenk and
Values are presented as mean ± SEM (n ¼ 6). ADP 8 mmol/l, Collagen 1.5 mg/ml, TRAP-6 30 mmol/l, and AA 1 mmol/l. Extract at 1 mg/ml. *p50.05 vs. negative control (saline 0.9%). AUC: area under the curve.
DOI: 10.3109/09537104.2014.898747
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(10 ± 5%, p40.05) 4 collagen (2 ± 2%, p40.05) as compared with the control. Therefore, strawberry extract in a concentrationdependently (0.1, 0.5, and 1 mg/ml) inhibited human platelet aggregation stimulated by AA and ADP (Figure 1). Thus, the platelet aggregation induced by AA in the presence of strawberry extract at 0.5 and 1 mg/ml was inhibited by 23 ± 5 and 65 ± 5% (p50.05), respectively. While platelet aggregation stimulated by ADP was inhibited by 23 ± 5, 39 ± 6, and 55 ± 4% at 0.1, 0.5, and 1 mg/ml (p50.05). Furthermore strawberry extract, both with AA- and ADP-induced platelet aggregation inhibited slope, AUC and lag time as compared with the control (p50.05).
Figure 1. Strawberry extract concentration-dependently inhibited platelet aggregation. Effect of strawberry extract on ADP (8 mmol/l) and AA (1 mmol/l) induced platelet aggregation. Results were expressed as % inhibition (mean ± SEM, n ¼ 6).
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After being subjected to 100 C, strawberry extract retained its ability to inhibit platelet activation induced by shear force, similar results were reported by Naemura et al. [24]. Therefore, the thermal stability of strawberry extract, unlike the thermal stability of Solanum lycopersicum [25], is dependent on the agonist used. Effects of strawberry extract on platelet sP-selectin, sCD40L, RANTES, and IL-1b It has been described in the last decade that the secretion of platelet pro-inflammatory molecules (sCD40L, RANTES, sP-selectin, and IL-1b) may play a pathogenic role in both the long-term atherosclerotic process, and in the triggering and propagation of acute coronary syndromes [26, 27]. In this study, we have demonstrated, for the first time that strawberry extract inhibited platelet inflammatory mediators of atherosclerosis (sP-selectin, sCD40L, RANTES, and IL-1b). In non-stimulated platelets, P-selectin, sCD40L, RANTES, and IL-1b release were minimal, whereas it was markedly enhanced after stimulation with thrombin (2 U/ml). Pretreatment of washed platelet with the strawberry extract (0.5–1 mg/ml) significantly inhibited thrombin-induced P-selectin expression. Concretely, thrombin-induced sP-selectin release was inhibited by 23 ± 4 (p50.05) and 37 ± 4% (p50.01), in the presence of strawberry extract at 0.5 and 1 mg/ml, respectively (Figure 2A). Moreover, as platelets are considered the major source of sCD40L in blood and sCD40L play a pivotal role in inflammation and atherosclerosis [28], we examined the effect of strawberry extract on platelet sCD40L release. As observed in Figure 2(B),
Figure 2. Effects of strawberry extract on release of sP-selectin, sCD40L, RANTES, and IL-1b from platelets. Washed platelets were pretreated with saline, ASA (acetylsalicylic acid) 0.3 mmol/l or strawberry extract (0.5–1 mmol/l) for 15 minutes at 37 C and then stimulated by thrombin (2 U/ml) for determination of sP-selectin (A), sCD40L (B), RANTES (C), and IL-1b (D). The graph depicts the mean ± SEM of n ¼ 6 experiments. *p50.05 and **p50.01 as compared with the thrombin activated group, analyzed by Student’s t-test.
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Figure 3. Strawberry extract inhibited arterial thrombosis formation. (A) Representative images of thrombus formation after laser irradiation in the saline control group (n ¼ 6); ASA, acetylsalicylic acid (200 mg/kg, n ¼ 6) and strawberry extract (200 mg/kg, n ¼ 6) to 60 minutes. (B) Time course changes of thrombus growth rate as described in Materials and methods section. I, intima; M, media; A, adventitia.
we found that strawberry extract significantly reduced thrombininduced sCD40L platelet release by 24 ± 3 (p50.05) and 43 ± 3% (p50.01) at concentrations of 0.5 and 1 mg/ml, respectively. It has been suggested that alteration of the level of the RANTES affects the progress of atherogenesis through the inflammatory pathway in patients with cardiovascular risk factors [29]. Moreover, IL-1b has been identified as a major mediator of platelet-induced activation and endothelial dysfunction [30]. In this study, the strawberry extract inhibited two important platelet mediators of inflammation, both RANTES and IL-1b. Thus, the strawberry extract at 1 mg/ml attenuated the effect of thrombininduced RANTES and IL-1b release by 41 ± 4 (p50.05) and 37 ± 4% (p50.01), respectively (Figure 2C and D). In this way, regular consumption of strawberries for 6 weeks may provide protection against high carbohydrate/fat meal-induced increase of inflammatory mediators (e.g. IL-1b) [31].
Effects of strawberry extract on arterial thrombus formation in vivo All animals in this study showed similar physiological values for rectal temperature before and after of thrombosis model among groups (data not shown).
Despite the benefits associated with current antiplatelet therapy, significant clinical limitations are associated with the use of aspirin and P2Y12 ADP receptor antagonist [32]. In this study, the strawberry extract presented inhibitory activity on human platelets aggregation and each constituent may possess multiply targets, and they may exert pleiotropic and synergistic effects. Thus, this prevents the access of the arachidonic aid to and the interactions of ADP with P2Y12 and with P2Y1, resulting in an inhibition of platelet thrombus formation under arterial flow [33–35]. It is known that strawberry is rich in polyphenols. We hypothesized that the mechanisms of antiplatelet action could be via the activation of PPARs, an increase of cAMP levels and inhibition of phospholipases C, NADPH oxidase, platelet isoprostanes, NF-kB and thromboxane A2, among others [36–38]. The dramatic inhibitory effects of the strawberry extract on platelet aggregation, and P-selectin, sCD40L, RANTES, and IL1b release supported the assessment of strawberry extract effectiveness to inhibit in vitro thrombus formation [39]. Taking into consideration all these findings, we provided further in vivo evidence of such in vitro-related strawberry extract- antithrombotic effects. To examine the in vivo antithrombotic activity of the strawberry extract, we evaluated the effects of the strawberry
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DOI: 10.3109/09537104.2014.898747
extract on laser-injured thrombus formation in mice mesenteric artery in vivo. As shown in Figure 3, in untreated mice (control), the mesenteric artery was totally blocked by a stable bulky thrombus at 20 minutes. In contrast, further analysis revealed that the time taken to form the artery thrombosis (42500 mm2) was drastically prolonged in strawberry extracttreated mice compared with mice receiving the same volume of vehicle. Thus, one intraperitoneally bolus injection of strawberry extract (200 mg/kg) 30 minutes before laser injury prevented thrombus formation over 60 minutes after laser injury, only achieving a maximum of occlusion of 35 ± 2% (p50.001 vs. control) (Figure 3). Although, in this study we used a different strawberry variety than the one published by other studies, the same antithrombotic effects have been identified [24]. Although antiplatelet drugs have proven to be beneficial in patients with clinical evidence of CVD, the outcome still remains poor. Currently, ASA is the gold standard for secondary prevention of stroke of vascular origin; however, it is frequently associated with serious adverse effects (internal bleeding and gastrointestinal adverse effects, among others) [6] and its effectiveness in primary prevention is still a matter of debate [40]. In this study, using a murine model of real-time thrombus formation, we demonstrate that the strawberry extract inhibited arterial thrombus growth at the same concentration as that of aspirin, a widely used antiplatelet agent as shown in the Figure 3. According to the previous results, aqueous fraction from Solanum lycopersicum (70 mg/kg) had a 20% reduction from baseline ex vivo platelet aggregation response in acute human study [41]. Therefore, according to the same antiplatelet effect in vitro and in vivo by strawberry it is possible to estimate that the amount of strawberry extract necessary in humans for proven antiplatelet effects is about 70 mg/kg. However, additional studies are needed to confirm the strawberry extract dose to observe acute and chronic antiplatelet effects in humans.
Conclusion In this study, we have demonstrated that aqueous extract from strawberries displayed a range of antiplatelet activities targeting different platelet activation responses in vitro and inhibition of thrombus formation. Based on these effects, it is possible to consider the strawberry as a functional food that could provide health benefits beyond basic nutrition.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This work was funded by the CONICYT REGIONAL/GORE MAULE/CEAP/R09I2001, Interdisciplinary Excellence Research Program on Healthy Aging (PIEI-ES), and supported by grant no. 1130216 (I. P., M. G., R. M., M. A., and J. C.) from Fondecyt, Chile.
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