Antiplatelet, anticoagulant, and fibrinolytic activity in ...

**NOTICE** This material may be protected by Copyright Law (Title 17 USC)

Original article 197

Antiplatelet, anticoagulant, and fibrinolytic activity in vitro of extracts from selected fruits and vegetables Constanza Torres-Urrutiaa, Luis Guzmańa, Guillermo Schmeda-Hirschmannb, Rodrigo Moore-Carrascoa, Marcelo Alarcońa, Luı´s Astudillob, Margarita Gutierrezb, Gilda Carrascoc, Jose´ A. Yuric, Eduardo Arandad and Ivań Palomoa A diet rich in fruits and vegetables is known to decrease the risk of cardiovascular disease. However, the information regarding the antithrombotic activity (antiplatelet, anticoagulant, and fibrinolytic) of fruits and vegetables is scarce. The aim of this study was to assess the antithrombotic activity of extracts from fruits and vegetables widely consumed in central Chile. The study included samples of 19 fruits and 26 vegetables, representative of the local diet. The extracts prepared from each sample included an aqueous (juice or pressed solubles) and/or methanol-soluble fraction. The extracts were evaluated for antiplatelet, anticoagulant, and fibrinolytic activity in vitro at a final concentration of 1 mg/ml. The antiplatelet activity was assessed by platelet aggregation inhibition; anticoagulant activity was measured by the prothrombin time (PT), diluted prothrombin time (dPT), activated partial thromboplastin time (APTT), kaolin clotting time (KCT), and thrombin time. The fibrinolytic effect was determined with the euglobin clot lysis time and fibrin plate methods. Extracts of green beans and tomatoes inhibited platelet aggregation induced by ADP and arachidonic acid, in a concentration-dependent manner. The methanolic extracts of grapes prolonged the PT and dPT. Finally, extracts of raspberry prolonged the APTT and also presented

Introduction Epidemiological evidence indicates that a diet rich in fruits and vegetables promotes health, decreasing the risk of cardiovascular diseases (CVDs) [1–4]. A Mediterranean diet, which is rich in the consumption of fruits and vegetables, clearly favors cardiovascular health. Worldwide, the consumption of fruits and vegetables by school children and adults is much lower than that recommended by WHO [5]. Considering this, the American Heart Association along with international agencies [6], and in our country, the Ministries of Health and Agriculture [7], and the Institute of Nutrition and Food Technology (INTA), have promoted the consumption of five or more fruits and vegetables daily [8,9]. High fat content diets can lead to an increase in the plasma levels of fibrinogen and cholesterol and at the same time, to a decrease in the fibrinolytic activity and clotting time. These changes may favor a pro-thrombotic state. 0957-5235 ß 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

fibrinolytic activity. In conclusion, from a screening that included a variety of fruits and vegetables, we found antiplatelet activity in green beans and tomatoes, anticoagulant activities in grapes and raspberries, whereas fibrinolytic activity was observed only in raspberries. Further investigations are necessary to advance in knowledge of the active compounds of these fruits and vegetables and their mechanisms of action. Blood Coagul Fibrinolysis 22:197–205 ß 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Blood Coagulation and Fibrinolysis 2011, 22:197–205 Keywords: anticoagulant activity, antiplatelet activity, antithrombotic activity, fibrinolytic activity, fruits, vegetables a Departamento de Bioquı´mica Clıńica e Inmunohematologıá, Facultad de Ciencias de la Salud, bInstituto de Quı´mica de Recursos Naturales, c Departamento de Horticultura, Facultad de Ciencias Agrarias, Universidad de Talca, Talca and dDepartamento de Hematologıá y Oncologıá, Escuela de Medicina, Pontificia Universidad Cato´lica de Chile, Santiago, Chile

Correspondence to Ivań Palomo, PhD, Departamento de Bioquı´mica e Inmunohematologıá Clıńica, Facultad de Ciencias de la Salud, Universidad de Talca, PO box 747, Talca, Chile Tel: +56 71 200493; fax: +56 71 200488; e-mail: [email protected] Received 10 October 2010 Accepted 21 December 2010

The contribution of several nutrients and nutraceuticals as well as antioxidants in fruits and vegetables is well known [10–12]. However, the antithrombotic activities of these products have been less studied [13,14]. Antiplatelet effect has been observed in several fruits, including black grapes (Vitis vinifera L.) [15], pineapple (Ananas comosus L. Merr.) [16], strawberry (Fragaria x ananassa L. Duch.) [17], and kiwi (Actinidia chinensis Planchon) [18] and vegetables such as garlic (Allium sativum L.) [19], onion (Allium cepa L.) [20], scallion (Allium schoenoprasum L.) [21], tomato (Solanum lycopersicon Mill.) [22], and melon (Cucumis melo L. var. inodorus) [23]. Anticoagulant effect has been described for pineapple [24] and Allium species [25]. Finally, fibrinolytic effect has been observed in kiwi [26] and pineapple [16]. The purpose of this study was to assess systematically and document the antiplatelet, anticoagulant, and fibrinolytic activities in vitro of extracts/juices from fruits and vegetables widely consumed in central Chile and Mediterranean countries. DOI:10.1097/MBC.0b013e328343f7da

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

198 Blood Coagulation and Fibrinolysis 2011, Vol 22 No 3

Materials and methods Fruits and vegetables extracts

Fruits and vegetables used in the study were purchased in the Central Regional Supply Talca, VII Region, Chile, at the time of harvest in March–April. The samples represent the average quality usually consumed in central Chile. Fruits with high water content were manually pressed to obtain a juice sample. A second sample from the fruits (150 g) as well as from all the vegetables was sliced/chopped and extracted with 150 ml of methanol by sonication (Transsonic 700/H; Elma-Hans Schmidbauer, Singen, Germany) for 5 min and then filtered. The filtrates, corresponding to a MeOH : water solution, were dried below 408C under reduced pressure, frozen, and lyophilized. The pressed fruit juice was lyophilized to calculate percentage w/w extraction yields. Both types of extracts were stored at 708C until use. The fruits and vegetables included in this study include common varieties cultivated in the country and are cited both by its scientific binomials and common names. Assays

The blood used for the experiments was obtained from healthy donors (60 volunteers, aged 20–25 years) who were informed about the study and signed an informed consent before starting the research. They had not ingested drugs for at least 10 days before the blood withdrawal. The protocol was authorized by the ethic committee of the Universidad de Talca in accordance with the Declaration of Helsinki. From the blood samples, platelet rich plasma (PRP) and platelet poor plasma (PPP) were prepared. Briefly, blood was collected over sodium citrate (3.2%) in a 9 : 1 v/v ratio. The PPP to be used in the anticoagulation assays was obtained by centrifuging the sample at 650g for 10 min and was stored at 708C. For the platelet aggregation assay, the PRP was obtained by centrifugation at 240g for 10 min and was immediately used for the experiments. Antiplatelet activity

The antiplatelet effect of the extracts was measured according to the method of Born [27] using a screening aggregometer consisting of a coagulometer (Clot1; RAL SA, Barcelona, Spain) to keep the platelets at 378C with stirring, and a spectrophotometer (Weather Plus; RAL SA) for transmittance readings. The positive results were validated in a classic aggregometer (platelet aggregation Profiler PAP-4; Bio-Data Corporation, Horsham, Pennsylvania, USA) at the Laboratory of Haemostasis and Thrombosis of the Pontificia Universidad Cato´lica de Chile, Santiago de Chile (courtesy of Dr Diego Mezzano). Briefly, 480 ml of PRP adjusted to a concentration of 2.5 105 platelets/ml with PPP were added to the reaction tube for 1 min and then added 20 ml of a solution of 26 mg/ml of crude extract (1 mg/ml final concentration), physiological saline (negative control), or acetylsalicylic acid 0.25 mg/ml (Sigma Chemical Co.,

St Louis, USA) as positive control. After incubating for 5 min, 20 ml of agonist (ADP 8 mmol/l or arachidonic acid 1 mmol/l; Sigma Chemical Co.) was added. Results are expressed as median percentage inhibition of platelet aggregation, respect to their negative controls. Anticoagulant activity

To evaluate the extrinsic pathway of coagulation, we measured prothrombin time (PT) and diluted thromboplastin time (dPT). To evaluate the intrinsic pathway, we used the following assays: activated partial thromboplastin time (APTT) and kaolin clotting time (KCT). For the final stage of coagulation, thrombin time was determined [28,29]. The tests were conducted in a coagulometer (Clot1, RAL SA, European Community). The PPP was preincubated for 4 min at 378C with 10 ml of a solution of 31 mg/ml of crude extract for APTT and PT; 26 mg/ml in the case of KCT; and 15 ml of a solution of 27.6 mg/ml of the extract for thrombin time to reach a final concentration of 1 mg/ml in all cases. Saline solution was used as negative control and heparin (12.4 UI/ml, final concentration) as positive control. The reagents used were calcium thromboplastin (Pacific Hemostasis, Middletown, Virginia, USA) for PT, calcium thromboplastin diluted 1/500 in physiological saline for dPT, cephaline (Pacific Hemostasis) for APTT, kaolin 0.01% for KCT, thrombin (Pacific Hemostasis) for thrombin time, and CaCl2 25 mmol/l for APTT, KCT, and thrombin time, respectively. The results of coagulation tests were expressed in seconds (s) as median value with minimum and maximum range. Fibrinolytic activity

To evaluate fibrinolytic activity, two methods were used: the euglobulin clot lysis time [30] and fibrin plate [31]. In the first method, in a reaction cuvette, the following were added: 11.4 ml of thrombin (Pacific Diagnostic, Middletown, Virginia, USA; 100 IU/ml), 88.6 ml of 20 mmol/l Tris-HCl buffer, pH 7.4, supplemented with either 20 ml of a solution of crude extract (28.5 mg/ml, 1 mg/ml final concentration in the reaction cuvette), Tris-HCl buffer as the negative control, or streptokinase 100 IU/ml (Aventis Behring, Marburg, Germany) as positive control. The reaction was initiated by adding 450 ml of euglobulin fraction and the reaction was followed in a spectrophotometer (Weather Plus, RAL, Barcelona) at 340 nm measuring every 30 s. The result was expressed as a percentage of fibrinolysis. The euglobulin fraction was obtained from citrated blood, which was centrifuged at 650g for 10 min to obtain plasma. Thereafter, 0.6 ml of plasma was transferred to a tube containing 5.4 ml of distilled water, and then 0.45 ml of acetic acid 0.25% was added. The mixture was incubated at 48C for 30 min and centrifuged at 315g for 5 min. The supernatant was discarded and the pellet was resuspended with 0.6 ml of 0.02 mol/l Tris-HCl buffer, pH 7.4.


Antithrombotic effect of fruits and vegetables Torres-Urrutia et al. 199

The fibrin plate method is used to form a layer of fibrin from the reaction of fibrinogen and thrombin in Tris-HCl buffer (pH 7.4) on a Petri dish. The mixture can be supplemented with plasminogen to assess the dependence of the reaction on this factor. The formed wells were loaded with 50 ml of solution at concentrations of 1, 10, 50, and 100 mg/ml of crude extract. The zones of fibrinolysis were observed as transparent halos around the wells, which were compared with the blank and controls. The result was expressed as diameter of the fibrinolysis halo. Statistical analysis

The nonparametric Kruskal–Wallis test included in the software SPSS 15.0 (SPSS Inc., Chicago, Illinois, USA) was used to analyze the results. P values less than 0.05 were considered significant.

Results Extracts yielding

The list of the selected fruits and vegetables as well as the percentage w/w extraction yields is presented in Table 1. For the juice, the solid content ranged roughly between 4 and 24%, whereas the methanol extracts presented a very high variability, depending on the species and variety. Antiplatelet activity

From the vegetables studied, green beans and tomatoes extracts presented significant antiplatelet activity. The methanol extract of green beans showed dose-dependent antiplatelet effect, inhibiting aggregation by approximately 90% at 1, 0.5 mg/ml and approximately 20% at 0.1 mg/ml, using ADP (4 mmol/l) as agonist (Fig. 1a); however, when ADP (8 mmol/l) was used, the percentage of platelet inhibition was approximately 50%, using 0.5 and 1 mg/ml of the extract. Additionally, when arachidonic acid was used as agonist, the extracts at 0.5 and 1 mg/ml slowed the platelet aggregation (Fig. 1). Under the same experimental conditions using arachidonic acid, acetyl salicylic acid inhibited platelet aggregation by 90–95%. For tomatoes (including hybrid grown outdoors and cluster), both aqueous and methanolic extracts showed dose-dependent platelet antiaggregant activity at concentrations more than 0.1 mg/ml, using ADP as agonist. The anti-aggregant activity of hybrid tomato grown outdoors is slightly higher, but not significantly different from tomato type cluster: at 1 mg/ml, the percentage mean inhibition of aqueous extract was approximately 34% and approximately 25%, respectively, whereas for methanolic extract, the figures were approximately 40.0% and approximately 32%, respectively (Table 2). Garlic, onions, the sapphire variety of red pepper, and scallion reduced aggregation by approximately 15% (Table 2). From the fruits studied, only plum and strawberry extracts showed a slight inhibitory effect on platelet

Scientific name, variety, and percentage (w/w) of extraction yields of the selected fruits and vegetables used in the study

Table 1

Species and variety Fruits Apple (Malus pumila Mill.) Galaxy Golden Granny Smith Royal Gala Scarlet Grapefruit (Citrus paradisi Macfad) Grapes (Vitis vinifera L.) Curtidurıá (rose) Paı´s (black) Red globe Sultanina Torontel Kiwi (Actinidia chinensis, Planchon) Pear (Pyrus communis L.) Abate´ Asia´tica type Agua type Plum (Prunus domestica L. var. Americana, Marshall) Quince (Cydonia oblonga Mill.) Raspberry (Rubus idaeus L.) Strawberry (Fragaria x ananassa L. Duch.) Vegetables Beans (Phaseolus vulgaris L.) Beetroot (Beta vulgaris L.) Broccoli (Brassica oleracea L.) Cabbage (Brassica oleracea var. capitata L.) Carrot (Daucus carota L.) Cauliflower (Brassica oleracea var. botrytis L.) Cucumber (Cucumis sativus L.) Eggplant (Solanum melongena L.) Garlic (Allium sativum L.) Melon (Cucumis melo L.) Pla´tano type Tuna type Onion (Allium cepa L.) Valenciana type Calderana type Pear melon (Solanum muricatum Aiton.) Scallion (Allium schoenoprasum L.) Spinach (Spinacea oleracea L.) Sweet pepper (Capsicum anuum L.) Fiuco type Sapphire type Swiss chard (Beta vulgaris subsp. cicla) Tomato (Solanum lycopersicum M.) Watermelon (Citrullus lanatus Thunb.)

Aqueous (%)

MeOH (%)

16.2 12.5 12.8 13.4 13.5 12.3

13.0 9.8 10.7 9.8 9.6 8.5

19.2 17.3 20.2 24.2 21.0 12.9

14.2 15.6 16.5 22.2 15.9 7.7

15.1 14.7 7.9 11.4

9.0 11.4 7.7 12.4

N/D 9.6 7.6

10.0 5.6 2.9

N/D N/D N/D N/D N/D N/D N/D N/D N/D

3.3 8.7 4.2 4.3 7.5 4.4 2.3 3.8 10.2

11.1 8.4

7.1 6.8

N/D N/D 8.4 N/D N/D

5.5 6.2 3.7 5.9 1.8

N/D N/D N/D 4.0 7.5

5.4 5.5 2.0 3.4 8.0

N/D, not determined.

aggregation (using ADP as agonist) of about 10% (Table 2). Anticoagulant activity

The aqueous extracts of the selected fruits did not show a significant effect on the extrinsic pathway (PT and dTP, Table 3). Only the methanolic extracts of grapes Sultanina (12.5 s) and Torontel (12.7 s) slightly lengthen the PT (negative control 12.3 s with a 11.6–13.0 s range). Moreover, the methanolic extracts of Curtidurıá (40.2 s) and Paı´s (40.5 s) grapes slightly lengthen dTP (negative control (40.1 s and a 38.2–42.9 s range; Table 3). Regarding the intrinsic pathway, both the aqueous and methanolic extracts of raspberry increased the APTT compared



Fig. 1

to the control (Table 4). On the other hand, fruit extracts showed no effects on the KCT (Table 4). Vegetable extracts did not increase PT or dPT, but the methanolic garlic fraction (108.8 s) slightly prolonged KCT (negative control 87.3 s with a 77.6–96.7 s range). Additionally, neither fruits nor vegetables extracts inhibited significantly the final phase of coagulation, assessed as thrombin time, respect to controls (not shown).

Fibrinolytic activity

Inhibitory effect of in-vitro platelet aggregation by methanolic extracts of green beans induced by the following: ADP 4 mmol/l, (a) and araquidonic acid 1 mmol/l (b). 1, negative control (saline); 2, 3, and 4: methanolic extracts of green beans at 0.1, 0.5, and 1 mg/ml, respectively.

From the fruit extracts studied, only raspberry presented a significant concentration-dependent fibrinolytic activity, assessed as euglobulin clot lysis time (Fig. 2). At a concentration of 1 mg/ml, it showed a slight effect, both the aqueous and methanolic fractions (8.1 and 10.6% of fibrinolysis, respectively). At 5 mg/ml, fibrinolytic activity in aqueous and methanolic fractions were 58.4 and 35.9%, respectively. The effect of this extract was also studied using the fibrin plate method. Fibrin plate supplemented with plasminogen presented fibrinolytic halos at a concentration of 50 and 100 mg/ml using aqueous and methanolic extracts. By contrast, the fibrin plate that was not supplemented with plasminogen did not produce fibrinolytic halos, neither in the wells filled with raspberry extracts (1, 10, 50, and 100 mg/ml) nor in the well loaded with streptokinase (250 IU/ml). Additionally, kiwi fruits presented a slight fibrinolytic activity in the aqueous fraction, although at a very high

Table 2 Inhibition of platelet aggregation induced by ADP and arachidonic acid-induced platelet aggregation by aqueous (Aqueous) and methanolic (MeOH) fruit and vegetables extracts in vitro at 1 mg/ml ADP Aqueous

Species and variety Fruits Apple (Malus pumila Mill.) Granny Smith Royal Gala Scarlet Grapefruit (Citrus paradisi Macfad) Plum (Prunus domestica L. var. Americana, Marshall) Raspberry (Rubus idaeus L.) Strawberry (Fragaria x ananassa L. Duch.) Vegetables Beans (Phaseolus vulgaris L.) Beetroot (Beta vulgaris L.) Cabbage (Brassica oleracea var. Capitata L Eggplant (Solanum melongena L.) Garlic (Allium sativum L.) Melon (Cucumis melo L.) Pla´tano type Tuna type Onion (Allium cepa L.) Valenciana Pear melon (Solanum muricatum Aiton.) Scallion (Allium schoenoprasum L.) Spinach (Spinacea oleracea L) Sweet pepper (Capsicum anuum L.) Fiuco type Sapphire Tomato (Solanum lycopersicum M.)

Arachidonic acid

0.8 10.3 18.8 13.6 38.8 0 27.4

MeOH

(0.6–1.4) (9.5–11.8) (16.7–19.0) (13.4–14.5) (35.8–38.9) (0.0–0.1) (26.8–29.4)

12.2 2.7 2.2 14.3 19.8 9.4 10.0

(11.8–12.6) (2.4–3.1) (2.1–3.0) (14.0–15.0) (19.3–20.1) (9.3–9.8) (9.3–10.7)

N/D N/D N/D N/D N/D

13.0 10.4 8.4 7.4 25.2

(11.9–13.3) (9.2–11.4) (8.1–9.5) (5.6–7.8) (25.1–27.9)

34.8 (32.7–35.6) 34.1 (34.0–35.9) N/D 9.0 (8.9–10.3) N/D N/D N/D N/D 25.4 (21.6–26.0)MMM

Aqueous

0 7.6 11.9 1.6 5.7 18.4 3.5

(0.0–0.6) (7.5–8.6) (11.5–12.4) (1.0–2.2) (5.6–6.6) (17.9–18.8) (3.4–3.7) N/D N/D N/D N/D N/D

39.4 (39.4–40.9) 41.7 (40.3–46.1)

5.9 (5.5–6.5) 9.0 (8.9–9.4)

6.0 15.0 25.4 30.1

(5.0–7.1) (13.7–18.2) (25.3–29.5) (30.0–38.1)

N/D 6.8 (6.6–6.9) N/D N/D

32.0 (30.7–33.3) 22.2 (21.9–24.5) 32.4 (27.6–42.4)MMM

N/D N/D 0 (0–0.8)

MeOH

3.7 0 0.7 0 0.3 8.6 0 63.6 0 0 0 0

(3.3–3.9) (0.0–0.9) (0.5–1.1) (0.0–0.5) (0.1–0.4) (8.1–9.0) (0.0–0.4) (62.5–64.7)M (0.0–0.6) (0.0–0.6) (0.0–1.2) (0.0–3.0)

0 (0.0–0.2) 1.3 (1.1–1.8) 0 27.8 5.8 0

(0.0–0.5) (25.6–28.3) (5.5–6.5) (0.0–1.3)

0.6 (0.4–1.0) 52.6 (51.7–52.9) 0 (0–1.6)

N/D, not determined. Results are expressed as median percentage inhibition of platelet aggregation, with respect to their negative controls. M, P < 0.05 vs. control, MMM 0.001 vs. control.


(12.3–12.6) (12.3–12.6) (12.5–12.8) (12.3–12.6) (12.3–12.6)

12.3 12.3 12.7 12.3 12.3

N/D N/D N/D 12.4 (12.1–12.5) 12.3 (12.1–12.5)

N/D 12.6 (12.6–12.9) N/D N/D


(12.3–12.6) (12.3–12.6) (12.3–12.6) (12.5–12.8)

(11.6–12.0) (11.6–12.0) (12.0–12.3) (12.0–12.3) (12.9–13.1) (12.5–12.8) (12.3–12.6) (12.9–13.1) (12.3–12.6)

11.7 11.7 12.1 12.1 13.0 12.7 12.3 13.0 12.3

(11.8–12.2) (12.0–12.3) (12.0–12.4) (11.5–12.0) (11.5–11.7) N/D 12.2 (12.1–12.5) 12.3 (12.2–12.6)

12.1 12.0 12.2 11.5 11.6

(12.2–12.6) (11.6–12.1) (11.5–11.9) (12.5–12.8) (12.7–13.1)

12.3 12.3 12.3 12.7

(11.6–11.8) (11.6–12.0) (11.6–11.8) (11.6–12.0) (11.6–12.0) (12.1–12.5) (12.0–12.3) (12.4–12.8)

11.6 11.7 11.6 11.7 11.7 12.2 12.1 12.5

12.5 11.6 11.5 12.6 12.8

(11.8–12.9) (12.1–12.5) (12.1–12.4) (11.9–12.4)

12.9 (12.8–13.1)

(12.3–12.6) (11.6–11.8) (11.6–11.8) (12.0–12.3) (12.0–12.3)

12.3 11.6 11.6 12.1 12.1

12.3 12.3 12.2 12.3

13.0 (12.9–13.1)

(12.4–12.6) (12.4–12.8) (12.4–12.8) (12.4–12.6)

12.4 12.5 12.5 12.4

Aqueous

N/D, not determined. The results are expressed in seconds (s) as median value with minimum and maximum range.

Fruits Apple (Malus pumila Mill.) Golden Royal Gala Scarlet Grapefruit (Citrus paradisi Macfad) Grapes (Vitis vinifera L.) Curtidurıá (rose) Paı´s (black) Red globe Sultanina Torontel Pear (Pyrus communis L.) Abate´ Asia´tica type Agua type Kiwi (Actinidia chinensis, Planchon) Plum (Prunus domestica L. var. Americana, Marshall) Quince (Cydonia oblonga Mill.) Raspberry (Rubus idaeus L.) Strawberry (Fragaria x ananassa L. Duch.) Vegetables Beans (Phaseolus vulgaris L.) Beetroot (Beta vulgaris L.) Broccoli (Brassica oleracea L.) Cabbage (Brassica oleracea var. capitata L.) Carrot (Daucus carota L.) Cauliflower (Brassica oleracea var. botrytis L.) Cucumber (Cucumis sativus L.) Eggplant (Solanum melongena L.) Garlic (Allium sativum L.) Melon (Cucumis melo L.) Tuna type Onion (Allium cepa L.) Valenciana type Pear melon (Solanum muricatum Aiton.) Scallion (Allium schoenoprasum L.) Spinach (Spinacea oleracea L) Sweet pepper (Capsicum anuum L.) Fiuco type Sapphire type Swiss chard (Beta vulgaris subsp. cicla) Tomato (Solanum lycopersicum M.) Watermelon (Citrullus lanatus Thunb.)

Control

Prothrombin time (s)

M

(12.1–12.9) (12.0–12.4) (12.1–12.6) (12.0–12.8)

(12.0–12.5) (11.8–12.8) (12.0–12.6) (12.0–12.7) (12.2–12.3) (12.1–12.3) (12.1–12.5) (12.5–13.1) (12.1–12.5)

(11.9–12.4) (11.9–12.5) (11.6–11.9) (12.0–12.5) (11.9–12.1) (12.0–12.3) (11.5–12.0) (12.1–12.5)

(12.4–12.8) (12.2–12.9) (12.3–12.6) (12.0–12.5) (12.2–12.8)

(11.9–12.5) (12.1–12.7) (11.9–12.1) (12.5–12.8)

P < 0.05 vs. control.

12.5 12.3 12.6 12.1 12.5

12.3 12.3 12.0 12.7

13.0 (12.7–13.2)

12.2 12.0 12.1 12.3 12.3 12.3 12.3 12.9 12.3

12.2 11.9 11.6 12.0 11.9 12.1 11.6 12.3

12.1 (12.0–12.9) 11.6 (11.5–12.5) 11.8 (11.8–12.7) 12.8M (12.5–13.4) 12.7M (12.6–12.8)

12.4 12.1 12.1 12.5

MeOH

(39.9–40.8) (39.1–40.9) (39.1–40.9) (39.1–40.9) (39.1–40.9) (39.1–40.9) (39.1–40.9) (39.1–40.9) (39.9–40.8)

(41.5–45.4) (39.1–40.9) (41.5–45.4) (41.5–45.4) (41.5–45.4) (39.9–40.8) (41.5–45.4) (41.5–45.4)

(36.9–39.4) (36.9–39.4) (36.9–39.4) (36.9–39.4) (36.9–39.4)

(36.9–39.4) (36.9–39.4) (36.9–39.4) (41.5–45.4)

40.1 40.1 39.7 40.1 42.9

40.1 39.7 40.1 39.7

(39.9–40.8) (39.9–40.8) (39.1–40.9) (39.9–40.8) (41.5–45.4)

(39.9–40.8) (39.1–40.9) (39.9–40.8) (39.1–40.9)

39.7 (39.1–40.9)

40.1 39.7 39.7 39.7 39.7 39.7 39.7 39.7 40.1

42.9 39.7 42.9 42.9 42.9 40.1 42.9 42.9

38.2 38.2 38.2 38.2 38.2

38.2 38.2 38.2 42.9

Control

Effect of aqueous and methanol extracts from fruits and vegetables 1 mg/ml, on prothrombin time and diluted prothrombin time

Species and variety

Table 3

(36.6–36.6) (35.5–37.6) (37.2–37.6) (39.6–40.9) (36.3–37.1)

(31.4–41.4) (37.5–39.2) (38.8–40.9) (36.7–38.4)

N/D N/D N/D 38.7 (37.6–38.9) 41.3 (40.6–42.3)

N/D 38.1 (38.1–38.3) N/D N/D

39.6 (38.9–40.2)


(37.2–38.8) (38.3–38.9) (38.7–39.0) (36.5–36.7) (36.4–38.5) N/D 36.7 (36.5–37.1) 36.6 (36.5–36.8)

37.8 38.3 38.7 36.5 38.0

36.5 36.0 37.4 40.4 37.0

37.5 37.8 39.4 37.6

Aqueous

Diluted prothrombin time (s)

(39.7–40.6) (39.4–40.3) (40.1–40.7) (36.9–40.3)

(38.8–39.8) (38.0–38.5) (39.6–40.2) (37.4–37.7) (38.2–39.4) (38.1–38.5) (38.4–38.9) (37.2–37.9) (39.0–40.0)

(40.6–41.6) (36.9–37.8) (40.2–40.8) (39.1–40.2) (38.3–39.7) (38.6–39.1) (38.7–39.3) (36.1–37.8)

39.0 40.3 37.6 38.6 41.2

39.9 39.4 39.4 38.2

(38.7–39.9) (40.2–41.0) (37.5–37.8) (38.4–38.7) (41.1–41.4)

(39.4–40.3) (38.8–39.4) (39.4–39.7) (38.0–38.2)

39.1 (38.0–39.3)

38.9 38.1 39.7 37.6 39.3 38.1 38.8 37.4 39.4

41.5 37.8 40.8 39.2 39.5 38.8 39.3 37.6

40.2M (39.6–40.3) 40.5M (39.9–41.0) 39.0 (38.7–39.7) 39.3 (37.8–40.6) 39.2 (39.2–40.0)

40.4 39.6 40.2 37.5

MeOH



(34.5–35.9) (35.1–36.9) (35.3–36.1) (34.5–35.9) (34.5–35.9) (35.1–36.9) (35.1–36.9) (35.3–36.1) (35.3–36.1) (35.3–36.1) (35.3–36.1) (35.3–36.1) (35.3–36.1) (35.4–36.6) (35.3–36.1) (34.5–35.8) (36.2–38.7) (34.5–35.9) (34.5–35.8) (39.1–41.0) (35.3–36.1) (39.1–41.0) (34.5–35.8) (34.5–35.8) (34.5–35.8) (34.5–35.9) (34.5–35.9) (34.5–35.9) (35.1–36.9) (34.5–35.8) (34.5–35.8) (34.5–35.8) (34.5–35.8) (34.5–35.9) (39.1–41.0) (34.5–35.8) (34.5–35.8) (39.1–41.0)

35.6 35.2 35.3 35.6 35.6 35.2 35.2 35.3 35.3 35.3 35.3 35.3 35.3 35.8 35.3 34.5 38.0 35.6 34.5 39.7 35.3 39.7 34.5 34.5 34.5 35.6 35.6 35.6 35.2 34.5 34.5 34.5 34.5 35.6 39.7 34.5 34.5 39.7

(34.8–37.6) (35.8–40.1) (38.3–39.8) (35.1–36.8) (35.2–36.4) (39.0–40.9)

(51.7–54.0) (35.3–38.2) (36.1–37.6) (36.1–37.6) (35.1–37.8) (39.7–42.1)

N/D N/D N/D N/D 34.7 (34.4–35.4) 40.9 (38.2–43.9) N/D

33.9 (33.5–34.0) 36.2 (35.9–37.4) N/D 37.8 (35.6–39.0)

N/D N/D N/D N/D N/D

N/D N/D N/D N/D N/D

35.6 (34.6–35.7) 34.9 (34.6–35.7) 36.6 (35.3–37.0) N/D 46.4M (39.9–50.2)

35.8 36.2 39.3 36.1 35.9 39.5

52.7 37.1 36.2 35.4 37.6 41.3

Aqueous

(33.8–34.8) (35.1–35.6) (36.2–38.0) (36.3–37.0) (35.5–37.9) (40.0–41.6)

(34.7–37.1) (34.2–37.3) (34.5–35.3) (34.5–35.3) (34.9–36.7) (39.7–41.1)

MeOH

P < 0.05 vs. control.

(24.6–35.4) (37.7–38.1) (36.8–41.1) (36.9–41.4) (32.8–35.7) (36.4–44.7) (38.4–38.8)

(33.6–35.3) (34.4–36.3) (39.6–42.8) (35.6–36.2)

(37.5–38.3) (35.0–36.8) (33.4–35.8) (32.8–38.1) (34.5–35.9)

(35.0–35.6) (37.1–38.1) (36.5–37.6) (34.1–36.2) (38.9–40.0)

M

35.1 38.1 37.3 39.2 34.1 39.9 38.5

35.1 35.2 40.7 35.9

38.0 35.3 33.7 36.2 35.7

35.4 37.5 36.8 34.9 39.6

37.2 (36.2–37.3) 34.4 (33.9–34.4) 37.0 (36.7–41.1) 44.6 (40.6–47.7) 54.3M (52.6–54.7)

34.2 35.1 36.8 36.5 36.2 41.5

35.6 36.0 35.0 34.6 35.0 40.1

N/D, not determined. The results are expressed in seconds (s) as median value with minimum and maximum range.

Fruits Apple (Malus pumila Mill.) Galaxy Golden Granny Smith Royal Gala Scarlet Grapefruit (Citrus paradisi Macfad) Grapes (Vitis vinifera L.) Curtidurıá (rose) Paı´s (black) Red globe Sultanina Torontel Kiwi (Actinidia chinensis, Planchon) Pear (Pyrus communis L.) Abate´ Asia´tica De Agua Quince (Cydonia oblonga Mill.) Raspberry (Rubus idaeus L.) Vegetables Swiss chard (Beta vulgaris subsp. cicla) Garlic (Allium sativum L.) Eggplant (Solanum melongena L.) Beetroot (Beta vulgaris L.) Broccoli (Brassica oleracea L.) Onion (Allium cepa L.) De guarda De verano Scallion (Allium schoenoprasum L.) Cauliflower (Brassica oleracea var. botrytis L.) Spinach (Spinacea oleracea L) Melon (Cucumis melo L.) Pla´tano Tuna Cucumber (Cucumis sativus L.) Pear melon (Solanum muricatum Aiton.) Sweet pepper (Capsicum anuum L.) Fiuco type Sapphire type Beans (Phaseolus vulgaris L.) Cabbage (Brassica oleracea var. capitata L.) Watermelon (Citrullus lanatus Thunb.) Tomato (Solanum lycopersicum M.) Carrot (Daucus carota L.)

Control

APPT (s)

87.3 87.3 96.7 96.0 77.6 96.7 96.0

77.6 77.6 96.0 96.0

87.3 87.3 87.3 96.0 96.0

96.0 87.3 96.0 96.7 87.3

85.2 85.2 85.2 77.6 96.7

85.2 96.0 85.2 85.2 85.2 85.2

85.2 85.2 85.2 85.2 85.2 77.6

(85.5–87.8) (85.5–87.8) (96.0–97.2) (92.2–98.0) (75.3–79.5) (96.0–97.2) (92.2–98.0)

(75.3–79.5) (75.3–79.5) (92.2–98.0) (92.2–98.0)

(85.5–87.8) (85.5–87.8) (85.5–87.8) (92.2–98.0) (92.2–98.0)

(92.2–98.0) (85.5–87.8) (92.2–98.0) (96.0–97.2) (85.5–87.8)

(84.8–85.8) (84.8–85.8) (84.8–85.8) (75.3–79.5) (96.5–97.3)

(84.8–85.8) (92.2–98.0) (84.8–85.8) (84.8–85.8) (84.8–85.8) (84.8–85.8)

(84.8–85.8) (84.8–85.8) (84.8–85.8) (84.8–85.8) (84.8–85.8) (75.3–79.5)

Control

(89.8–92.5) (91.5–92.7) (90.2–93.6) (95.5–96.2) (89.6–94.0) (70.6–74.8)

N/D N/D N/D N/D 77.2 (75.0–83.1) 91.2 (90.6–91.9) N/D

72.9 (70.3–76.1) 69.5 (66.9–73.5) N/D 92.0 (81.9–94.1)

N/D N/D N/D N/D N/D

N/D N/D N/D N/D N/D

94.8 (94.4–100.0) 94.1 (93.1–95.4) 87.3 (85.7–89.4) N/D 85.4 (84.1–87.4)

93.5 (92.3–99.7) 97.1 (97.1–100.4) 93.5 (90.5–93.7) 100.9 (98.4–101.0) 98.9 (94.9–99.6) 91.8 (88.5–92.0)

91.9 92.3 90.9 96.1 90.2 74.1

Aqueous

KCT (s)

Effect of aqueous and methanol (MeOH) extracts from fruits y vegetables 1 mg/ml on activated partial thromboplastin time and kaolin clotting time

Species and variety

Table 4

(91.5–94.2) (93.5–97.5) (82.0–85.1) (67.1–79.3) (92.4–95.1)

(92.1–94.8) (95.5–98.6) (90.9–93.5) (90.4–94.2) (98.6–100.8) (89.1–96.2)

(90.6–95.6) (91.8–96.3) (91.4–94.4) (92.5–97.6) (95.0–99.8) (68.8–80.1)

(75.8–78.2) (76.3–78.1) (88.8–95.7) (96.2–99.8) 89.2 (88.6–90.0) 80.7 (80.0–80.8) 101.3 (98.6–101.4) 70.3 (69.8–71.7) 76.8 (75.1–80.5) 91.3 (89.6–92.7) 96.2 (96.1–96.4)

76.9 76.8 90.0 99.7

92.8 (91.8–106.9) 92.3 (91.7–93.1) 96.2 (86.1–97.2) 96.4 (91.6–96.4) 106.0 (100.6–108.2)

93.6 (92.7–96.3) 108.6M (100.3–120.9) 84.2 (83.7–86.1) 86.3 (85.0–87.5) 94.0 (92.8–96.8)

92.5 93.9 82.8 71.8 93.9

92.8 97.2 91.7 93.8 99.5 91.5

91.4 92.0 92.9 94.9 97.4 70.4

MeOH




Fig. 2

Percentage of fibrinolysis

100% 90% 80% 70% 60%

1 mg/ml

50%

5 mg/ml

40% 30% 20% 10% 0% Water extract

Methanolic extract

Positive control

Fibrinolytic effect of methanolic and aqueous extracts of raspberry at 1 and 5 mg/ml, using the euglobin clot lysis. The positive control represents the activity of streptokinase 100 IU/ml.

concentration (10 mg/ml). The vegetable extracts evaluated at 1 mg/ml using the technique of euglobulin clot lysis time did not show fibrinolytic effect in vitro.

Discussion Mortality from CVD is associated with high prevalence of cardiovascular risk factors [32]. This requires efforts aimed to reduce the prevalence of risk factors, especially those known as the modified diet and sedentary lifestyle, as they are involved in the reduction of other modifiable factors. In this regard, there is need to promote consumption of fruits and vegetables, thereby reducing the prevalence of risk factors mentioned above [33]. The antioxidant effects of fruit and vegetables have been well studied [34,35], but other activities such as antithrombotic effect should be investigated as well [14]. With the aim of contributing to the knowledge of these properties, we studied the antiplatelet, anticoagulant, and fibrinolytic activities of aqueous and methanolic extracts of some fruits and vegetables widely consumed in Chile as well as in Mediterranean climate countries. The in-vitro antiplatelet activity of the fruit extracts studied, inducing aggregation with ADP and arachidonic acid, showed that extracts of plum and strawberries have a mild antiaggregatory effect. In the literature, several fruits have been reported to present antiplatelet effect [13]. In aqueous and methanolic extracts of strawberry, a mild antiplatelet effect was found with ADP as agonist. Ellagic acid and flavonoids present in strawberries have been reported to inhibit the activity of cyclooxygenases [36]. Moreover, filtrates of different varieties of strawberries have shown a significant antiplatelet effect in vitro and in vivo [17]. Kiwi fruit has shown an inhibitory effect in vitro and ex vivo of platelet aggregation induced by ADP and collagen [18]. Bromeline, a compound found in pineapple, has been observed to inhibit platelet aggregation induced by ADP and TRAP-6 in vitro [16].

Finally, it was found that daily consumption for more than a week of black grape juice inhibits platelet aggregation induced by collagen [15]. It has been postulated that flavonoids can inhibit enzymes such as cyclooxygenase and phosphodiesterase [37,38]; and other studies have found that juice of black grape and red wine increase the production of nitric oxide in platelets and endothelial cells [39,40]. In the present work, the methanolic extracts of some vegetables (garlic, onion, scallion, melon, pepper) showed a weak antiplatelet activity, whereas tomatoes showed a mild platelet aggregation with ADP. A relevant inhibition was found for green beans using ADP as agonist. The inhibitory effect on platelet aggregation exhibited by the methanol extracts of green beans is presented for the first time. As several of the horticultural products investigated are consumed after cooking, new studies are needed to compare the effect of extracts after processing with the information obtained using raw plant material. In-vitro studies of aqueous extract of raw and boiled garlic and onion on platelet aggregation induced by collagen have shown varying degrees of platelet inhibition [20]. The antithrombotic properties of garlic and onion have been attributed to organosulphur compounds [41–43]. It has also been reported that the aqueous extract of melon shows inhibitory effect on platelet aggregation induced by several agonists [23]. In different varieties of tomatoes, inhibition of platelet aggregation induced by ADP has been reported, but no effect was observed with arachidonic acid, thus suggesting that these extracts act on events preceding the cyclooxygenase pathway [22,44,45]. In our study, both types of tomatoes studied (hybrid grown outdoors and cluster) showed mild concentration-dependent antiplatelet activity when the aggregation was induced by ADP and to a lesser extent by collagen. It has been



suggested that the antioxidant lycopene, found in high concentrations in tomatoes, and heat-stable components of low molecular weight such as adenosine and others, could be responsible for this antiplatelet effect [22,44,45]. In general, anticoagulant activity was not observed in fruit extracts both in extrinsic and intrinsic pathways, as well as in the final stage. It has been reported that bromelin, a proteolytic enzyme of pineapple, has antithrombin activity in vitro, at concentrations above 10 mg/ml [24]. Among the vegetables, the methanolic extract of garlic and green beans showed a mild anticoagulant activity in tests that assess the intrinsic pathway. In the literature, the anticoagulant effect has been described in garlic and onions [25,46,47]. Aqueous and methanolic extracts of raspberry were able to lyse fibrin in the euglobulin clot lysis test at concentrations above 1 mg/ml, an effect that appears to be dependent on the activation of plasminogen (fibrin plate test). It has been demonstrated in vivo that the consumption of berries, which belongs to the family of Rosaceae like raspberries, enhances fibrinolysis in murine models of thrombosis [48]. In turn, Jung et al. [26] found effects of a methanol:water kiwi fruit extract. The differences of the present report with previous studies regarding presence or absence of antithrombotic activities in fruits and vegetables could be partly due to the varieties studied, the conditions of cultivation, the platelet agonists used, and concentrations assessed. In conclusion, we found some antithrombotic activity in fruits and vegetables that may have an important impact on human health. Further studies are necessary to establish the compounds and mechanisms that account for these effects.

Acknowledgements The present study was supported by the Research Program of Cardiovascular Disease Risk Factors (PIFRECV), Universidad de Talca, Chile. We would like to thank Dr Daniel R. Gonzalez for his critical review of the manuscript.

References 1 2 3 4

5

6

Rimm EB. Fruit and vegetables: building a solid foundation. Am J Clin Nutr 2002; 76:1–2. OMS: Estrategia Mundial sobre Re´gimen Alimentaria, Actividad Fı´sica y Salud. 57 Asamblea Mundial de Salud; 2004 (Geneva). WHO. Diet, nutrition and the prevention of chronic disease. Report of a joint WHO/FAO expert consultation. Geneva: WHO; 2003. Bazzano LA, He J, Ogden LG, Loria CM, Vupputuri S, Myers L, Whelton PK. Fruit and vegetable intake and risk of cardiovascular disease in US adults: the first National Health and Nutrition Examination Survey Epidemiologic Follow-up Study. Am J Clin Nutr 2002; 76:93–99. Villalobos P, Rojas A, Leporati M. Chile as an agricultural and food power: commitment to nutrition and population health. Rev Chil Nutr 2006; 33:232– 237. Liu S, Manson JE, Lee IM, Cole SR, Hennekens CH, Willett WC, Buring JE. Fruit and vegetable intake and risk of cardiovascular disease: the Women’s Health Study. Am J Clin Nutr 2000; 72:922–928.

7 8 9

10

11

12

13

14

15

16 17

18

19

20

21

22 23

24 25

26

27 28

29 30

31 32 33 34 35

Fletcher B, Lamendola C. Insulin resistance syndrome. J Cardiovasc Nurs 2004; 19:339–345. Lansbury AJ, Grant PJ, Catto AJ. Atherothrombotic risk factors in subjects with a family history of stroke. Cerebrovasc Dis 2002; 14:153–160. Mills JD, Mansfield MW, Grant PJ. Factor XIII-circulating levels and the Val34Leu polymorphism in the healthy male relatives of patients with severe coronary artery disease. Thromb Haemost 2002; 87:409–414. De Angelis RC. New concepts in nutrition: considerations on the connection diet-health. Arquivos de Gastroenterologia 2001; 38:269– 271. Palomo GI, Gutie´rrez CM, Astudillo SL, Rivera SC, Torres UC, Guzmań JL, et al. Antioxidant effect of fruits and vegetables of central region of Chile. Revista Chilena de Nutricioń 2009; 36:152–158. Yuri JA, Neira A, Quilodran A, Motomura Y, Palomo I. Antioxidant activity and total phenolics concentration in apple peel and flesh is determined by cultivar and agroclimatic growing regions in Chile. J Food Agri Environ 2009; 7:3–4. Torres C, Guzmań L, Moore-Carrasco R, Palomo I. Antithrombotic effect, a not well known characteristic of fruits and vegetables. Revista Chilena de Nutricioń 2008; 35:10–17. Palomo I. Efectos antitrobo´ticos de las frutas y hortalizas. Dieta Mediterrańea. Prevencioń de las Enfermedades Cardiovasculares. In: Palomo I, Leiva E, Va´squez M, editors. Editorial Universidad de Talca, Talca; 2007. p. 173. Keevil JG, Osman HE, Reed JD, Folts JD. Grape juice, but not orange juice or grapefruit juice, inhibits human platelet aggregation. J Nutr 2000; 130:53–56. Glaser D, Hilberg T. The influence of bromelain on platelet count and platelet activity in vitro. Platelets 2006; 17:37–41. Naemura A, Mitani T, Ijiri Y, Tamura Y, Yamashita T, Okimura M, Yamamoto J. Antithrombotic effect of strawberries. Blood Coagul Fibrinolysis 2005; 16:501–509. Duttaroy AK, Jorgensen A. Effects of kiwi fruit consumption on platelet aggregation and plasma lipids in healthy human volunteers. Platelets 2004; 15:287–292. Cavagnaro PF, Camargo A, Galmarini CR, Simon PW. Effect of cooking on garlic (Allium sativum L.) antiplatelet activity and thiosulfinates content. J Agric Food Chem 2007; 55:1280–1288. Ali M, Bordia T, Mustafa T. Effect of raw versus boiled aqueous extract of garlic and onion on platelet aggregation. Prostaglandins Leukot Essent Fatty Acids 1999; 60:43–47. Chen JH, Chen HI, Tsai SJ, Jen CJ. Chronic consumption of raw but not boiled Welsh onion juice inhibits rat platelet function. J Nutr 2000; 130:34–37. Dutta-Roy AK, Crosbie L, Gordon MJ. Effects of tomato extract on human platelet aggregation in vitro. Platelets 2001; 12:218–227. Altman R, Rouvier J, Weisenberger H. Identification of platelet inhibitor present in the melon (Cucurbitacea cucumis melo). Thromb Haemost 1985; 53:312–313. Hawkey C, Howell M. Orally induced fibrinolysis: a critical assessment of pineapple protease. Thromb Diath Haemorrh 1964; 12:382–390. Kim GN, Lee MK, Park I. Acid and heat stability of the anticoagulative activity of an onion extract. Biosci Biotechnol Biochem 2002; 66:859– 861. Jung KA, Song TC, Han D, Kim IH, Kim YE, Lee CH. Cardiovascular protective properties of kiwifruit extracts in vitro. Biol Pharm Bull 2005; 28:1782–1785. Born GV. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 1962; 194:927–929. Palomo I, Munõz B, Retamales E, Hidalgo P, Panes O, Artigas C, et al. Estudio de Laboratorio de las Enfermedades Hemorragı´paras. Hematologıá, Fisiopatologıá y Diagno´stico. In: Palomo I, Pereira J, Palma J, editors. Editorial Universidad de Talca; 2005. Lafan M, Manning R. Investigation of haemostasis. In Dacie and Lewis practical haemostasis. Elsevier, Churchill Livingstone. Chapter 16; 2006. Boudjeltia KZ, Cauchie P, Remacle C, Guillaume M, Brohee D, Hubert JL, et al. A new device for measurement of fibrin clot lysis: application to the euglobulin clot lysis time. BMC Biotechnol 2002; 2:8. Raftery AT. Method for measuring fibrinolytic activity in a single layer of cells. J Clin Pathol 1981; 34:625–629. O’Donnell CJ, Elosua R. Cardiovascular risk factors. Insights from Framingham Heart Study. Rev Esp Cardiol 2008; 61:299–310. Araya H, Lutz M. Functional and healthy foods. Rev Chil Nutr 2003; 30:8–14. Avello M, Suwalsky M. Free radicals, natural antioxidants and protection mechanisms. Atenea (Concepc) 2006; 494:161–172. de Angelis RC. New concepts in nutrition: considerations on the connection diet-health. Arq Gastroenterol 2001; 38:269–271.



36 37 38

39 40

41

42

Hannum SM. Potential impact of strawberries on human health: a review of the science. Crit Rev Food Sci Nutr 2004; 44:1–17. Silva J, Riguld J, Cheynier V, Cheminat A, Moutounet M. Procyanidin dimers and trimers from grape seeds. Phytochemistry 1991; 30:1259–1264. Laughton MJ, Evans PJ, Moroney MA, Hoult JR, Halliwell B. Inhibition of mammalian 5-lipoxygenase and cyclo-oxygenase by flavonoids and phenolic dietary additives. Relationship to antioxidant activity and to iron ion-reducing ability. Biochem Pharmacol 1991; 42:1673–1681. Folts JD. Potential health benefits from the flavonoids in grape products on vascular disease. Adv Exp Med Biol 2002; 505:95–111. Freedman JE, Parker C, Li L, Perlman JA, Frei B, Ivanov V, et al. Select flavonoids and whole juice from purple grapes inhibit platelet function and enhance nitric oxide release. Circulation 2001; 103:2792–2798. Augusti K. Therapeutic and medicinal values of onions and garlic. In Rabinowitch HD, Brewster JL editors. Onions and allied crops. Boca Raton FL: CRC Press; 1990. vol 3, pp. 94–108. Lawson LD, Ransom DK, Hughes BG. Inhibition of whole blood plateletaggregation by compounds in garlic clove extracts and commercial garlic products. Thromb Res 1992; 65:141–156.

43

44

45

46

47

48

Lancaster J, Collin H. Presence of alliinase in isolated vacuoles and of alkylcysteine sulphoxides in the cytoplasm of bulbs of onion (Allium cepa). Plant Sci Lett 1981; 22:169–176. Lazarus SA, Garg ML. Tomato extract inhibits human platelet aggregation in vitro without increasing basal cAMP levels. Int J Food Sci Nutr 2004; 55:249–256. O’Kennedy N, Crosbie L, Whelan S, Luther V, Horgan G, Broom JI, et al. Effects of tomato extract on platelet function: a double-blinded crossover study in healthy humans. Am J Clin Nutr 2006; 84:561–569. Goldman IL, Kopelberg M, Debaene JE, Schwartz BS. Antiplatelet activity in onion (Allium cepa) is sulfur dependent. Thromb Haemost 1996; 76:450–452. Yamada K, Naemura A, Sawashita N, Noguchi Y, Yamamoto J. An onion variety has natural antithrombotic effect as assessed by thrombosis/ thrombolysis models in rodents. Thromb Res 2004; 114:213–220. Yamamoto J, Naemura A, Ura M, Ijiri Y, Yamashita T, Kurioka A, Koyama A. Testing various fruits for antithrombotic effect: i. Mulberries. Platelets 2006; 17:555–564.
