The antithrombotic effects of onion filtrates in rats and mice

doi:10.4236/health.2011.36055

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Vol.3, No.6, 319-325 (2011)

doi:10.4236/health.2011.36055

Health

The antithrombotic effects of onion filtrates in rat s and

mice

Kanae Hyodo1, Izumi Horii1, Masaru Nishino2, John C Giddings3, Junichiro Yamamoto1*

1Laboratory of Physiology, Faculty of Nutrition, Kobe Gakuin University and Cooperative Research Center of Life Sciences, Kobe

Gakuin University, Kobe, Japan; *Corresponding Author: [email protected]

2Hyogo Prefectural Technology Center for Agriculture, Forestry and Fisheries, Awaji Agricultural Technology Institute, Awaji, Japan;

3Wales College of Medicine, Cardiff University, Cardiff, UK.

Received 10 February 2011; revised 29 March 2011; accepted 1 April 2011.

ABSTRACT

The prevention of arterial thrombotic disease

has a high priority in developed countries. An

inappropriate diet is known to enhance the risk

for acute thrombotic events, and a regular diet

with proven antithrombotic effects might be a

beneficial way to prevent disease. The present

study was undertaken as part of a series of in-

vestigations to examine various vegetables and

fruits for antithrombotic activity, and to add to

the previously reported data on different onion

varieties produced in the northern part of Japan

(Hokkaido). For this purpose, a sophisticated

method to assess shear-induced platelet reac-

tivity/thrombolysis in vitro (The Global Throm-

bosis Test, GTT) was used to screen 5 different

varieties o f on ion p roduced in the middle part of

Japan (Awaji Island). The different onion varie-

ties demonstrated a variable effect on thrombo-

sis, and one particular variety, designated

ONA-03, appeared to exert an antithrombotic

effect. Another variety, ONA-01, appeared to

have prothrombotic activity by inhibiting spon-

taneous thrombolytic activity. The especially

effective variety was further investigated using

an in vivo, laser-induced thrombosis model in

mice. The heat stable antithrombotic activity in

vitro and in vivo w as demonstrated to be due to

antiplatelet activity. The present findings added

to the list of antithrombotic fruits and vegeta-

bles.

Keywords: Cardiovascular Dise ase; Stroke;

Atherothrombosis; Onion; Quercetin

1. INTRODUCTION

The prevention of “lifestyle-related atherothrombotic

diseases” such as myocardial infarction and stroke has

become an important and urgent social task in many

developed countries. Studies have provided clear evi-

dence that an inappropriate diet, such as the Western-

style high fat diet, plays a causative role in the patho-

genesis and clinical outcome of thrombotic diseases [1].

The so-called French Paradox and red wine hypothe-

sis [2,3] has prompted many laboratory studies on anti-

thrombotic fruits and vegetables, and epidemiological

studies have provided evidence that intake of fresh fruits

and vegetables could help to prevent cardiovascular dis-

ease and stroke [4-7].

Platelets play a pivotal role in arterial thrombotic dis-

eases. Platelet-function in vitro is commonly assessed

using platelet aggregometry, which measures platelet

aggregation induced by various chemical agonists. In

clinical practice, however, beneficial tests for the diag-

nosis and treatment of patients with thromboembolic

disorders remain to be fully defined. Tests using native,

non-anticoagulated blood in the presence of physiologi-

cal shear force are likely to be much more relevant to the

in vivo environment than those using anticoagulated

blood and chemical platelet agonists [8-9]. We have

demonstrated that shear-induced platelet reactivity tests

in vitro, using non-anticoagulated blood, significantly

correlate with the in vivo helium-neon (He-Ne) laser-

induced thrombosis model in animals [10].

We have established in our laboratory a shear-induced

platelet-rich thrombosis/coagulation test (Haemosta-

tometry), a novel, commercially available shear-induced

platelet-rich thrombosis/thrombolysis method (The Global

Thrombosis Test, GTT) and a He-Ne laser-induced

thrombosis model to investigate specific fruits and

vegetables for antithrombotic activity. We have shown

that different varieties of tomato, onion and strawberry

can be classified according to shear-induced platelet-rich

thrombotic activity (Haemostatometry), and that varie-

ties of mulberry and carrot can be classified on this basis

K. Hyodo et al. / Health 3 (2011) 319-325

320

together with spontaneous thrombolytic activity (GTT)

[11-15]. We have also demonstrated using Haemosta-

tometry, that one onion variety cultivated in the northern

part of Japan (Hokkaido) was notably antithrombotic [12]

but the availability of this product remains limited. In the

present study we have expanded our studies on the anti-

thrombotic effects of onions using potentially more

readily available onion varieties produced in the central

area of Japan (Awaji).

2. MATERIALS AND METHODS

2.1. Animals

Male Wistar ST rats, at least 13 weeks old, and male

C57BL/6 mice, 10 weeks old were purchased one week

before use (Japan SLC Co. Ltd., Hamamatsu, Japan).

Rats were fed a standard solid chow (MF, Oriental Yeast

Co. Ltd., Osaka, Japan) and mice were similarly fed with

standard solid chow (MF, Japan Clea CO. Ltd., Tokyo,

Japan). Animals were allowed tap water ad libitum, and

were maintained in compliance with the “Guiding Prin-

ciples for the Care and Use of Animals in the field of

Physiological Sciences,” published by Physiological

Society of Japan. The protocol was approved by the

Animal Experiment Committee of Kobe Gakuin Univer-

sity. Animals were sacrificed using Somnopentyl fol-

lowing the experimental procedures.

2.2. Onions

Five varieties of onion, designated ONA-01, ONA-02,

ONA-03, ONA-04 and ONA-05, were sown and har-

vested in August on the same test field of the Hyogo

Prefectural Technology Center for Agriculture, Forestry

and Fisheries, Awaji Agricultural Technology Institute,

Awaji, Japan.

2.3. Preparation of Onion Filtrate

Six brown skin peeled bulbs per each variety were

graded to a standard size using a plastic grader at room

temperature to avoid inter-individual variation. The juice

was centrifuged (3000 rpm, 15min, 4℃) and the super-

natant was filtered (FP30/5.0 CN-S, 5.0 μm, Whatman

PLC, Kent, UK). Clear filtrates were stored at –80℃

before use.

2.4. In Vitro Assessment of Shear-Induced

Platelet Reactivity and Spontaneous

Thrombolytic Activity using the

Global Thrombosis Test (GTT)

The technique has been described in detail elsewhere

[15-16]. The instrument was purchased from Montrose

Diagnostics Ltd., London, UK (www.globalthrombosis.com).

Figures 1 and 2 illustrate the principles of the technique.

A flat segment created along the inner wall of a conical

Figure 1. Schematic illustration of the GTT apparatus.

Figure 2. Schematic diagram showing the principle of the

GTT.

K. Hyodo et al. / Health 3 (2011) 319-325

321321

plastic tube forms the basis of the technique. When per-

fectly round steel ball-bearings are placed into the coni-

cal tube, the flat segment prevents the spheres from oc-

cluding the lumen. Blood is added, flows through the

narrow gaps and exits in droplets into an adjacent col-

lecting tube. The latter is trans-illuminated by a light

emitter, and a sensor opposite the light source generates

a signal whenever a drop of blood interrupts the light

path. In essence, the instrument detects the time interval

(d, sec) between consecutive blood drops. At the start,

blood flow is rapid and hence (d) is small. Subsequently,

the flow rate gradually decreases and hence (d) increases.

When the actual (d) exceeds 15 seconds (occlusion-d),

the instrument displays “Occlusion Time (OT)”, which is

the time elapsed from the detection of the first drop of

blood until (occlusion-d). Later, flow is completely ar-

rested. Eventually, due to thrombolysis, flow is restored

as indicated by the further detection of blood droplets.

There is also an arbitrarily pre-set (d) (200 seconds) for

lysis measurement (lysis-d). When (d) between the last

drop before and the first drop after occlusion exceeds

this (lysis-d), the instrument displays “Lysis Time (LT)”.

Hence, lysis time is calculated as follows: LT = (time of

first drop with d > (lysis-d)) – (time of last drop with d <

(lysis-d)). Blood flows by gravity at 37oC through the

narrow gaps formed between the larger ball bearing and

the inner wall of the tube, and the resulting high shear

stress (175 dyne/cm2) activates platelets. These activated

platelets remain single, since the very short transit time

and high shear prevent aggregation. In contrast, in the

space distal to the large ball bearing, i.e. between the

two ball bearings, the low shear and turbulent flow favor

large platelet aggregate formation. Furthermore, in this

space between the ball bearings, activated platelets gen-

erate thrombin and initiate blood coagulation. Flow then

carries these fibrin-stabilised platelet aggregates into the

lower gaps where they are captured, resulting in occlu-

sion and arrest of flow. Increased or decreased OT indi-

cates inhibition or enhancement of platelet reactivity,

respectively. Increased or decreased LT indicates inhibi-

tion or enhancement of spontaneous thrombolysis, re-

spectively. Measurements were made six times (n = 6) in

each sample.

Animals were fasted overnight but allowed water ad

libitum. Blood was obtained from the abdominal aorta

30 minutes after anesthesia with sodium pentobarbital

（Somnopentyl, 64.8 mg/ml, Kyoetsu Seiyaku Co. Ltd.,

Tokyo, Japan）diluted 5 times with saline (65 mg/kg,

intramuscularly). Non-anticoagulated blood was mixed

with saline (1:1). 3.6 ml of the diluted blood and 0.4 ml

of onion filtrate or saline (control) (blood: filtrate = 9:1)

were mixed in a syringe by inversion and the mixture

was applied to GTT tube.

2.5. In Vivo Assessment of the

Antithrombotic Effects Using

the Laser-Induced Thrombosis

Test in Mice Carotid Arteries

The He-Ne laser-induced platelet-rich thrombosis

method has been previously described in detail [17,18].

Mice were anaesthetised with Somnopentyl (65 mg/kg,

intramuscularly). A polyethylene tube (PE10, Becton

Dickinson and Company, New Jersey, USA) was placed

into the left femoral artery to inject dye and the carotid

artery (450 - 500 μm in diameter) was exposed by inci-

sion. The mouse was placed on a specially adapted mi-

croscope stage (Olympus Model BH-2, Olympus Co.

Ltd., Tokyo, Japan) and Evans blue dye (30 mg/kg,

Merck, Darnstadt, Germany) was injected intra-arterially.

The centre of the exposed carotid artery was irradiated

with a laser beam (Model Neo-50MS, 25 mW under an

objective lens, Neoark Co. Ltd., Osaka, Japan). Throm-

bus formation at the site of irradiation was monitored

under epi-illumination and simultaneously recorded on

videotape using a CCD camera (Model TMC-7, Take-

naka System Co. Ltd., Kyoto, Japan).

2.6. Oral Ad ministration of Onion Filtrates

to Mice

The vegetable filtrate or saline only (control) was ad-

ministered through a gastric tube at 15.4 ml/kg (un-

heated filtrate) and 15.4 or 30.8 ml/kg (heated filtrate).

Half volume of filtrate or saline (7.7 ml/kg (unheated

filtrate) and 7.7 or 15.4 ml/kg (heated filtrate) was re-

peatedly given at 30 minute interval as previously de-

scribed [11]. The mouse was then anaesthetised and the

thrombosis experiments commenced 90 min after the

second oral administration. Antithrombotic or prothrom-

botic effects were assessed by estimating total thrombus

size.

2.7. Calculation of Thrombus Size

Details of this technique have been described else-

where [18]. Images of thrombus formation were com-

puter-analysed at intervals of ten seconds. The area of

thrombus was delineated and the mass of thrombus cal-

culated by multiplying gray scale and area using Image J

software (Image Processing and Analysis Java version

1.30, National Institutes of Health, Maryland, USA).

Thrombotic status was expressed as the total sum of

thrombus mass after the first 10 minutes of irradiation.

2.8. Statistical Analysis

Data from GTT were analysed by repeated ANOVA

(General Linear Model), followed by the multiple com-

parison test (Dunnett), data from the laser-induced

K. Hyodo et al. / Health 3 (2011) 319-325

322

thrombosis experiments by the unpaired t-test. Values

were expressed as means ± SEM. P < 0.05 was consid-

ered as the limit of significance. Analyses were per-

formed using Statistical Package Unistat 5.6 Light

(London, UK).

3. RESULTS

3.1. Effects of Raw Filtrates from Onion

Varieties on Shear-Induced Platelet

Thrombosis and Spontaneous

Thrombolysis in Vitro

The results are shown in Table 1. The filtrate from

ONA-01 did not affect occlusion time (OT) but signifi-

cantly inhibited spontaneous thrombolytic activity (LT),

indicating that this variety had prothrombotic activity.

ONA-02, ONA-04 and ONA-05 did not affect signifi-

cantly either OT or LT. In contrast, the undiluted filtrate

from ONA-03 significantly prolonged OT but did not

affect LT, indicating that this particular variety had anti-

thrombotic activity both in vitro and in vivo.

3.2. Heat Stability of Antithrombotic Effect

of ONA-03

The antithrombotic effects of ONA-03 in vivo, were

re-examined after the raw filtrate was heated in boiling

water for 10 minutes. The filtrate was cooled to room

temperature and the OT and LT were measured. The

results are shown in Table 2. Similar results to those

using unheated filtrate were obtained. The heated and

undiluted (x1) filtrate significantly prolonged OT but

had no significant effect on LT, again suggesting anti-

thrombotic activity in vivo.

Table 1. Effects of raw filtrates from five onion varieties on shear-induced platelet thrombosis (occlusion time, OT)

and spontaneous thrombolysis (lysis time, LT).

Variety Dilution Occlusion time Lysis time

ONA-01 control 307.9 ± 9.9 989.7 ± 70.9

× 1 265.0 ± 6.3 1520.3 ± 110.9**

× 3 327.7 ± 12.1 983.3 ± 103.4

× 10 311.5 ± 20.7 1024.3 ± 46.5

ONA-02 control 295.6 ± 4.7 855.3 ± 39.6

× 1 260.4 ± 16.2 1022.5 ± 90.2

× 3 289.7 ± 14.2 972.4 ± 76.9

× 10 289.1 ± 16.9 841.7 ± 57.9

ONA-03 control 329.1 ± 12.9 944.8 ± 24.8

× 1 457.4 ± 36.1** 977.3 ± 102.7

× 3 352.0 ± 22.5 935.3 ± 99.7

× 10 344.7 ± 16.5 900.6 ± 55.4

ONA-04 control 287.0 ± 20.7 985.0 ± 48.5

× 1 272.9 ± 25.1 1042.8 ± 80.7

× 3 294.0 ± 25.5 895.5 ± 52.2

× 10 299.5 ± 18.1 913.3 ± 78.5

ONA-05 control 304.0 ± 18.2 976.0 ± 32.4

× 1 367.1 ± 37.6 1032.4 ± 102.3

× 3 341.9 ± 19.4 891.8 ± 26.6

× 10 311.7 ± 14.9 889.3 ± 56.7

**: P < 0.01

Table 2. Effects of heat treatment on the inhibitory activity of ONA-03 in shear-induced platelet thrombosis.

Variety Dilution factor of filtrate Occlusion time Lysis time

ONA-03 saline (control) 302.7 ± 12.5 1066.7 ± 66.7

× 1 446.0 ± 19.4** 1249.6 ± 41.3

× 3 345.6 ± 9.3 981.5 ± 75.0

× 10 331.5 ± 12.7 959.2 ± 69.7

**: P < 0.01

K. Hyodo et al. / Health 3 (2011) 319-325

323323

3.3. Antithrombotic Activity of Orally

Administered ONA-03

Unheated or heated onion filtrate was orally adminis-

tered to mice and the antithrombotic effect was assessed

using the He-Ne laser-induced thrombosis test on ex-

posed carotid arteries. Results are shown in Figure 3.

Unheated and undiluted (x1) filtrate from ONA-03 sig-

nificantly inhibited He-Ne laser-induced thrombosis (A).

The same volume of heated filtrate did not inhibit

thrombosis (B) but administration of twice the volume

significantly inhibited thrombus formation (C). This

inconsistency between the in vitro GTT test and the in

vivo laser-induced thrombosis test might have been be

due to different sensitivity of the methods or the effects

of intestinal absorption. Nevertheless, the results dem-

onstrated that the filtrate from ONA-03 was relatively

heat stable.

4. DISCUSSION

Arterial platelet-rich thrombotic diseases are a social

problem in many countries, and reliable tests to assess

thrombotic tendency could be critically important in

attempts to prevent these diseases. Various methods have

been developed, including agonist-induced platelet ag-

gregation tests using anticoagulated whole blood or

platelet rich plasma. Biomarkers of coagulation and fi-

brinolysis have also been devised [19-24]. It is widely

appreciated, however, that thrombus formation in vivo is

governed by interactions between blood flow, blood

components and the blood vessel wall as proposed by

Virchow, and the pathophysiological relevance of these

tests to thrombotic status in vivo is not clear. Alterna-

tively, innovative tests, in which non-anticoagulated

blood is used and platelets are activated by shear force

under flow, have been proposed [8,9,15]. Clinical and

experimental studies have suggested that these tests may

be more relevant to thrombotic status in vivo [8-10,16].

We have utilized in vivo tests of this nature to charac-

terize various fruits and vegetables on the basis of anti-

thrombotic activity. In particular, Haemostatometry, the

GTT and the He-Ne laser-induced thrombosis test [17,

18] have enabled classification into different subgroups

[11-14].

In an earlier study we used Haemostatometry and the

He-Ne laser-induced thrombosis model to identify a

specific antithrombotic onion variety produced in the

northern part of Japan [12]. Distribution of this variety

was limited, however, and further studies were devised

to examine onion varieties from central Japan (Awaji

Island). These varieties were potentially more available

than others, and were classified using the GTT in place

of Haemostatometry.

The principle aim of our current series of investiga-

tions is to broadly identify fruits and vegetables with

antithrombotic activity. In addition, thrombus evolution

is largely determined by a balance between pro-aggregatory

and procoagulant mechanisms on the one hand, and fi-

brinolytic processes on the other [25], and the relation-

ship between the concentration of specific antithrom-

botic substances and overall antithrombotic activity of

fruits and vegetables remains to be defined. In this re-

spect, quercetin is reported to be important in inhibiting

collagen-stimulated platelet aggregation [26-28], but a

high concentration of quercetin in onions does not nec-

essarily mean that onions are antithrombotic, and we

Figure 3. Antithrombotic activity of unheated and heated onion filtrates from ONA-03 n: number

of measurements; *: P < 0.05, **: P < 0.01.

K. Hyodo et al. / Health 3 (2011) 319-325

324

have previously demonstrated that there was no signifi-

cant correlation between quercetin concentration of on-

ions and overall antiplatelet activity using shear-induced

platelet function tests in vitro (Haemostatometry; 12).

Furthermore, the fibrinolytic properties of onions have

been the focus of some antithrombotic studies [29] but

significant fibrinolytic activity was not evident in any of

the five varieties of onions used in the present investiga-

tions. Moreover, for clinical purposes it is important to

note that the amounts of onion extracts given to animals

in the present studies would be approximately equivalent

to 40 kg onion given to a person with a body weight 70 kg.

This is clearly impracticable, and further studies are re-

quired to identify and isolate antithrombotic substances

that could be scientifically important and lead to signifi-

cant clinical benefit. Detailed analysis of this nature is

outside the scope of the present studies, but nevertheless,

our characterization of particular prothrombotic and an-

tithrombotic fruits and vegetables could lead to the de-

velopment of novel therapeutic products to help to pre-

vent arterial thrombotic diseases.

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