Platelets, December 2005; 16(8): 469–473
1
2
3
1
ENSBANA, Dijon, France, and
2
Provexis Limited, Aberdeen, Scotland, UK,
3
Department of Nutrition, Faculty of Medicine, University of Oslo, Norway
(Received 12 November 2004; revised 22 February 2005; accepted 15 March 2005)
Abstract
Effect of aqueous extract of several herbs on human platelet aggregation in vitro was investigated. Out of 28 herbs/ nutriceuticals investigated, camomile, nettle alfalfa, garlic and onion exhibited most significant anti-platelet activity ( 45% inhibition). Aqueous extracts of alfalfa, fresh nettle, and camomile inhibited ADP induced-platelet aggregation by 73, 65 and 60%, respectively, compared with control ( P < 0.05). Camomile and alfalfa inhibited collagen-induced platelet aggregation by 84 and 65%, respectively, but nettle could not inhibit collagen-induced aggregation. In contrast, nettle was the most potent inhibitor (66%) of whole blood aggregation induced by collagen, followed by alfalfa (52%), and camomile
(30%) compared with control ( P < 0.05). None of these three herbs however could inhibit arachidonic acid or thrombin induced platelet aggregation. Camomile and alfalfa strongly inhibited thromboxane B
2 synthesis induced by ADP or collagen, but nettle had no effect. Alfalfa and nettle increased cGMP levels in platelets by 50 and 35%, respectively, compared with the control (1.85
0.23 nM) ( P < 0.005). All these data indicate that camomile, nettle and alfalfa have potent anti-platelet properties, and their inhibitory actions are mediated via different mechanisms.
Keywords: Platelets, herbs, Platelet aggregation, camomile, nettle, alfalfa, human, cGMP thromboxane B
2
, nutriceuticals, whole blood aggregation, herbal extract
Introduction
Activation of blood platelets plays a crucial role not only in haemostasis but also in pathological development of several arterial disorders, including strokes, and myocardial infarction [1–3]. There is increasing evidence that acute clinical manifestations of coronary atherosclerotic disease are caused by plaque disruption and subsequent platelet-thrombus formation [4–6]. It is documented that platelets also participate in tumor progression, allergic inflammation, and non-allergic responses by producing inflammatory substances like cytokines and eicosanoids. Thus platelet activity may play a major role in the development of several diseases as well as in the stability of atherosclerotic plaques [4]. In support of the pathophysiological role of platelets, platelet inhibitory drugs such as aspirin reduce the incidence of myocardial infarction, stroke and death from cardiovascular disease (CVD) in secondary prevention trials [7–10]. Therefore, today, the research is oriented toward the discovery of new compounds that can inhibit platelet aggregation reversibly and without any side effect.
The anti-thrombotic property of dietary nutrients and non-nutrients of plants and vegetables origin including tomatoes and kiwifruits have been described [2, 11, 12]. Herbs have been used as medical treatments since the beginning of civilization and some derivatives (e.g., aspirin, reserpine, and digitalis) have become mainstays of human pharmacotherapy [13, 14]. For cardiovascular diseases, herbal treatments have been used in patients with congestive heart failure, systolic hypertension, angina pectoris, atherosclerosis, cerebral insufficiency, venous insufficiency, and arrhythmia [14, 15]. With the high prevalence of herbal use in the world today, further research is necessary to elucidate the pharmacological activities of the many herbal remedies now being used to treat CVD. Very little information
Correspondence: Professor Asim K. Duttaroy, Faculty of Medicine, Department of Nutrition, University of Oslo P.O. Box 1046, Blindern, N-0316 Oslo,
Norway. Tel: 47 2285 1547. Fax: þ 47 2285 1341. E-mail: a.k.duttaroy@medisin.uio.no
ISSN 0953–7104 print/ISSN 1369–1635 online ß 2005 Taylor & Francis
DOI: 10.1080/09537100500129540

470 S. Pierre et al.
is available on the anti platelet property of herbs. The purpose of this study is to investigate systematically the anti-platelet activity of several herbs using in vitro aggregation methods (platelet-rich plasma and whole blood aggregation). The herbs were chosen in this study on the basis of their reported beneficial effects in CVD and other diseases [13, 14]. In this paper we report anti-aggregatory activity of aqueous extracts of several herbs.
Subjects
Healthy subjects (aged 20–35 years) were recruited after assessment of their medical and dietary history.
The study protocol was approved by the Joint Ethical
Committee of the Grampian Health Board, and the
University of Aberdeen, Scotland, UK. Exclusion criteria were the presence of overt vascular, haematological or respiratory disease, hypertension, infection, frequent consumption of drugs which affect platelet function (e.g., aspirin, paracetamol, ibuprofen, steroids), habitual consumption of omega-3 fatty acid supplements.
Materials and methods
Materials
ADP, collagen, and arachidonic acid were obtained from Helena Biosciences (USA). Bovine serum albumin fraction V fatty acid free was purchased from ICN Biomedical (UK). Human fibrinogen, and all other reagents used were purchased from Sigma
Chemical (UK). cGMP and thromboxane B
2 assay kits were purchased from Amersham Pharmacia
Biotech (UK).
Herbs
The following herbs were purchased from Grampian
Health Foods (Aberdeen, UK).
Lime flower, peppermint, nettle, lemon grass, camomile were the products of Heath & Heather (UK). Feverfew, hawthorn berry, passiflora, ginseng, liquorice root, mistletoe, yarrow, motherwort, prickly ash, lily of the valley, devils claw, horse chestnut, rosemary, marigold, ginger, and garlic were from Herbs of
Grace (Newmarket, UK). Dandelion and black tea were produced by Salus (Warrington, UK) and
Tetley (UK), respectively. In some cases, fresh plants
(alfalfa, origan, mint, thyme, sage, and nettle) were also used.
Platelet aggregation in platelet-rich plasma
Overnight fasted venous blood was collected from volunteers who had not taken any medication for at least 14 days before donation. Blood was collected through siliconized needles into plastic syringes: coagulation was prevented by mixing 9 vol of blood with 1 vol sodium citrate (final concentration,
13 mM). Platelet-rich plasma (PRP) was obtained by centrifugation of samples for 15 min at 200 g .
The supernatant platelet-rich plasma (2–3 10
8 cells/ml) was collected. Platelet counts were performed using a Baker cell Counter.
Platelet aggregation was monitored on a Packs-4 aggregometer (Helena Biosciences, USA) at a constant stirring speed of 1000 rpm at 37 C, as described before [16]. Since maximal amplitude of aggregation of PRP was obtained with either of
2 m g/ml collagen, 10 m M ADP, 500 m g/ml arachidonic acid, these concentrations of agonists were used and aggregation was followed over 10 min. PRP (450 m l) was incubated with 50 m l of pH 7.4 adjusted extracts for 15 min at 37 C prior to the addition of an aggregating agent. Controls were run in parallel replacing extract with 50 m l of PBS. Inhibition of platelet aggregation is expressed as the decrease in the area under the curve compared with the control.
Effect of herbal extracts on thrombin-induced platelet aggregation was investigated in washed platelet suspensions. Their preparation from PRP was carried out as described previously [16].
Preparation of herbal extracts
Herbs (2 g) were boiled in 20 ml distilled water for
3 min for isolation of water soluble fraction. The boiled suspension was then clarified by centrifugation at 2000 g for 10 min. Concentration of the aqueous extract was then determined by weighing of the 5-ml freeze dried sample. The extract was then stored at 20 C. The dried extracts were then dissolved in an appropriate volume of phosphatebuffered saline (PBS) to produce final concentration of 10 mg/ml. The pH of these herbal extracts was adjusted to 7.4 immediately prior to the aggregation studies. In some cases, fresh plants such fresh plants alfalfa, origan, mint, thyme, sage, and nettle were also used. These plants (2 g) were homogenized and then the aqueous extract was prepared as mentioned above. Extracts of onions, garlic, and ginger were prepared as above without boiling.
Whole blood aggregation
Blood taken from healthy subjects and mixed with sodium citrate, was incubated at 37 C in a water bath during the course of the experiment. A
Chronolog whole blood aggregometer was used for these experiments [17]. Typically, 500 m l of blood and 500 m l of PBS were placed in a plastic cuvette kept at 37 C. Herbal extract (final concentration,
1 mg/ml) was added and pre-incubated for 15 min before the addition of collagen (1 m g/ml). The average of several aggregations in the absence and presence of herbal extract was determined so that the

percentage inhibition described before [17].
Thromboxane B
2
(TxB
2
) assay
TxB
2
, the stable metabolite of TxA
2 was estimated using the Amersham TxB
2
EIA assay kit, as described before [16, 17]. Briefly, at the end of platelet aggregation in the absence and presence of extracts (1 mg/ml), 400 m l post-aggregation plasma were acidified to pH 3 and then applied to a conditioned Amprep C-2 column. The TxB
2 was eluted using methylformate and then assayed according to the manufacturer’s instructions, as described [16, 17].
could be calculated, as
Inhibitory effect of aqueous extracts of herbs
Table I. Effect of various herbs and plant extracts
(1 mg/ml) on human platelet aggregation in PRP induced by ADP (10 m M).
Herbs
Alfalfa
Black tea
Camomile
Dandelion leaves
Devils Claw
Feverfew
Garlic
Ginseng
Ginger
Hawthorn berry
Horse chestnut
Lemon grass
Licorice root
Lily of the valley
Lime Flower
Marigold
Mint
Mistletoe
Mother wort
Nettle (powder)
Nettle (fresh)
Onion
Origan
Passiflora
Peppermint
Prickly ash
Rosemary
Thyme
Yarrow
Each experiment was done in triplicate, on each of
10 volunteers.
% Inhibition
73.1
12.2
5.2
1.9
60.8
8.2
0
7.2
2.3
5.1
1.9
90.5
12.2
1.2
2.5
6.3
3.2
7.4
1.3
4.2
2.3
2.5
0.9
5.3
3.2
6.2
2.4
20 5
1.9
1.0
0
0
2.7
2.1
45.2
5.3
65.4
9.3
71.2
11.9
0
1.3
2.1
25.9
4.4
0
0
0
12.3
5.5
471 cGMP assay cGMP was measured both in plasma and platelets, since cGMP produced in the platelets can be released into the plasma. Aliquots (450 m l) of PRP were incubated with extracts (1 mg/ml) for 15 min at 37 C. A control was also incubated with 50 m l of saline. The samples were then centrifuged at
8000 g for 5 min. Supernatant plasma (150 m l) was kept in order to measure the plasma cGMP. The platelet pellet was washed in saline. The pellet was resuspended in 300 m l of ice-cold trichloroacetic acid and kept on ice for 1 h to lyse. After this time, samples were centrifuged at 8000 g for 5 min and the platelet pellet was discarded. PPP and platelet extracts were washed four times with five volumes of water saturated with diethyl ether and then dried, using a freeze dryer. After having resuspended samples in assay buffer, the EIA procedure was performed in the 96-coated wells plate using protocol supplied with the kit.
Statistical analysis
Results are presented as the mean SEM. Results were analysed by the Student’s t -test. Values for the area under the curve for platelet aggregation in the absence and in the presence of herbal extract were evaluated by a computer assisted program. Other statistical analyses were performed using ANOVA where appropriate, values were considered to be significantly different when P < 0.05.
Results
Effect of extracts on platelet aggregation in PRP
Since the amounts of the material extracted from these herbs were highly variable, the dried extracted material was dissolved in an appropriate volume of
PBS to produce final concentration of 10 mg/ml for each herb. Table I shows the inhibitory effect of aqueous extracts (final concentration, 1 mg/ml) of
28 herbs/nutriceuticals on human platelet aggregation in vitro. Results were expressed as the percent inhibition of aggregation in response to 10 m M ADP compared with control. Among these extracts used in this study, camomile, nettle (fresh and powdered), alfalfa, garlic, and onion were the most effective against ADP induced platelet aggregation, inhibiting platelet aggregation by more than 45% ( P < 0.05).
The maximum inhibitory effect (90%) was observed with garlic, followed by alfalfa, (73%), onion (71%), fresh nettle (65%), and camomile (60%), whereas peppermint, and lime flower inhibited platelet aggregation by 25 and 20%, respectively. Other herbs, such as feverfew, hawthorn, dandelion leaves, devils claw, ginseng, licorice root, lily of the valley, mint, marigold, mistletoe, motherwort, origan, passiflora, prickly ash, rosemary, thyme, and black tea had little or no effect.
Since several studies have been already published on the anti-platelet properties of garlic and onions [2,
18, 19], in this study we concentrated only on alfalfa, camomile, and nettle. Table II shows the dose dependant inhibition of ADP induced platelet aggregation by aqueous extracts of these three herbs. At
0.66 mg/ml, alfalfa, and fresh nettle inhibited 54–61% platelet aggregation, whereas camomile inhibited platelet aggregation by only 29%. Effects of these

472 S. Pierre et al.
Table II. Dose-dependent inhibition of ADP (10 m M)-induced platelet aggregation in PRP by three herbal extracts.
Herbs
Alfalfa
Concentration
(mg/ml)
% Inhibition of aggregation
0.30
0.66
1.0
23.2
5.5
61.4
8.5
73.1
10.4
Camomile
0.30
0.66
1.0
1.44
7.3
2.6
29.1
4.5
60.5
10.7
65.3
8.5
Nettle
0.30
0.66
1.0
1.44
39.1
54.5
65.1
67.4
8.7
6.2
11.2
9.8
Experiments were performed as described in the Methods section.
PRP was aggregated by agonists as described in the presence and absence of varying concentrations of herbal extracts. Each experiment was done in triplicate, on each of 10 volunteers.
Table III. Effects of herbal extracts on TxB
2 synthesis in platelets in response to ADP and collagen.
Agonist
ADP (10 m M)
ADP (10 m M)
ADP (10 m M)
ADP (10 m M)
Collagen (2 m g/ml)
Collagen (2 m g/ml)
Collagen (2 m g/ml)
Collagen (2 m g/ml)
Herbs
(1.0 mg/ml)
–
Alfalfa
Camomile
Nettle
–
Alfalfa
Camomile
Nettle
TxB
2 synthesis (ng/ml)
28.0
3.5
16.6
4.1*
19.8
2.8*
27.5
2.1
79.3
8.5
37.3
7.5*
41.0
6.2*
82.0
3.4
Experiments were performed as described in the Methods section.
TxB
2 was measured after PRP was aggregated by agonists as described. Each experiment was done in triplicate, on each of
10 volunteers. * P < 0.05 compared with the respective control.
Collagen-induced TxB
2 synthesis (79.3
8.5 ng/ml) was also inhibited by 53 and 48% by alflalfa
(37.3
7.5 ng/ml) and camomile (41.0
6.2 ng/ml), respectively. However, nettle did not inhibit thromboxane synthesis either induced by ADP or collagen.
three herbs on platelet aggregation response to two other widely used agonists (arachidonic acid and collagen) were also investigated. Both camomile and alfalafa inhibited collagen-induced platelet aggregation by 80 and 60%, respectively compared with control ( P < 0.005), whereas nettle had no inhibitory effect. Alfalfa, camomile, and nettle had very little or no effect on arachidonic acid-induced platelet aggregation (less than 8%). In addition, these herbs failed to inhibit thrombin-induced aggregation of washed platelets.
Effects of herbs on cGMP synthesis cGMP levels in platelets and plasma were determined following incubation of extracts (1 mg/ml) with PRP (0.5 ml). Alfalfa and nettle increased synthesis of cGMP by 50 9.4 and 32 5.3% significantly compared with the control (1.85
0.23 nM) ( P < 0.005, n ¼ 4), whereas camomile had no inhibitory effect.
Effects of herbal extracts on whole blood aggregation
Nettle, alfalfa, and camomile at 1 mg/ml inhibited collagen-induced whole blood aggregation by
60 12.4, 50 8.6, and 30 8.4%, respectively,
( P < 0.05, n ¼ 5) compared with a corresponding control.
Effects of herbs on TxB
2 synthesis in Platelets
To determine whether the inhibitory effect extracts on platelet aggregation was due to the decreased synthesis of TxA
2
, levels of TxB
2
, the stable breakdown product of TxA
2
, were measured in post aggregated plasma. Table III shows effects of these herbs on TxB
2 synthesis. TxB
2 was measured in the plasma after platelet aggregation induced by ADP or collagen in the presence and absence of these extracts. Both camomile and alfalfa inhibited TxB
2 synthesis in response to ADP and collagen, with a great inhibitory effect observed for collagen induced thromboxane synthesis (48–53%) compared with
ADP (30–41%), P < 0.05. ADP-induced TxB
2 synthesis was inhibited by 41 and 30% by alfalfa (16.6
4.1 ng/ml) and camomile (19.8
2.8 ng/ml), respectively, compared with control (28.0
3.5 ng/ml).
Discussion
Of the extracts investigated, garlic, onions, camomile, nettle and alfalfa proved to be the most potent inhibitors of platelet aggregation in vitro, after incubation with PRP for 15 min. Alfalfa, and camomile, and nettle inhibited ADP-induced platelet aggregation in PRP in a dose-dependent manner.
In contrast to alfalfa and camomile, nettle could not inhibit collagen-induced platelet aggregation, indicating that nettle may have different mechanism of action. Despite its inability to inhibit collageninduced platelet aggregation in PRP, nettle had a greater anti-aggregating activity in whole blood induced by collagen. These differences in activity and effectiveness of nettle in PRP and whole blood are not known at present but may be attributed to differences in their mode of action or metabolism in different blood cell types and platelets. This was clearly observed by the fact that thromboxane synthesis in platelets was inhibited by camomile and alfalfa but not by nettle. Nettle inhibited ADP induced platelet aggregation but not by collagen, indicating its mechanism of action is independent of thromboxane pathway. However, when using arachidonic acid as the agonist, very little or no inhibition

was observed by any of these three herbs, suggesting that platelet cyclooxygenase pre se is not inhibited by these extracts. Inhibition at upstream of cyclooxygenase at the release of arachidonic acid from membrane phospholipids is therefore more likely the possibility. However, further investigations are required for better understanding the mode of actions of these extracts.
Both nettle and alfalfa appear to inhibit platelets via the modulation of cyclic nucleotides. Both these herbs increased cGMP levels in platelets. cGMP is known to inhibit platelet aggregation [20]. Taken together, these data indicate that alfalfa, camomile, and nettle inhibited platelet aggregation via different mechanisms. It is important to note that the aggregation of platelets was performed in vitro, in a different environment than the blood stream. Studies on human volunteers would be required to ascertain their effects in vivo. Nevertheless, our study shows that consuming these herbs may be beneficial in
CVD. Alfalfa is commonly used as diuretic, is rich in minerals has anti-inflammatory properties, and may reduce cholesterol levels [13, 14]. Nettle also acts as diuretic and has anti-arthritis and anti-inflammatory effects, while camomile is used as a sedative, and has anti hypertensive and anti-bacterial properties
[13, 14]. In the current study, hitherto unknown we demonstrate that camomile, nettle and alfalfa have anti-platelet property.
In conclusion, camomile, nettle and alfalfa are equally effective at inhibiting platelet aggregation in vitro. Our data for the first time thus provide in vitro evidence that these three herbs may have potential for inhibiting platelet activation and platelet mediated events in CVD. Further investigation of the effects of individual herbs on in vivo platelet function, is required in order to elucidate their role in the relationship between herbs and the risk of CVD.
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