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Phytomedicine 11 (2004) 596601


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Novel antiplatelet and antithrombotic activities of essential oil from Lavandula hybrida Reverchon grosso
V. Ballabenia, M. Tognolinia, M. Chiavarinia, M. Impicciatorea, R. Brunib, A. Bianchib, E. Barocellia,*
" Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche Applicate, Universita di Parma, Parco Area delle Scienze 27/A, 43100 Parma, Italy b " Dipartimento di Biologia Evolutiva e Funzionale, Universita di Parma, Parco Area delle Scienze 27/A, 43100 Parma, Italy
a

Received 10 November 2003; accepted 8 January 2004

Abstract
Lavender extracts are known to produce several mild effects at central and peripheral level. However, no studies are so far available about the potential effects of lavender essential oil on the hemostatic system. In this work, we demonstrated antiplatelet properties of lavender oil towards platelet aggregation induced by arachidonic acid, U46619, collagen and ADP (IC50=51, 84, 191 and 640 mg/ml, respectively) on guinea-pig platelet rich plasma (PRP) and its ability to destabilize clot retraction (IC50=149 mg/ml) induced by thrombin on rat PRP. Furthermore, antithrombotic properties were studied in an in vivo model of pulmonary thromboembolism induced by intravenous injection of a collagenepinephrine mixture in mice subacutely treated with lavender oil. In this model, lavender oil (100 mg/kg/day os for 5 days) signicantly reduced thrombotic events without inducing prohemorrhagic complications at variance with acetylsalicylic acid used as reference drug. Finally, main components of the oil were studied in vitro in order to assess their antiplatelet effects, but none of them possessed an activity comparable to the oil itself. These results provide the rst experimental evidence of lavender oils antiplatelet/antithrombotic properties which could be due to a synergistic effect of its components. r 2004 Elsevier GmbH. All rights reserved.
Keywords: Lavandula hybrida; Lamiaceae; Essential oil; Platelet aggregation; Clot retraction; Bleeding; Pulmonary thromboembolism

Introduction
Lavender is one of the most used aromatic plants in the world and both extracts and essential oils from various Lavandula spp. are traditionally used to treat diseases such as epilepsy and migraine and to reduce spasms in colic pain (Cavanagh and Wilkinson, 2002). Extensive studies have been carried out on chemical
*Corresponding author. Tel.: +39-0521-905090; fax: +39-0521905091. E-mail address: barocell@unipr.it (E. Barocelli). 0944-7113/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2004.01.002

composition of Lavandula spp. essential oil, being fragrance a prominent factor in its commercial value denition. Recently, laboratory tests demonstrated its anticonvulsant properties on pentylenetetrazole-induced convulsions in mice and showed a moderate sedative effect (Gilani et al., 2000). Preliminary clinical trials with small samples of patients indicate lavender as a possible adjuvant treatment of pain, anxiety and mild depression (Louis and Kowalski, 2002; Akhondzadeh et al., 2003) and as a suitable approach to improve psychological well-being, through a positive mood change in the angerfrustration test (Morris, 2003). Components of

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Lavandula angustifolia essential oil, ()-linalool and linalyl acetate, showed antiinammatory properties in rats (Peana et al., 2002) and ()-linalool alone induced a signicant antinociceptive effect, reducing inammatory and thermal pain in mice (Peana et al., 2003). In vitro studies demonstrated that lavender oil might produce spasmolytic effect most likely increasing cAMP intracellular concentration (Lis-Balchin and Hart, 1999). Since some antiaggregants are known to enhance intraplatelet cAMP concentration, in this work we investigate the antiplatelet/antithrombotic properties of lavender oil. It is worth noting that the prevention of thrombogenesis has become one of the most important targets in the prophylaxis and therapy of cardiocirculatory disorders with thromboembolic complications (Fitzgerald, 2001). The different antiplatelet agents currently used for this purpose proved to be effective in the prevention of thromboembolic disease, but they are not free from side effects such as gastric erosion (aspirin) and agranulocytosis (ticlopidine) or show a poor separation between therapeutic efcacy and hemorrhagic complications (glycoprotein IIb/IIIa receptor inhibitors) (Van De Graaff and Steinhubl, 2001). This evidence supports the continuous efforts in the development of new antiplatelet/antithrombotic agents of synthetic or natural origin. In this work, we evaluated the properties of lavender oil in vitro towards platelet aggregation induced by various agents (ADP, arachidonic acid, collagen and the stable thromboxane receptor agonist U46619) and its effect on clot retraction induced by thrombin. Antithrombotic effects of a subacute treatment with lavender oil were studied in vivo in a model of pulmonary thromboembolism induced in mice and acetylsalicylic acid (ASA) was used as reference compound. Finally, the essential oil was analyzed and its main components were studied in vitro in order to assess their role in lavender oil effects.

Chemical analysis
Essential oil samples were analyzed using a Fisons (Rodano, Milano, Italy) 9130-9000 gas-chromatograph equipped with an FID detector and an MEGA SE52 (Mega, Legnano, Italy) column (i.d. = 0.32 mm, length 30 m, lm thickness= 0.15 mm). Operating conditions: injector temperature 280 C; FID temperature 280 C, Carrier (Helium) 2 ml/min, split ratio 1:40. Oven temperature was initially 45 C, then raised to 100 C (1 C/min ), then raised to 250 C (5 C/min ) and nally held at 250 C for 10 min. One microliter of oil dissolved in CH2Cl2 was injected. GSMS analysis was performed on a Hewlett Packard HP5890 series II plus GC equipped with a HPMS 5989b mass spectrometer using EI. The MS conditions were as follows: ionization voltage, 70 eV; emission current, 40 mA; scan rate, 1 scan/s; mass range, 35300 Da; ion source temperature, 200 C. Identication of compounds was based on relative retention times (Adams, 2001), matching with NBS MS library and comparison of fragmentation patterns with literature data [11]. The oil composition is reported in Table 1.

Table 1. Chemical composition and retention indices of the constituents of the essential oil of Lavandula hybrida Reverchon Grossoa Compounds a-Pinene Camphene b-Pinene b-Myrcene Hexyl acetate 1,8-Cineole cis-Ocimene trans-Ocimene Linalool Octen-3-ol Acetate Camphor Hexyl isobutanoate 4-Terpineol a-Terpineol Hexyl butanoate Linalyl acetate Lavandulyl acetate Neryl acetate Geranyl acetate b-Caryophyllene Farnesene Caryophyllene Oxide a-Bisabolol Total identied
a b

KIb 941 955 974 990 1010 1034 1037 1049 1099 1122 1048 1153 1176 1191 1194 1260 1291 1363 1384 1421 1455 1585 1690

% 0.1 0.1 0.2 1.3 0.1 5.8 0.2 0.5 33.4 0.3 7.6 0.2 2.1 1.0 0.4 36.2 3.0 0.7 1.4 0.6 0.6 0.6 0.2 96.6

Materials and methods


Plant material
Lavandula hybrida Reverchon cv. Grosso plants were cultivated by La Gachore, Puimoisson, Plateau de Valensole, Alpes de Haute Provence, France. A voucher specimen of the plant was deposited (Voucher no. OPR1) at the Herbarium of ofcinal plants, University of Parma Botanical Garden, Parma, Italy. The essential oil was obtained from stream distillation of fresh owers few minutes after their machine harvesting, using the same harvesting container as an industrial large-scale steam distiller (yield 1.55%). Essential oil characteristics were in accordance with literature data.

Compounds listed in order of elution from an SE52 column. Kovats indices were calculated against n-alkanes on a SE52 column.

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Materials and animals


Arachidonic acid and collagen for in vitro tests were purchased from Menarini (Firenze, Italy). ADP, thrombin, acetylsalicylic acid (ASA), calf collagen type III for in vivo experiments, epinephrine bitartrate, sodium citrate, dimethyl sulfoxide (DMSO), Triton, CH2Cl2 and ferric chloride were obtained from Sigma (St Louis, USA). U46619 was from Caymanchem (MI, USA). ASA as lysine acetylsalicylate use for in vivo experiments was from Carlo Erba (Milano, Italy). The experiments were performed applying experimental procedures supervised and approved by the " Ministero della Sanita (D.Lgs 116/92). Male guinea pigs (400500 g) and Wistar rats (150200 g) were used for in vitro assays; male Swiss mice (2025 g from Charles River) were used for antithrombotic tests.

assay was performed according to Davidson and Henry (1974). Briey, 450 ml aliquots of the above platelet suspension were added to siliconized glass tubes and incubated 10 min with 5 ml solvent (DMSO) or compound under study at 37 C. Fibrin clot retraction was induced by addition of 50 ml thrombin 20 U/ml. Pictures were taken every 15 min for 1 h using a digital camera. Quantication of clot retraction was performed measuring clot area by means of the NIH Image 1.67e software. Data were expressed as percentage of retraction=(area t0area t)/area t0 100. Where t0 was the area of the clot 2 min after thrombin addition and t was the area at the test time (15, 30, 45 and 60 min).

In vivo assays
Animal treatment Male Swiss mice were orally treated for 5 days, once a day, with 100 mg/kg lavender oil or ASA. Control animals received only vehicle (methocel 0.5%). Animals body weight, behavior and physical appearance were recorded daily.

Blood manipulation
Blood from male guinea pig or Wistar rat was obtained by cardiac puncture after CO2 euthanasia, collected in plastic tubes and anticoagulated with sodium citrate 3.8% 1 part citrate:9 part blood. After centrifugation for 15 min at 180 g to obtain platelet rich plasma (PRP), the remaining blood was centrifuged again 10 min at 2000 g to obtain platelet poor plasma (PPP).

In vitro assays
Platelet aggregation studies PRP from guinea pig was used to perform aggregation in the Aggrecorder PA 3220 (Menarini, Firenze) following Borns turbidimetric method (Born, 1962). Aggregation was recorded as the percent change in light transmission: the baseline was set using PRP and maximal transmission using PPP. PRP was preincubated at 37 C for 5 min with solvent (DMSO, nal concentration 0.5%) or the compound under study before the addition of the platelet aggregatory agent. Maximal aggregation was induced stimulating platelets with 3 mM ADP, 50 mM Arachidonic acid, 5 mg/ml collagen or 1 mM U46619. Tests were performed within 3 h to avoid platelet inactivation. The effects of test compounds and aspirin were expressed as percent inhibition compared with control samples. DMSO at 0.5% did not interfere with platelet aggregation.

Acute pulmonary thromboembolism A modication of Di Minnos method was used (Di Minno, 1983). Pulmonary acute thromboembolism was induced in mice by rapid intravenous injection in the tail vein of a mixture of 12 mg/kg collagen and 1 mg/kg epinephrine in order to induce about 80% of paralysis in the control group. The occurrence of paralyzed animals was recorded for 5 min after thrombotic mixture injection. The loss of the righting reex was considered as indication of paralysis.

Bleeding The tail transection bleeding was determined modifying Dejanas method (Dejana et al., 1979). Briey, tails of lightly anesthetized mice were transected at 2 mm from the tip and immersed in 1 ml of 37 C saline for 2 min. Red blood cells were lised by adding 20 ml of triton 5% and absorbance of the solution was read at 560 nm (LKB, Ultraspec 4050). The amount of haemorrhage was estimated by linear regression analysis of a standard curve constructed from known volumes of mouse blood.

Clot retraction assay


PRP from rats was diluted with tyrode buffer (137 mM NaCl, 20 mM Hepes, 5.6 mM glucose, 1 mM MgCl2, 2.7 mM KCl, 3.3 mM NaH2PO4, pH=7.4) to obtain a nal concentration of 200.000 plts/ml and the Statistical analysis All the results are expressed as means7standard error of the mean (SEM). Differences between groups were analyzed by w2-test for pulmonary thromboembolism and by Mann-Whitney test for bleeding assay.

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Results
In vitro assays
Lavender essential oil showed antiplatelet effects on the aggregation induced by all the platelet stimulants used on guinea pig PRP. Lavender oil completely blocked the aggregation induced by arachidonic acid, U46619 and collagen and almost completely inhibited the aggregation induced by ADP (74% inhibition) (Fig. 1). Furthermore, the oil displayed its highest potency towards arachidonic acid induced aggregation showing a 2-, 4- and 13-fold lower potency against U46619, collagen and ADP respectively (Table 2). In order to better evaluate the active principles of the oil, the antiplatelet effect of main components (linalool, linalyl acetate, camphor and 1,8 cineol) was studied. As shown in Table 2, a complete inactivity up to 1 mg/ml was detected for the most abundant component linalyl acetate. Instead, linalool prevented collagen and ADP-

induced aggregation whereas camphor exhibited antiplatelet effect against arachidonic acid and U46619. The active principle 1,8 cineol completely inhibited aggregation stimulated by U46619 with low potency. None of these effects was comparable to those produced by the phytocomplex. ASA, used as reference compound for these studies, showed, as expected, a great efcacy towards arachidonic acid and collagen-induced aggregation and had little or no effect on ADP or U46619 induced aggregation up to 1 mM concentration. Clot retraction is a physiological event which requires the presence of activated brinogen receptor aIIbb3 on the platelet surface, bound to extracellular brinogen or brin and anchored to intracellular actin and myosin. Contraction of actin/myosin laments within the platelet places tension on the brin clot, causing it to contract (Prevost et al., 2003). Lavender oil, as well as linalyl acetate, completely inhibited clot retraction induced by thrombin in rat PRP at a concentration as low as 270 mg/ml (Fig. 2). Linalool and 1,8 cineol partially inhibited clot retraction beginning from 900 mg/ml. Camphor, up to 270 mg/ml, was inactive and it was not tested at higher concentration because of its low solubility. ASA was ineffective up to 2700 mg/ml (Fig. 2).

In vivo assays
Initial body weight ranged from 21 to 27 g for the mice assigned to vehicle or treated group. All the animals survived. Growth as well as behavior and physical appearance of treated animals did not differ from controls throughout the study. Animals subacutely treated for 5 days with saline, 100 mg/kg/day os of ASA or 100 mg/kg/day os of lavender oil, underwent intravenous shot injection of a collagenepinephrine mixture. 13 out of 14 (93%) saline

Fig. 1. Antiplatelet effect of lavender oil towards aggregation induced by 3 mM ADP, 1 mM U46619, 1 mg/ml collagen or 50 mM arachidonic acid (AA) in guinea pig PRP. Each point represents the mean of six individual experiments, standard errors of the mean are indicated as vertical bars.

Table 2. In vitro antiplatelet potency of lavender oil and its main components on guinea-pig PRP against Arachidonic acid (AA), U46619, collagen and ADP AA50 mM Compounds Lavender oil Linalool Linalyl acetate 1,8 cineol Camphor ASA IC50 51 (4953)
a c c

U46619 1 mM IC50 84 (53133)


a a

Collagen 1 mg/ml IC50 191 (163226) 380 (225640)


a a b

ADP 3 mM IC50 640 (495826) 790 (5301153)


a c b c

134 (79227) 10 (714)

677 (593774) 96 (80114)


a

11 (816)

The IC50 values are expressed as mg/ml and condence limits are indicated in brackets. a Inactive up to 1000 mg/ml. b Inactive up to 250 mg/ml and insoluble at higher concentrations. c IC50 not calculable, because maximal inhibition is lower than 50%.

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Discussion
In this work, we provide the rst experimental evidence of lavender oil antiplatelet/antithrombotic properties. The essential oil is able to inhibit platelet aggregation induced by all the agonists used in the study, showing a higher efcacy than the reference drug ASA, which was poorly or no effective toward ADPand U46619-induced aggregation. It is known that drugs acting on a common step of the aggregation pathway, such as cAMP formation or brinogen receptor interaction, are able to reduce aggregation induced by many agonists (Coller et al., 1995; Hirose et al., 2000). Lavender oil shows a similar broad spectrum antiplatelet effect; its exact mechanism of action is presently uncertain even if an ASA-like activity can be ruled out. However, the present data indicate that there is a synergistic antiplatelet effect among the components of the oil since they singularly show lower potency and efcacy than the whole lavender oil where their presence ranges from 36% to 5%. An analogue consideration could be done for clot retraction even if the main component of the phytocomplex, linalyl acetate, seems to primarily contribute to lavender oil effect being as potent as the whole essential oil. Not only inhibition of platelet aggregation but also clot retraction are critical factors for the activity of antithrombotic drugs. Indeed the inhibition of retraction destabilizes clots making them weaker and more likely to be degraded by the brinolytic system (Collet et al., 2001). In pulmonary thromboembolism in mice, lavender oil exhibits higher antithrombotic activity than ASA and we speculated that this nding may be due to the ability of the phytocomplex to decrease both platelet aggregation and clot retraction, at variance with ASA. A further favorable feature of lavender oil with respect to the reference drug is the lack of any prohemorrhagic property which is a frequent complication of antiplatelet medications (Van De Graaff and Steinhubl, 2001). The dissociation between antithrombotic and bleeding effect, already described for other antithrombotic drugs (Wong et al., 2002), is related to a mechanism not yet claried. Furthermore, a preliminary indication of a good tolerability arises from the lack of adverse effects on body weight, physical appearance and behavior in subacutely lavender oil-treated mice according to recent data on linalool or linalyl acetate subchronic toxicity studies (Letizia et al., 2003a, b) These results extending further the wide range of effects described for Lavandula angustifolia essential oil encourage other studies in order to elucidate the mechanisms of action involved in the antithrombotic action of lavender oil and to better dene the signicance of this effect with respect to the various biological activities of this aromatic plant.

Fig. 2. Inhibition of clot retraction. Doseresponse curves of lavender oil, 1,8 cineol, linalool, linalyl acetate, camphor and the reference drug aspirin (ASA) on clot retraction induced by thrombin on rat PRP after 60 min from induction. Each point represents the mean of six individual experiments, standard errors of the mean are indicated as vertical bars.

Fig. 3. Acute pulmonary thromboembolism induced by i.v. shot injection of a mixture of 12 mg/kg collagen and 1 mg/kg epinephrine in mice, treated for 5 days with vehicle alone, ASA (100 mg/kg/day os) or lavender oil (100 mg/kg/day os). Every column represents the total number of animals used for each treatment closed section indicating paralyzed animals and open section indicating not paralyzed animals. # p 0:051 and po0:01 by w2 test compared to control.

treated animals were paralyzed compared to 2 out of 11 (18%) of lavender-treated mice. Treatment with ASA reduced thrombotic events (p 0:051), accounting for 6 out of 10 (60%) paralyzed animals (Fig. 3). The evaluation of bleeding in mice is a useful tool in revealing the prohemorrhagic activity of the pharmacological treatments affecting mainly primary hemostasis. The mean amount of blood lost from control animals in 2 min was 6.472.5 ml. Mice treated with 100 mg/kg/day os of lavender oil showed no signicant difference from control losing 10.874.9 ml of blood. Animals treated with ASA (100 mg/kg/day os), experienced a hemorrhage of 14.478.9 ml, showing a signicant difference from control groups.

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