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Phytomedicine 11 (2004) 596–601

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

Received 10 November 2003; accepted 8 January 2004

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 collagen–epinephrine mixture in mice subacutely treated with lavender oil. In this model, lavender oil (100 mg/kg/day os for 5 days) significantly 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 first experimental evidence of lavender oil’s 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

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: (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 definition. 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 anger–frustration test (Morris, 2003). Components of

0 0.15 mm).2 1. film thickness= 0. Carrier (Helium) 2 ml/min. . but they are not free from side effects such as gastric erosion (aspirin) and agranulocytosis (ticlopidine) or show a poor separation between therapeutic efficacy and hemorrhagic complications (glycoprotein IIb/IIIa receptor inhibitors) (Van De Graaff and Steinhubl. This evidence supports the continuous efforts in the development of new antiplatelet/antithrombotic agents of synthetic or natural origin. Milano.7 1.6 0. Essential oil characteristics were in accordance with literature data. split ratio 1:40. / Phytomedicine 11 (2004) 596–601 597 Lavandula angustifolia essential oil. Alpes de Haute Provence. Since some antiaggregants are known to enhance intraplatelet cAMP concentration. showed antiinflammatory properties in rats (Peana et al. The different antiplatelet agents currently used for this purpose proved to be effective in the prevention of thromboembolic disease. 2001). Italy) column (i. mass range. Puimoisson. Identification of compounds was based on relative retention times (Adams. Plateau de Valensole. matching with NBS MS library and comparison of fragmentation patterns with literature data [11]. 200 C. 2001).6 0.6 Materials and methods Plant material Lavandula hybrida Reverchon cv. 1 scan/s.4 36. Kovats indices were calculated against n-alkanes on a SE52 column.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 identified 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. Italy. Chemical composition and retention indices of the constituents of the essential oil of Lavandula hybrida Reverchon ‘‘Grosso’’a Compounds a-Pinene Camphene b-Pinene b-Myrcene Hexyl acetate 1.8 0. 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.3 7. Oven temperature was initially 45 C. 70 eV. Compounds listed in order of elution from an SE52 column. Chemical analysis Essential oil samples were analyzed using a Fisons (Rodano. emission current.6 0.2 3.0 0.55%). Parma. The essential oil was obtained from stream distillation of fresh flowers few minutes after their machine harvesting.d. then raised to 100 C (1 C/min ). GS–MS analysis was performed on a Hewlett Packard HP5890 series II plus GC equipped with a HPMS 5989b mass spectrometer using EI. then raised to 250 C (5 C/min ) and finally held at 250 C for 10 min. Legnano. One microliter of oil dissolved in CH2Cl2 was injected. Finally.6 0.. 40 mA.2 0. ion source temperature. = 0. using the same harvesting container as an industrial large-scale steam distiller (yield 1.2 96.1 0. 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..1 0.5 33. France. 2001).ARTICLE IN PRESS V. University of Parma Botanical Garden. A voucher specimen of the plant was deposited (Voucher no.2 2.1 1. Operating conditions: injector temperature 280 C. Table 1. The oil composition is reported in Table 1. Ballabeni et al. reducing inflammatory and thermal pain in mice (Peana et al.3 0. collagen and the stable thromboxane receptor agonist U46619) and its effect on clot retraction induced by thrombin. 2002) and (–)-linalool alone induced a significant antinociceptive effect. Italy) 9130-9000 gas-chromatograph equipped with an FID detector and an MEGA SE52 (Mega. scan rate. In vitro studies demonstrated that lavender oil might produce spasmolytic effect most likely increasing cAMP intracellular concentration (Lis-Balchin and Hart. in this work we investigate the antiplatelet/antithrombotic properties of lavender oil. the essential oil was analyzed and its main components were studied in vitro in order to assess their role in lavender oil effects.4 0.32 mm.1 5. we evaluated the properties of lavender oil in vitro towards platelet aggregation induced by various agents (ADP. FID temperature 280 C. In this work. arachidonic acid. 2003). 35–300 Da. Grosso plants were cultivated by La Gachore. 1999). (–)-linalool and linalyl acetate. OPR1) at the Herbarium of officinal plants. The MS conditions were as follows: ionization voltage.4 0. length 30 m.

8% 1 part citrate:9 part blood. Italy). In vivo assays Animal treatment Male Swiss mice were orally treated for 5 days. behavior and physical appearance were recorded daily. male Swiss mice (20–25 g from Charles River) were used for antithrombotic tests. assay was performed according to Davidson and Henry (1974). / Phytomedicine 11 (2004) 596–601 Materials and animals Arachidonic acid and collagen for in vitro tests were purchased from Menarini (Firenze. Where t0 was the area of the clot 2 min after thrombin addition and t was the area at the test time (15. 50 mM Arachidonic acid. PRP was preincubated at 37 C for 5 min with solvent (DMSO.5%). 2. 1 mM MgCl2. acetylsalicylic acid (ASA). 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. Maximal aggregation was induced stimulating platelets with 3 mM ADP. Male guinea pigs (400–500 g) and Wistar rats (150–200 g) were used for in vitro assays. once a day. 20 mM Hepes. Red blood cells were lised by adding 20 ml of triton 5% and absorbance of the solution was read at 560 nm (LKB. Quantification of clot retraction was performed measuring clot area by means of the NIH Image 1. 1962). Italy). final concentration 0. Animals body weight. Control animals received only vehicle (methocel 0. Aggregation was recorded as the percent change in light transmission: the baseline was set using PRP and maximal transmission using PPP. epinephrine bitartrate. Fibrin clot retraction was induced by addition of 50 ml thrombin 20 U/ml. Acute pulmonary thromboembolism A modification of Di Minno’s method was used (Di Minno. Bleeding The tail transection bleeding was determined modifying Dejana’s method (Dejana et al. dimethyl sulfoxide (DMSO). Triton. pH=7. 5 mg/ml collagen or 1 mM U46619. Clot retraction assay PRP from rats was diluted with tyrode buffer (137 mM NaCl. 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.3 mM NaH2PO4. The amount of haemorrhage was estimated by linear regression analysis of a standard curve constructed from known volumes of mouse blood. 45 and 60 min). In vitro assays Platelet aggregation studies PRP from guinea pig was used to perform aggregation in the Aggrecorder PA 3220 (Menarini. collected in plastic tubes and anticoagulated with sodium citrate 3. Differences between groups were analyzed by w2-test for pulmonary thromboembolism and by Mann-Whitney test for bleeding assay.5%) or the compound under study before the addition of the platelet aggregatory agent. 30. Blood manipulation Blood from male guinea pig or Wistar rat was obtained by cardiac puncture after CO2 euthanasia.7 mM KCl.5% did not interfere with platelet aggregation. ASA as lysine acetylsalicylate use for in vivo experiments was from Carlo Erba (Milano. ADP. CH2Cl2 and ferric chloride were obtained from Sigma (St Louis. sodium citrate. 5. The effects of test compounds and aspirin were expressed as percent inhibition compared with control samples.67e software. Briefly. Ultraspec 4050). U46619 was from Caymanchem (MI. 1979). 1983). The occurrence of paralyzed animals was recorded for 5 min after thrombotic mixture injection. USA). ..ARTICLE IN PRESS 598 V.Lgs 116/92). Tests were performed within 3 h to avoid platelet inactivation. DMSO at 0. 3. Pictures were taken every 15 min for 1 h using a digital camera. Firenze) following Born’s turbidimetric method (Born. Briefly. 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. Ballabeni et al. After centrifugation for 15 min at 180 g to obtain platelet rich plasma (PRP). thrombin. The experiments were performed applying experimental procedures supervised and approved by the " ‘‘Ministero della Sanita’’ (D. Data were expressed as percentage of retraction=(area t0area t)/area t0 Â 100. calf collagen type III for in vivo experiments. the remaining blood was centrifuged again 10 min at 2000 g to obtain platelet poor plasma (PPP). with 100 mg/kg lavender oil or ASA.4) to obtain a final concentration of 200. USA).000 plts/ml and the Statistical analysis All the results are expressed as means7standard error of the mean (SEM).6 mM glucose. The loss of the righting reflex was considered as indication of paralysis.

Lavender oil completely blocked the aggregation induced by arachidonic acid. 1 mM U46619. standard errors of the mean are indicated as vertical bars.. causing it to contract (Prevost et al. showed. In vitro antiplatelet potency of lavender oil and its main components on guinea-pig PRP against Arachidonic acid (AA). None of these effects was comparable to those produced by the phytocomplex. ASA.8 cineol completely inhibited aggregation stimulated by U46619 with low potency.8 cineol partially inhibited clot retraction beginning from 900 mg/ml. as expected. a Inactive up to 1000 mg/ml. underwent intravenous shot injection of a collagen–epinephrine mixture. ASA was ineffective up to 2700 mg/ml (Fig. as well as linalyl acetate. up to 270 mg/ml. completely inhibited clot retraction induced by thrombin in rat PRP at a concentration as low as 270 mg/ml (Fig. a great efficacy towards arachidonic acid and collagen-induced aggregation and had little or no effect on ADP or U46619 induced aggregation up to 1 mM concentration. 2). In vivo assays Initial body weight ranged from 21 to 27 g for the mice assigned to vehicle or treated group. The active principle 1. collagen and ADP respectively (Table 2). Furthermore. Animals subacutely treated for 5 days with saline. Clot retraction is a physiological event which requires the presence of activated fibrinogen receptor aIIbb3 on the platelet surface. Contraction of actin/myosin filaments within the platelet places tension on the fibrin clot. . the oil displayed its highest potency towards arachidonic acid induced aggregation showing a 2-. Lavender oil. collagen and ADP AA50 mM Compounds Lavender oil Linalool Linalyl acetate 1. 2003). U46619. a complete inactivity up to 1 mg/ml was detected for the most abundant component linalyl acetate. c IC50 not calculable. Ballabeni et al. 1. All the animals survived. linalool prevented collagen and ADP- induced aggregation whereas camphor exhibited antiplatelet effect against arachidonic acid and U46619.and 13-fold lower potency against U46619. Each point represents the mean of six individual experiments. 1). 2). 13 out of 14 (93%) saline Fig. Camphor. Linalool and 1. / Phytomedicine 11 (2004) 596–601 599 Results In vitro assays Lavender essential oil showed antiplatelet effects on the aggregation induced by all the platelet stimulants used on guinea pig PRP.8 cineol) was studied. bound to extracellular fibrinogen or fibrin and anchored to intracellular actin and myosin.ARTICLE IN PRESS V. Table 2. was inactive and it was not tested at higher concentration because of its low solubility. b Inactive up to 250 mg/ml and insoluble at higher concentrations. In order to better evaluate the active principles of the oil. linalyl acetate. camphor and 1. 4. Growth as well as behavior and physical appearance of treated animals did not differ from controls throughout the study. Antiplatelet effect of lavender oil towards aggregation induced by 3 mM ADP. 100 mg/kg/day os of ASA or 100 mg/kg/day os of lavender oil.8 cineol Camphor ASA IC50 51 (49–53) a c c U46619 1 mM IC50 84 (53–133) a a Collagen 1 mg/ml IC50 191 (163–226) 380 (225–640) a a b ADP 3 mM IC50 640 (495–826) 790 (530–1153) a c b c 134 (79–227) 10 (7–14) 677 (593–774) 96 (80–114) a 11 (8–16) The IC50 values are expressed as mg/ml and confidence limits are indicated in brackets. 1 mg/ml collagen or 50 mM arachidonic acid (AA) in guinea pig PRP. U46619 and collagen and almost completely inhibited the aggregation induced by ADP (74% inhibition) (Fig. the antiplatelet effect of main components (linalool. Instead. used as reference compound for these studies. because maximal inhibition is lower than 50%. As shown in Table 2.

/ Phytomedicine 11 (2004) 596–601 Discussion In this work. a preliminary indication of a good tolerability arises from the lack of adverse effects on body weight. # p ¼ 0:051 and ÃÃ po0:01 by w2 test compared to control. Indeed the inhibition of retraction destabilizes clots making them weaker and more likely to be degraded by the fibrinolytic system (Collet et al. linalyl acetate.5 ml. 2001).8 cineol. Acute pulmonary thromboembolism induced by i. linalyl acetate. Hirose et al. 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 define the significance of this effect with respect to the various biological activities of this aromatic plant. Fig. 1995. Animals treated with ASA (100 mg/kg/day os). shot injection of a mixture of 12 mg/kg collagen and 1 mg/kg epinephrine in mice. . An analogue consideration could be done for clot retraction even if the main component of the phytocomplex. 3).478. experienced a hemorrhage of 14. Ballabeni et al. ASA (100 mg/kg/day os) or lavender oil (100 mg/kg/day os).. camphor and the reference drug aspirin (ASA) on clot retraction induced by thrombin on rat PRP after 60 min from induction.472. 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. linalool. 2000). which was poorly or no effective toward ADPand U46619-induced aggregation. 2.. is related to a mechanism not yet clarified. 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. The mean amount of blood lost from control animals in 2 min was 6. However. In pulmonary thromboembolism in mice.. showing a significant difference from control groups. 2003a. 1... Inhibition of clot retraction. Mice treated with 100 mg/kg/day os of lavender oil showed no significant difference from control losing 10. 2001). lavender oil exhibits higher antithrombotic activity than ASA and we speculated that this finding may be due to the ability of the phytocomplex to decrease both platelet aggregation and clot retraction. at variance with ASA. treated for 5 days with vehicle alone. Not only inhibition of platelet aggregation but also clot retraction are critical factors for the activity of antithrombotic drugs. showing a higher efficacy than the reference drug ASA. seems to primarily contribute to lavender oil effect being as potent as the whole essential oil.ARTICLE IN PRESS 600 V.9 ml. Treatment with ASA reduced thrombotic events (p ¼ 0:051). the present data indicate that there is a synergistic antiplatelet effect among the components of the oil since they singularly show lower potency and efficacy than the whole lavender oil where their presence ranges from 36% to 5%. 3. accounting for 6 out of 10 (60%) paralyzed animals (Fig. 2002). such as cAMP formation or fibrinogen receptor interaction. Each point represents the mean of six individual experiments.9 ml of blood. its exact mechanism of action is presently uncertain even if an ASA-like activity can be ruled out.874.v. Furthermore. Lavender oil shows a similar broad spectrum antiplatelet effect. It is known that drugs acting on a common step of the aggregation pathway. The dissociation between antithrombotic and bleeding effect. standard errors of the mean are indicated as vertical bars. Fig. treated animals were paralyzed compared to 2 out of 11 (18%) of lavender-treated mice. already described for other antithrombotic drugs (Wong et al. Every column represents the total number of animals used for each treatment closed section indicating paralyzed animals and open section indicating not paralyzed animals. Dose–response curves of lavender oil. The essential oil is able to inhibit platelet aggregation induced by all the agonists used in the study. we provide the first experimental evidence of lavender oil antiplatelet/antithrombotic properties. The evaluation of bleeding in mice is a useful tool in revealing the prohemorrhagic activity of the pharmacological treatments affecting mainly ‘‘primary’’ hemostasis. are able to reduce aggregation induced by many agonists (Coller et al.

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