PHYTOTHERAPY RESEARCH

Phytother. Res. 16, 398–399 (2002)

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.979

SHORT COMMUNICATION
Human Platelet Aggregation Inhibitors from
Thyme (Thymus vulgaris L.)

Kenji Okazaki,1* Kazuyoshi Kawazoe2 and Yoshihisa Takaishi2

1
Kagawa Prefectural Fermentation and Food Experimental Station, 1351-1 Nouma Uchinomi-cho, Souzu-gun, Kagawa 761-4421, Japan
2
Faculty of Pharmaceutical Sciences, The University of Tokushima, 1-78 Sho-machi, Tokushima 770-8505, Japan

Two antiaggregant compounds, thymol (compound 1) and 3,4,3',4'-tetrahydroxy-5,5'-diisopropyl-2,2'-

dimethylbiphenyl (compound 2) were isolated from the leaves of thyme (Thymus vulgaris L.). The struc-
tures were determined by 1H-, 13C-NMR and mass spectra (MS) studies. These compounds inhibited
platelet aggregation induced by collagen, ADP, arachidonic acid (AA) and thrombin except that com-
pound 2 did not inhibit platelet aggregation induced by thrombin. Copyright # 2002 John Wiley &
Sons, Ltd.
Keywords: thyme; antiplatelet activity; Thymus vulgaris L.; Labiatae.

INTRODUCTION observed over 5 min in the presence of a vehicle without

Thyme (Thymus vulgaris L.) is originally circum-
Mediterranean and has been used commonly as a culinary
herb for adding flavour. Recently several biological
activities of thyme have been investigated such as
antimicrobial (Ueda et al., 1982), antioxidative, (Miura
and Nakatani, 1989) and deodorizing activity (Nakatani
et al., 1989).
Platelet aggregation is an important reaction to stop
bleeding but it is considered that excessive platelet
aggregation causes thrombosis and arterioscleosis. Re-
cently, the antiaggregant activities of some spices were
investigated (Block et al., 1986; Srivastava, 1993;
Takenaga et al., 1987).
We have studied the antiaggregant activity of several
culinary herbs and reported that thyme strongly inhibited
platelet aggregation induced by collagen (Okazaki et al.,
1998).
In this paper, we describe the details of the isolation of
active compounds from thyme and their antiaggregant
activity.
extract, and antiaggregant activity was defined as the
percentage reduction of this response by the extract.
Isolation of the active compound. 2 kg of commercially
dried thyme was extracted with methanol (MeOH) for 1
day at room temperature. The extracts were filtered and
the residue re-extracted three times. All filtrates were
combined and evaporated to dryness. These MeOH
extracts were partitioned between water and ethyl acetate
(EtOAc) to obtain the EtOAc fraction (74 g). The water
fraction phase was partitioned by adding n-butanol
(BuOH) to give a BuOH fraction (55 g) and a water
fraction (150 g). The EtOAc fraction, which showed the
strongest antiaggregant activity, was applied to a silica
gel column and eluted with n-hexane (Hex)–
EtOAc = 3:1, 2:1, 1:1, 1:2, EtOAc–MeOH = 9:1, 7:3,
and MeOH 100%. Active components were eluted in
fraction 1. Fraction 1 was rechromatographed on silica
gel with stepwise elution (Hex–EtOAc = 95:5, 9:1, 8:2)
and the active fractions were collected (4.5 g). Finally,
purification was carried out by preparative high-perfor-
mance liquid chromatography (HPLC) equipped with a

MATERIALS AND METHODS

Antiaggregant activity. Antiaggregant activity was

investigated according to a method previously reported
(Okazaki et al., 1998). The platelet aggregation was
determined by turbidimetry. After incubation of a
mixture of platelet-rich plasma and samples at 37 °C for
2 min with stirring, 20 mL of a platelet stimulating agent
was added. The maximal aggregation response was
defined as the maximum change in light transmission

* Correspondence to: Dr K. Okazaki, Kagawa Prefectural Fermentation and

Food Experimental Station, 1351-1 Nouma Uchinomi-cho, Souzu-gun,
Kagawa 761-4421, Japan. Figure 1. Structures of compound 1 and compound 2.

Received 8 May 2000

Copyright # 2002 John Wiley & Sons, Ltd. Accepted 7 August 2000
 PLATELET AGGREGATION INHIBITORS FROM THYME
Table 1. The effects of compound 1 and compound 2 on human platelet aggregation induced by collagen, ADP, AA and
thrombin
Test sample Collagen (2.0 mg/mL) ADP (2.0 mM)
Compound 1 520
Compound 2 531
Each ®gure indicates the IC50 values (mM).
a
Inhibits 25% aggregation.
b
Inhibits 21% aggregation.
silica gel column with a solvent system of Hex–EtOAc
(8:2) to give the active compound 1 and further
rechromatographed with Hex–EtOAc(7:3) to give com-
pound 2.
RESULTS AND DISCUSSION
The structures of these compounds were determined by
1
H-, 13C-NMR and MS studies. Compounds 1 and 2 were
confirmed to be thymol and 3,4,3',4'-tetrahydroxy-5,5'-
diisopropyl-2,2'-dimethylbiphenyl, respectively (Fig. 1).
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Copyright # 2002 John Wiley & Sons, Ltd.
399
AA (800 mM) Thrombin (0.19 U)
1333a 0.53 332
636b 18.3 no inhibition
These NMR spectra were identical to those previously
reported (Miura et al., 1989).
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inhibited platelet aggregations induced by almost all of
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Phytother. Res. 16, 398–399 (2002)