Inhibition of platelet aggregation and 5-HT release by extracts of Australian plants used traditionally as headache treatments

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Abstract

To identify potential migraine therapeutics, extracts of eighteen plants were screened to detect plant constituents affecting ADP induced platelet aggregation and [14C]5-hydroxytryptamine (5-HT) release. Extracts of the seven plants exhibiting significant inhibition of platelet function were reanalysed in the presence of polyvinyl pyrrolidone (PVP) to remove polyphenolic tannins that precipitate proteins. Two of these extracts no longer exhibited inhibition of platelet activity after removal of tannins. However, extracts of Crataegus monogyna, Ipomoea pes-caprae, Eremophila freelingii, Eremophila longifolia, and Asteromyrtus symphyocarpa still potently inhibited ADP induced human platelet [14C]5-HT release in vitro, with levels ranging from 62 to 95% inhibition. I. pes-caprae, and C. monogyna also caused significant inhibition of ADP induced platelet aggregation. All of these plants have been previously used as traditional headache treatments, except for C. monogyna which is used primarily for protective effects on the cardiovascular system. Further studies elucidating the compounds that are responsible for these anti-platelet effects are needed to determine their exact mechanism of action.

Introduction

Migraine and tension-type headache are complex neurological disorders that are economically and socially debilitating, and highly prevalent worldwide. Migraine is characterised by a unilateral pulsating headpain that is moderate to severe, and usually associated with symptoms such as nausea, vomiting, photophobia and phonophobia (Lance, 1993, Dalessio, 1994). In contrast, tension-type headache is generally characterised by a band like type of pain that is bilateral and of mild to moderate severity (Silberstein, 1992, Dalessio, 1994).

Biochemical studies provide significant evidence that serotonin (5-HT) plays a role in the pathogenesis of migraine (Ferrari and Saxena, 1993, Peroutka, 1993). It is undoubtedly one of the major targets for most migraine drugs (Deliganis and Peroutka, 1991, Silberstein, 1992). Although migraine is well documented both pathophysiologically and biochemically, aetiological mechanisms are still relatively unclear. It is a problem that may be solved by molecular genetic studies currently undertaken in our laboratory and others (Nyholt et al., 1998a, Nyholt et al., 1998b). Indeed, recent studies show involvement of a calcium channel gene associated with neuronal 5-HT release in familial hemiplegic migraine (FHM) (Ophoff et al., 1996). Currently, prophylactic treatments for migraine include calcium channel blockers, 5-HT2 receptor antagonists and beta blockers (Amery, 1983, Solomon, 1985). In addition, abortive treatments such as 5-HT1D/1A receptor agonists and non-steroidal anti-inflammatory drugs are also available (Humphrey et al., 1990, Silberstein and Lipton, 1994). Unfortunately, many of these treatments are non-specific and not always effective.

Studies of traditional plant treatments have confirmed the incidence of headache and perhaps migraine among native peoples worldwide (Russo, 1992). Phytotherapeutics have offered alternative sources of therapy for some migraine sufferers, and similarly have also provided some additional information about the pathogenesis of migraine. Tanacetum parthenium, more commonly known as feverfew, from the Compositae family, is a European medicinal plant that has been used for centuries for relief of migraine (Groenewegen et al., 1992). Clinical trials appear to indicate that feverfew taken prophylactically has beneficial effects in patients who suffer from migraine (Johnson et al., 1985, Murphy et al., 1988). In vitro studies have also shown that feverfew can potently inhibit platelet aggregation and 5-HT release induced by a number of different agonists such as ADP, arachidonic acid (AA), thrombin and collagen. Furthermore, there is evidence to indicate that feverfew can inhibit 5-HT secretion induced by low concentrations of the calcium ionophore A23187, and by phorbol esters which activate protein kinase C (Heptinstall et al., 1985, Groenewegen and Heptinstall, 1990). The major active component of feverfew has been identified as parthenolide, which is a sesquiterpene lactone containing an α-methylene butyrolactone moiety that can interfere with sulphydryl groups, and platelet function (Groenewegen et al., 1986).

Previous research suggests that platelet activity as well as platelet storage of 5-HT is altered in migraine patients D’Andrea et al., 1989, D’Andrea et al., 1995, Lingjaerde and Monstad, 1986), and that most drugs used for treatment of migraine are capable of interfering with this system (Addonizio et al., 1986, Pukhal’skaya, 1993, Taylor et al., 1992, Vinge et al., 1988). Thus, the ability of parthenolide to affect platelet function is likely to be an important feature of feverfew’s efficacy in the prophylactic treatment of migraine. Since mutations in a gene that codes for a neuronal calcium channel have been found in the rare FHM sub-type of migraine, it is possible that a similar phenomenon for platelet 5-HT transport might co-exist, or could contribute to other forms of migraine. Levels of cytosolic calcium regulate platelet activation and several reports have likened the release of 5-HT from platelet stores to neuronal synaptic release of 5-HT, suggesting that the platelet is a suitable model for studying serotonergic metabolism (D’Andrea et al., 1995).

In this study we have implemented a bioassay that has been previously used to demonstrate platelet effects of feverfew, to detect plant extracts that modulate platelet aggregation and 5-HT release (Groenewegen and Heptinstall, 1990). This method allows a preliminary identification of plant species that could contain constituents with potential anti-migraine applications. Since it has been suggested that primary headache could be a continuum between tension-type headache and migraine (Silberstein, 1992), we chose to screen primarily Australian medicinal plants traditionally used for treating headache and possibly migraine in this study.

Section snippets

Preparation of plant extracts

Seventeen plants were identified and collected in South East Queensland, and then sent to the Queensland herbarium for confirmation of species identification. A sample of C. monogyna was supplied by a medicinal plant grower in Victoria, and was further identified by Dr. R. Spencer at the National Herbarium of Victoria. Identification of A. latescens was confirmed by the Northern Territory Herbarium. Voucher specimens are deposited in the Genomics Research Centre in the School of Health Science,

Results

PRP was incubated for 3 min with 100 μl of plant extract derived from each of the tested eighteen plants, and upon addition of ADP (final 10 μM), percentage 5-HT release and percentage platelet aggregation were measured simultaneously. An average of 94% of the total [14C]5-HT present within the PRP was taken up by platelets, in all blood samples that were tested (as determined from blank measurements). Treatment of platelets with ADP (10 μM) resulted in an average release of 36% of [14C]5-HT in

Discussion

When screening crude extracts of plants, with a goal to identify constituents that selectively affect platelet mechanisms, it is beneficial to identify those which may be active due to non-specific interference by agents such as tannins. We have found that when performing assays that measure platelet aggregation and 5-HT release, PVP is a useful tool for identifying extracts with these characteristics.

In this study, we have identified several plant species that contain agents which modulate

Acknowledgments

The authors would like to thank the Queensland Herbarium, the National Herbarium of Victoria and the Northern Territory Herbarium for identification of plant specimens. We are grateful to Melanie Carless for her assistance and also to Jan Glazebrook and Robert Downs for their help with identification and collection of plant samples. We would also like to acknowledge the contribution of the Australian aboriginal people to the knowledge and understanding of Australian plants.

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