The essential oils obtained by steam distillation from the aerial parts of 104 specimens of eight Helichrysum species and distinct chemical profiles were analysed by GC-MS. The oil of Helichrysum orientale notably contained four linear hydrocarbons, including nonacosane (11.1%), and also caryophyllene epoxide (4.4%), while the oil of H. heldreichii was dominated by E-caryophyllene (38.5%). By contrast, the oil of H. italicum ssp microphyllum was characterised by b-selinene (17.2%) and g-curcumene (13.7%), while that of H. doerfleri had a mixture of four eudesmol isomers (31.4%). H. amorginum and H. italicum contained geraniol (32.1% and 35.5%), geranyl acetate (20.7% and 14.7%) and neryl acetate (17.5% and 7.2%) as major contributors. H. barrelieri, contained mainly sesquiterpenes with E-caryophyllene (15.6%) and b-elemene (13.1%) being the major ones. H. taenari was dominated by geraniol (50%) and camphene (18.6%). Analysis of oils before and after anthesis showed some significant quantitative differences. In H. doerfleri carvacrol increased noticeably, while in H. italicum ssp microphyllum the content of a-pinene reduced from 8.2% to a trace. Besides the taxonomically recognized species several populations of intermediate morphs with distinct chemical profiles from different compartments of Greece were analyzed.

In the framework of our chemical and biological investigations on the volatile metabolites of Greek endemic and Mediterranean plant species (Petrakis and Roussis 1997; Roussis et al., 1995) we recently were able to collect and study specimens of 8 Helichrysum species, from the 10 species which are native to Greece.

The members of this genus are herbs or dwarf shrubs, often lanate or tomentose; leaves are alternate, simple entire; bracts are numerous white or coloured and flowers are yellow (Clapham, 1976).

Along with the taxonomically recognised Helichrysum species a significant number of evolved hybrids is also found (Jahn and Schoenfelder, 1995). The individual morpho-anatomical characteristics of these hybrids in several cases can be deceiving and can lead to wrong assignments to recognized taxa. The high degree of genotypic variability, observed in a number of Helichrysum species, is reflected in the biochemical variability, which is usually studied at the levels of terpene composition and isozyme variation. The volatile constituents, particularly monoterpenes, have been extensively studied since it has been demonstrated that the monoterpene composition, besides some environmental variabilities, is dependent upon the plant's genotype and can be used for taxonomic purposes (Harborne, 1977; Poulose and Croteau, 1978; Cane, 1990).

Very little is known on the chemistry of the volatile metabolites of Helichrysum species grown in Greece (Chinou et al., 1996; 1997). We describe here the first detailed analysis of essential oils in Greek Helichrysum species.

Field Sampling and Vouchers

Plant material were collected during the period of May 1997 - May 1998 from the islands of Crete, Melos, Corphu, Koufonisi, Kithyra and the coast of Porto Germeno in Attiki. Voucher specimens have been deposited at the Herbarium of the Laboratory of Pharmacognosy, University of Athens and the Herbarium of MAICh.

Chemical analyses

3-12 harvested plants from each population and phenological stage were separately steam distilled for 3 hrs in a Clevenger modified apparatus with a water cooled oil receiver, to reduce overheating artefacts.

GC-MS analysis

The GC analyses were performed on a Varian 3300 Gas Chromatograph equipped with an on column injector and a flame ionisation detector as well as on a Hewlett Packard 5973-6890 GC-MS system operating in EI mode. The identification of constituents was based on comparison of the R_t values and mass spectra with those obtained from authentic samples and/or the NIST/NBS and Wiley library spectra. The quantification of the components was performed on the basis of their GC peak areas, without corrections for response factors.

Statistical analysis

Throughout this study only population means are shown though original data of terpene profiles of individual plants are actually analysed. In this report cluster analysis of standardised terpene data was employed. The linkage strategy was the ward's variance minimising method and the distance was the Euclidean distance (Dunn and Everitt, 1982).

A total of 104 plants, representing the eight species and five chemical profiles under examination, were selected for this study. The plants were collected from natural stands and the aerial parts of the individuals were steam distilled to afford the equivalent number of essential oils that were analyzed by GC and GC-MS. More than one hundred compounds were detected and quantified in the investigated oils (Table 1). The constituents were identified on the basis of their mass spectra, retention indices and/or by comparison with authentic compounds.

Because the harsh conditions of the steam distillation have in the past been suspected as a source of artefacts, the head space volatiles of a number of Helichrysum specimens were collected and analysed separately. The comparison of the chemical profiles obtained by both techniques showed that the steam distillation did not produce any artefacts and thus the study was subsequently safely carried out using the essential oils.

The analyses showed that the oils were dominated by sesquiterpenes and linear chain aliphatic hydrocarbons (Table 1). It is worth noting that in literature reports on other Helichrysum species the main constituents of the oils were monoterpenes. Very little is known on the chemistry of the volatile metabolites of Helichrysum species grown in Greece (Chinou et al., 1996; 1997).

Although all investigated oils contain a similar array of constituents, the relative contribution of them varies significantly resulting in clear and characteristic chemical profiles for each species. Based on the terpenes with the highest contribution distinct chemical profiles for the investigated Helichrysum species and intermediate morphs were recognized.

Statistical analysis

In order to gain a better insight in the phylogenetic relationships of the genus and examine the suitability of their volatile metabolites for further biosysthmatic studies, the chemical constituents of the investigated species were submitted to a cluster analysis. Cluster analysis can accommodate for the relationships between taxa since it reveals not only the structure of the naturally grouping of Helichrysum species but also the appropriateness of characters other than morphological ones used in this study (Abbott et al., 1985). The results of the analysis produced a hierarchical order shown in the following dendrogram (Fig.1) that is not congruent with the conventional phylogenetic relationships among the species studied.

REFERENCES

1. Cane E.D. (1990): Enzymatic formation of Sesquiterpenes, Chem Rev. 90, 1089-1103.

2. Chinou I., Demetzos C., Harvala C., Roussakis C. and Verbist J.F. (1994): Cytotoxic and antibacterial labdane-type diterpenes from the aerial parts of Cistus incanus subsp. creticus, Planta Medica, 60, 34-36.

3. Chinou I., Roussis V., Perdetzoglou D. and Loukis A. (1996): Chemical and biological studies on two Helichrysum species of Greek origin, Planta Medica 62, 377-379.

4. Chinou I., Roussis V., Perdetzoglou D., Tzakou O. and Loukis A. (1997): Chemical and antibacterial studies of two Helichrysum species of Greek origin, Planta Medica 63, 181-183.

5. Clapham A.R. (1976): Flora Europaea, eds. Tutin T.G., Heywood V.H., Burges N.A., Moore D.M., Valentine D.,H., Walters S.M. and Weebb D.A., Cambridge University Press, Cambridge, London, New York, Melbourne 4, 128.

6. Dunn G. and Everitt B.S. (1982): An Introduction to Mathematical Taxonomy, Cambridge University Press, London.

7. Harborne J.B. (1977): Chemosystematics and coevolution, Pure and Appl. Chem. 49, 1403-1421.

8. Harborne J.B and Turner B.L. (1984): Plant Chemosystematics, Academic Press, London, New York, Tokyo.

9. Jahn R. and Schonfelder P. (1995): Exkursionsflora für Kreta, Verlag Eugen Ulmer, Stuttgart, Germany.

10. Petrakis P.V. and Roussis V. (1997): Evolution in Mediterranean climate region, TREE 12, 113.

11. Poulose A.J. and Croteau R. (1978): Biosynthesis of aromatic monoterpenes, Arch. Biochem. Biophys. 187, 307-314.

12. Roussis V., Petrakis P.V., Ortiz A. and Mazomenos B. (1995):Volatile constituents of five Pinus species grown in Greece, Phytochemistry 39, 357-361.

13. Roussis V., Tsoukatou M., Chinou I. and Ortiz A. (in press): Chemical and biological studies of two Helichrysum species growing in the Balearic islands (Part III), Planta Medica.