Melaleuca teretifolia, a Novel Aromatic and Medicinal Plant from I. Southwell, M. Russell and R.L. Smith Wollongbar Agricultural Institute Wollongbar, NSW, 2477 J. Day The Paperbark Co. Claremont, WA, 6010 J.J. Brophy School of Chemistry University of New South Wales Sydney, NSW, 2052 Keywords: Banbar, 1,8-cineole, citral, chemical variation, essential oil, marsh honey, Myrtaceae, myrtle Abstract The essential oils of the leaves and twigs of two chemotypes of Melaleuca teretifolia Endl growing wild in Western were investigated by GC and GC/MS. One chemical form which yielded 0.2% on a fresh weight basis was found to be rich in 1,8-cineole (84.0%) with α-pinene (1.8%), β-pinene (1.2%), limonene (3.1%), terpinen-4-ol (1.8%) and α-terpineol (3.3%) as the only other significant constituents. The lemon form gave a much higher yield (1.5%) of an oil rich in neral (29.1%), geranial (38.8%), and myrcene (9.8%) with significant concentrations of limonene (1.0%), citronellal (1.0%), terpinen-4-ol (3.4%), geraniol (2.1%), nerol (1.1%), Z-isocitral (1.6%) and E-isocitral (2.4%). The potential commercial value of these oils as medicinal and aromatic plant products is discussed. INTRODUCTION Melaleuca teretifolia Endl. is a medium to tall (2-5 m) myrtaceous shrub with long (3-8 cm) narrow (2 mm) curved leaves and usually small white or cream flowers. Commonly known as Banbar or Marsh Honey Myrtle, this tea tree, native to southwestern, grows in moist, poorly drained soils (Elliot and Jones, 1993) in a similar environment to the commercially important M. alternifolia of the east coast (Southwell and Lowe, 1999). This species has not, to our knowledge, been previously investigated for essential oils. This communication describes the essential oil yield and composition of two chemical forms of this species with vastly different oil concentrations and volatile chemical constituents. MATERIALS AND METHODS Plant Material Leaf material of the 1,8-cineole chemovar (BJL & TRL 1711 lodged at the n National Botanical Gardens, Canberra) was collected 6.3 km WNW of Mogumber on the road to Regans Ford, Western at 31 02 02 S and 115 59 24 E. Leaf material of the citral chemovar (Ian Southwell RP01-155 lodged at the n National Botanical Gardens, Canberra) was collected 10 km NW of Harvey (33 02 S, 115 52 E) at Richardson Rd, Harvey, Western. Isolation of Volatiles Fresh leaves and terminal stems of the 1,8-cineole and citral chemovars were hydrodistilled in a Clevenger-type apparatus for three hours to produce 0.2 and 1.5% v/w essential oil from five and one trees respectively. Confirmation of the uniformity of the latter chemovar was obtained from the micro-ethanolic extraction (Baker et al., 2000; Southwell, 1999; Southwell et al., 1995) of eight additional trees and the bulk distillation Proc. WOCMAP III, Vol. 3: Perspectives in Natural Product Chemistry Eds. K.H.C. Başer, G. Franz, S. Cañigueral, F. Demirci, L.E. Craker and Z.E. Gardner Acta Hort. 677, ISHS 2005 79
of leaf from 30 or 40 trees in a partially filled commercial sized (0.6 tonne) still. Composition of Volatiles Area percent concentrations of constituents were on an (a) Shimadzu GC6 AMP gas chromatograph, a SMAD electronic integrator, an SP1000 [85 m x 0.5 mm] SCOT column programmed from 50 C to 225 C at 3 C/min, using helium carrier gas for the 1,8-cineole variety and a (b) Hewlett Packard 6890 gas chromatograph, HP Chemstation v A.06.03 (509) and autosampler and an Alltech AT35 60 m x 0.25 mm, 0.25 µm film thickness, mid polarity FSOT column with hydrogen (55 cms/sec) as carrier gas, injection port (split 1:50) at 200 C, flame ionisation detector at 300 C and temperature programming from 60 C (3 min) to 240 C at 9 C/min for the citral variety. GC/MS investigations were performed similarly using a (c) VG Quattro mass spectrometer operating at 70eV ionisation energy, a DB-Wax [60 m x 0.32 mm x 0.25 µm] column programmed from 35 C to 220 C at 3 C/min, with helium as carrier gas for the cineole variety and a (d) Hewlett Packard 6890 instrument fitted with an HP5-MS 29.5 m x 0.25 mm, 0.25 µm film thickness, FSOT column with helium (36 cm/sec) as carrier gas, injection port (split 1:50) at 250 C, mass selective detector (HP 5973) at 250 C (source) and 150 C (quad) with transfer line 280 and ion source filament voltage of 70eV, for the citral chemovar. Retention indices were measured with respect to n-alkane standards on column (d) above. Component identifications were made on the basis of mass spectral fragmentation, retention time comparison with authentic constituents and mass spectral and retention matching with commercial (NIST, Wiley and Adams) and in house libraries. RESULTS AND DISCUSSION The components present at greater than 0.1% identified in the leaf oils of the two known chemotypes of Melaleuca teretifolia were determined by gas chromatography (GC) and are shown in Table 1. For the 1,8-cineole chemical variety (Fig. 1) the only significant components other than 1,8-cineole (mean 84.0%, range 81-87%), present at greater than one percent were α-pinene (mean 1.8%), β-pinene (mean 1.2%), myrcene (mean 1.0%), limonene (mean 3.1%), terpinen-4-ol (mean 1.8%) and α-terpineol (mean 3.3%). For the citral chemical variety (Fig. 2), concentrations of neral of 28.2-30.0% and geranial of 38.7-38.8% indicate an oil with a citral content of approximately 68% with a significant amount of myrcene (mean 9.8%, range 6.3-13.3%). Other components present at greater than one percent were the monoterpene hydrocarbon limonene (0.3-1.7%), the alternative lemon constituent citronellal (0.2-1.7%), the tea tree alcohols terpinen-4-ol (0.1-6.7%) and α-terpineol (0.1-1.6%) and the rose alcohols citronellol (0.6-1.2%), geraniol (1.7-2.5%) and nerol (0.2-2.0%). In addition, the commonly occurring citral congeners (Southwell et al., 2000), Z-isocitral (syn isoneral) (1.3-1.8%), E-isocitral (syn isogeranial) (2.1-21.7%) and exo-isoctral (0.3-0.5%) were also present. These oil results and the extract percentages indicate greater variation within the citral variety than the cineole variety. Of the approximately 230 Melaleuca species described (Southwell and Lowe, 1999) very few have been reported as containing lemon constituents. Two of the lemon varieties of M. citroleons have been reported to contain up to 36 and 42% citral in the volatile oil (Brophy, 1999; Brophy et al., 1989; Brophy and Clarkson, 1989; Brophy and Doran, 1996) and a third 17% with high concentrations of citronellol (30%) and citronellyl acetate (21%) (Southwell and Wilson, 1993). This species also has a 1,8- cineole rich (34-50%) oil chemical variety with terpinolene (10-20%) the second most abundant constituent (Brophy, 1999; Brophy et al., 1989; Brophy and Clarkson, 1989; Brophy and Doran, 1996). M. stipitata also yielded a lemon oil containing 43.5% citral (Brophy, 1999) along with quantities of 1,8-cineole (5.3%), terpinen-4-ol (10.4%), α- and γ-terpinene (3.4 and 5.8% respectively) and α-terpineol (5.4%). M. alsophila is a third Melaleuca species containing a lemon scented chemotype (Brophy, 1999). This 80
chemotype contains 0.1-0.6% (fresh weight) of oil with neral (2-10%), geranial (2-19%) and terpinen-4-ol (23-32%) as the major components. The other chemotype of this species is rich in α-pinene (8-65%) and 1,8-cineole (15-66%) with oil yield 0.04-0.1% (Brophy, 1999). The oil composition and oil yield of M. teretifolia now described indicates the richest Melaleuca source for citral and also illustrates a unique chemotaxonomic association with substantial quantities of myrcene. The quantitative variability in the volatiles, particularly with respect to terpinen-4-ol content, suggests that selection for or against terpinen-4-ol could produce specific anti-microbial or perfumery oils respectively. CONCLUSION Although the cineole content of chemovar A indicates that this variety is a good source of an 80-85% cineole eucalyptus-type oil, the low oil yield of the five trees investigated (mean 0.17%) suggests that this provenance could never be a viable source of commercial 1,8-cineole. The higher oil yield of 1.5% for the citral chemical variety, B, indicates potential for commercial development as a lemon oil or as an alternative source of citral. Although not matching the alternative n citral sourced from Backhousia citriodora, the citral form of Melaleuca teretifolia (Southwell et al., 2000). provides an oil equivalent to the traditional lemongrass and Litsea cubeba lemon oils. Consequently some 10,000 seedlings of this form have been planted to explore their commercial potential. ACKNOWLEDGEMENTS The authors gratefully acknowledge, Lyn Craven and Brendan Lepschi, n National Botanical Gardens Herbarium, for plant identification and collection. Literature Cited Baker, G.R., Lowe, R.F. and Southwell, I.A. 2000. Comparison of oil recovered from tea tree leaf by ethanol extraction and steam distillation. J. Agri. Food Chemistry 48:4041-4043. Brophy, J.J. and Clarkson, J.R. 1989. The essential oils of four chemotypes of Melaleuca citrolens. J. and Proc. Roy. Soc. N.S.W. 122:11-18. Brophy, J.J. and Doran, J.C. 1996. Essential oils of Tropical Asteromyrtus, Callistemon and Melaleuca species; In search of interesting oils with commercial potential. ACIAR Monograph No. 40, ACIAR, Canberra. Brophy, J.J. 1999. Potentially Commercial Melaleucas. Ch. 16. In: I.A. Southwell and R.F. Lowe (eds.), Tea Tree, the Genus Melaleuca; 9:247-274. In: R. Hardman (ed.), Series Medicinal and Aromatic Plants - Industrial Profiles, Harwood Academic Publishers, Amsterdam. Brophy, J.J., Boland, D.J. and Lassak, E.V. 1989. Survey of the leaf essential oils of Melaleuca and Leptospermum species from tropical. p.193-203. In: D.J. Boland (ed.), Trees for the Tropics - Growing n multipurpose trees and shrubs in developing countries, ACIAR Monograph No. 10, Canberra. Elliot, W.R. and Jones, D.L. 1993. Encyclopaedia of n Plants Suitable for Cultivation. Lothian Publishing Co., Melbourne. 6:369. Southwell, I.A. and Wilson, R.W. 1993. The potential for tea tree oil production in northern. Acta Hort. 331:223-227. Southwell, I.A. 1999. Tea Tree Constituents. Ch. 2. In: I.A. Southwell and R.F. Lowe (eds.), Tea Tree, the Genus Melaleuca, 9:29-62. In: R. Hardman (ed.), Series Medicinal and Aromatic Plants - Industrial Profiles, Harwood Academic Publishers, Amsterdam. Southwell, I.A. and Lowe, R.F. (eds.). 1999. Tea Tree: The Genus Melaleuca. 300p. Vol 9. In: R. Hardman (ed.), The series Medicinal and Aromatic Plants - Industrial Profiles, Harwood Academic Press, Amsterdam. Southwell, I.A., Maddox, C.D.A. and Zalucki, M.P. 1995. Metabolism of 1,8-cineole in tea tree (Melaleuca alternifolia and M. linariifolia) by pyrgo beetle (Paropsisterna 81
tigrina). J. Chemical Ecology 21:439-453. Southwell, I.A., Russell, M., Smith, R.L. and Archer, D.W. 2000. Backhousia citriodora F. Muell. (Myrtaceae), a superior source of citral. J. Essent. Oil Res. 12:735-741. Tables Table 1. Percentage composition of the leaf oils of the 1,8-cineole (A) and citral (B) chemotypes of Melaleuca teretifolia. Peak No. Compound Retention Index a 1,8-Cineole chemovar.a b Citral Chemovar.B c 1 α-pinene 937 1.8 0.6 2 β-pinene 979 1.2 0.3 3 6-methyl-5-hepten-2-one 990-0.4 4 dehydro-1,8-cineole 992-0.3 5 Myrcene 993 1.0 9.8 6 p-cymene 1028 0.8-7 limonene 1031 3.1 1.0 8 1,8-cineole 1035 84.0 0.5 9 linalool 1101 tr 0.3 10 exo-isocitral 1150-0.4 11 citronellal 1157-1.0 12 cis-isocitral 1169-1.6 13 terpinen-4-ol 1185 1.8 3.4 14 trans-isocitral 1187-2.4 15 α-terpineol 1196 3.3 0.9 16 citronellol 1233 0.1 0.9 17 nerol 1233-1.1 18 neral 1249-29.1 19 geraniol 1259-2.1 20 geranial 1278-38.8 21 citronellyl acetate 1356 0.1-22 geranyl acetate 1385 0.1 0.6 23 allo-aromadendrene 1478 0.1-24 spathulenol 1596 0.3-25 caryophyllene oxide + globulol 1603 0.3-26 viridiflorol 1612 0.1 - Mean Oil Yield (%) 0.2 1.5 No. of samples 5 2 a Determined on column (d) Determined on columns (a) and (c) Determined on columns (b) and (d) [see Experimental] 82
Figures Fig. 1. Total ion current gas chromatogram of the 1,8-cineole chemotype A of Melaleuca teretifolia. Fig. 2. Total ion current gas chromatogram of the citral chemotype B of Melaleuca teretifolia. 83