FLAVOUR AND FRAGRANCE JOURNAL, VOL. 11,35-41(1996)

Volatile Leaf Oils of some South-western and Southern Australian Species of the Genus Eucalyptus. Part VII. Subgenus Symphyomyrtus, Section Exsertaria

C. M. Bignell and P. J. Dunlop Department of Chemistry, University of Adelaide, South , SM5, Australia

J. J. Brophy Department of Organic Chemistry, University of New South Wales, Sydney, NSW, 20S2, Australia

J. F. Jackson Department of Viticulture, Oenology and Horticulture, Waite Agricultural Research Institute, University of Adelaide, South Australia, 5005, Australia

The volatile leaf oils of Eucalyptus seeana Maiden, E. bancrofrii (Maiden) Maiden, E. parramattensis C. Hall, E. amplifolia Naudin, E. tereticornis J. Smith, E. blakelyi Maiden, E. dealbata A. Cunn. ex. Schauer, E. dwyeri Maiden & Blakely, E. vicina L. A. S. Johnson & K. D. Hill, E. flindersii Boomsma, E. camaldulensis Dehnh. var camaldulensis, E. camaldulensis Dehnh. var. obtusa Blakely, E. rudis Endl., E. exserta F. Muell. and E. gillenii Ewart & L. R. Kerr, isolated by vacuum distillation, were analysed by GC-MS. Most species contained a-pinene (1.5-14%), 1,&cineole (0-81%), p-cymene (O.6-28%) and aromadendrene/terpinen-4-01 (0.6-24%) as principal leaf oil components.

KEY WORDS Eucalyptus seeana Maiden; Eucalyptus bancrofrii (Maiden) Maiden; Eucalyptus parramattensis C. Hall; Eucalyptus amplifolia Naudin; Eucalyptus tereticornis J. Smith; Maiden; A. Cunn. ex. Schauer; Maiden & Blakely; Eucalyptus vicina L. A. S. Johnson & K. D. Hill; Eucalyptusflindersii Boomsma; Eucalyptus camaldulensis Dehnh. var. camaldulensis; Eucalyptus camaldulensis Dehnh. var. obtusa Blakely; Eucalyptus rudis Endl.; Eucalyptus exserta F. Muell.; Eucalyptus gillenii Ewart & L. R. Kerr; ; leaf essential oil composition; torquatone; mono-and sesquiterpenoids; GC-MS

INTRODUCTION vicina L. A. S. Johnson & K. D. Hill, E. dealbata A. Cunn. ex. Schauer, E. amplijolia Naudin and Continuing our investigation of the volatile leaf E. parramattensis C. Hall, occur mainly in New oils of indigenous Australian eucalypts'-6 we South Wales while four others, E. blakelyi have examined the oils of 15 of the species Maiden, E. rereticornis J. Smith, E. bancroftii belonging to section Exsertaria of the Eucalyptus (Maiden) Maiden and E. seeana Maiden, occur in subgenus Symphyomyrtus (see system M. I. H. New South Wales and Queensland. Finally two Brooker and D. A. Kleinig).' Locations of indi- species, E. camaldulensis Dehnh. var. camaldu- vidual species are listed in Table 1. lensis and E. camaldulensis Dehnh. var. obtusa Two of these, E. flindersii Boomsma and E. Blakely, are widespread along the water courses gillenii Ewart & L. R. Kerr, are native to South in most parts of eastern (var. camaldulensis), Australia, while E. rudis Endl. occurs only in central and Western Australia (var. obrusa). South-westem Australia with E. exserra F. Muell. More detailed descriptions of the occurrence of being a native of South-eastem Queensland. Five these eucalypts have been given elsewhere.7*8 other species, E. dwyeri Maiden & Blakely, E. To our knowledge the oils of E. see an^,^ E.

CCC 0882-5734/%/010035-U7 Received 21 February I995 0 1996 by John Wiley & Sons, Ltd. Accepted 18 April 1995 36 C. M. BIGNELL ETAL.

Table 1. Oil yields from Eucalyprus species, Section Exsertaria"

Oil yield Species and locality wt% (dry weight) Eucalyptus seeana Maiden 0.37 Waite arboretum, South Australia E. bancroftii (Maiden) Maiden 0.92 Waite arboretum, South Australia E. parramattensis C. Hall 1.02 Waite arboretum, South Australia E. amplifolia Naudin 0.39 Waite arboretum, South Australia E. tereticornis J. Smith 0.62 Waite arboretum, South Australia E. blakelyi Maiden 1.13 Black Mountain, Canberra E dealbura A. Cunn. ex. Schauer 1.77 Wittunga arboretum, South Australia E. dwyeri Maiden & Blakely 2.34 Waite arboretum, South Australia E. vicina L.A.S. Johnson & K.D. Hill 1.19 Manara Hill. New South Wales E. flindersii Boomsma 0.78 Waite arboretum, South Australia E. camaldulensis Dehnh. var. camaldulensis 0.30 Waite arboretum, South Australia E. camaldulensis Dehnh. var. obtusa Blakely 1.50 Ormiston Gorge, Northern Territory E. rudis Endl. 0.73 Waite arboretum, South Australia E. exserra F. Muell. 0.35 Waite arboretum, South Australia E. gillenii Ewert & L.R. Kerr 0.64 Waite arboretum, South Australia

The specimens for these species were authenticated by Mr M.I.H. Brooker, Australian National Herbarium or Dean Nicolle, Valley Orchids, South Australia.

bancroftii,' E. parramattensis,' E. am lijolia, lo The dry powder was then vacuum distilled so that E. tereticornis,'* '-' E. blakelyi, g-14 E. the leaf oil condensed on to a gold-plated copper dealbata,9"3 E. dwyeri,I2 E. camaldulensis var. rod maintained at approximately -75°C. camaldulen~is,'~~'~*'~E. camaldulensis var. Complete details of this procedure have been obtusa,'* E. rudis' and E. e~serta'"~"~have been published previously.l7 All oils obtained were investigated previously. colourless to pale yellow liquids which floated on water. Table 1 lists the oil yields (wt%, dry weight) for the fifteen species studied. EXPERIMENTAL Analytical gas chromatography (GC) was car- ried out on a Shimadzu GC6 AMP gas chromato- For each species samples of clean, mature leaves graph. A glass SCOT column of SPlOOO (85 m X were picked from over ten positions on a single 0.5 mm) which was programmed from 65°C to tree and, after drying and freezing with liquid 225°C at 3"C/min was used with helium carrier nitrogen, were reduced to a fine powder using a gas. The GC integrations of the peaks were per- stainless steel Waring blender (Model no. SS110). formed on a SMAD electronic integrator. GC VOLATILE LEAF OILS OF EUCALYPTUS SPECIES 37

analyses were also performed with a HP5890 cineole and 997 s for torquatone. Torquatone was Series I1 unit operated in conjunction with a found to be present in almost all oil samples; HP3396 Series I1 integrator. The 'on-column' in- when this was not the case a sufficient amount was jection technique was used with a SGE BP20 added to the oil solution to obtain that reference capillary column of (25 m X 0.33 mm i.d., and point. (A sample of pure torquatone was kindly film thickness 0.5 pm). The carrier gas was hydro- supplied by Dr Emilio Ghisalberti, Chemistry gen with an inlet pressure of 25 kPa: the flow rate Department, University of Western Australia.) was 2.0 cm3/min. The oven was programmed to The normalized retention times of the column rise from 80°C to 220°C at 5"/min, and the inlet were identified with oil components analysed pre- temperature set to 83°C and increased at the same viously by GC-MS for over 75 eucalyptus species: rate as the column. Using these conditions and a many of these results have been published.'-6 2.0 p1 sample of a 0.2% solution of oil in purified All GC analyses were performed in duplicate dry ether essentially all the components were re- and the retention times and percentage composi- corded by the integrator in 31 minutes. GC-MS tions of each component averaged. Duplicate was performed on a VG Quattro mass spec- times were discarded if they differed by more trometer operating at 70 eV ionization energy. than one second. Components which contributed The GC column in this case was a DB-Wax (60 less than 0.06% to the final analyses were not m X 0.32 mm). Compounds were identified by considered (an arbitrary but practical decision). comparison of their GC retention indices to Figure 1 is a typical chromatogram (average areas known compounds and by comparison of their (log scale) versus normalized times) obtained mass spectra either with known compounds or from the oil of an E. camaldulensis var. camaldu- published lensis tree estimated to be over 100 years in age. Only three of the species (E. parrumaftensis,E. rereticornis and E. dwyeri) were analysed with GC-MS. The oil components of the rest were RESULTS AND DISCUSSION identified using normalized retention times. For this purpose the column was calibrated by assum- Freshly isolated oils obtained by vacuum distilla- ing times for three markers, l,&cineole, octa- tion of powdered leaves from single trees were decane (OD added to the ether) and torquatone. analysed by GC-MS and GC. It was necessary to The raw retention times were first normalized to powder the leaves to rupture the oil glands. If 525 s for OD, and times before and after OD unpowdered leaves are used absolutely no oil adjusted by assuming linearity and using 99 s for is obtained. It appears that the membranes

50 I I I I I I] 5c I 0.5 0.05

5- - 0.5 - 0.05 -

NORMALISED RETENTION TIMES

Fig. 1 Normalized chromatogram (see text) for Eucalyptus curnaldulensis Dehnh. var. camaldulensis: areas (log scale) versus normalized retention times in seconds Table 2. Compounds identified and their percentage occurrence (10.05%) in the leaf oils of Eucalyprus species, Section Exsertaria" mW

.-e -u u 2 2 -A5 -, -.9 E $ k5 5X P No. Compound di) d

1 a-Pinene 4.08 3.98 1.44 4.61 1.51 1.78 2.19 8.74 1.78 12.10 3.99 14.72 0.59 4.76 8.01 2 a-Fenchene ------0.06 ------0.08 - 3 Camphene - 0.10 - - - - - 0.11 - - - 0.08 - 0.22 0.07 4 p-Pinene 0.08 - 0.08 0.16 0.12 0.07 - 3.54 0.20 0.53 0.15 0.21 - 0.77 6.29 5 Sabinene - - - 0.83 ------0.11 - 6 Myrcene 0.20 - - 0.27 0.28 - - 0.08 - 0.20 0.06 - 0.08 - 7 a-Phellandrene 0.98 0.13 - 0.29 - 0.08 ------3.40 - 8 a-Terpinene - - 0.06 1.89 1.45 ------9 Limonene 2.17 1.14 0.75 2.14 2.06 3.05 - 1.78 2.43 4.44 0.80 3.56 0.29 1.31 2.39 - 1.79 - 10 f3-Phellandrene - - - - 0.12 ------P 61.89 74.15 50.68 m 11 1.8-Cineole 10.10 77.19 71.50 24.27 8.45 67.24 80.67 68.14 64.12 48.67 - 2.49 0.08 0.06 12 y-Terpinene 4.76 - - 0.18 0.14 0.07 - - 5.13 0.16 0.24 - - 5 0.07 0.10 0.08 13 p-irons-Ocimene - 0.08 0.09 ------0.08 - - z 14 24.89 0.90 0.64 0.81 27.74 3.02 1.07 0.63 0.78 4.78 21.88 0.67 1.69 0.79 0.32 p-Cymene 0.16 - - r I5 Terpinolene - 0.39 - - 0.13 - - 0.13 - - - - - 0.34 - 1.34 - - - 0.28 - h 16 Isoamyl isovalerate - - - - '3 17 0.12- 0.06 - - - - - 0.07 0.14 0.08 - - L a-p-Dimethyl styrene - 0.48 - 18 a-Cubebene - - - 0.35 0.60 ------? 0.14 - 0.09 - - 0.24 - - 0.06 - 0.08 19 a-Copaene - - - - - 20 0.10 - - 2.78 - - - - 0.06 - - - 0.66 - a-Gurjunene - 0.37 - - 21 Camphor ------22 - - - 0.96 - - 0.70 - - CldilSO 0.15- 0.67 - - - 0.88 - - - 23 trans-p-Menth-2-en-1-01 0.11 ------0.48- 0.39 1.36 3.75- 0.37 0.89 1.34 0.66 24 Pinocawone 0.21 0.86 0.67 0.50 0.06 - - - 0.42 - 0.22 0.15 1.23 0.43 25 Fenchol - 0.16 0.09 ------0.12 0.10 - - 0.26 26 p-Elemene - - 1.22 - - - - 0.11 - 4.65 - - - - 0.15 - - 0.15 0.21 - 27 p-Caryophyllene 1.43 - - - - 28 (Terpinen4ollAromadendrene) 0.57 1.68 6.35 23.97 2.70 1.97 0.91 1.84 3.92 2.92 3.79 3.27 2.17 0.45 10.53 29 a-Bulnesene - - 0.26 0.84 - - - 0.08 - 0.10 - 0.12 0.07 0.06 0.29 30 CIS& - - - - - 0.17 ------31 Myrtenal 0.18 0.07 0.10 0.07 - - - 0.31 0.08 0.09 - - - - 0.12 32 cis-p-Menth-2-en-1-01 - - - 0.72 - - - 0.32 - 1.01 - - 0.18 0.30 33 allo- Aromadendrene 0.52 0.48 1.76 4.74 0.25 0.45 - 0.70 0.95 0.95 2.16 0.77 3.42 0.28 2.58 34 rrans-Pinocarveol 0.43 2.25 1.55 1.43 0.09 1.26 1.33 3.01 8.57 0.90 0.51 2.92 - 2.79 2.11 0.47 35 CisHz4 ------36 a-Humulene 0.55 ------37 6-Terpineol - 0.14- - - 1.33 - 0.13 - 0.12 - - - 0.11- 0.13 38 Cryptone 0.67 0.13 - 14.95 - - 0.09 0.20 - 14.06 - 0.07 - - - - 39

22 82 00 00

2% 8 00 10

4 I 1121 I I I I I I I I I I I

0-I- N-!? 000

0Fl I

-00hl 2 lo 40 C. M. BIGNELL ETAL. surrounding the leaf oils guard their contents with for terpinen-4-01, those components in the great tenacity. The results for 15 species of sec- table between the dashed lines are the tricyclic tion Exsertaria are listed in Table 2. The principal sesquiterpenes together with the bicyclic bicyclo- components in each oil were the monoterpenes a- germacrene. As found in Part VI, it appears that pinene (1.5-15%), P-pinene (0.1-6.3%), 1,8- the process of hydrodistillation partially or almost cineole (0-81%) and p-cymene (0.6-28%). completely converts bicyclogermacrene into There were smaller amounts of limonene (0- tricyclic sesquiterpene products. These trans- 4.4%) and y-terpinene (0.1-5.1'3'0). Apart from formations have been noted previou~l~~-*~and 178-cineole, the main oxygenated monoterpenes possible mechanisms suggested. In such transfor- detected were pinocarvone (0.1-3.8%) and trans- mations one would expect the total concentration pinocarveol (O.l-8.6%). of bicyclogermacrene plus all the sesquiterpenes The principal sesquiterpenes encountered in to be conserved. That this is not the case is seen these species were the hydrocarbons bicyclo- by inspection of the total percentages at the bottom germacrene (0.1-6770)~allo-aromadendrene (0- of Table 3. 4.7%) and the related alcohols, globulol (0.2- The results of some previous studies of the oils 6%), viridiflorol (0.2-1.8%) and spathulenol (0- of E. seeana,' E. bancroftii,' E. parramattensis,' 16%), as well as y-eudesmol (0.1-0.5%), a- E. amplifolia,'" E. tereticornis,' E. dealhata,' E. eudesmol(0-1.5%) and P-eudesmol(O-2.9%). It carnaldulensis var. camaldulensis,'6 E. rudis' and was not possible to resolve the sum of the concen- E. exserta' are not very detailed, so that the trations (0.6-24%) of the components aromaden- results of this study are in substantial agreement drene and terpinen-4-01. The aromatic ketone with the data in these publications. Because of the torquatone was detected in all species studied transformations during hydrodistillation reported (0.1-0.7%). here and in Part VI of this study' we do not expect In Part VI of this series we indicated that the our results to agree with the more detailed composition of the volatile leaf oils, obtained 1.12.14. IS reported for the above species from the vacuum and hydrodistillation tech- using hydrodistillation techniques. In particular niques, may differ quite markedly. Once again, Zrira et af. ,13 using hydrodistillation, have recently we have found a similar effect in this study for oils reported oil analyses for E. camaldulensis, E. isolated from the same sample of leaves of E. exserta, E. tereticonsis, E. blakelyi and E. deal- rudis (see Table 3). Before distillation the leaves bata which differ markedly from the results of this were powdered in an SS-WaringBlender. l7 Except study.

Table 3. Oils obtained using two different techniques to extract oil from Eucalyptus rudis Emil. Vacuum Hydrodistillation Distillation C.M.B./P.J.D.

a-Pinene 0.6 0.9 a-Phellandrene 3.1 1.6 1.8-Cineole 2.5 8.2 p-Cymene 1.7 5.6

( AromadendrcnelTerpinen-4-01) 2.2 4.7 0110- Aromadendrene 3.1 2.5 Viridiflorene 0.9 5. I Bicyclogermacrene 67.4 11.0 Globulol 0.7 7.7 Viridiflorol 0.6 4.6 Spathulenol 6.4 21.5

y-Eudesrnol 0.1 0.3 a-Eudesmol 0.1 0.4 P-Eudesmol 0.2 0.5 Other unidentified CI5Hz60alcohols I .2 7.9 Total percentages (between the two dashed lines) of bicyclogermacrene 81.6 51.1 and tricyclic sesquiterpenes VOLATILE LEAF OILS OF EUCALYPTUS SPECIES 41

Acknowledgements -The authors thank Mr Ian Brooker, Eucalypts, Especially in Regard ro their Essential Oils, 2nd Australian National Herbarium, and Dean Nicolle, Valley edn, Government Printer, Sydney (1920). Orchids, South Australia, for identifying the species and 10. R. 0. Hellyer and H. H. G. McKern, Ausr. 1. Chem., 19, helpful discussions. We are grateful to Professor Harold 1541 (1966). Woolhouse. Director of the Waite Agricultural Research 11. M. P. Shiva, G. S. Paliwal and K. Chandra, Indiun Institute, and Dr Jennifer Gardner, Curator of the Waite Forester, 110, 23 (1984). Arboretum, for their interest in this study. This work was 12. J. J. Brophy in D. J. Boland, J. J. Brophy and A. P. N. supported in part by a grant from the Australian Research House, Eucalyptus Leaf Oih, lnkata Press, Melbourne Council to P. J. D. and J. F. J., and a grand from the Austra- (1991). lian Council for International Agricultural Research 13. S. S. Zrira, B. B. Benjilali, M. M. Fechtal and H. H. (ACIAR) to J. J. B. Richard, 1. Essenr. Oil Res., 4, 259 (1992) 14. M. Holeman, M. Rombourg, M. Fechtal, J. P. Gonichon and G. Lassaigne, Plantes mkdicinales et phytothhapie, 4, 311 (1987). 15. J. C. Doran and J. J. Brophy, New Foresfs,4, 25 (1990). REFERENCES 16. A. Gandini, Ann. Chim., 26, 344 (1936). 17. R. B. Inman, P. J. Dunlop and J. F. Jackson in Modern 1. Part I. C. M. Bignell, P. J. Dunlop, J. J. Brophy and J. F. Methods of Analysis New Series, ed. H. F. Linskens Jackson, Flavour and Fragr. J., 9, 113 (1994). and J. F. Jackson, Vol. 12, p. 201 Springer-Verlag, 2. Part 11. C. M. Bignell, P. J. Dunlop, J. J. Brophy and J. Heidelberg (1991). F. Jackson, Flavour and Fragr. J., 9, 167 (1994). 18. S. R. Heller and G. W. A. Milne, EPAlNIH Mass 3. Part 111. C. M.Bignell, P. J. Dunlop, J. J. Brophy and J. Spectral Data Base, US Government Printing Office. F. Jackson, Fluvour and Fragr. J., 9, 309 (1994). Washington, DC (1978, 1980, 1983). 4. Part IV. C. M. Bignell, P. J. Dunlop, J. J. Brophy and J. 19. E. Stenhagen, S. Abrahamsson and F. W. McLafferty, F. Jackson, Flavour and Fragr. J., 10, 85 (1995). Registry of Mass Spectral Data, John Wiley, New York 5. Part V. C. M. Bignell, P. J. Dunlop, J. J. Brophy and J. (1974). F. Jackson, Flavour and Fragr. J., 10,313. 20. A. A. Swigar and R. M. Silverstein, Monoterpenes, 6. Part VI. C. M. Bignell, P. J. Dunlop, J. J. Brophy and J. Aldrich, Milwaukee, WI (1981). F. Jackson, Flavour and Fragr. J., 10, 359. 21. K. Nishimura, N. Shinoda and Y. Hirose, Tetrahedron 7. M. 1. H. Brooker and D. A. Kleinig. A Field Guide to rhe Letters, 36, 3097 (1969). Eucalypls, Vol. 2, lnkata Press, Melbourne (1990). 22. R. Tressl, K.-H. Engel, M. Kossa and H. Koppler. 1. 8. M. I. H. Brooker and D. A. Kleinig, A Field Guide ro rhe Agric. Food Chem. 31, 892 (1983). Eucalypts, Vol. 1 (rev. edn), lnkata Press, Melbourne 23. J. Garnero and R. Tabacchi in Capillary Chromatography (1!@0). in Essential Oil AM~YS~S,ed. P. Sandra and C. Bicchi. p. 9. R. T. Baker and H. G. J. Smith, A Research on rhe 362, Heutig, New York (1987).