FLAVOUR AND FRAGRANCE JOURNAL, VOL. 11,145-151 (19%)

Volatile Leaf Oils of some South-westem and Southern Australian Species of the Genus Eucalyptus. Part MI. A. Subgenus Eudesmia: B. Subgenus Symphyomyrtus: (a) Section Exsertaria; (b) Series Globulares

C. M. Bigell and P. J. Dunlop Department of Chemistry, University of Adelaide, 5005, Australia

J. J. Brophy Department of Organic Chemistry, University of New South Wales, Sydney, NSW 2052, Australia

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

The volatile leaf oils of Eucalyptus gamophylla F. Muell.; E. gongylocarpa Blakely; E. eudesmioides F. Muell. subsp. eudesmioides; E. tetragona (R.Br.) F. Muell.; E. erythrocorys F. Muell.; E. gittinsii Brooker & Blaxell; E. ebbanoenis Maiden; six specimens of E. camaldulensis Dehnh. var. camaldulensis; E. globulus Labill. subsp. glob- ulus; E. globulus subsp. bicostata Maiden, Blakely & J. Simm.; E. globulus subsp. maidenii F. Muell.; and E. glob- ulus subsp. pseudoglobulus Naudin ex. Maiden; isolated by vacuum distillation, were analysed by GC and by GC-MS. Many species contained a-pinene (1.0-47.2%), P-pinene (0-0.9%), 1.8-cineole (0.2-68.8%), p-cymene (0.2-20.1 %), aromadendrene (0-lS.O%), bicyclogermacrene (0-46.6%) and spathulenol (0.1 -29.0%) as pnnci- pal leaf oil components: all species contained torquatone.

KEY WORDS Eucalyptus gamophylla F. Muell.; Eucalyptus gongylocarpa Blakely; Eucalyptus eudesmioides F. Muell. subsp. eudesmioides; Eucalyptus tetragona (R.Br.) F. Muell.; Eucalyptus erythrocorys F. Muell.; Eucalyptus gittinsii Brooker & Blaxell; Eucalyptus ebbanoensis Maiden; Eucalyptus camaldulensis Dehnh. var. camaldulensis; Eucalyptus globulus Labill. subsp. globulus; Eucalyptus globulus subsp. bicostata Maiden, Blakely & J. Simm.; Eucalyptus globulus subsp. maidenii F. Muell.; Eucalyptus globulus subsp. pseudoglobulus Naudin ex. Maiden; E. camaldulensis clones; ; leaf essential oil composition; torquatone; mono- and sesquiterpenoids; GC-MS

INTRODUCTION most part are native to southern Australia, are listed in Table 1. A more detailed description of Continuing our investigation of indigenous the occurrence of these eucalypts has been given Australian eucalypts' we have examined the leaf elsewhere?*4 oils of 12 species; seven from subgenus Eudesmia To our knowledge analyses of the oils of E. and five from subgenus Symphyomyrtus: six eudesrnioides (barkoil): E. tetr~gona,6.~E. camal- specimens of E. carnaldulensis var. carnaldulensis d~lensis;-'~E . globulus subsp. glob~lus~'~*'~~'~ belonging to section Exsertaria and four species E. globulus subs bi~ostata,'"'~E. globulus to series Globulares (see system of M. I. H. subsp. maidenii,** 8~1g3~16 and E. globulus subsp. Brooker and D. A. Kleinig' and Flora of Aus- pseudoglobulus" have been published pre- tral$). Locations of all species, which for the viously."

ccc 0882-5734/%/03014547 Received 3 August 1995 0 19% by John Wiley & Sons, Ltd. Accepted 20 September 1995 146 C. M. BIGNELL ETAL

Table 1. Oil yields from several Eucalyprus species. A. Subgenus Eudesmia, B. Subgenus Symphyomyrtus: (a) Section Exsertaria; (b) Series Globulares" Species and locality Oil yield (wt%: dry weight) Subgenus EudesmiabL. D. Pryor & L. A. S. Johnson Eucalyptus gamophylla F. Muell. 2.61 MacDonnell Ranges, Northern Territory E. gongylocarpa Blakely 1.93 Waite arboretum, South Australia E. eudesmioides F. Muell. subsp. eudesmioides 1.31 Waite arboretum, South Australia E. rerragona (R.Br.) F. Muell. 0.08 Waite arboretum, South Australia E. eryrhrocorys F. Muell. 0.54 Waite arboretum, South Australia E. gittinsii Brooker & Blaxell 0.33 Currency Creek arboretum, South Australia E. ebbanoensis Maiden 1.81 Waite arboretum, South Australia

Section Exsertaria Benth. E. camaldulensis Dehnh. var. camaldulensis 0.32 Waite arboretum, South Australia E. camaldulensis Dehnh. var. camaldulensis 0.73 Bolivar arboretum, South Australia (a clone ECLAl from Lake Albacutya, Victoria) E. camaldulensis Dehnh. var camaldulensis 1.43 Bolivar arboretum, South Australia (a clone ECLA6 from Lake Albacutya. Victoria) E. camaldulensis Dehnh. var camaldulensis 0.86 Bolivar aboretum, South Australia (a clone AGl from Lake Agnes, Victoria) E. camaldulensis Dehnh. var camaldulensis 0.19 Bolivar arboretum, South Australia (a clone HlNl from Lake Hindmarsh, Victoria) E. camaldulensis Dehnh. var camaldulensis 0.07 Bolivar arboretum, South Australia (a clone SWI from Swanport. South Australia)

Series Globulares Bhkely E. globulus Labill. subsp. globulus 1.83 Adelaide Parklands, South Australia E. globulus subsp. bicosrata Maiden, Blakely & J. Simm 0.77 Wittunga arboretum, South Australia E. globulus subsp. maidenii F. Muell. 1.63 Waite arboretum, South Australia E. globulus subsp. pseudoglobulus Naudin ex. Maiden' 1.55 Mt. Drummer, Victoria (S37"34'1E149"21')

~~ The specimens for these species were authenticated by Mr. M. I. H. Brooker, Aus- tralian National Herbarium or Dean Nicolle, Valley Orchids, South Australia. 'Unpublished. 'These specimens were selected by Mr M. 1. H. Brooker and Mr. A. Slee. National Herbarium. Canberra.

EXPERIMENTAL oil yields (wt%, dry weight) for the twelve species studied. As described previ~usly'~the leaf oils were vac- Analytical gas chromatography (GC) was car- uum distilled to yield colourless to pale yellow ried out on a Shimadzu GC6 AMP gas chromato- liquids which floated on water. Table 1 lists the graph. A glass SCOT column of SPlOOO (85 m X VOLATILE LEAF OILS OF EUCALYPTUS SPECIES 147

0.5 mm) which was programmed from 65°C to Two additional unidentified components, X 225°C at 3"C/min was used with helium carrier and Y, were detected in E. erythrocorys. The gas. The GC integrations of the peaks were per- following mass spectra were recorded: formed on a SMAD electronic integrator. GC analyses were also performed with a HP5890 Component X,m/z (YO):194 (2) 152 (2), 137 (3), Series I1 unit operated in conjunction with a 135 (13), 134 (32), 119 (32), 109 (ll), 93 (40), 92 HP3396 Series I1 integrator. The 'on-column' (96), 91 (loo), 79 (31), 77 (20), 59 (13), 43 (67). injection technique was used with a SGE BP20 Component Y, m/z (YO):153 (4), 152 (40), 121 capillary column (25 m X 0.33 mm i.d., film (77), 109 (loo), 93 (46). 91 (50), 79 (87), 77 (24). thickness 0.5pm). The carrier gas was hydrogen 67 (25), 59 (23), 43 (70). with an inlet pressure of 25 kPa: the flow rate was 2.0 cm3/min. The oven was programmed to rise from 80°C to 220°C at 5"C/min, and the inlet RESULTS AND DISCUSSION temperature set to 83°C and increased at the same rate as the column. Using these conditions Freshly isolated oils obtained by vacuum distilla- and a 1.0 p1 sample of 0.4% solution of oil in tion of powdered leaves from single trees were purified dry ether essentially all the components analysed by GC and by GC-MS. The results for were recorded by the integrator in 31 min. the 12 species are listed in Table 2; only those GC-MS was also performed on a VG Quattro components with concentrations greater than mass spectrometer operating at 70 eV ionization 0.05% are reported. The principal components in energy. The GC column in this case as a DB- the oils were the monoterpenes a-pinene Wax (60 m X 0.32 mm). Compounds were identi- (1.0-47.2%), limonene (0.3-7.3%), 1S-cineole fied by their GC retention indices to known (O.2-68.8%) and p-cymene (0.2-20.1 YO).Apart compounds and by comparison of their mass from 1,&cineole, the main oxygenated monoter- spectra with either known compounds or pub- penes detected were trans-pinocarveol(O-2.7%), lished spectra.18-Zo a-terpineol(O-23.5%) and pinocarvone (0-2.8%). Only four of the species (E. rerragona, E. The principal sesquiterpenes encountered in erythrocorys E, camaldulensis and E. globulus these species were aromadendrene (0-15.0%), subsp. pseudoglobulus) were analysed with allo-aromadendrene (0.3-3.7%) and bicycloger- GC-MS. The oil components of the rest were macrene (0-46.6%), and the related alcohols, identified using normalized retention times. For globulol (0.4-7.3%). viridiflorol (0.1-1.9%) and this purpose the column was calibrated by spathulenol (0.1 -29.0%), as well as y-eudesmol assuming times for three markers, 1,8-cineole, (0-4.9%), a-eudesmol(0-6.7%) and P-eudesmol octadecane (OD added to the ether) and tor- (0-12.5%). The aromatic ketone torquatone was quatone. The raw retention times were first detected (0.1-5.5%) in all the 12 species. normalized to 525 s for OD, and times before The mass spectra and retention times of the and after OD adjusted by assuming linearity and two unidentified components of E. erythrocorys using 99 s for cineole and 997 s for torquatone. on a polar column suggest they may be either Torquatone was found to be present in almost all monoterpene diacetates or monoacetates of oil samples; when this was not the case a suf- monoterpene diols. When this species was ficient amount was added to the oil solution to hydrodistilled the yield of component X obtain that reference point. The normalized decreased to approximately 5% while Y com- retention times of the column were identified pletely disappeared. with oil components analysed previously by In Table 2 the six trees belonging to section GC-MS for over 75 Eucalyptus species: some of Exsertaria are all E. camaldulensis var. camal- these results have been published.' dulensis, which occur naturally in the Murray- All GC analyses were performed in duplicate Darling river system.* The last five are clones and the retention times and percentage compo- from trees which occur in Victoria and South sitions of each component averaged. Duplicate Australia. As noted by Doran and Brophy," times were discarded if they differed by more when studying trees of E. camaldulensis var. than 1 s. Components which contributed less obrusa, the sesquiterpene concentrations are than 0.06% to the final analyses were not con- inversely proportional to those of the mono- sidered (an arbitrary but practical decision). terpene cineole. NO - - PI'I oso - SI'O EZO 9L'O - €9'0 SSO- WO- 81'0 90'0 120- 9E.O - ZS'O- pz'- I - - 90'0 - 11.0 - - LZO LOO - - 90'0 60'0 - 80'0 - 80'0 S80 60'0 180 260 PE'O - LE'O - wo SI'O €1'0 6E'O LE'O wo ------€8'0 ------ES'O EE'O - - 90'0- - - 01'0 99'0- 82'0- - - - SO1 - - 11'0- - - WI - - 60'0 LOO - 90'0 - - - PI'O - SI'O ZZ'O- - - - WO 6E'O EP'O - EE'O W'O - EL'I ZS'O SZ'O 80'0 IL'O 11'0 - - 11'0. - 01'0 mo PI'O - LE'O - 90'0 LOO 11'0 80'0

mo - M'O 11'0 - 800 90'0 ------60'0 - - - - - 92'0 - 62'0 0E.O - "0 LOO ZZ'O - 51'0 - - - - ZL'I - - - - - 60'0 - - - 80'0 - - - - 90'0 - - - - - ZI'O ------LE'O 91'0 - - wo 09'1 WZI 58'8 82'0 9L'O ZE'O II'OZ LP'Z 60'0 16'2: SI'O €20 ZZE 80'0 ------SI'O 8L'O ZE'O ZZZ - €1.0 06'0 80'1 LZO - €8.1 - ZLO - 91'0 ------IP'ZS 19P EL6Z 61'99 86PS S8LP ZP'Z- WE 819 OZ'O 90'ES PL'ZE PLS EE'Z 0sI ZI'E 121 n.9 WE 101 ZCI z9 I ZS'E PZE WE 62'0 ri ------OZO - - - 80'0 Li - 01'0 PZ'O - - - - PL'I - - SE'Z EZ.0 9E'O LZO 95'0 IZO 82'0 IS0 PI'O ES'O €1'0 KO IE'O PI'O - - - 08'0 - - - - 9E'O EE'O NO 60'0 SE'O LE'O OZO zz'o 8E'O SI.0 9ZO $80 PZO LZZI %O 101 WPI 90's 16'9 6P'E 6€9Z 6P'P 98'1 196 SI'LP 65'1 eueliasxa :a eysapng:v 32 Terpinen-4-01 1.21 ------2.61 - 1.21 0.68 2.57 3.05 - - - - 33 Aromadendrene 4.83 1.06 6.00 12.57 4.05 7.47 14.96 - 12.10 6.45 1.15 7.17 - 12.31 5.57 12.04 1.16 34 aBulnesene 0.17 - 0.12 0.36 0.11 0.21 0.40 - 0.53 0.30 - 0.27 - 0.40 0.22 0.32 - 35 cis-p-Menth-2- en-1-01 - 0.06 1.24 0.10 0.07 0.08 0.37 0.69 0.07 0.11 0.15 0.13 36 Myrtenal ------0.17 - - - - 0.09 - - - 0.1 1 37 db-Aromadendrene 2.86 1.79 1.20 1.75 0.64 1.49 1.35 2.21 3.40 2.45 0.48 3.73 2.75 2.90 1.XI 2.95 0.28 38 tronr-Pinocarveol - 1.75 0.28 2.30 2.03 1.55 0.56 0.36 1.27 0.13 0.95 0.08 0.66 2.14 2.23 2.73 39 &Terpineol 0.33 ------0.22 - 0.12 - - 0.17 0.34 0.22 40 Humulene 0.10 0.14 - - 0.23 - - - - 0.55 41 Cryptone - 15.36 - 0.10 5.87 9.81 - 42 Cir-Piperitol - 0.12 ------0.07 43 Ned 0.38 0.08 0.33 0.43 0.38 0.28- 0.66 0.59 0.53 0.12- 0.40 0.30 - - 44 Viridaorene 1.28 0.08 0.88 0.41 - 0.60 1.04 - 0.26 - - 0.37 - - 0.08 - 45 a-Terpineol - 0.31 1.35 0.74 23.49 0.76 0.55 0.35’ 1.02 1.74 1.06 0.73 0.85 3.65 1.05 0.84 46 Born01 0.26 0.24 1.65 0.08 0.17 - 0.08 0.10 - 0.06 0.07 ? 47 Verbenone - - 0.13 - - 0.54 0.06 - - - 0.10 - 0.18 2 48 P-Selinene 0.22 0.48 - 0.21 - 0.27 0.35 0.27 - 0.19 0.28 49 a-Selinene ------0.08 - - - 0.31 E;; 50 Amuurokne - 0.12 0.17 0.24 - 0.12 0.31 0.10 0.1 1 - - 0.18 0.51 0.40 - in 51 Piperitone - - - - - 0.26 ------52 Bicyclogermacrene 46.55 2.18 4.69 4.04 21.29 - - 0.06- 0.40 1.20 - - - G 53 cawone - - - - - 0.37 0.06 - - 0.13- 0.23 - 0.16 0.15 0.31 54 tronr-Piperitol 0.16 - - 0.24 0.11 0.55 0.09 0.18 0.16 0.30 0.41 0.14 0.07 0.11 - i? 55 &Cadinenc 052 0.69 0.29 0.70 0.37 0.15 0.36 0.37 0.13 0.38 0.21 0.42 0.17 0.27 0.25 0 56 y-cadinene 0.75 ------0.13 57 cuminal - - - - 2.50 - - - - C 58 Cadina-1,Qdiene - 0.27 0.36 - - 0.39 0.22 - - - - 2 59 Myrted - 0.09 - - 0.20 - - - 0.11 0.23 0.08 0.11 P 60 fronr-p-Mentha-1(7),8- - 5 dien-2-01 0.20 0.28 0.45 0.69 0.30 0.19 0.32 0.18 0.97 0.94 0.59 Y 61 Calamenene - - 0.50 - - 0.15 0.22 - - - - s rA 62 trm-p-Mentha-l,& w dien-691 - 0.15 - 0.12 0.16 0.12 - 0.52 0.14 - 0.13 0.08 0.15 0.12 0.25 0.25 0.45 m 63 p-Cymen-gOl 0.10 - 2.46 0.11 - 0.12 0.15 0.82 0.22 0.21 - 0.46 0.68 0.15 0.09 0.13 0.16 64 cir-p-Mentha-1,8- c1 dien4-01 0.06 0.07 0.08 0.07 0.07 65 cis-p-Mentha- 1(7),8- dien-2-01 0.10 - 0.06 0.52 0.34 - 0.41 0.33 0.11 0.07 0.24 1.12 1.05 66 Calacorene ------0.06 0.15 - - - 0.06 - - - 67 Palustrol 0.17 0.06 0.1 1 0.40 0.14 0.17 0.35 0.14 0.21 0.13 - 0.21 0.17 0.18 0.12 0.16 68 Caryophyllene oxide - - 0.24 69 B-Phenylethyl - propionate 0.12 - - - - 0.09 - 0.14 - 0.09 0.12 0.21 0.17 0.21 - 70 Ci~Ha0 0.27 0.06 0.11 0.36 0.28 0.21 0.12 1.02 0.22 0.14 0.73 0.92 0.17 0.13 0.18 71 C,,I-I~O 0.22 0.06 0.48 1.91 0.25 0.91 1.07 0.15 0.98 0.57 0.69 0.61 1.05 0.61 0.98 72 CISHaO 0.36 0.23 0.15 0.47 0.16 0.25 0.21 0.35 0.31 0.23 0.45 0.53 0.25 0.15 0.23 73 CI5H240 - - 0.1 1 - - - - 0.20 - - 0.07 0.17 - - - 74 ci,I-Iao 0.39 0.11 0.18 0.60 0.49 0.38 0.20 - 0.37 0.22 0.22 0.08 0.26 0.20 0.22 c2 a8 n

Y) K E. gamophylla F. Muell.

Y) x 5. gongylocarpa Blakely

W 5. eudesmioides F. Muell. E ,ubsp.eudesmioides I 5. tetragona (R.Br) b :. Muell.

9 z 5. erythrocorys F. Muell. wY) E. gittinsii P Brooker & Blaxell

E. ebbanoensis Maiden

ui 9 E. camaldulensir Dehnh. 0 var. camaldulensis

W I" E. camaldulensu Dehnh. o! var. camaldulensis (ECLAl) 8 E. camaldulensis Dehnh. 0 var. camaldulensis (ECLA6) W E. camaldulensis Dehnh. 3 var. camaldulensis (AGl) 8 E. camaldulensis Dehnh. in war. camaldulensb (HN1)

00 E. camaldulensis Dehnh. c var. camaldulensis (SW1)

W P W E. globulus Labill. iubsp. globulus 8 E. globulus subsp. bicosrata bo Maiden , Blakely & J. Simm

3 E. globulus subsp. 0 maidenii F. Muell. P E. globulus subsp. b pseudoglobulus Naudin ex. Maiden

'7Y13113N918 'W '3 0s 1 VOLATILE LEAF OILS OF EUCALYPTUS SPECIES 151

Our oil analyses agreed, when comparison 5. A. Blumann. N. Michael and D. E. White, 1. Chem. SOC., was possible, with those of some previous 778 (1953). workers5.68.9.11.13-16 b 6. E. M. Watson, J. Roy. SOC.W. Ausr., 22.113 (1935136). ut not with other^:'*^^*'^ 7. J. J. Brophy and E. V.Lassak, Flavour and Fragr. J., 6, in general many more components were identi- 265 (1991). fied. 8. R. T. Baker and H. B. Smith, A Research on the Eucu- lypts, Especially Oils, 2nd edn. Government Printer, Sydney (1920). 9. A Gandini, Ann. Chim., 26,344 (1936). Acknowledgements-The authors thank Mr Ian Brooker, S. S. Zrira, B. B. Benjilali, M. M. Fenchtal and H. H. Australian National Herbarium, and Dean Nicolle, Valley Richard, J. Essent. Oil. Res.. 4,259 (1992). Orchids, South Australia, for identifying the species and ll. J. C. Doran and J. J. Brophy, in Tropicul Red Gum-A helpful discussions. We are grateful to Professor Harold Source of l,8-Cineole-rich Eucalyptus Oil. New Forests, Woolhouse, Director of the Waite Agricultural Research 4,25 (1990). Institute, and Dr Jennifer Gardner. Curator of the Waite 12. E. Dellacassa, P. Menkndez, P. Moyna and E. Soler. Arboretum, for their interest in this study. This work was Flavour und Frugr. 1..5.91 (1990). supported in part by a pant from the Australian Research 13. J. J. Brophy, in Eucalyptus Leaf Oils, eds (D. J. Boland, Council to P. J. D. and J. F. J., and a grant from the J. J. Brophy and A. P. N. House. lnkata Press, Mel- Australian Council for International Agricultural Research bourne (1991). (ACIAR) to J. J. B. A sample of pure torquatone was kindly 14. A. R. Penfold and F. R. Momson, J. Proc. Roy. SOC. supplied by Dr Ernilio Ghisalberti, Chemistry Department, NSW.,64,210(1930). University of . 15. E. Gildemeister and F. Hoffmann, Die Atherischen ole. Vol. 6,4th edn. Akademie-Verlag, Berlin (l%l). 16. B. N. Rutowski and I. W. Winogradowa, Truns. Sci. Chem. Phum fnst. (Moscow), 39-68 (1927). REFERENCES 17. R. B. Inman, P. J. Dunlop and J. F. Jackson, in Modern Methods of Analysis New Series, eds H. F. Linskens Part XI. C. M. Bignell, P. J. Dunlop, J. J. Brophy and J. F. and J. F. Jackson Vol. 12, pp. 201-219. Springer-Verlag. Jackson, Flavour und Frugr.- 1. 11, 107 (1996) and earlier Berlin (1991). Parts cited therein. 18. S. R. Heliir and G. W. A. Milne, EPANIH Mass M. I. H. Brooker and D. A. Kleinig, A Field Guide to the Spectral Duta Ease. U. S. Government Printing Office, Eucalypts, Vol. 2. lnkata Press, Melbourne (1990). Washington D. C. (1978,1980,1983). G. M. Chippendale, Floru of Australia, Vol. 19. Australian 19. E. Stenhagen, S. Abrahamsson and F. W. McLafferty, Government Publications Service, Canberra (1988). Registry of Mars Spectrul Duta. Wiley. New York (1974). M. I. H. Brooker and D. A. Kleinig, A Field Guide ro the 20. A. A. Swigar and R. M. Silverstein, Monoterpenes. Eucalypts. Vol. 1 revised edn. Inkata Press, Melbourne Aldrich, Milwaukee WI (1981). (1990).