FLAVOUR AND FRAGRANCE JOURNAL, VOL. 11.95-100 (1996)

C. M. BigneU and P. J. Dunlop Department of Chemistry. University of Adelaide, South Australia. 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, Wai!e Agricultural Research Institute, University of Adelaide, South Australia. 5005, Australia

The volatile leaf oils of cylindriflora Maiden and Blakely, E. erythronema Turcz var. erythronema, E. erythronema Turcz var. marginata (Benth.) Domin, E. dielsii C.A. Gardner, E. stricklandii Maiden, E. carnei C.A. Gardner, E. kruseana F. Muell., E. brachyphylla C.A. Gardner, E. ewartiana Maiden, E. orbijolia F. Muell., E. websteriana Maiden, E. crucis Maiden subsp. cruck, E. caesia Benth. subsp. caesia and E. caesia, Benth. subsp. magna Brooker and Hopper, isolated by vacuum distillation, were analysed by GC-MS and by GC. All species contained a-pinene (5.1-49.6%), l$-cineole (4.2-75.1%), p-cymene (0.2-23.5%) and aromadendrene/terpinen-4-ol (0-26.8%) as principal leaf oil components.

KEY WORDS Eucalyptus cylindriflora Maiden and Blakely ; Eucalyptus erythronema, Turcz var. erythronema; Eucalyptus erythronema Turcz var. marginata, (Benth.) Domin; Eucalyptus dielsii C.A. Gardner; Maiden; C.A. Gardner; Eucalyptus kruseana, F. Muell. ; Eucalyptus brachyphylla C. A. Gardner; Maiden; F. Muell; Maiden; Maiden subsp. crucis; Benth. subsp. caesia; Eucalyptus caesia Benth. subsp. magna, Brooker and Hopper; ; leaf essential oil composition; torquatone; mono- and sesquiterpenoids; GC-MS

INTRODUCTION All of these species are native to southern West- em Australia. A more detailed description of the Continuing our investigation of indigenous Aus- occurrence of these eucalypts have been given tralian eucalypts’ we have examined the leaf oils elsewhere.* To our knowled e the oils of E. of fourteen species belonging to Subgenus Sym- erythr~nema,~E. srricklandii! E. caesia,6 and phyomyrtus, Section Bisectaria: four from Series E. orbifolia’ have been investigated previously. Elongatae, two from Series Stricklandiae, two from Series Kruseanae and six from Series Orbi- foliae (see system M. I. H. Brooker and D. A. EXPERIMENTAL Kleinig2 and , Vol. 193.) Lo- cations of individual species are listed in Table 1. Samples of clean, mature leaves were picked from

CCC 0882-5734/%/020095-06 Received 15 March 1995 0 1996 by John Wiley & Sons, Ltd. Accepted 20 June 1995 % C. M. BIGNELL ET AL.

Table 1. Oil yields from the Eucalyprw species. Series Elongatae, Series Strick- landiae, Series Kruseanae and Series Orbifoliae" Oil yield Species and locality wt% (dry weight) Series Elongatae Blnkely E. cylindripora Maiden & Blakelyb 1.46 North of Esperence. (S33"08'49"/E1229xi'290) E. eryrhronema Turcz. var. erythronema 2.36 Waite Arboretum, South Australia E. eryrhronema Turcz. var. mrginara (Benth.) Domin 1.70 Waite Arboretum, South Australia E. dielsii C. A. Gardner 4.11 Waite Arboretum. South Australia Series Stricklandiae Bmokef E. srricklandii Maiden 2.81 Waite Arboretum, South Australia E. carnei C. A. Gardner 1.70 Waite Arboretum. South Australia Series Kruseanae Chippendale E. kruseana F. Muell. 0.94 Waite Arboretum, South Australia E. brachyphylla C. A. Gardner 1.69 Waite Arboretum, South Australia Series Orbifolie Brooker and Hopper Eucalyprus ewarriana Maiden 1.48 Valley Orchids Arboretum, South Australia E. orbifolio F. Muell. tr Waite Arboretum, South Australia E. websreriana Maiden 1.38 Waite Arboretum, South Australia E. crucis Maiden subsp. crucis 2.17 Waite Arboretum, South Australia E. caesia Benth. subsp. caesia 0.79 Waite Arboretum, South Australia E. caesia Benth. subsp. magna Brooker & Hopper 0.48 Valley Orchids 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 Aus- tralia. bA botanical voucher specimen (DN1103) has been deposited at the South Australian Herbarium in Adelaide by Dean Nicolle. 'An unpublished Series. over ten sites on single trees and, after drying and ried out on a Shimadzu GC6 AMP gas chromato- freezing with liquid nitrogen, were reduced to a graph. A glass SCOT column of SPlOOO (85 m x fine powder using a stainless steel Waxing blender 0.5 mm) which was programmed from 65°C to (model no. SS110). The dry powder was then 225°C at 3"C/min was used with helium carrier vacuum-distilled so that the leaf oil condensed on gas. The GC integrations of the peaks were to a gold-plated copper rod maintained at ap- performed on a SMAD electronic integrator. GC proximately -75°C. Complete details of this analyses were also performed with a HP5890 procedure have been published previously. All Series I1 unit operated in conjunction with a oils obtained were colourless to pale yellow liquids HP3396 Series I1 integrator. The 'on-column' which floated on water. Table 1 lists the oil yields injection technique was used with a SGE BP20 (wt% ,dry weight) for the fourteen species studied. capillary column of (25m x 0.33mm i.d., and Analytical gas chromatography (GC)was car- film thickness 0.5 pm). The carrier gas was hydro- VOLATILE LEAF OILS OF EUCALYPTUS SPECIES 97 gen with an inlet pressure of 25 kPa: the flow rate Component B, m/z (%), 280 (M+,4) 224 (13), 223 was 2.0cm3/min. The oven was programmed to (loo), 208 (4), 193 (5), 165 (lo), 135 (4), 107 (6), rise from 80°C to 220°C at 5"/min, and the inlet 91 (lo), 77 (8), 57 (20). temperature set to 83°C and increased at the same rate as the column. Using these conditions a 2.0 Torquatone, m/z (%), 280 (M+,7), 224 (12), 223 pI sample of 0.2% solution of oil in purified dry (loo), 208 (4), 193 (5), 165 (12), 139 (ll), 125 ether essentially all the components were re- (18), 97 (23), 79 (%), 77 (33), 43 (82). corded by the integrator in 31 minutes. GC-MS was performed on a VG Quattro mass spectro- meter operating at 70eV ionization energy. The RESULTS AND DISCUSSION GC column in this case was a DB-Wax (60m x 0.32 mm). Compounds were identified by their Freshly isolated oils obtained by vacuum-distil- GC retention indices to known compounds and lation of powdered leaves from single trees were by comparison of their mass spectra either with analysed by GC-MS and by GC. The results for known compounds or published spectra.Y-" four species of series Elongatae, two species of Only three of the species (E. erythronema var. series Stricklandiae, two species of series Kru- marginata, E. caesia subsp. caesia and E. caesia seanae and six species of series Orbifoliae are subsp. magna) were analysed with GC-MS. The listed in Table 2; only those components with oil components of the rest were identified using concentrations greater than 0.05% are reported. normalized retention times. For this purpose the The principal components in the oils were the column was calibrated by assuming times for monoterpenes a-pinene (5.1-49.6%), limonene three markers, 1,&cineole, octadecane (OD (0.3-2.8%), l,&cineole (4.2-75.1%) and p- added to the ether) and torquatone.6 The raw cymene (0.2-23.5%). Apart from 1,8-cineole, the retention times were first normalized to 525 s for main oxygenated monoterpenes detected were OD, and times before and after OD adjusted by trans-pinocarveol (0.1-12.2%), a-terpineol (0.1- assuming linearity and using 99s for cineole and 4.5%) and pinocarvone (0-4.5%). 997 s for torquatone. Torquatone was found to be present in almost all oil samples; when this was not the case a sufficient amount was added to the Me oil solution to obtain that reference point.* The normalized retention times of the column were identified with oil components analysed previously by GC-MS for over 75 Eucalyptus species: some of these results have been published. ' All GC analyses were performed in duplicate and the retention times and percentage compo- sitions of each component averaged. Duplicate Me0 0 times were discarded if they differed by more than one second. Components which contri- buted less than 0.06% to the final analyses were Component A not considered (an arbitrary but practical de- cision). Me The principal ions in the mass spectra of I the unknown components A and B and of tor- quatone are: MeO*oMe Component A, m/z (%), 266 (M+,5), 224 (2), 223 (lOO), 208 (4). 193 (7), 165 (14), 135 (5), 107 Mewc4 (6),91 (14), 77 (13), 43 (50). Me0 0 *A sample of pure torquatone was kindly supplied by Dr Emilio Ghisalberti. Chemistrv*. Deoartment. Universitv of

Western Australia. Comnonent~~ B Table 2. Compounds identifiedand their percentageOccurrence (>0.05%) in the leaf oils of the Eucalyptusspecies"

Series: Elongatae Stricklandiae Kruseanae Orbifoliae 1 a-Pinene 20.37 10.31 8.04 49.57 20.14 33.24 22.37 17.87 5.14 6.49 15.85 16.31 13.92 6.59 2 Camphene 0.06 - - - - 0.06 ------3 4-Methylpent-2-yl acetate - - - - 0.37 - - 0.72 - - - - 4 p-Pinene 0.41 0.30 0.36 0.79 0.48 0.20 0.61 0.35 0.50 0.68- 0.29 0.62- 0.44 0.11 5 Sabinene - - 1.35 ------0.08 - - - - 6 Myrcene 0.07 - 1.06 0.40 - 0.32 0.22 - 0.27 0.24 0.07 7 a-Phellandrene 0.32 0.09 0.68 0.44 - 0.11 0.23 - - 1.31 - - - 8 Limonene 0.49 0.21 0.83 2.57 1.07 3.57 1.81 1.55 2.44 2.80 1.64 1.41 0.37 0.29 9 1.8-Cineole 33.91 56.57 13.02 33.56 13.95 45.38 50.95 57.95 75.10 65.91 33.83 62.33 4.24 5.42 10 a-cis-Ocimene - - - - - 0.06 ------11 y-Terpinene - - 0.07 0.14 - 0.22 0.15 0.09 0.26 0.28 9.77 0.07 12 p-truns-Ocimene 0.08 0.07 ------13 p-Cymene 1.48 1.33 12.95 0.67 0.21 0.99 2.02 1.40 0.65 1.57 23.44 0.76 0.53 0.26 Terpinolene - - - - - 0.12 - - 0.08 - 0.88 - 14 Isoarnyl isovalerate 15 - 0.16 - - - 0.10 0.38 0.12 0.28 Dehydrocineole - 0.08 - - - - - 16 a-Cubebene - 17 - 0.41 0.09 0.15 - - 0.12 0.07 Bicycloelemene ------0.11 0.06- 18 a-Copaene 19 0.22 0.06 - 0.21 0.11 0.06 0.14 0.08 - 0.11 0.28- 0.20 0.12 20 a-Campholenic aldehyde 0.16 - - - 0.07 - - - 0.19 0.12 0.17 21 a-Gujunene 0.13 - 0.11 0.30 0.14 0.39 - - 0.06- - - 22 Camphor ------0.07 23 Linalol - - 0.59 ------trans-p-Menth-2-en-1-01 0.25 0.25 0.07 0.31 0.18 24 Pinocawone ------25 4.49 3.49 0.09 - 0.88 0.09 0.90 1.26 0.36 0.60 1.12 1.84 2.43 Fenchol 0.14 26 p-Gurjunene - - 0.09 0.10 0.13 0.14 0.07 - 27 p-Elemene 0.11 ------28 p-Caryophyllene - 0.08 - 0.11 0.27 0.19 - - - 2.03 1.53 29 1.96 - 4.44 0.36 - - 0.11 - 0. I0 - - - - 30 ( AromadendrenelTerpinen-4-01) 5.56 4.02 1.42 2.06 4.95 3.14 0.22 0.49 0.78 2.59 0.97 26.81 16.37 31 a-Bulnesene 0.13 0.09 - - 0.15 0.07 - - - - - 0.69 0.50 0.15 32 C1SH24 - - 0.07 - - - - 33 Myrtenal 0.07 - 0.29 - - 0.06 - 0.12 - - 34 C15H24 - 0.06 ------0.06 - 35 cis-p-Menth-2-en-1-01 ------0.07 36 allo-Aromadendrene 1.21 0.94 1.46 0.33 1.41 0.58 0.15 0.20 0.13 0.30 - 0.28 4.47 2.93 37 rrans-Pinocaweol 12.23 10.06 0.12 0.84 2.32 0.36 3.03- 4.42 0.53- 0.48 1.08 0.83 3.98 0.27 38 6-Terpineol 0.06 0.06 - - - - 0.09 0.08 0.17 0.21 0.06 0.09 - - 39 Cryptone 0.22 0.08 4.41 - 0.11 - - 0.07 - 0.13 0.41 - 40 CisHz4 - - - 0.08 0.06 0.17 - 0.12 0.35 0.08 0.16 0.12- 41 cis-Piperitol - - 0.06 - - - - - 42 Viridiflorene 0.23 - 0.07 0.22 0.19 ------43 a-Terpineol 0.19 - - 0.42 0.38 - 0.78 0.49 5.26 0.67 0.94 0.16 44 Borneo1 0.21 0.14 0.06 0.18 0.21 0.09 0.17 - 0.08 - 0.07- 0.30 45 Verbenone 0.13 0.34 - - - - - 0.10 - - 46 f3-Selinene 0.25 - - 0.10 - - 0.18 - - - - - 47 a-Selinene 0.09 0.20 - - 0.13 0.16 - 0.14 0.11 0.32 0.57 0.47 48 AMuurolene 0.18 0.22 0.07 0.09 0.07 0.11 0.13 - - - - 0.19 0.10 49 Piperitone - - - - 0.06 0.07 - 0.69 0.16 0.06 - - - 50 Bicyclogermacrene 1.90 0.33 4.55 0.68 - 0.09 0.87 - 5.77 - - - 51 Carvone 0.10 0.13 - - 0.08 0.08 - - 0.10 - - - - - 52 trans-Piperitol 0.07 - - - - 0.17 0.11 0.27 - - - - 53 &Cadinene 0.62 0.08 - 0.17- 0.15 0.14 0.07 0.06 0.16 0.15 - 0.07 0.23 - 54 CisHz - - 0.12 ------55 Myrtenol 0.26 0.24 0.18 - - 0.07 0.11 - - 0.11 56 Cadina-1.4-diene - - - 0.15 - - 0.22 - 0.20 - 57 nans-p-Mentha-l(7),8dien-2-01 0.47 0.62 0.19 0.13 - 0.19 0.11 0.11- 0.16 - - 0.17 58 Calamenene 0.18 ------0.20 - - 59 nans-p-Mentha-1,8-dien-6-01 0.29 0.17 0.10 0.12 - 0.10 0.09 0.11 - 0.10 0.17 60 p-Cymen-8-01 - 0.08 0.25 - - - 0.06- 0.10 0.21 0.09 0.10 - 61 Geraniol - 0.06 - - - - 62 CLSHXO - - - - - 0.18 - - - 0.07 - 63 cis-p-Mentha-1(7),8-dien-2-01 0.51 0.51 0.06 - 0.24 - 0.16 0.10 0.07 0.27 64 Calacorene 0.09 0.09 0.11 0.15 - 0.06 0.06 0.12 65 C15H260 ------0.09 - 66 C15H260 0.09 0.12 0.09 0.06- 0.11 0.16- 0.19 67 Palustrol - - 0.14 - - - - 68 Caryophyllene oxide 0.32 6.12 - 69 p-Phenylethyl propionate - - 0.20 - - 0.20- 0.19- 70 Cl5HxO 0.11 0.09 0.24 0.14 0.06- - 0.25 0.21 71 C15HmO 0.36 0.35 - 0.08 - 0.07 2.33 0.13 72 C15H260 - - 0.57 0.28 - - - 1.63 73 C15HZ6O 0.13 0.10 0.21- 2.57 0.19 0.07 0.09 0.36 0.39 74 C15HZ60 - 0.45 - - - - 75 ClSH260 0.18 0.11 0.07 - 0.11 - - 0.07 0.32 0.36 76 C15HZO 0.16 - - - - - 0.11 0.08 L - - - 0.09 77 Globulol 1.66 1.84- 0.32 0.52 1.88 1.42 0.12 0.35 0.20 0.14 0.22 6.64- 5.60 78 Viridiflorol 0.33 0.56 0.24 0.14 0.49 0.41 0.10 0.10 0.11 0.12 0.08 0.97 0.89 79 C15h60 0.07 - 0.08 - - - - - 0.06 - - 0.18 - fB C15H260 0.13 0.22 0.46 0.07 4.32 0.12 0.07 0.12 0.06 0.07 0.72 0.38 81 C15HZO - - - - 0.11 ------0.32 82 C15H260 0.25 0.19 0.08 0.42 0.34 - 0.07 0.05 0.06- 0.06 - 0.77 0.74 83 Spathulenol 1.19 0.56 19.71 0.14 0.17 - 0.35 0.16 0.08 0.48 - 0.12 0.42 0.19 84 y-Eudesmol 0.10 - 0.23 - 0.10 0.58 0.41 - 0.07 0.40 0.50 0.05 - 85 &Cadino1 0.07 0.09 0.12 0.06 0.29 - - 0.29 - - 86 CISHMO 0.07 - - - - - 0.15 - - - - - 87 a-Eudesmol 0.09 0.20 0.19 0.78 0.11 0.42 1.97 0.76 0.25 0.30 1.80- 1.56 0.20 0.10 88 @-Eudesmol 1.19 1.51 - 1.19 0.15 0.55 6.38 3.67 0.55 0.86 2.79 5.04 0.49 0.11 89 CisHz40 - - 0.23------90 C15H240 0.09 0.09 0.15- - 0.06 0.06 0.18 0.17 0.12 0.17 91 ComponentA - - 0.21 - - - 0.08 - 0.63 1.43 92 Component B - - 0.42 - - 0.18 - - - 0.27 0.99 93 Torquatone 0.18 0.50- 0.64 21.17 0.84- 0.09 1.48 0.39 0.36 0.08 0.07 17.69 28.93 Total percentages: 96.3 97.3 87.4 98.0 85.1 96.2 %.3 96.9 94.5 97.2 %.7 96.0 95.5 82.7 'A dash in a column indicatesa percentage composition between0% and 0.05%. - 100 C. M. BIGNELL ETAL.

The principal sesquiterpenes encountered in REFERENCES the species of these three series were aflo- aromadendrene (0-4.5%) and bicyclogermacrene 1. Part VIII. C. M. Bignell. P. J. Dunlop. J. J. Brophy and (0-5.8%), and the related alcohols, globulol J. F. Jackson, Flavour and Fragr. J.. 11, 43 (1996), and (0-6.6%), viridiflorol (0-1 .O%) and spathulenol earlier parts cited therein. 2. M. I. H. Brooker and D. A. Kleinig. A Field Guide lo the (0-19.7%), as well as y-eudesmol (0-0.6%), a- Eucalyprs, Vol. 2. Inkata Press. Melbourne (1990). eudesmol(O.l-2.0%) and p-eudesrnol 0-6.4%). 3. G. M. Chippendale, Flora of Australia, Vol. 19, Aust. The aromatic ketone torquatone6 (2,4,6- Govt. Publ. Service. Canberra (1988). trimethoxy-3,5-dimethyl- 1-(3-methylbutyroyl)- 4. E. M.Watson. J. Roy. SUC.W. Aust.. 28.247 (1941-42). 5. J. J. Brophy and E. V. Lassak. Flavour and Fragr. 1.. 6. benzene) was detected (0-28.9%) in all but one 265 (1991). of the fourteen species. In addition two com- 6. R. C. Bowyer and P. R. Jefferies. Aust. 1. Chem.. 12.442 ponents, A and B were detected. On the basis of (1959). their mass spectra the tentative structures A and 7. J. J. Brophy and E. V. Lassak, 1. Pruc. Roy. Suc. N.S. W., 119, 103 (1980). B analogous to torquatone are proposed. 8. R. B. Inman. P. J. Dunlop and J. F. Jackson in Modern Our oil analyses agreed with those of previous Methodr of Amlysis. New Series, Vol. 12. ed. H. F. worker^^-^ but in general many more component Linskens and J. F. Jackson. p. 201. Springer-Verlag. compounds were identified. Heidelberg (1991). 9. S. R. Heller and G. W. A. Milne. EPAlNlH Mass Spec- tral Data Base, US. Government Printing Office. Acknowledgements-The authors thank Mr Ian Brooker. Washington, DC (1978. 1980, 1983). Australian National Herbarium, and Dean Nicolle, Valley 10. E. Stenhagen. S. Abrahamsson and F. W. McLafferty. Orchids, South Australia, for identifying the species and help Registry of Mass Spectral Data. John Wiley. New York ful discussions. We are grateful to Professor Harold Wool- (1974). house, Director of the Waite Agricultural Research Institute. 11. A. A. Swigar and R. M. Silverstein, Monoterpenes, and Dr Jennifer Gardner, Curator of the Waite Arboretum, Aldrich. Milwaukee, W1 (1981). for their interest in this study. This work was supported in part by a grant from the Australian Research Council to P.J.D. and J.F.J., and a grant from the Australian Council for Inter- national Agricultural Research (ACIAR) to J.J.B.