FLAVOUR AND FRAGRANCE JOURNAL, VOL. 12, 433±437 (1997)

Aromatic of Tropical Central Africa. Part XXX. Studies on Volatile Leaf Oils of 10 Species of Eucalyptus Naturalized in RwandaÃ

The reÁ se Molangui,1 Chantal Menut,1 Philippe Bouchet,1 Jean-Marie BessieÁ re2 and Jean-Bosco Habimana3 1Laboratoire de Chimie Organique Physique, Universite de Montpellier II, 34095 Montpellier CEDEX 5, France 2Laboratoire de Phytochimie, ENSCM, 34053 Montpellier CEDEX 1, France 3Institut de Recherche Scienti®que et Technologique, B.P. 227 Butare, Rwanda

The leaf essential oils of ten species of Eucalyptus () naturalized in Rwanda were analysed by GC and GC±MS coupling on two capillary columns of di€erent polarity (OV101 and Carbowax 20 M). About 60 compounds have been identi®ed. The major constituents of these oils are widespread monoterpenes (pinenes, 1,8-cineole, limonene, piperitone, terpinyl acetate, etc.: 65.3±99.1%), except for E. fastigata Deane & Maiden oil, which contains mainly sesquiterpenes (56.5%) of which the eudesmols (37.0%) make a large contribution. # 1997 John Wiley & Sons, Ltd.

Flavour Fragr. J. 12, 433±437 (1997) (No. of Figs: 0 No. of Tables: 4 No. of Refs: 22)

KEY WORDS: Eucalyptus anceps (R. B. ex Maiden) Blakely; Eucalyptus calophylla R. Br. ex Lindley; Eucalyptus cornuta Labill.; Eucalyptus dives Schauer; Eucalyptus diversicolor F. Muell.; Eucalyptus fastigata Deane & Maiden; Eucalyptus gummifera (Sol. ex Gaertner); R. Baker; Eucalyptus pilularis Smith; Eucalyptus piperita Smith; Myrtaceae; essential oil composition; monoterpenes; 1,8-cineole; eudesmols; piperitone; terpinyl acetate

INTRODUCTION (Gaertn.) K. D. Hill and L. A. S. Johnson and E. callophylla becomes Corymbia calophylla With a view to selecting individuals suitable for (Lindl.) K. D. Hill & L. A. S. Johnson. essential oil production, we have continued the screening of Eucalyptus essential oils from Central EXPERIMENTAL Africa.2±4 In this paper we present the results of our analyses of sample oils obtained from 10 species Material and Extraction grown in the Ruhande arboretum (Rwanda): The 10 species examined originate from the . Two species belong to the subgenus Corymbia Ruhande arboretum (Butare, Rwanda). For (C): E. calophylla R. Br. ex Lindley and each species, 200±300 g of leaves were collected E. gummifera (Sol. ex Gaertner). in January 1993. The botanical identi®cation of . Five species to the subgenus Monocalyptus the material collected was carried out by (M): E. dives Schauer, E. fastigata Deane & L. Ntezurubanza and voucher specimens were Maiden, E. oreades R. Baker, E. pilularis Smith deposited at the National Herbarium of Butare. and E. piperita Smith. After air-drying, the plant material was sub- . The others are met in the subgenus Symphyo- jected to hydrodistillation using a Clevenger-type myrtus (S).5 apparatus. The essential oils were obtained with an extraction yield varying between 0.2% and 3.2% It is worth noting that the subgenus Corymbia (w/w, calculated on a dry basis). has been recently separated from Eucalyptus and elevated to the status of genus;6 consequently, Gas±Liquid Chromatography E. gummifera is now Corymbia gummifera The compounds were ®rst tentatively identi®ed à For Part XXIX in this series, see Ref. 1. by peak enrichment and by their GC retention Correspondence to: Chantal Menut. indices on two fused-silica capillary columns

CCC 0882±5734/97/060433±05$17.50 Received 11 July 1996 # 1997 John Wiley & Sons, Ltd. Accepted 19 September 1996 434 T. MOLANGUI ET AL.

Table 1. Botanical identity and essential oil yields of 10 Eucalyptus species grown in Rwanda

Subgenus Section Series Species Code5 Yield (%) Corymbia (C) Rufaria (CA) Gummiferae (CAF) E. calophylla R. Br. ex Lindley CAFUF 0.3 E. gummifera Sol. ex Gaertner CAFUA 0.2 Monocalyptus (M) Renantheria (MA) Obliquae (MAK) E. oreades R. Baker MAKDA 1.0 E. fastigata Deane & Maiden MAKCB 0.4 Pilulares (MAI) E. pilularis Smith MAIAA 0.2 Piperitae (MAT) E. dives Schauer MATEB 3.2 E. piperita Smith MATHA 1.3 Symphyomyrtus (S) Bisectaria (SI) Cornutae (SIC) E. cornuta Labill. SICBA 1.6 Dumaria (SL) Dumosae (SLE) E. anceps (R. B. ex Maiden) Blakely SLE: IB 3.1 Transversaria (SE) Diversicolores (SEB) E. diversicolor F. Muell. SEB: A 0.2

(25 m  025 mm i.d. coated with OV101 and Table 2. Chemical composition of leaf oils of the two 25 m  0:22 mm i.d. coated with Carbowax Eucalyptus species belonging to the subgenus Corymbia 20 M), using a chromatograph (Shimadzu GC-14 Constituent Content (%) A) equipped with a Shimadzu C-R4A Chromato- pac Integrator. Detector and injector temperatures E. callophylla R. E. gummifera Sol. were set at 2508C and 2108C respectively; the oven Br. ex Lindley ex Gaertner temperature was programmed from 50 to 2008C at E†-Hex-2-enal Ð 2.0 58C min1, with nitrogen as the carrier gas. Z†-Hex-3-enol 1.5 1.4 The percentage compositions were obtained n-Hexanol 0.6 Ð a-Thujene 0.3 1.0 from electronic measurements using ¯ame ioniza- a-Pinene 46.0 4.8 tion detection without taking relative response Sabinene Ð 0.7 factors into account. b-Pinene 0.6 37.6 Z†-Hex-3-enyl acetate 0.1 2.4 Myrcene 0.1 1.0 Gas±Liquid Chromatography±Mass Spectrometry a-Phellandrene 0.1 0.5 All the samples were then analysed by GC±MS, a-Terpinene 0.2 2.0 p-Cymene 7.1 3.1 using a Hewlett-Packard capillary GC±quadrupole Limonene 0.2 Ð MS System (Model 5970) ®tted with a 25 m  b-Phellandrene Ð 2.0 0:23 mm i.d. fused-silica column coated with DB-1 1,8-Cineole 7.8 1.3 programmed as follows: 608C (1 min), 60±2508C g-Terpinene 17.0 4.0 (58C min1). Helium was used as carrier gas at a Terpinolene 0.7 2.1 1 Terpinen-4-ol 2.2 12.1 ¯ow rate of 0.9 ml.min ; the mass spectrometer a-Terpineol 1.5 4.3 was operated at 70 eV. Bornyl acetate 0.5 Ð The identi®cation of the compounds was based Terpinyl acetate Ð 1.2 on a comparison of retention indices and mass Aromadendrene 0.6 0.5 allo-Aromadendrene 0.2 0.5 spectra with those of authentic samples and with Bicyclogermacrene 0.3 1.2 7;8 literature data. d-Cadinene 0.1 1.0 Spathulenol 0.3 1.0 Globulol 1.2 3.4 RESULTS AND DISCUSSION epi-Globulol 0.7 3.4 Cubenol Ð 0.5 The botanic identity and the oil yields (a range of g-Eudesmol 0.5 0.1 b-Eudesmol 0.5 Ð 0.2±3.2%) of the 10 Eucalyptus species are given in a-Eudesmol 0.5 0.2 Table 1. These results con®rm the important yield Farnesols‡ 5.6 Ð variation between species commonly found in ‡ Eucalyptus; the highest oil content was found Correct isomers not determined. in E. dives and the lowest in E. calophylla and E. pilularis. respectively the species belonging to the sub- Chemical composition of the di€erent samples genera Corymbia, Monocalyptus and Symphyo- are given in Tables 2, 3 and 4, which group myrtus.

# 1997 John Wiley & Sons, Ltd. Flavour and Fragrance Journal, Vol. 12, 433±437 (1997) VOLATILE LEAF OILS OF EUCALYPTUS SPECIES FROM RWANDA 435

Table 3. Chemical composition of leaf oils of the ®ve Eucalyptus species belonging to the subgenus Monocalyptus

Constituent Content (%)

E. oreades E. dives E. fastigata E. pilularis E. piperata R. Baker Schauer Deane & Maiden Smith Smith Diisopropyl ketone ± ± 1.6 ± ± E†-Hex-2-enal ± ± 0.5 2.0 0.1 E†-Hex-2-enol ± ± 0.6 4.3 0.2 Isobutyl isobutyrate ± ± 1.0 ± ± a-Thujene 1.6 2.6 0.5 1.2 0.5 a-Pinene 0.3 0.3 15.5 9.0 1.3 b-Pinene ± ± 0.1 1.3 0.5 Myrcene 1.0 1.5 ± 0.1 0.6 a-Phellandrene 13.4 12.4 0.1 2.0 ± a-Terpinene 3.2 1.0 ± ± ± p-Cymene 13.0 14.4 0.3 31.1 9.3 Limonene ± 0.5 2.0 3.7 15.0 b-Phellandrene 14.5 2.2 ± ± ± 1,8-Cineole 0.5 ± 1.0 2.4 52.0 E†-b-Ocimene 0.2 0.2 ± 1.0 ± g-Terpinene 0.5 0.5 ± 1.1 ± Terpinolene 1.0 1.4 ± 0.5 0.1 Linalol 1.6 1.0 3.0 ± 0.6 Thujanols 10.3 1.6 ± 1.3 3.1 Terpinen-4-ol 5.5 6.1 0.2 9.3 2.4 a-Terpineol 1.5 1.2 1.0 1.3 8.1 Piperitols(cis ‡ trans) 3.3 ± ± ± 1.0 Piperitone 0.3 52.2 ± ± 0.5 Thymol ± ± 0.5 ± ± Benzyl butyrate ± ± 1.0 ± ± Phenylethyl isobutyrate ± ± 0.2 ± ± Aromadendrene ± ± 2.3 0.5 ± a-Humulene ± ± 0.6 0.6 0.7 Bicyclogermacrene 1.0 ± ± ± ± d ‡ g† Cadinenes 1.0 ± ± 1.3 ± Elemol ± ± 0.7 ± ± Palustrol ± ± 0.6 ± ± Spathulenol 0.3 ± 3.3 6.0 0.1 Caryophyllene oxide ± ± ± 2.1 ± Globulol ± ± 3.3 1.0 0.3 epi-Globulol ± 1.7 1.0 0.3 Humulene oxide ± ± ± 1.0 ± g-Eudesmol 1.8 ± 11.0 ± 0.3 Bulnesol ± ± 2.0 ± ± b-Eudesmol 1.7 ± 18.0 ± 0.3 a-Eudesmol 1.5 ± 8.0 ± 0.6 Farnesol ‡ ± ± 5.6 ± ± Lepidozenal? ± ± ± 3.1 ± ‡ Correct isomer not determined.

Most of the analysed volatile oils are made up of . 1,8-Cineole for E. cornuta (77.0%) and monoterpenes (65.3±99.1%), except for E. fasti- E. piperita (52.0%). gata extract which contains a majority of sesqui- . Piperitone for E.dives (52.2%). terpenes (56.5%) with large amounts of eudesmols . Terpinyl acetate for E. diversicolor (46.0%). (37.0%). . p-Cymene for E. pilularis (31.1%). Seven samples are distinguished by the presence The species presented in this paper have been of a major compound: previously analysed more or less completely; the . a-Pinene for E. calophylla (46.0%). most recent studies concern E. anceps,9;10 E. dives,9 . b-Pinene for E. gummifera (37.6%). E. diversicolor,11 E. oreades9 and E. piperita12

# 1997 John Wiley & Sons, Ltd. Flavour and Fragrance Journal, Vol. 12, 433±437 (1997) 436 T. MOLANGUI ET AL.

Table 4. Chemical composition of leaf oils of the three (75%), E. camphora subsp. aquatica (33%), Eucalyptus species belonging to the subgenus Symphyomyrtus E. dawsonii (61%), E. quadrangulata (50%) and 9 Constituent Content (%) E. wilcoxii (35%). The sample of E. piperita examined gave an oil which contains mainly E. anceps E. cornuta E. diversicolor (R. B. ex Maiden) Labill. F. Muell. 1,8-cineole (52.0%) and di€ers from the `typical' oil Blakely rich in piperitone (40±50%) but is comparable to the `variety A' described by Penfold and Morrison,18 a-Thujene 3.1 0.1 ± a-Pinene 0.5 9.5 1.0 with, however, a much higher 1,8-cineole content. Myrcene 1.5 0.1 ± The European pharmacopoeia requires, for Euca- a-Phellandrene 14.0 0.1 ± lyptus essential oils used for medicinal purposes, a a-Terpinene 2.0 ± ± minimum of 70% of 1,8-cineole and an a-phellan- p-Cymene 16.0 2.0 16.0 19 Limonene 0.7 2.0 6.2 drene content of less than 1%. The essential oil b-Phellandrene 17.3 ± obtained from E. cornuta acclimatized in Rwanda, 1,8-Cineole ± 77.0 1.0 which contains 77.0% of 1,8-cineole and 0.1% of g-Terpinene 0.5 0.2 17.1 a-phellandrene, may be commercialized without any Terpinolene 1.0 ± 0.5 further recti®cation treatment. Linalol 1.1 ± a ‡ b†-Pinene oxide ± 0.5 ± Studies on biological activity have been carried Thujanols 20.0 ± ± out, among others, on volatile extracts of Terpinen-4-ol 6.0 0.3 2.1 E. calophylla, E. diversicolor and E. piperita grown a-Terpineol 2.0 1.2 7.4 in Morocco.20;21 In particular, a sample of cis‡trans†-Piperitols 6.3 ± 20 Piperitone 6.7 0.1 ± E. diversicolor tested by Hmamouchi et al., with Terpinyl acetate ± ± 46.0 a mean content of 27.7% in 1,8-cineole and 16.4% in terpinyl acetate, revealed an appreciable anti- fungal and antibacterial activity. essential oils, the others having been examined No correlation may be clearly deduced from the prior to 1950. All screened samples can be related species' botanical classi®cations and their oil to chemotypes already described in literature, those chemical compositions except for the high content from E. anceps, E. fastigata and E. oreades being of pinenes in the essential oils of the two repre- the most complex, without any compound reaching sentatives of the subgenus Corymbia (E. calophylla 20.0%. and E. gummifera). Boland and Brophy22 have First reported by Penfold and Morrison,13 also mentioned this diculty in establishing a E. dives is known to exist in several chemical relationship between the chemical characteristics or physiological forms.12;14 The one we examined of species and their place within the genus. is a piperitone variant (52.2%),15 whose oil may Nevertheless, these authors noticed that if oil be commercialized and used for manufacture yields are moderate to high (> 1%), 1,8-cineole is of crystalline †-, employed both as a most likely the major component. If the oil yield is ¯avouring agent and as an additive to various low, then a-pinene is usually the principal compo- medicinal preparations. nent. Among the oils we have studied, this Terpinyl acetate is the main constituent of tendency has been con®rmed only for E. cornuta the essential oil of E. diversicolor (46.0%), which and E. piperita extracts. can be produced for perfumery purposes.16 This preliminary screening would be followed In a sample of the same species, Dellacassa by a systematic study of the individual variation et al.11 have detected terpinyl acetate in a lower of essential oil yield and chemical composition, proportion (31.0%) and 1,8-cineole in a higher in function of the vegetative cycle. Already, proportion (35.0%) than us. These compounds E. cornuta, E. dives and E. diversicolor from have been found to be 11.0% and 29.7% respect- Rwanda seem to be the most attractive for the ively in the essential oil from a Moroccan variety production of the essential oil for use in industry. studied by Zrira et al.17 The essential oil isolated from E. fastigata is dominated by a; b; g†-eudesmols (37%). This chemo- REFERENCES variety may be classi®ed with other Eucalyptus 1. Part XXIX. C. Eyele Mve -Mba, C. Menut, J. M. BessieÁ re, species characterized by high content of eudesmols: G. Lamaty, L. Nze Ekekang and J. Denamganai, J. Essent. E. beyeri (63%), E. camphora subsp. camphora Oil Res., 9, 369 (1997).

# 1997 John Wiley & Sons, Ltd. Flavour and Fragrance Journal, Vol. 12, 433±437 (1997) VOLATILE LEAF OILS OF EUCALYPTUS SPECIES FROM RWANDA 437

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# 1997 John Wiley & Sons, Ltd. Flavour and Fragrance Journal, Vol. 12, 433±437 (1997)