Extensive Study on the Minor Constituents of the Essential Oil of Eucalyptus dives Schau. Type

Journal of Essential Oil Research: JEOR, Nov/Dec 2006 by Weber, Berthold, Hartmann, Beate, Stöckigt, Detlef, Kohlenberg, Birgit, Et al

Abstract

An oil of the piperitone type of Eucalyptus dives produced from trees cultivated in South Africa was analyzed by GC and GC/MS. Seventy-three constituents were characterized with piperitone ranging from 29.2% to 64.9%, depending on whether the oil was produced from dried or fresh leaves. Two novel constituents such as p-menth-5-en-2-one and p-menth-6-en-3-one were characterized as minor constituents of the oil; the former being found in nature for the first time.

Key Word Index

Eucalyptus dives Myrtaceae, essential oil composition, piperitone, p-menth-5-en-2-one, p-menth-6-en-3-one.

Introduction

Trees of the species Eucalyptus dives can be differentiated into four chemotypes (type, and var. A, B, and C) depending on their chemical composition (1,2). Eucalyptus dives Schau, type, which is rich in piperitone, α-phellandrene and p-cymene, grows naturally in the coastal regions of Victoria and New South Wales (3), Australia, but is also cultivated in Central and South Africa. In the past, different investigations were done on the volatile leaf oil (4-7), which is important in the flavor and fragrance industry as a raw material source, e.g. piperitone was used as a starting material for the production of thymol and menthol (8,9). During our studies on the essential oil of Eucalyptus dives Schau, type we became interested in the determination of the minor constituents. We identified 73 components by GC/MS analysis and isolated two components of which one at least has not been reported from a natural source before.

Experimental

Plant material and oil isolation: The leaves were harvested from trees of the species Eucalyptus dives Schau, type in South Africa, where these trees are cultivated by Busby Essential oils (PTY) LTD, Lothair, South Africa. A commercial sample obtained by hydrodistillation of the fresh leaves was analyzed. In addition, a sample prepared by hydrodistillation (5 h) of dried leaves (50 g) as a slightly yellow oil (3.5 g) was characterized. For reasons of comparison a slurry of dried leaves (50 g) was stirred for 8 h in Et^sub 2^O. After filtration and evaporation of the solvent 3.1 g of a slightly yellow oil were obtained and analyzed.

Oil analysis: GC/MS analyses of the oil were performed using Agilent s GC 6890 (60 m DB-Wax, film 0.32 pm, 0.25 mm) with KAS 4 and a Hewlett Packard MSD 5973N (70 eV ionization energy, EI mode). The column temperature was programmed from 60°-240°C at 4°C/min and He was used as carrier gas. For preparative gas chromatography a Gerstel MCS-system HP5980 Series gas Chromatograph equipped with a KAS 3 injector and a flame ionization detector (300°C) was used. Preparative separations were performed on a wide bore DB-WAX fused silica column (30 m × 0.53 mm d^sub f^ = 1.0 µm). The column temperature was programmed from 60°-230°C at 3°C/min. Helium carrier gas was used with a column flow of 5 mL/min.

Mass spectra of the isolated substances were acquired using Thermo Finnigan's SSQ 7000 mass spectrometer operating at 70 eV ionization energy and a DB-I column (60 m x 0.25 mm, film thickness 0.25 µm).

GC/FTIR were recorded on a Biorad Tracer System, consisting of a Hewlett Packard 5890 Series II gas Chromatograph (DB-1, 60 m x 0.25 mm, film thickness 0.25 µm; 60°-240°C at 4°C/min, carrier gas: He), a 575C FTIR spectrometer and a tracerunit. NM R spectra were recorded on a Varian ^sup Unity^INOVA with 400 MHz (^sup 1^H-NMR) or 100 MHz (^sup 13^C-NMR) in CDCl^sub 3^ using TMS as internal standard. Chemical shifts of ^sup 13^C-atoms were obtained from gHSQC and gHMBC indirect detection experiments.

Oil fractionation: Compound [1] and [2] were isolated starting with fractional distillation of the essential oil under reduced pressure followed by preparative gas chromatography as described above.

Spectral data: p-Menth-5-en-2-one [1]: ^sup 1^H-NMR (main isomer, CDCl^sub 3^): δ = 0.91 (d, 6.8 Hz, 3H, 10-H), 0.91 (d, 6.6 Hz, 3H, 9-H), 1.14 (d, 7.3 Hz, 3H, 7-H), 1.73 (dqq, 2.1, 6.7, 6.7 Hz, 1H, 8-H), 2.34-2.54 (m, 3H, 3-H, 4-H), 2.89-2.97 (m, 1H, 1-H), 5.65 (ddd,, 1.9,2.8,9.9 Hz, 6-H), 5.70-5.74 (m, 1H, 5-H). GC/MS (70 eV) m/z (%): 41(14), 53(7), 67(14), 68(8), 79(10), 81(22), 95 (100), 110(48), 152(14). GC/FTIR υ (cm^sup -1^) = 3022, 2961, 2932, 2873, 1713, 1464, 1368, 1318, 1140, 796, 724. ^sup 1^H-NMR (minor isomer, CDCl^sub 3^): δ = 0.896 (d, 6.6 Hz, 3H, 10-H), 0.901 (d, 6.7 Hz, 3H, 9-H), 1.19 (d, 7.3 Hz, 3H, 7-H), 1.68 (dqq, 2.1, 6.7, 6.7 Hz, 1H, 8-H), 2.34-2.54 (m, 3H, 3-H, 4-H), 2.79-2.88 (m, 1H, 1-H), 5.69 (ddd,, 1.6, 3.1, 9.9 Hz, 6-H), 5.73-5.79 (m, 1H, 5-H). GC/MS (70 eV) m/z (%): 41(14), 53(7), 67(13), 68(7), 79(10), 81(18), 95, (100), 110(43), 152(14). GC/FTIR υ (cm^sup -1^) = 3025, 2962, 2933, 2873, 1711, 1466, 1368, 1332, 1147. ^sup 13^C-NMR (diastereomeric mixture, CDCl^sub 3^): δ = 16.0/17.2 (q, C-8), 19.3/19.3 (q, C-10), 19.3/19.3 (q, C-9), 32.2/32.4 (d, C-8), 41.0/41.2 (t, C-3), 43.3/43.5 (d, C-1), 44.3/44.6 (d, C-4), 129.9/130.3 (d, C-5), 131.1/131.2 (d, C-6), 213.2/213.6 (s, C-2). p-Menth-6-en-3-one [2]: ^sup 1^H-NMR (CDCl^sub 3^): δ = 0.90 (d, 6.7 Hz, 3H, 10-H), 0.92 (d, 6.7 Hz, 3H, 9-H), 1.70 (dd, 1.0, 1.7 Hz, 3H, 7-H), 2.11 (dqq, 6.7, 6.7, 6.7 Hz, 8-H), 2.17-2.28 (m, 2H, 4-H, 5H^sub a^), 2.39-2.50 (m, 1H, 5-H^sub b^), 2.72 (dm, 20.3 Hz, 1H, 2-H^sub a^), 2.83 (dm, 20.3 Hz, 1H, 2-H^sub b^), 5.52 (m, 1H, 6-H). ^sup 13^C-NMR (CDCl^sub 3^): δ = 19.5 (q, C-10), 21.0 (q, C-9), 22.3 (q, C-1), 25.7 (d, C-8), 28.0 (t, C-5), 44.0 (t, C-2), 53.2 (d, C-4), 118.9 (d, C-6), 131.5 (s, C-1), 210.3 (s,C-3). GC/MS (70 eV) m/z (%):41(26), 53(12), 67(38), 68(76), 69 (100), 79(13), 81(20), 82(23), 83(27), 84(29), 95 (25), 109(31), 110(67), 134(7), 137(7), 152(28). GC/FTIR υ (cm^sup -1^) = 3042, 2960, 2931, 2872,1713, 1443, 1381,1369, 1214, 1154, 786.