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Journal of the Brazilian Chemical Society

Print version ISSN 0103-5053On-line version ISSN 1678-4790

J. Braz. Chem. Soc. vol.29 no.2 São Paulo Feb. 2018

http://dx.doi.org/10.21577/0103-5053.20170156 

Articles

Enantiomeric Ratio Changes of Terpenes in Essential Oils from Hybrid Eucalyptus grandis × E. tereticornis and its Pure Species

Cecilia Naspi1 

Agustín A. Costa1 

Alejandro Lucia1 

Paola G. Audino1 

Hector M. Masuh1  * 

1Centro de Investigaciones de Plagas e Insecticidas (UNIDEF/CONICET), Juan Bautista de La Salle 4397 (B1603ALO) Villa Martelli, Buenos Aires, Argentina

ABSTRACT

Some Eucalyptus species produce oils with biological activities and the effect of their interspecific hybridization on the enantiomeric composition of terpenes has not been reported. The enantiomeric excesses of monoterpenes in the essential oil of Eucalyptus grandis × E. tereticornis and its parental taxa were determined by gas chromatography-mass spectrometry (GC-MS), and to resolve coelutions problems by preparative high performance liquid chromatography (HPLC) and GC-MS with two columns in series. The essential oil composition of the hybrid presented qualitative and quantitative differences with the composition of its parental taxa. Great differences were found for the enantiomeric ratio in monoterpene alcohols among the three essential oils. Our results suggest that the enantiomeric analysis can be a reliable method for the study of how the interspecific hybridization can module the enantiomeric chemical profile in Eucalyptus essential oils. These results suggest the use of interspecific hybridization to improve or expand the source of bioactive compounds.

Keywords: essential oils; enantiomers; Eucalyptus hybrid

Introduction

The genus Eucalyptus (Myrtaceae) is native to Australia, comprises about 800 species and is now one of the most widely cultivated plants in the world due to many favorable characteristics such as high growth rates and wide adaptability to soils and climate.1 Most of Eucalyptus species produce oils, some with biological activities of great importance for pest control, but few of them are being exploited for commercial uses.1,2 In forest plantations, hybrid breeding is gaining more interest as a potential tool to improve tree performance, growth rate, propagation, pulp yield and resistance to pests and diseases.3 Even though the essential oils from Eucalyptus have been widely studied in different species including hybrids there is a lack of information about the enantiomers proportions.3-8

The analysis of enantiomers is an issue of interest in the study of natural products, flavors, pharmacology and organic chemistry. It is well known that both enantiomers of a same molecule may have different biological activities mainly due to differences at the molecular recognition by a protein receptor.9 Terpene enantiomers can differ in the way they are perceived as odorants, not only because of their characteristic aromatic notes but also due to their different odor threshold.10 The analysis of enantiomers in an essential oil can be useful to determine their authenticity in those cases in which one of its components is present in a high enantiomeric purity, or exists in a specific enantiomeric ratio.11,12 Moreover, the determination of chiral compounds can be used for defining chemotypes in aromatic plants and for establishing their geographical origin.13 In addition to this, the enantiomeric ratio of terpenes can vary depending on the part of the plant in study.14

Many different chiral stationary phases based on permethylated cyclodextrins have been reported for their capability to solve different enantiomeric pairs in plant volatiles and essential oils.12,13,15,16

Due to the complexity of the composition of some essential oils and the similarity in chemical structure of their components, some terpenes usually coelute in gas chromatography (GC). To solve this problem, high performance liquid chromatography (HPLC) may be a useful alternative as a pre-fractionation technique. In fact, essential oil characterization by HPLC analysis has previously been reported based on both direct and reverse phase chromatography and UV, fluorescence, diode array and mass spectrometric detection.17-19 In consequence, a semi-preparative separation by HPLC for its later analysis by gas chromatography could be suitable for terpene characterization in essential oils.20,21

The objective of this work was to use this combination of techniques to determine the enantiomeric composition for the main chiral monoterpenes in the essential oil of the hybrid Eucalyptus grandis × E. tereticornis and its relationship with E. tereticornis Smith and E. grandis W. Hill ex Maiden. The effect of the interspecific hybridization on the enantiomeric composition of terpenes has not been reported up to the present.

Experimental

Plant material

Eucalyptus grandis W. Hill ex Maiden, Eucalyptus tereticornis Smith and the hybrid Eucalyptus grandis × E. tereticornis were used for this study. The plant samples were obtained from an experimental field located at 34° 33' 42'' S, 58° 30' 39'' W (Centro de Investigaciones de Plagas e Insecticidas (CIPEIN), Villa Martelli, Buenos Aires, Argentina). The experimental field was built by two months old seedlings. The Eucalyptus species were acquired in Forest Nurseries, registered at INASE-SENASA (National Seeds Institute of the Republic of Argentina-National Service of Agriculture Sanity and Quality). E. tereticornis, E. grandis and the hybrid E. grandis × E. tereticornis were purchased at Paul Forestal, Forest Nursery (No. INASE J/5188, San Isidro, Buenos Aires Argentina).

Essential oil extraction

Fresh leaves (two years old) were submitted to steam distillation in our lab using a modified Clevenger-type apparatus. The extraction was performed for 70 min, time at which the yield remained constant.4 Five independent extractions, consisting of a mixture of nine individuals, were performed for each Eucalyptus species. After extraction, the essential oil was separated from water and dried over anhydrous sodium sulfate. The five extractions were pooled to obtain one sample per species. The essential oils were maintained under -4 °C in argon atmosphere until use.

Chemical standards

All chemical standards were purchased from Sigma-Aldrich® Argentina: (S)-(-)-α-pinene (98%), (R)-(+)-α-pinene (98%), (R)-(+)-camphene (80%), (S)-(-)-camphene (80%), (S)-(-)-β-pinene (98%), (R)-(+)-β-pinene (98%), α-terpinene (85%), p-cymene (99%), (S)-(-)-limonene (96%), (R)-(+)-limonene (97%), 1,8-cineole (99%), γ-terpinene (97%), linalool (97%), (S)-(-)-borneol (90%), (S)-(+)-4-terpineol (96%), α-terpineol (99%), caryophyllene oxide (99%).

Essential oils chemical composition

It was combined the gas and high performance liquid chromatography (GC and HPLC) to determine and quantify the chemical composition of the essential oils of the Eucalyptus species (Figure 1). The first step was to analyze the composition of essential oils by GC using two columns, DB-5 and DB-WAX, and two detectors, mass spectrometry (MS) and flame ionization detector (FID) (Figure 1a, Essential oil composition by gas chromatography-mass spectrometry analysis section). To solve the enantiomers, it was used the GC with a chiral column, CycloSil-b, and the detectors mentioned above (Figure 1b), Enantiomeric analysis of Eucalyptus essential oils section). Preparative HPLC was performed to solve problems of coelution of certain molecules (Figure 1c), Fractionation of the essential oil by high performance liquid chromatography section). Finally, the fractions obtained with preparative HPLC were analyzed by GC using a non-polar column, DB-5MS, and two columns in tandem, DB-WAX + CycloSil-β and an MS as detector (Figures 1d and 1e, HPLC fraction analysis by GC-MS section).

Figure 1 Representative diagram of techniques and methodologies used to separate pure compounds and enantiomers and finally, to determine the proportion of the (+) and (-) enantiomers in the Eucalyptus essential oils. (a) Essential oil composition by gas chromatography-mass spectrometry analysis section; (b) Enantiomeric analysis of Eucalyptus essential oils section; (c) Fractionation of the essential oil by high performance liquid chromatography section; (d, e) HPLC fraction analysis by GC-MS section. 

Essential oil composition by gas chromatography-mass spectrometry analysis

The essential oils were analyzed in a Shimadzu GC-17A gas chromatograph interfaced to a Shimadzu quadrupole mass spectrometer (GCMS-QP5050A), and equipped with a DB-5MS column (J&W Scientific, Agilent Technologies, 30 m × 0.25 mm i.d., 0.25 μm film thickness). The GC conditions were programmed as follows: injector and detector temperature were 250 and 260 °C, respectively. Initial oven temperature was held at 60 °C for 3 min, then increased to 100 °C (2 °C min -1) and finally up to 240 °C (7 °C min -1) with a final hold time of 3 min. Helium (99.995%) was used as carrier gas at a flow rate of 1.6 mL min -1 (constant column pressure of 100 kPa). The essential oil samples were diluted in hexane (1 mg mL -1), and 0.3 µL were injected with a split ratio of 1:30. The essential oils solutions were also analyzed in GC-MS with a polar DB-WAX column (J&W Scientific, Agilent Technologies, 30 m × 0.32 mm i.d. × 0.25 μm film thickness). The temperature ramp started at 40 °C (3 min hold time) and then increased up to a final temperature of 200 °C (3 °C min -1) with a final hold time of 3 min. Column inlet pressure was set up at 35 kPa (53.6 cm s -1 linear velocity), flow column was 2.2 mL min -1 with a split ratio of 1:10 (injection volume: 0.5 µL). Injector and detector were set at 240 and 245 °C, respectively.

The mass spectra were recorded between 45 and 350 amu, with ionization energy of 70 eV. Quantification with FID was performed using normalized relative peaks areas obtained from the GC chromatograms. Individual components were identified by different methods: (i) mass spectra obtained from each compound were compared against Wiley mass spectral database ver. 7 and NIST database; (ii) comparison of retention times with those of authentic compounds (Sigma-Aldrich®); (iii) comparison of linear retention indexes obtained by co-injection of authentic compounds with a homologous series of n-alkanes (C9-C17, Sigma-Aldrich) in DB-5MS, with those reported in bibliography; (iv) comparison of linear retention indexes obtained in DB-WAX column with those available in bibliography.22-26 Quantitation analyses were performed on a Shimadzu GC-2014 with a flame ionization detector and using a DB-WAX column (J&W Scientific, Agilent Technologies, 30 m × 0.32 mm i.d., 0.25 μm film thickness). Nitrogen was used as carrier gas. The column pressure and flow, temperature program, injector and detector temperatures were equal as GC-MS analysis with DB-WAX column. The analysis for each essential oil was performed in triplicate.

Enantiomeric analysis of Eucalyptus essential oils

The essential oils were analyzed in a Shimadzu GC-17A gas chromatograph interfaced with a Shimadzu quadrupole mass spectrometer (GCMS-QP5050A). The chiral column used was CycloSil-β that consists of 30% heptakis (2,3-di-O-methyl-6-O-t-butyl dimethylsilyl)-β-cyclodextrin in DB-1701 as stationary phase (J&W Scientific, Agilent Technologies, 30 m × 0.25 mm i.d., film thikness 0.25 μm). The GC column was initially programmed at 70 °C (isothermal for 5 min), then up to 83 °C (2 °C min -1), immediately up to 85 °C (0.5 °C min -1) and finally up to 200 °C (5 °C min -1) with a final hold time of 5 min. The carrier gas was helium (99.995%) using a split ratio of 1:30, a constant pressure of 100 kPa, a linear velocity of 45.6 cm s -1 and flow rate of 1.5 mL min -1. The samples were diluted in hexane (1 mg mL -1) and 0.5 μL were injected. The injector and detector temperatures were 235 and 240 °C, respectively. The ionization energy was 70 eV, and mass scan was performed between 45 and 280 amu. For the identification of individual components, retention times were compared with those of authentic compounds and their mass spectra were analyzed using database libraries (Wiley mass spectral database version 7; NIST database). The elution order of each enantiomeric pair was assigned by comparison of retention time with those of authentic standards and confirmed with previous reports.13,16,27 Linear retention indexes were obtained by co-injection of authentic standards with a homologous series of n-alkanes in GC-MS using a different temperature ramp (70 °C, 5 min isothermal; 3 °C min -1 up to 200 °C hold time 5 min) with the other chromatographic conditions having the same value as described before.

Enantiomeric excesses (ee) were calculated from peak areas obtained from GC-FID analyses with a Shimadzu GC-2014 chromatograph using nitrogen as carrier gas, 100 kPa column pressure, 32.1 cm s -1 linear velocity and 1.28 mL min -1 of column flow. The oven temperature program was identical as described before. The enantiomeric excess is expressed as percentage, i.e., [(predominant enantiomer - minor enantiomer)/(predominant enantiomer + minor enantiomer)] × 100.

Fractionation of the essential oil by high performance liquid chromatography

Analytical conditions for HPLC analysis of essential oils

Chemical analysis was performed in a Shimadzu high performance liquid chromatograph consisting of two pumps LC-8A (A and B), an FCV-130AL reservoir selection valve, an SIL-10AF autosampler, a CBM-20A communication bus module, an SPD-M20A photodiode array detector and an FRC-10A fraction collector. An analytical reverse phase column was used (GraceSmart RP 18, 250 × 4.6 mm i.d., 5 μm particle size) at a constant flow rate of 0.8 mL min -1. HPLC analysis was performed with different gradients of acetonitrile (pump A, ACN) and water (pump B): initially a linear gradient from 30 to 50% of ACN (23 min), then an isocratic step (2 min), then a linear gradient from 50 to 60% of ACN (25 min) and finally a linear gradient from 60 to 80% of ACN (20 min). After each analysis, the column was equilibrated to the initial conditions for 10 min with a linear gradient back to the initial conditions: ACN:H2O (30:70). Essential oils samples were diluted in isopropanol (2 mg mL -1) and 5 µL were injected. The chromatogram was acquired using a photodiode array detector (wavelength 190-800 nm) at room temperature (25 °C) using a slit width of 1.2 nm and band width of 1 nm. UV spectra obtained for each peak were compared with reported data.19 All proceedings and chromatograms analyses were performed with LC Solution Software (version 1.23, Shimadzu Corporation, Japan).

Semi-preparative fractionation of the essential oil by HPLC

Chromatographic conditions and solvent gradient were the same as in the analytical analyses. The essential oil samples were diluted in isopropanol (100 mg mL -1) and 5 µL were injected. The HPLC chromatograph was programmed on the basis of slope (µV s -1) and level (µV) to collect each peak in a different vial. The semi-preparative chromatographic procedure was performed five times; each fraction corresponding to the same peak was collected together in a same vial. The fractions were extracted once with 0.2 mL of hexane; the organic layers were dried under anhydrous sodium sulfate and stored at -4 °C until their analysis. The assignment of each peak identity was based on the comparison of the retention times with authentic standards when available and by the analysis of each fraction obtained from HPLC by GC-MS. Maximum absorption wavelengths were also corroborated with reported data (Tables 1-3).19

Table 1 Composition of HPLC fractions from E. grandis 

HPLC fraction Retention time / min Method of identificationa λmax / nm Compound assignment LRIb Percentage in fraction / %
1 15.28 n.i. - n.i. - -
2 15.87 B,D < 200 trans-carveol 1220 67.0
3 17.54 B,D < 200 trans-pinocarveol 1130 89.6
4 18.16 B,D < 200 trans-pinocarveol 1130 3.8
A,B,C,D borneol 1167 7.7
A,B,C,D α-terpineol 1192 88.5
5 19.31 A,B,C,D < 200, 225 (sh), 255 borneol 1167 36.1
A,B,C,D 4-terpineol 1176 24.6
A,B,C,D α-terpineol 1192 39.4
6 21.02 A,B,C,D < 200, 239, 271 1,8-cineole 1029 10.2
A,B,C,D linalool 1101 46.4
A,B,C,D borneol 1167 11.2
A,B,C,D 4-terpineol 1176 5.1
A,B,C,D α-terpineol 1192 3.7
7 21.72 A,B,C,D < 200, 242 1,8-cineole 1029 19.7
B,D fenchol 1116 59.9
8 22.23 A,B,C,D < 200, 244 (sh), 275 1,8-cineole 1029 73.7
B,D fenchol 1116 21.6
9 22.75 A,B,C,D < 200, 256 (sh), 278 1,8-cineole 1029 64.5
B,D fenchol 1116 20.6
A,B,C,D timol 1286 10.1
10 23.52 - - n.i. - -
11 37.95 B,D < 200 spathulenol 1580 44.7
12 42.85 A,B,C,D < 200, 213 (sh), 265 p-cymene 1021 98.5
13 54.57 A,B,C,D < 200 γ-terpinene 1054 99.0
14 55.38 A,B,C,D < 200 camphene 944 7.3
A,B,C,D limonene 1025 62.3
A,B,C,D γ-terpinene 1054 30.4
15 56.85 A < 200 β-pinene - -
16 61.9 A,B,C,D < 200 α-pinene 929 95.3

n.i.: not identified; sh: shoulder.

aMethod of identification: A: comparison with authentic standard in HPLC; B: comparison of MS spectrum against spectral databases (Wiley-Nist-Adams); C: retention time and LRI comparison with authentic standard in GC-MS analysis; D: LRI comparison with DB-5MS column bibiliographic data;

bLRI: linear retention index on DB-5MS.

Table 2 Composition of HPLC fractions from E. tereticornis 

HPLC peak Retention time / min Method of identificationa λmax / nm Compound assignment LRIb Percentage in fraction / %
1 15.25 B,D < 200, 215, 209 (sh) cuminol 1291 80.5
2 16.08 B,D 200 (sh), 230 cryptone 1182 85.5
3 18.19 B,D < 200 cryptone 1182 3.9
A,B,C,D α-terpineol 1192 89.5
4 18.83 B,D < 200, 211 (sh), 240 trans-p-menth-2-en-1-ol 1139 57.0
A,B,C,D α-terpineol 1192 29.1
5 19.32 A,B,C,D < 200 4-terpineol 1176 99.0
6 21.13 A,B,C,D < 200 linalool 1101 46.3
B,D cis-p-menth-2-en-1-ol 1121 5.4
A,B,C,D 4-terpineol 1176 30.7
7 21.63 A,B,C,D < 200 linalool 1101 99.0
8 22.64 A,B,C,D < 200 1,8-cineole 1029 79.7
9 24.59 B,D 200, 225 (sh), 259 cuminaldehyde 1243 98.6
10 36.38 B,D < 200 spathulenol 1580 8.0
B,D isospathulenol 1634 69.4
11 37.84 B,D < 200 spathulenol 1580 96.8
12 42.75 A,B,C,D < 200, 213 (sh), 265 p-cymene 1021 86.4
13 45.58 A,B,C,D < 200, 243 (sh), 277 caryophyllene oxide 1585 85.9
14 50.23 B,D < 200 sabinene 968 99.5
15 51.60 B,D < 200, 207 (sh), 223 β-myrcene 987 92.2
16 53.86 B,D < 200, 245 (sh), 266 β-myrcene 987 16.2
A,B,C,D α-terpinene 1013 31.9
A,B,C,D γ-terpinene 1054 10.3
- n.i. - 41.5
17 54.61 B,D 199 (sh), 230 β-phellandrene 1026 93.7
A,B,C,D γ-terpinene 1054 6.3
18 55.19 B,D < 200, 219 (sh), 235 α-phellandrene 1005 27.2
A,B,C,D 249 (sh), 263 limonene 1025 15.1
B,D β-phellandrene 1026 57.7
19 56.78 B,D < 200 α-tujene 922 13.2
A,B,C,D β-pinene 973 0.4
A,B,C,D α-phellandrene 1005 14.6
A,B,C,D β-phellandrene 1026 52.8
20 61.58 A,B,C,D < 200 α-pinene 929 99.3

n.i.: not identified; sh: shoulder.

aMethod of identification: A: comparison with authentic standard in HPLC; B: comparison of MS spectrum against spectral databases (Wiley-Nist-Adams); C: retention time and LRI comparison with authentic standard in GC-MS analysis; D: LRI comparison with DB-5MS column bibliographic data;

bLRI: linear retention index on DB-5MS.

Table 3 Composition of HPLC fractions from E. grandis × E. tereticornis 

HPLC peak Retention time / min Method of identificationa λmax / nm Compound assignment LRIb Percentage in fraction / %
1 15.95 B,D < 200 trans-carveol 1220 40.4
- n.i. - 26.9
2 17.59 B,D < 200 trans-pinocarveol 1130 89.6
3 18.22 B,D < 200 trans-pinocarveol 1130 11.1
A,B,C,D borneol 1167 3.9
A,B,C,D α-terpineol 1192 84.0
4 19.36 B,D < 200 trans-pinocarveol 1130 2.9
A,B,C,D borneol 1167 27.9
A,B,C,D 4-terpineol 1176 40.7
A,B,C,D α-terpineol 1192 28.6
5 21.52 A,B,C,D < 200 1,8-cineole 1029 98.0
A,B,C,D linalool 1101 0.3
A,B,C,D fenchol 1116 1.2
A,B,C,D α-terpineol 1192 0.1
6 42.32 A,B,C,D < 200, 213 (sh), 266 p-cymene 1021 98.5
7 51.18 - - n.i. - -
8 54.78 A,B,C,D < 200 limonene 1025 70.8
9 55.54 A,B,C,D < 200 limonene 1025 40.5
A,B,C,D β-pinene 973 49.5
10 61.42 A,B,C,D < 200 α-pinene 929 99.3

n.i.: not identified; sh: shoulder.

aMethod of identification: A: comparison with authentic standard in HPLC; B: comparison of MS spectrum against spectral databases (Wiley-Nist Adams); C: retention time and LRI comparison with authentic standard in GC-MS analysis; D: LRI comparison with DB-5MS column with those of bibliographic data;

bLRI: linear retention index on DB-5MS.

HPLC fraction analysis by GC-MS

HPLC fraction analysis by GC-MS with non-polar column

The organic extracts obtained after organic partition of HPLC fractions were analyzed by GC-MS. Chromatographic conditions were similar as described for essential oils analyses: 1.5 µL of each organic fraction were injected in DB-5MS column, using the same temperature ramp program described before, with a split ratio of 1:2 instead. The components in each fraction were identified by: (i) comparison of mass spectra against database (Wiley mass spectral database ver. 7, NIST database); (ii) comparison of linear retention indexes, using a homologous series of n-alkanes (C9-C17, Aldrich) with those of bibliography.22-24

HPLC fraction analysis with chiral GC-MS

The enantiomeric analysis of limonene in E. grandis was performed on fraction 14 obtained from the HPLC fractionation. Analytical conditions were similar as described in Enantiomeric analysis of Eucalyptus essential oils section, using a split ratio of 1:2 instead. The enantiomeric excesses for limonene and β-pinene for E. tereticornis and the hybrid were calculated using a chromatographic system consisting of two columns connected in series. This system consisted of a gas chromatograph (Shimadzu, GCMS-QP5050A) equipped with a DB-WAX column (column 1, J&W Scientific, Agilent Technologies, 30 m × 0.32 mm i.d. × 0.25 μm film thickness) connected in series to a CycloSil-β (column 2, J&W Scientific, Agilent Technologies, 30 m × 0.25 mm i.d., film thickness 0.25 μm) using a column connector (Hewlett-Packard 5041-2174 pk/10). The injector and interface temperatures were set at 240 and 245 °C, respectively. The carrier gas was helium (99.995%), using a constant column pressure of 100 kPa, column flow 0.8 mL min -1 (linear velocity 23.2 cm s -1). The fractions were analyzed using a 1:2 split ratio and 1.5 µL of injection volume. Column oven was initially programmed at 60 °C (isothermal for 5 min), increased at 220 °C (3 °C min -1) with 3 min of final hold time. Chemical assignments were done by comparison of the retention times with available standards and by mass spectrum comparison against database libraries22 (Wiley mass spectral database ver. 7, NIST database). Limonene enantiomers were analyzed in fraction 18 and β-pinene in fraction 19 in E. tereticornis; meanwhile, in E. grandis×E. tereticornis, limonene was analyzed in fraction 11. To improve detection, the lectures of signals were performed in SIM (selected-ion monitoring) mode with m/z 68 for limonene (base peak C5H8 +) and m/z93 for β-pinene (base peak, C7H9 +).

Results and Discussion

Initially, we analyzed the chemical composition of Eucalyptus essential oils by GC-MS/FID with two columns (DB-5MS and DB-WAX). The analysis by GC-MS/FID allowed the identification of most of the terpenes in the essential oil, showing that the main components were monoterpenes (Table 4).

Table 4 Chemical composition of Eucalyptus essential oils analyzed by gas chromatography 

No. Compound LRI Method of identificationc Aread / %
DB-5MSa DB-WAXb EGr ETe EGrETe
1 α-thujene 922 1016 A,C - 1.78 -
2 α-pinene 929 1012 A,B,C 48.89 6.46 25.71
3 camphene 944 1047 A,B,C 0.61 - 0.21
4 sabinene 968 1108 A,C - 1.06 -
5 β-pinene 973 1091 A,B,C 0.41 0.43 2.28
7 β-myrcene 987 1153 A,C - 0.98 -
8 α-phellandrene 1005 1147 A,C - 6.45 -
9 a-terpinene 1013 1163 A,B,C - 0.38 -
10 p-cymene 1021 1250 A,B,C 10.82 15.78 0.53
11 limonene 1025 1184 A,B,C 4.08 2.22 4.60
12 β-phellandrene 1026 1190 A,C - 18.28 -
13 1,8-cineole 1029 1193 A,B,C 12.90 13.30 58.17
14 γ-terpinene 1054 1230 A,B,C 3.59 1.42 -
15 linalool 1101 1548 A,B,C 0.25 0.80 -
16 fenchol 1116 1572 A,C 1.04 - -
17 cis-p-menth-2-en-1-ol 1121 1552 A,C - 0.80 -
18 campholenaldehyde 1123 1465 A,C 0.90 - -
19 trans-pinocarveol 1136 1637 A,C 1.33 - 0.65
20 trans-p-menth-2-en-1-ol 1139 1617 A,C - 0.51 -
21 borneol 1167 1685 A,B,C 2.03 - 0.36
22 4-terpineol 1176 1589 A,B,C 0.34 4.22 0.21
23 cryptone 1182 1635 A,C - 4.48 -
24 α-terpineol 1192 1686 A,B,C 4.96 1.41 1.71
25 cuminaldehyde 1243 1749 A,C - 1.27 -
26 phellandranal 1278 1685 A,C - 0.44 -
27 spathulenol 1580 2105 A,C < 0.1 7.07 -
28 caryophyllene oxide 1585 1944 A,B,C - 0.78 -
29 isospathulenol 1634 2207 A,C - 0.51 -
Monoterpene hydrocarbon 68.40 55.24 33.33
Oxygenated monoterpene 23.75 27.23 61.10
Sesquiterpene hydrocarbon - - -
Oxygenated sesquiterpene 0.1 8.36 -
Total 92.25 90.83 94.43

LRI: linear retention index in

aDB-5MS;

bDB-WAX;

cmethod of identification: A: mass spectrum comparison against library (NIST-Wiley-Adams); B: comparison of retention time and LRI with authentic standard; C: LRI comparison with those of bibliography;

drelative percentage of total area in the chromatogram. EGr: Eucalyptus grandis; ETe: Eucalyptus tereticornis; EGrETe: Eucalyptus grandis × E. tereticornis.

In Eucalyptus grandis, monoterpene hydrocarbons (68.40%) constituted the main components of the essential oil; the most abundant was α-pinene (48.89%), followed by p-cymene (10.82%) and limonene (4.08%). Oxygenated monoterpenes (24.64%) were also present, including 1,8-cineole (12.90%), α-terpineol (4.96%) and borneol (2.03%). A low quantity of the sesquiterpene spathulenol was found in this essential oil. These results are in concordance with those reported previously.4,28

The essential oil of E. tereticornis is characterized by the presence of monoterpene hydrocarbons (55.24%), oxygenated monoterpenes (28.34%) and a small amount of oxygenated sesquiterpenes (8.36%). The most abundant terpenes were 1,8-cineole (13.30%), p-cymene (15.78%) and β-phellandrene (18.28%). Previous reports of E. tereticornis composition informed similar proportion of 1,8-cineole, but higher quantities of α-pinene.29,30 Besides, some essential oils with low quantity of 1,8-cineole were also reported.7,31 This discrepancy in the chemical composition could be attributed to the existence of different chemotypes influenced by climate conditions. The monoterpene hydrocarbon β-phellandrene was present in higher concentration than previously reported.4,7,30,32 In this essential oil, some oxygenated sesquiterpenes were also found: spathulenol (7.07%) was the most abundant and has previously been found in E. tereticornis.7,32 Some minor components, such as limonene, cryptone, 4-terpineol, α-terpineol and cuminaldehyde are commonly found in this essential oil.4

The analysis of the essential oil of the hybrid E. grandis× E. tereticornis showed an enrichment in the oxygenated monoterpene, 1,8-cineole (58.17%) compared to the parents composition. This enrichment, as consequence of interspecific hybridization, was previously found in other studies.4,28 Limonene (4.60%) was found in a similar proportion than in E. grandis; meanwhile, β-pinene (2.28%) was more abundant respect to the pure species in accordance with results previously reported.5 The α-pinene (25.71%) is present in an intermediate concentration with respect to the parental composition. The hybridization resulted in a lack of sesquiterpenes and there was not novel biosynthesis of terpenes in the hybrid in accordance to literature.8,33

In addition to this initial analysis, we analyzed the enantiomeric composition of some monoterpenes by using GC-MS/FID with a CycloSil-β capillary column (Enantiomeric analysis of Eucalyptus essential oils section). As a result, eight enantiomeric pairs of monoterpenes were determined: α-pinene, camphene, β-pinene, limonene, linalool, 4-terpineol, borneol and α-terpineol based on their presence in at least two of the essential oils. The order of elution for each enantiomer was assigned by comparison with authentic standards and is in concordance with those reported in bibliography using equivalent stationary phases.9,12,13,16 The elution orders of (+) and (-) enantiomers of linalool and α-terpineol were obtained from previous reports with equivalent column (Table 5).9,13

Table 5 Enantiomeric excess and predominant enantiomer of chiral monoterpenes in Eucalyptus essential oils using CycloSil-β column in GC-FID 

Enantiomeric compound LRI Enantiomeric excess (predominant enantiomer)a / %
E. grandis E. tereticornis E. grandis × E. tereticornis
(S)-(-)-α-Pinene 977 89.03 (+) 47.21 (+) 84.81 (+)
(R)-(+)-α-Pinene 980
(S)-(-)-Camphene 1010 74.30 (+) b 43.27 (+)
(R)-(+)-Camphene 1015
(R)-(+)-β-Pinene 1025 34.72 (-) 50.18 (-)a 83.84 (-)
(S)-(-)-β-Pinene 1031
(S)-(-)-Limonene 1059 61.48 (+) 29.15 (+)a 5.10 (+)a
(R)-(+)-Limonene 1075
(R)-(-)-Linalool 1219 75.06 (+) 82.13 (+) b
(S)-(+)-Linalool 1226
(S)-(+)-4-Terpineol 1301 64.53 (+) 82.57 (-) 11.2 (-)
(R)-(-)-4-Terpineol 1308
(S)-(-)-Borneol 1344 88.56 (+) b 20.6 (-)
(R)-(+)-Borneol 1348
(S)-(-)-α-Terpineol 1352 66.10 (+) 31.08 (+) 7.20 (-)
(R)-(+)-α-Terpineol 1362

aEnantiomeric excess calculated by prefractionation with HPLC and analysis with double column system (DB-WAX+CycloSil-β);

bmeans not present in the essential oil. LRI: Linear retention index relative to an homologous series of n-alkanes; enantiomeric excess is expressed as percentage and predominant enantiomer and showed in parenthesis.

At this moment, we were able to determine the enantiomeric excess of six enantiomeric pairs: α-pinene, camphene, linalool, 4-terpineol, borneol and α-terpineol. However, β-pinene and limonene still coeluted with other components, thus interfering with their resolution (Figure 1). In these cases, when the samples are complex, it is common to observe coelution of two or more pure or enantiomeric components, resulting in an overlap of signal peaks observed in the total ion chromatogram. For example, (i) in the analysis of E. tereticornis essential oil, the retention time of (+)-β-pinene coincided with sabinene (unknown enantiomer), while (+)-limonene coeluted with β-phellandrene and 1,8-cineole. (ii) In E. grandis× E. tereticornis high peak areas of 1,8-cineole also overlapped the signal of (+)-limonene (Figure 1c).

To solve these coelutions of components we used the pre-fractionation by HPLC and these fractions generated were characterized by GC-MS with a DB-5MS column or two columns in a series (DB-WAX + CycloSil-β). In the last case, DB-WAX column was connected to the injector port and the CycloSil-β column was connected to the detector.

As a result of HPLC analysis in reverse phase, the monoterpene alcohols eluted first, followed by sesquiterpene alcohols and finally monoterpene hydrocarbons. Despite this, p-cymene, an aromatic oxygenated monoterpene, eluted after spathulenol but prior to caryophyllene oxide, both oxygenated sesquiterpenes. Cryptone was the only ketone terpene found, present only in E. tereticornis, and eluted in second place after the aromatic alcohol cuminol. As it is observed in the HPLC traces, many peaks were partially resolved not only for monoterpene alcohols but also for monoterpenic hydrocarbons (Tables 1-3). Many monoterpene hydrocarbons have such a similar structure that they are not properly resolved even in an analytical run. Our results showed the coelution of limonene with camphene and α-phellandrene. These findings have also been reported in reverse phase HPLC.19

In the fractionation of Eucalyptus tereticornis oil by HPLC, 1,8-cineole was separated from β-phellandrene and limonene, and sabinene was separated from β-pinene. These compounds, 1,8-cineole and sabinene, were determined by GC-MS with the DB-5MS column (HPLC fraction analysis by GC-MS with non-polar column section). The HPLC fractions of the E. tereticornis essential oil containing limonene (which coelutes with β-phellandrene) and β-pinene were used for the chiral analysis with the DB-WAX + CycloSil-β columns (HPLC fraction analysis with chiral GC-MS section). In this case, the first column (DB-WAX) could separate limonene (KI 1184) versus β-phellandrene (KI 1190). The enantiomeric excess of limonene in E. grandis and E. grandis × E. tereticornis was calculated through the analyses of the fraction obtained by HPLC with the columns in a series, too (Table 5).

Furthermore, the components of Eucalyptus essential oils were present in their both enantiomeric forms, and their proportion is very important as their bioactivity may be different. Figure 2 shows the proportion of enantiomers for each component determined. In all the compounds evaluated both enantiomers (+) and (-) were found. Linalool, camphene and borneol were not present in at least one of three species analyzed. The proportion of (+) and (-) enantiomers of α-pinene, β-pinene and limonene was similar among E. grandis, E tereticornis and the hybrid. On the other hand, the proportion of the enantiomers of 4-terpineol and α-terpineol from the E. grandis×E. tereticornis was intermediate between the proportions found in its parentals.

Figure 2 Percentage of each enantiomer (+) and (-) for each component in its respective essential oil (E. grandis, E. tereticornis and E. grandis×E. tereticornis). The total amount of each component ((+) and (-) enantiomer) in the essential oil was considered 100% ((+) + (-) = 100%). The grey bar shows the proportion of (-) enantiomer and the black bar the (+) enantiomer. 

Conclusions

This paper provides an exhaust chemical composition analysis of the essential oils of Eucalyptus grandis, E. tereticornis and the hybrid. Combined methodologies like preparative HPLC and GC with columns in a series were useful to resolve coelution problems. The essential oil composition of the hybrid presented qualitative and quantitative differences with the composition of its parental taxa. Furthermore, differences in the enantiomeric proportions of optically active components were also observed.

Finally, our results suggest that the enantiomeric analysis of terpenes can be a reliable method to study how the interspecific hybridization can module the chemical profile in Eucalyptus essential oils as potential sources of bioactive compounds.

Acknowledgments

This investigation was financially supported by Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, Argentina) PICT 0797-2008.

References

1 Brooker, I. In Eucalyptus: The Genus Eucalyptus (Medicinal and Aromatic Plants - Industrial Profiles); Coppen, J. J. W., ed.; Taylor & Francis Inc.; London, UK, 2002, ch. 1.1. [ Links ]

2 Batish, D. R.; Singh, H. P.; Kohli, R. K.; Kaur, S.; For. Ecol. Manage. 2008, 256, 2166. [ Links ]

3 Varshney, V. K.; Pandey, A.; Thoss, V.; Kumar, A.; Ginwal, H. S.; Ann. For. Res. 2011, 55, 53. [ Links ]

4 Lucia, A.; Licastro, S.; Zerba, E.; Masuh, H.; Entomol. Exp. Appl. 2008, 129, 107. [ Links ]

5 Baptista, E. B.; Zimmermann-Franco, D. C.; Lataliza, A. A. B.; Raposo, N. R. B.; Rev. Soc. Bras. Med. Trop. 2015, 48, 746. [ Links ]

6 Elaissi, A.; Medini, H.; Khouja, M. L.; Simmonds, M.; Lynen, F.; Farhat, F.; Chemli, R.; Harzallah-Skhiri, F.; Chem. Biodiversity 2011, 8, 352. [ Links ]

7 Dellacassa, E.; Menéndez, P.; Moyna, P.; Soler, E.; Flavour Fragrance J. 1990, 5, 91. [ Links ]

8 Farah, A.; Fechtal, M.; Chaouch, A.; Biotechnol., Agron., Soc. Environ. 2002, 6, 163. [ Links ]

9 Tkachev, A. V.; Russ. Chem. Rev. 2007, 76, 951. [ Links ]

10 Ravid, U. In Selected Topics in the Chemistry of Natural Products; Ikan, R., ed.; World Scientific Publishing: Singapore, 2008, ch. 6. [ Links ]

11 Bicchi, C. D.; Amato, A.; Rubiolo, P.; J. Chromatogr. A 1999, 843, 99. [ Links ]

12 Ruiz del Castillo, M. L.; Blanch, G. P.; Herraiz, M.; J. Chromatogr. A 2004, 10, 87. [ Links ]

13 Bisht, D.; Chanotiya, C. S.; Rana, M.; Semwal, M.; Ind. Crops Prod. 2009, 30, 422. [ Links ]

14 Borg-Karlson, A. K.; Lindström, M.; Norin, T.; Persson, M.; Valterová, I.; Acta Chem. Scand. 1993, 47, 138. [ Links ]

15 König, W. A.; Gehrcke, B.; Icheln, D.; Evers, P.; Dönnecke, J.; Wang, W.; J. High Resolut. Chromatogr. 1992, 15, 367. [ Links ]

16 Pragadheesh, V. S.; Yadav, A.; Singh, M.; Chanotiya, C. S.; Nat. Prod. Commun. 2013, 8, 221. [ Links ]

17 Ross, M. S. F.; J. Chromatogr. A 1978, 160, 199. [ Links ]

18 Newbery, J. E.; Lopez de Haddad, M. P.; Charlwood, K. A.; Anal. Chim. Acta 1983, 147, 387. [ Links ]

19 Turek, C.; Stintzing, F. C.; Anal. Bioanal. Chem. 2011, 400, 3109. [ Links ]

20 Chamblee, T. S.; Clark Jr., B. C.; Radford, T.; Iacobucci, G. A.; J. Chromatogr. A 1985, 330, 141. [ Links ]

21 Buiarelli, F.; Cartoni, G. P.; Coccioli, F.; Leone, T.; J. Chromatogr. A 1996, 730, 9. [ Links ]

22 Adams, R. P.; Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th ed.; Allured Publishing Corporation: Carol Stream, Illinois, USA, 2007. [ Links ]

23 Kim, T. H.; Lee, S. M.; Kim, Y. S.; Kim, K. H.; Oh, S.; Lee, H. J.; Food Chem. 2003, 83, 151. [ Links ]

24 Asuming, W. A.; Beauchamp, P. S.; Descalzo, J. T.; Dev, B. C.; Dev, V.; Frost, S.; Ma, C. W.; Biochem. Syst. Ecol. 2005, 33, 17. [ Links ]

25 Goodner, K. L.; LWT - Food Sci. Technol. 2008, 41, 951. [ Links ]

26 Lee, S. J.; Umano, K.; Shibamoto, T.; Lee, K. G.; Food Chem. 2005, 91, 131. [ Links ]

27 Liberto, E.; Cagliero, C.; Sgorbini, B.; Bicchi, C.; Sciarrone, D.; Zellner, B. D.; Mondello, L.; Rubiolo, P.; J. Chromatogr. A 2008, 1195, 117. [ Links ]

28 Lucia, A.; Gonzalez Audino, P.; Seccacini, E.; Licastro, S.; Zerba, E.; Masuh, H.; J. Am. Mosq. Control Assoc. 2007, 23, 299. [ Links ]

29 Batista-Pereira, L. G.; Fernandes, J. B.; Corrêa, A. G.; Silva, M. F. G. F.; Vieira, P. C.; J. Braz. Chem. Soc. 2006, 17, 555. [ Links ]

30 Kaur, S.; Singh, H. P.; Batish, D. R.; Kohli, R. K.; J. Med. Plants Res. 2011, 5, 4788. [ Links ]

31 Ogunwande, I. A.; Olawore, N. O.; Adeleke, K. A.; Konig, W. A.; J. Essent. Oil Res. 2003, 15, 297. [ Links ]

32 Pino, J. A.; Marbot, R.; Quert, R.; García, H.; Flavour Fragrance J. 2002, 17, 1. [ Links ]

33 Farah, A.; Fechtal, M.; Chaouch, A.; Ann. For. Sci. 2002, 59, 445. [ Links ]

Received: May 7, 2017; Accepted: August 31, 2017

*e-mail: hmasuh@gmail.com

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