Chromatographia

, Volume 69, Issue 1, pp 145–150

GC Comparative Analysis of Leaf Essential Oils from Two Myrtle Varieties at Different Phenological Stages

Authors

    • Aromatic and Medicinal Plants UnitBiotechnologic Center Borj-Cedria Technopark
  • Baya Mhamdi
    • Aromatic and Medicinal Plants UnitBiotechnologic Center Borj-Cedria Technopark
  • Brahin Marzouk
    • Aromatic and Medicinal Plants UnitBiotechnologic Center Borj-Cedria Technopark
Full Short Communication

DOI: 10.1365/s10337-008-0818-9

Cite this article as:
Aidi Wannes, W., Mhamdi, B. & Marzouk, B. Chroma (2009) 69: 145. doi:10.1365/s10337-008-0818-9

Abstract

The essential oils obtained from leaves of two Myrtus communis varieties (baetica and italica), growing wild in North Tunisia, were investigated by GC and GC–MS at their different phenological stages. The highest essential oil yield was observed at the flowering stage with 0.6% (w/w) for italica and 0.4% (w/w) for baetica and 49 compounds were identified. The main essential oil leaf compounds of both myrtle varieties, belonging to the monoterpene class, were α-pinene, 1,8-cineole, limonene and linalool and their percentages showed significant changes during the phenological stages.

Keywords

Gas chromatographyEssential oil compositionPhenological cycleMyrtus communis var. baetica and italica

1 Introduction

Myrtle (Myrtus communis L.) is an evergreen sclerophyll perennial shrub belonging to the Myrtaceae family growing spontaneously throughout the Mediterranean area [1]. In Tunisia, it develops under Quercus suber L. and Q. faginea Lamk. forests in humid and sub-humid bioclimatic stages [2, 3]. However, in Cap Bon and Tunisian Dorsal, myrtle is found in association with Pinus halepensis Mill, Juniperus phoenicea L., Ceratonia siliqua L. and with shrubs such as Pistacia lentiscus L. [4]. Two myrtle varieties are described in old local flora: M. communis var. italica L. and M. communis var. baetica L. [3] which present the same vegetative characters. The morphological difference between the two varieties is the larger size of baetica fruits and leaves.

Myrtle is very aromatic because of the high essential oil content in its leaf, flower and fruit glands. Leaf essential oil has been employed for its antimicrobial, tonic and balsamic properties [5, 6] and was used in culinary, pharmaceutical, therapeutical, industrial and cosmetic fields. In addition, it was the subject of many investigations [718] because of the variability of its composition. All these studies have not specified the myrtle variety and there are only a limited number of publications concerning the essential oil composition of M. communis var. italica L. and M. communis var. baetica L. [19, 20].

In the present work, we compare the chemical composition of leaf essential oils isolated from two Tunisian myrtle varieties at their different phenological stages.

2 Experimental

2.1 Plant Material

Myrtle aerial parts were collected at the fruiting, vegetative and flowering stages respectively in January, April and July 2007 from North Tunisia. In particular, M. communis var. italica was gathered in Jbal Stara of the Haouaria region and M. communis var. baetica in Jbal Er-Rimel of the Bizerte region. These two stations were characterized by calcareous soil and sub-humid bioclimate. The sampling was done by a randomized collection of 15–20 shrubs in an area of about 200 m2 for each variety. Collection always took place in the same geographic area to avoid variability. Only the leaves were analyzed and they were isolated manually from the aerial parts in our laboratory to obtain a weight of 500–700 g of leaves for each variety. Full details of leaf collection data are provided in Table 1. The plant material was botanically characterized by Prof. Abderrazak Smaoui (Botanist at the Biotechnology Center in Borj-Cedria Technopark, Tunisia) and according to the morphological description presented in Tunisia flora [3].
Table 1

Collection data for the myrtle plants used in this study (2007)

Myrtle variety

Location in North Tunisia

Phenological stage

Month

Temperature (°C)

Humidity (%)

Rainfall (mm)

Myrtus communis var. baetica

Bizerte (Jbal Er-Rimel)

Fruiting

January

12.6

88

57.8

Vegetative

April

16.3

88

45.0

Flowering

July

26.5

73

1.6

M. communis var. italica

Houaria (Jbal Stara)

Fruiting

January

14

82

55.9

Vegetative

April

17

82

41.0

Flowering

July

26.9

75

0

2.2 Essential Oil Extraction

At every phonological stage, three lots of 100 g of leaves from each variety were separately hydrodistilled during 3 h (time fixed after a kinetic survey during 30, 60, 90, 120, 150, 180 and 210 min). The hydrodistillation was performed by a simple laboratory Quik-fit apparatus which consisted of a 2,000 mL steam generator flask, a distillation flask, a condenser and a receiving vessel. The volatile compounds of essential oils were collected from the distillate in diethyl ether using a liquid–liquid isolation. In order to quantify essential oils and their constituents, an internal standard, 6-methyl-5-hepten-2-one was used. Essential oil yields were then estimated on the basis of the dry weight of the plant material.

2.3 Chromatographic Analysis

2.3.1 Gas Chromatography (GC-FID)

Essential oils were analyzed by gas chromatography using a Hewlett-Packard 6890 apparatus (Agilent Technologies, Palo Alto, CA, USA) equipped with a flame ionization detector (FID) and an electronic pressure control injector. A polyethylene glycol capillary column (HP Innowax: 30 m × 0.25 mm, 0.25 µm film thickness) and a 5% diphenyl, 95% dimethylpolysiloxane capillary column (HP-5: 30 m × 0.25 mm, 0.52 µm film thickness) were used; the flow of the carrier gas (N2) was 1.6 mL min−1. The split ratio in the injector was 60:1 and 0.4 µL of each sample injected. Analysis was performed using the following temperature program: oven temps isotherm at 35 °C for 10 min, from 35 to 205 °C at the rate of 3 °C min−1 and isotherm at 205 °C during 10 min. Injector and detector temperature were held at 250 and 300 °C respectively.

2.3.2 Gas Chromatography–Mass Spectrometry (GC–MS)

An HP 5890 series II coupled to an HP 5972 mass spectrometer (Agilent Technologies) with electron impact ionization (70 eV) and an HP-5MS capillary column (30 m × 0.25 mm coated with 5% phenyl methyl silicone, 95% dimethylpolysiloxane, 0.25 µm film thickness) were used. The column temperature was programmed to rise from 50 to 240 °C at a rate of 5 °C min−1; transfer line temperature was 250 °C. The carrier gas was helium with a flow rate of 1.2 mL min−1 and a split ratio of 60:1. Scan time and mass range were 1 s and 40–300 m/z respectively.

2.4 Compounds Identification

Essential oil components identification was assigned by comparison of their retention indices relative to (C8–C22) n-alkanes with those in the literature or to the authentic compounds available in our laboratory. Further identification was made by matching their recorded mass spectra with those stored in the Wiley/NBS mass spectral library of the GC–MS data system and other published mass spectra [21]. Quantitative data were obtained from the electronic integration of the FID peak areas.

2.5 Statistical Analyses

Data were subjected to statistical analysis using the statistical program package STATISTICA (Statsoft, 1998). Percentage of each volatile compound was the mean of three experiments ± SD. The one-way analysis of variance (ANOVA) followed by Duncan multiple range test were employed and the differences between individual means were deemed to be significant at < 0.05.

3 Results and Discussion

3.1 Essential Oil Yield

Leaf essential oil yields (based on dry weight) at fruiting, vegetative and flowering stages were 0.18, 0.11 and 0.41% (w/w) for baetica and 0.20, 0.14 and 0.61% (w/w) for italica (Table 2). The italica leaf essential oil yields were significantly higher than those of baetica at all these stages. The phenological dynamics of the essential oil yield in both varieties were similar. The maximum yield recorded was for the flowering stage with 0.61% for italica and 0.41% for baetica. According to Jerkovic et al. [14] and Jamoussi et al. [16], these results showed a strong correlation between the extraction yield and the phenological stage of the plant with a maximum biosynthesis of essential oil at the flowering period. However, [22] reported that the leaf essential oil yield of myrtle did not vary significantly with seasons.
Table 2

Essential oil yields and composition (%) of leaf from two myrtle varieties at different phenological stages

  

Phenological stages

Variety

Fruiting stage

Vegetative stage

Flowering stage

Essential oil yield (% on the basis of dry matter weight)

baetica

0.18 ± 0.1Bb

0.11 ± 0.01Bc

0.41 ± 0.05Ba

italica

0.20 ± 0.08Ab

0.14 ± 0.01Ac

0.61 ± 0.01Aa

Volatile compound*

RIa

RIb

Identificationc

% Compositiond

(E)-2-Hexenal

850

1,219

GC–MS, Co-GC

baetica

0.04 ± 0.03Aa

nd ± 0.00Ab

0.07 ± 0.13Aa

italica

nd ± 0.00Aa

nd ± 0.00Aa

0.4 ± 0.35Aa

(Z)-3-Hexenol

855

1,370

GC–MS, Co-GC

baetica

0.15 ± 0.19Aab

0.03 ± 0.03Ab

0.32 ± 0.07Aa

italica

0.03 ± 0.01Aa

0.11 ± 0.09Aa

0.06 ± 0.03Ba

Hexanol

865

1,354

GC–MS, Co-GC

baetica

nd ± 0.00Aa

nd ± 0.00Aa

0.07 ± 0.05Aa

italica

nd ± 0.00Aa

nd ± 0.00Aa

0.06 ± 0.02Aa

Tricyclene

924

1,014

GC–MS

baetica

0.13 ± 0.01Ba

0.20 ± 0.04Aa

0.10 ± 0.08Aa

italica

0.21 ± 0.02Aa

0.17 ± 0.14Aab

0.01 ± 0.01Bb

α-Thujene

928

1,035

GC–MS

baetica

0.19 ± 0.30Ab

0.19 ± 0.15Ab

1.34 ± 0.45Aa

italica

0.69 ± 0.38Aa

0.21 ± 0.23Aa

0.58 ± 0.05Bb

α-Pinene

939

1,032

RI, MS, Co-GC

baetica

49.14 ± 2.60Aa

26.65 ± 0.3Bb

29 ± 0.57Bb

italica

31.04 ± 0.53Bc

40.10 ± 0.69Ab

58.05 ± 5.39Aa

Camphene

954

1,076

RI, MS

baetica

0.04 ± 0.02Aa

nd ± 0.00Aa

0.01 ± 0.01Aab

italica

0.01 ± 0.01Aa

0.03 ± 0.04Ba

nd ± 0.00Aa

Sabinene

975

1,132

RI, MS

baetica

0.02 ± 0.01Aa

0.01 ± 0.01Aa

nd ± 0.00Ab

italica

nd ± 0.00Aa

0.01 ± 0.02Aa

nd ± 0.00Aa

β-Pinene

980

1,118

RI, MS, Co-GC

baetica

1.69 ± 0.29Ba

1.03 ± 0.56Bb

1.56 ± 0.22Ba

italica

4.51 ± 0.36Ab

4.06 ± 0.15Ab

6.5 ± 0.56Aa

Myrcene

991

1,174

RI, MS

baetica

0.03 ± 0.02Aa

0.21 ± 0.08Aa

0.01 ± 0.03Aa

italica

nd ± 0.00Aa

0.05 ± 0.30Aa

nd ± 0.00Aa

α-Phellandrene

1,006

1,176

RI, MS

baetica

0.05 ± 0.01Aa

0.15 ± 0.07Aa

0.15 ± 0.09Ba

italica

nd ± 0.00Ba

0.07 ± 0.12Aa

nd ± 0.00Aa

δ-3-Carene

1,011

1,159

RI, MS

baetica

0.03 ± 0.02Ab

0.19 ± 0.11Aa

0.11 ± 0.11Aab

italica

0.08 ± 0.07Aa

0.12 ± 0.05Aa

0.22 ± 0.02Aa

α-Terpinene

1,018

1,188

RI, MS

baetica

0.24 ± 0.17Aa

0.26 ± 0.46Aa

0.06 ± 0.11Ab

italica

nd ± 0.00Bb

0.49 ± 0.41Aa

0.18 ± 0.11Ab

p-Cymene

1,026

1,280

RI, MS, Co-GC

baetica

0.13 ± 0.14Aa

0.47 ± 0.42Aa

0.12 ± 0.12 Ba

italica

0.16 ± 0.06Ab

0.29 ± 0.24Ab

0.68 ± 0.09Aa

Limonene

1,030

1,203

RI, MS, Co-GC

baetica

7.37 ± 0.97Ab

15.44 ± 0.72Aa

16.6 ± 0.93Aa

italica

6.43 ± 0.38Ab

8.72 ± 0.46Ba

0.11 ± 0.00Bc

1,8-Cineole

1,033

1,213

RI, MS, Co-GC

baetica

25.25 ± 1.97Ba

13.67 ± 0.30Bb

23.50 ± 0.56Aa

italica

30.77 ± 0.31Aa

23.66 ± 0.45Ab

21.67 ± 4.11Ab

E-β-Ocimene

1,050

1,266

RI, MS

baetica

0.12 ± 0.05Aa

0.41 ± 0.42Aa

0.16 ± 0.05Aa

italica

0.13 ± 0.11Aa

0.48 ± 0.35Aa

0.17 ± 0.15Aa

γ-Terpinene

1,062

1,255

RI, MS

baetica

0.19 ± 0.01Aa

0.50 ± 0.40Aa

0.44 ± 0.12Aa

italica

nd ± 0.15Ab

0.24 ± 0.24Aa

0.03 ± 0.03Bb

cis-Linalool oxide

1,074

1,450

RI, MS

baetica

0.01 ± 0.01Bb

0.13 ± 0.08Aa

0.01 ± 0.01Ab

italica

0.10 ± 0.02Aa

0.10 ± 0.04Aa

0.02 ± 0.02Ab

trans-Linalool oxide

1,088

1,475

RI, MS

baetica

0.01 ± 0.01Aa

0.05 ± 0.02Aa

0.02 ± 0.03Aa

italica

0.06 ± 0.04Aa

0.05 ± 0.03Aa

0.02 ± 0.00Aa

Terpinolene

1,092

1,290

RI, MS, Co-GC

baetica

0.19 ± 0.05Aa

0.29 ± 0.27Aa

0.38 ± 0.14Aa

italica

0.10 ± 0.08Aa

0.30 ± 0.24Aa

0.37 ± 0.19Aa

Linalool

1,098

1,553

RI, MS, Co-GC

baetica

2.69 ± 1.62Bb

9.17 ± 0.52Aa

11.55 ± 0.97Aa

italica

14.18 ± 0.28Aa

6.46 ± 0.52Bb

2.45 ± 0.33Bc

Borneol

1,165

1,719

RI, MS

baetica

0.15 ± 0.12Aa

0.25 ± 0.03Aa

0.11 ± 0.01Aa

italica

0.05 ± 0.01Aa

0.05 ± 0.03Ba

0.01 ± 0.00Bb

Terpinene-4-ol

1,178

1,611

RI, MS, Co-GC

baetica

0.26 ± 0.06Bb

0.58 ± 0.04Aa

0.11 ± 0.01Bc

italica

0.61 ± 0.18Aa

0.67 ± 0.41Aa

0.36 ± 0.15Aa

p-Cymene-8-ol

1,183

1,864

RI, MS, Co-GC

baetica

0.05 ± 0.00Ab

0.23 ± 0.01Aa

0.13 ± 0.08Ab

italica

0.07 ± 0.01Aa

0.02 ± 0.01Bb

0.03 ± 0.01Ab

α-Terpineol

1,189

1,709

RI, MS, Co-GC

baetica

2.38 ± 0.17Bc

5.28 ± 0.32Aa

3.39 ± 0.44Ab

italica

3.34 ± 0.28Aa

3.04 ± 0.58Ba

0.82 ± 0.18Bb

Myrtenol

1,194

1,804

RI, MS, Co-GC

baetica

0.13 ± 0.06Aab

0.47 ± 0.31Aa

0.05 ± 0.01Ab

italica

0.11 ± 0.05Aa

0.05 ± 0.04Aa

0.02 ± 0.01Ab

Nerol

1,228

1,797

RI, MS, Co-GC

baetica

0.09 ± 0.11Ab

1.18 ± 0.46Aa

0.11 ± 0.06Aa

italica

0.07 ± 0.04Aa

0.07 ± 0.06Ba

0.04 ± 0.00Aa

cis-Carveol

1,247

1,861

RI, MS

baetica

0.03 ± 0.00Ab

0.20 ± 0.05Aa

0.12 ± 0.08Aab

italica

0.04 ± 0.01Aa

0.02 ± 0.01Bb

0.01 ± 0.00Ab

Geraniol

1,255

1,857

RI, MS, Co-GC

baetica

0.81 ± 0.09Ab

1.89 ± 0.10Aa

0.40 ± 0.15Ac

italica

0.80 ± 0.07Aa

0.41 ± 0.03Bb

0.20 ± 0.07Ac

Linalyl acetate

1,257

1,556

RI, MS, Co-GC

baetica

0.32 ± 0.24Ab

0.21 ± 0.01Ab

1.22 ± 0.92Aa

italica

nd ± 0.00Bc

1.19 ± 0.12Ba

0.72 ± 0.00Ab

Bornyl acetate

1,295

1,597

RI, MS

baetica

0.01 ± 0.01Ab

0.25 ± 0.18Aa

0.01 ± 0.01Ab

italica

0.04 ± 0.01Aa

0.13 ± 0.10Aa

0.03 ± 0.02Aa

Tridecane

1,300

1,300

RI, MS, Co-GC

baetica

0.12 ± 0.01Ab

0.01 ± 0.00Ac

0.18 ± 0.04Aa

italica

nd ± 0.00Ba

0.01 ± 0.01Aa

0.06 ± 0.01Ba

Myrtenyl acetate

1,335

1,707

RI, MS, Co-GC

baetica

0.04 ± 0.03Bb

0.13 ± 0.00Aa

0.20 ± 0.17Aa

italica

0.18 ± 0.06Aa

0.05 ± 0.06Ab

0.04 ± 0.01Ab

α-Terpenyl acetate

1,344

1,706

RI, MS

baetica

0.17 ± 0.00Ab

1.49 ± 0.67Aa

0.38 ± 0.33Ab

italica

0.04 ± 0.01Bb

0.18 ± 0.15Bab

0.36 ± 0.17Aa

Eugenol

1,356

2,186

RI, MS, Co-GC

baetica

0.05 ± 0.03Ab

0.49 ± 0.62Aa

0.07 ± 0.04Ab

italica

0.10 ± 0.05Aa

0.22 ± 0.15Aa

0.07 ± 0.04Aa

Geranyl acetate

1,383

1,765

RI, MS, Co-GC

baetica

1.98 ± 0.14Ab

5.26 ± 0.78Aa

1.25 ± 0.21Ab

italica

0.56 ± 0.08Bb

1.61 ± 0.53Ba

1.34 ± 0.42Aab

Neryl acetate

1,385

1,733

RI, MS, Co-GC

baetica

0.15 ± 0.03Aa

0.50 ± 0.40Aa

0.17 ± 0.09Aa

italica

0.06 ± 0.01Aa

0.09 ± 0.07Aa

0.12 ± 0.04Aa

Methyl eugenol

1,401

2,030

RI, MS, Co-GC

baetica

0.86 ± 0.07Ab

2.32 ± 0.48Aa

0.53 ± 0.13Ab

italica

0.93 ± 0.07Aa

0.75 ± 0.18Ba

0.38 ± 0.09Ab

β-Elemene

1,391

1,600

RI, MS

baetica

0.44 ± 0.04Aa

0.08 ± 0.04Ab

0.22 ± 0.11Aa

italica

0.04 ± 0.02Ba

0.07 ± 0.08Aab

0.01 ± 0.01Ba

β-Caryophyllene

1,419

1,612

RI, MS

baetica

0.09 ± 0.11Ab

0.76 ± 0.01Aa

0.26 ± 0.10Aa

italica

0.31 ± 0.16Ba

0.32 ± 0.33Aa

0.06 ± 0.03Ab

α-Humulene

1,454

1,687

RI, MS

baetica

0.02 ± 0.02Aa

0.20 ± 0.25Aa

0.06 ± 0.08Aa

italica

0.05 ± 0.00Aa

0.17 ± 0.12Aa

0.12 ± 0.09Aa

Allo-Aromadendrene

1,474

1,661

RI, MS

baetica

0.09 ± 0.11Aa

0.27 ± 0.41Aa

0.18 ± 0.04Aa

italica

0.33 ± 0.08Aa

0.12 ± 0.03Ab

0.05 ± 0.00Ab

Germacrene-D

1,480

1,726

RI, MS

baetica

0.26 ± 0.05Ab

1.19 ± 0.52Aa

0.98 ± 0.08Ab

italica

0.25 ± 0.06Aa

0.22 ± 0.21Aa

0.27 ± 0.13Aa

Thiophene

1,501

2,033

RI, MS

baetica

0.81 ± 0.04Bb

3.85 ± 0.41Aa

0.52 ± 0.12Ab

italica

1.78 ± 0.34Aa

1.54 ± 0.57Ba

0.57 ± 0.25Ab

Geranyl 2-methybutyrate

1,562

1,880

RI, MS

baetica

0.18 ± 0.02Aa

0.44 ± 0.22Aa

0.32 ± 0.23Aa

italica

0.21 ± 0.05Aa

0.33 ± 0.34Aa

0.01 ± 0.00Aa

Spathulenol

1,576

2,144

RI, MS

baetica

0.04 ± 0.03Aa

0.19 ± 0.14Aa

0.06 ± 0.03Aa

italica

0.07 ± 0.03Aa

0.09 ± 0.06Aa

0.01 ± 0.00Ba

Caryophyllene oxyde

1,581

2,008

RI, MS

baetica

0.09 ± 0.02Ab

0.50 ± 0.03Aa

0.08 ± 0.08Ab

italica

0.16 ± 0.15Aa

0.08 ± 0.06Ba

0.01 ± 0.00Aa

Nonadecane

1,900

1,900

RI, MS, Co-GC

baetica

0.71 ± 0.07Ab

1.43 ± 0.15Aa

0.24 ± 0.22Ac

italica

0.44 ± 0.21Aa

0.69 ± 0.80Aa

0.04 ± 0.02Aa

Total identified

 

baetica

99.35 ± 0.61Aa

98.75 ± 0.56Ab

97.46 ± 0.05Ac

 

italica

99.25 ± 1.43Aa

98.05 ± 0.44Ab

97.28 ± 1.20Ac

*Components are listed in order of elution in apolar column (HP-5); RIa, RIb: Retention indices calculated using respectively an apolar column (HP-5) and polar column (HP-Innowax); c: RI: retention indices relative to C8-C22 n-alkanes on the (HP-Innowax), MS = mass spectrum, Co-GC = co-injection with authentic compound; d: The percentage composition was calculated from the chromatograms obtained on the HP-Innowax column; nd: not detected; values followed by the same capital letter in the columns and small letter in the rows did not share significant differences at 5% (Duncan test)

3.2 Essential Oil Composition

The leaf essential oil composition of the two myrtle varieties at different phonological stages are summarized in Table 2. Forty-nine compounds, according for 92.5–99.7% of the total essential oils, were identified. α-Pinene was the major compound of leaf essential oils from the two myrtle varieties during the phenological cycle but it reached a maximum at the fruiting stage for baetica (49.14%) and at the flowering stage for italica (58.05%). Tunisian myrtle essential oil is α-pinene chemotype; this is in agreement with the results published by Chalchat et al. [11], Jamoussi et al. [16] and Messaoud et al. [17]. The same chemotype was detected in other geographic areas like France [11], Iran [23], Italy [15] and Lebanon [11].

Other predominant compounds were present like 1,8-cineole, limonene and linalool. Their percentages changed irregularly during the different phenological stages. So, at the fruiting stage, leaf essential oil presented important percentages of 1,8-cineole (25.25%), limonene (7.37%) and linalool (2.69%) in baetica and 1,8-cineole (30.77%), linalool (14.18%) and limonene (6.43%) in italica. At the vegetative stage, leaf essential oil contained more limonene (15.44%) than 1,8-cineole (13.67%) and linalool (9.17%) in baetica but more 1,8-cineole (23.66%) than limonene (8.72%) and linalool (6.46%) in italica. At the flowering stage, leaf essential oils of the two myrtle varieties were rich in 1,8-cineole with 23.50% for baetica and 21.67% for italica. This compound was followed by limonene (16.60%) and linalool (11.55%) in baetica while by linalool (2.45%) and limonene (0.11%) in italica. In agreement with Jerkovic et al. [14] and Weyerstahl et al. [23], there are significant seasonal variations in the main constituents of myrtle essential oils which could be due to genetic dynamics and environmental factors such as geography, temperature, rainfall, day length, nutrients, etc. These factors influence the plant biosynthetic pathways and consequently the relative proportion of the main compounds.

Our results are different to those described by Gauthier et al. [19, 24] who indicated that the major components of Morocco leaf essential oil were myrtenyl acetate (23.6%), 1,8-cineole (21%) and linalool (9%) for baetica and 1,8-cineole (25%), myrtenyl acetate (22%) and α-pinene (21%) for italica at the vegetative stage. Concerning the other stages, it is difficult to compare our results to those of Gauthier et al. [19, 24] because they only distillated the italica leaves and fruits together at the fruiting stage as well as leaves and flowers at the flowering stage. Moreover, only few investigations were found to be on the seasonal variation of myrtle essential oil and without variety mention. Jerkovic et al. [14] reported that myrtenyl acetate (13.5–30.7%), 1,8-cineole + limonene (12.6–29.8%), linalool (10.8–18.3%) and α-pinene (6.6–16.4%) were the five predominant components in Croatian myrtle leaf essential oils during the phenological cycle. These same major components were observed by Gardeli et al. [22] but with different proportions and the absence of limonene; namely the Greek myrtle leaf essential oil contained more myrtenyl acetate (23.7–39%) and 1,8-cineole (12.7–19.6%) but less linalool (7–15.8%) and α-pinene (10.1–11.6%). The myrtenyl acetate was also the chemotype of Spanish [8] and Moroccan [11] myrtle essential oils whereas in the present study, this compound was detected only in low percentages that did not pass 0.5% at all phenological stages of each variety. Flamini et al. [15] identified 82 constituents in the Italian myrtle leaf essential oils of two different localities. The authors reported that the type of soil played an important role in the essential oil composition variability. So, α-pinene and limonene were identified in greater amounts in plants growing on calcareous soils while linalool, linalyl acetate and trans-myrtanol acetate were detected at higher percentages on siliceous soils. This is not the case of our study because the two localities contained calcareous soils.

3.3 Chemical Class Characterization of Leaf Essential Oil

The chemical class characterization of the leaf essential oils from both varieties (Table 3) showed that monoterpene hydrocarbons and oxygenated monoterpenes were the main classes in baetica with respectively 60.44 and 36.23% at the fruiting stage and 51.02 and 43.63% at the flowering stage. They were also the predominant classes of italica essential oils at the vegetative and flowering stages formed 55.39 and 66.87% of monoterpenes hydrocarbons and 40.82 and 29.37% of oxygenated monoterpenes. However, at the fruiting stage, italica essential oil presented more oxygenated monoterpenes (54.12%) than monoterpene hydrocarbons (43.39%) like baetica essential oil at the vegetative stage with 48.62 and 46.00%. In fact, there were significant variations in these two main class distributions during the phenological stages which could be related to changes in the plant physiological behaviour facing to biotic and abiotic factors. It is worth mentioning the presence of sesquiterpene hydrocarbons, oxygenated sesquiterpenes, aliphatic hydrocarbons, alcohols and aldehydes as minor essential oil chemical classes of the two myrtle leaf varieties at all stages.
Table 3

Proportions of leaf volatile compound classes from two myrtle varieties at different phenological stages

Chemical class

Variety

Phenological stages

Fruiting stage

Vegetative stage

Flowering stage

Monoterpene hydrocarbons

baetica

60.44 ± 4.18Aa

46.00 ± 1.58Bc

51.02 ± 0.72Bb

italica

43.39 ± 1.14Bc

55.39 ± 1.64Ab

66.87 ± 3.85Aa

Oxygenated monoterpenes

baetica

36.23 ± 3.50Bc

48.62 ± 0.05Aa

43.63 ± 0.04Ab

italica

54.12 ± 2.53Aa

40.82 ± 1.54Bc

29.37 ± 6.00Bb

Sesquiterpene hydrocarbons

baetica

0.89 ± 0.17Ab

2.45 ± 0.17Aa

1.72 ± 0.42Ab

italica

0.99 ± 0.42Aa

0.92 ± 0.27Ba

0.53 ± 0.27Aa

Oxygenated sesquiterpenes

baetica

0.13 ± 0.06Ab

0.70 ± 0.15Ab

1.15 ± 0.12Aa

italica

0.24 ± 0.84Aa

0.17 ± 0.15Ba

0.02 ± 0.01Aa

Aliphatic hydrocarbons

baetica

0.83 ± 0.07Ab

1.48 ± 0.16Aa

0.43 ± 0.25Ac

italica

0.44 ± 0.26Ba

0.70 ± 0.97Aa

0.11 ± 0.03Aa

Alcohols

baetica

0.15 ± 0.19Aab

0.04 ± 0.02Ab

0.40 ± 0.13Aa

italica

0.40 ± 0.01Aa

0.11 ± 0.12Aa

0.11 ± 0.05Aa

Aldehydes

baetica

0.04 ± 0.03Aa

nd ± 0.00Aa

0.07 ± 0.14Aa

italica

nd ± 0.00Aa

nd ± 0.00Aa

0.38 ± 0.3Aa

Others

baetica

0.85 ± 0.12Ac

1.25 ± 0.23Ab

2.54 ± 1.72Ba

italica

0.74 ± 0.11Ac

1.96 ± 0.57Ab

2.72 ± 1.20Aa

Values followed by the same capital letter in the columns and small letter in the rows did not share significant differences at 5% (Duncan test); nd non detected

4 Conclusion

The leaf essential oil composition of the two M. communis varieties at different phenological stages generally presented the same predominant monoterpene components but with significant different proportions. 1,8-Cineole, limonene and linalool proportions presented the most remarkable changes in the two essential oils during the different phenological stages of this plant. These variations could be due to both genetic and environmental factors.

Acknowledgments

The authors wish to thank Prof. Mohamed Hammami and Mr. Imed Chraief, from the Medicine University of Monastir (Tunisia) for performing GC–MS analyses.

Copyright information

© Vieweg+Teubner | GWV Fachverlage GmbH 2008