Chemical Composition of Moroccan Argania spinosa leaf Essential Oils Isolated by Supercritical CO2, Microwave and Hydrodistillation

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Journal of Essential Oil Bearing Plants

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Chemical Composition of Moroccan Argania

spinosa leaf Essential Oils Isolated by Supercritical CO

₂

, Microwave and Hydrodistillation

Abdelaziz El Amrani, José Antonio Cayuela Sánchez & Jamal Jamal Eddine

To cite this article: Abdelaziz El Amrani, José Antonio Cayuela Sánchez & Jamal Jamal Eddine (2015) Chemical Composition of Moroccan Argania spinosa leaf Essential Oils Isolated by Supercritical CO₂, Microwave and Hydrodistillation, Journal of Essential Oil Bearing Plants, 18:5, 1138-1147, DOI: 10.1080/0972060X.2014.977563

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Chemical Composition of Moroccan Argania spinosa leaf Essential Oils Isolated by Supercritical CO₂, Microwave and Hydrodistillation

Abdelaziz El Amrani ¹*, José Antonio Cayuela Sánchez ² and Jamal Jamal Eddine ¹

1 Laboratoire Synthèse, Extraction et Etude Physico-Chimique des Molécules Organiques, Université Hassan II de Casablanca, Faculté des Sciences

Ain -Chock, B.P. 5366 Maarif, Casablanca, Morocco

2 Instituto de la Grasa-CSIC, Department of Physiology and Technology of Plant Products. Avda. Padre García Tejero, 4 41012 Sevilla, Spain

Abstract: The air-dried leaves of Moroccan Argania spinosa were subjected to hydrodistillation (HD), to microwave (M) extraction and to supercritical fluid extraction (SFE) using CO₂. To the best of our knowledge, it is the first time that we report the microwave (M) and supercritical fluid extraction (SFE) for Argania spinosa. The essential oils obtained were analysed by GC and GC-MS. The main component for HD and SFE essential oils was cubenol (35.7- 42.1 %), while the 1-epi-cubenol (21.8 %) dominated M essential oils. A total of 57 compounds, amounting 95 % of the oil, were identified. In addition, thirty six components of our A.

spinosa essential leaf oil have not been previously described in the Argania spinosa essential oil in the literature.

Key words: Argania spinosa, essential oil composition, hydrodistillation, microwave, Sapotaceae, supercritical fluid extraction.

Introduction

The argan tree (Argania spinosa L. Skeels) is a tropical plant, which belongs to the Sapotaceae family and is endemic in south western Morocco

1,2. It has demonstrated an irreplaceable ecological function for centuries in Morocco, protecting the soil against erosion, maintaining soil fertility and guaranteeing most of the dietary needs of small scale farmers ³. Thus, argania preservation and cultivation has become an important crop for the maintenance of agriculture activity in this part of Morocco. Several bilateral projects and local programmes have promoted the establishment of argania to orient the utilization of the plant for multiple purposes ^4,5.

The argania tree is exploited essentially for its fruits ^6-8. The endosperm seed of fruit constitutes

a good potential source of edible oil for human consumption and endowed with important medicinal properties decreasing cholesterol level, stimulation of vascular circu-lation ^9-12. Argan oil is also widely incorporated in many cosmetic products such as antiacne agents ^13,14.The Argan oil helps in alleviating stress and speeding up healing. The essential oils have also cosmetic properties and can be used in skincare and hair care products. The oils have known for antiviral, antifungal and antiseptic properties ^13-15.

Several studies have been engaged on the chemical characterization of argan oil ^15-17 and secondary metabolites from leaf extract ^18-20 in the last decade. However, a few authors have studied the volatiles and the chemical composition of A.

spinosa essential oils obtained only by hydro- ISSN Print: 0972-060X ISSN Online: 0976-5026

*Corresponding authors (Abdelaziz El Amrani)

TEOP 18 (5) 2015 pp 1138 - 1147 1138

Downloaded by [Gazi University] at 02:46 29 October 2015

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distillation (HD) from the leaves ^21-23and the fresh and dried pulp of the fruit ²⁴.

The supercritical fluid extraction (SFE) of some plants, at the optimum conditions, was studied and describedby many authors ^25-29 and can be high- lighted the review presented by Chen and Ling ³⁰ concerning SFE in the preparation and analysis of different Chinese herbal medicine. The authors mentioned the SFE parameters and the analytical method of each plant. In addition, the supercritical fluid extraction has been applied to isolate the essential oil from Rosmarinus officinalis leaves

31, from Syzygium aromaticum L. and Cinna- momum zeylanicum ³², from Calendula officinalis L. and Aloysia tryphylla ³³. Otherwise, the study of the oil obtained using microwave extraction (M) was reported by many authors ^34-36. Recently, M was used for isolation of the essential oil from the Moroccan Rosmarinus eriocalix by Elamrani et al. ³⁷and SFE was used for isolation of the oil from the Moroccan Ammi visnaga by Zrira et al.

38. However, no information could be found regarding the use of M and SFE for isolation of the oil from A. spinosa leaves. The distillation method has traditionally been applied for the recovery of essential oils from plant materials.

One of the disadvantages of the distillation method is that essential oils undergo chemical alterations and the heat sensitive compounds can easily be destroyed. Therefore, the quality of the essential oil extracts is significantly impaired. The other method applied for oil recovery from plant materials uses organic solvent extraction, which has limitations with regard to the loss of valuable volatiles during vacuum evaporation of solvent, and difficulty in obtaining solvent-free extracts.

These disadvantages can be avoided if supercritical fluids are used to carry out the extraction.

Supercritical fluid extraction (SFE) has received increased attention recently, mainly in the environmental field, where analysis must be fast and accurate ³⁸.

Microwave extraction technique involves heating at reflux the sample in a solvent or a mixture of several appropriate solvents. In all cases, focused microwave can considerably reduce the time extraction with high accuracy and excellent reproducibility. In addition, with regard

to a system operating at atmospheric pressure, this technique has great flexibility and a great ease of use ³⁷.

In continuation of our investigation of Moroc- can aromatic flora, the object of this work is to study and to examine particularly the influence of isolation methods on chemical composition of the leaves of A. spinosa essential oil. We were interested to the comparison among HD, M and SFE.

Experimental Plant Material

The samples of A. spinosa leaves were collected in January 2010 from Essaouira regions (600 km south of Rabat). In Morocco, tree forestry, fruit and forage, arganier covers currently 870000 Ha (Around 21 million trees). About 14 % of the national forest area space to arganier is essentially extends into the territory of the provinces of Essaouira (130000 Ha.). The plant material was identified by Prof. Dr. M. Rejdali from the Agronomic Institute and Veterinary Hassan II, Rabat (Morocco). Certified voucher specimens have been deposited in the Herbarium of the Department of Botany and Ecology at the Agronomic Institute and Veterinary Hassan II, Rabat (Morocco) and in the Chemistry Department at the Faculty of Science Ain-chock, Casablanca (Morocco) under voucher number AS 12012011.

In the sampling area, we have chosen a parcel at random of about 200 m x 200 m. On this parcel, we have randomly chosen 5 sites with a surface of 20 m x 20 m (Fig. 1). In each place, 10 individual trees samples situated inside were chosen. We collected from each tree about 1 kg of leaves at the same stage of development in order to a reliably comparison of data. In total, 50 trees were used. In each place, from these 10 individual trees, we made a heterogeneous sample of leaves (about 4 Kg). Therefore we took in total 5 main heterogeneous samples (leaves), which underwent different extraction methods.

Extraction of leaf essential oil

After air-drying in the shade for a week, the air-dried leaves of the plant material were

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subjected to HD, M and to SFE.

The essential oils were extracted from the dried plant material by HD using a Clevenger-type apparatus according to the European Pharma- copoeia ³⁹. The essential oils were stored at 20^oC in the dark until analysis. The oils obtained were light yellow.

The M was realised with a Toshiba microwave.

The consumed power is 1250 W and the frequency of the microwave is 2450 MHz. 10 g of the leaves placed in 50 ml of hexane, were exposure to the radiation for 5 min. Those were the best conditions of optimization for extraction. The solvent used was of the highest purity available (Merck). At the end of the exposure, the solution was filtered and the solvents were submitted to evaporation.

The oils obtained were light red.

The SFE was carried out in 10 mL stainless steel extraction cells, loaded with a 2 g sample, using an Isco SFX2-10 extractor. Pure CO₂was passed into the cell as a supercritical fluid using an Isco Model 260D syringe pump. Extraction conditions of 3000 psi and 50°C were maintained for 5 minutes under static conditions. Dynamic extraction was then carried out at a CO₂flow of 0.5 to

1.0 mL/min for 60 min by allowing the fluid to first pass through the cell and then through a stainless steel tube (88 mm x 5 mm) packed with Porapk Q (400 mg) which had been previously extracted with 10 % dichloromethane in pentane.

The physicochemical characteristics of the oils such as specific gravity, refractive index, optical rotation and solubility in alcohol were also determined according to the AFNOR ⁴⁰ standards at 20°C.

Oil analysis

The different A. spinosa essential oils obtained were analysed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC- MS). The GC analysis was carried out using a Hewlett-Packard HP 5980 gas chromatography apparatus equipped with FID and two capillary columns DB-5 and CW20M (25 m x 0.25 mm, film thickness 0.25 μm). Analytical conditions were: injector and detector temperature 240 and 260°C respectively, oven temperature programmed from 50 to 250°C at 4°C/min. Iso- thermal temperature at 250°C for 30 min; carrier gas 1 ml N2/min. Relative concentrations were Figure 1. Planning for sampling

Abdelaziz El Amrani et al., / TEOP 18 (5) 2015 1138 - 1147 1140

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calculated using peak areas as given by HP 3396A integrator, without correction for response factors.

We have identified most constituents by comparison of their GC Kovats retention indices (R.I.), determined with reference to an homologous series of C₈-C₃₀ n-alkanes and with those of authentic commercial standards available in the authors laboratory.

The GC-MS analysis was done using a Hewlett- Packard HP 5980 Series II gas chromatograph equipped with HP-5 capillary column (25 m x 0.3 mm; film thickness 0.25 μm) and an HP 5772A mass selective detector. Analytical conditions were: injector and detector temperatures: 240 and 260°C respectively. The oven temperature is programmed from 50 to 250°C at 4°C/min, then isothermal at 250°C for 10 min; the carrier gas was 2 ml He/min; we used an ionisation mode with electronic impact at 70 eV. The constituents were identified by the combination of retention index data and mass spectra data using NBS library and other literature data ^41,42.

Statistical analysis

The percentage composition of the isolated essential oils was used to determine the relationship between the different samples by cluster analysis using Numerical Taxonomy Multivariate Analysis System (NTSYS-pc software, version 2.2, Exeter Software, Setauket, New York). For cluster analysis, correlation coefficient was selected as a measure of similarity among all accessions, and the Unweighted Pair

Group Method with Arithmetical Averages (UPGMA) was used for cluster definition. The degree of correlation was evaluated according to Pestana and Gageiro ⁴³ and classified as very high (0.9-1), high (0.7-0.89), moderate (0.4-0.69), low (0.2-0.39) and very low (<0.2).

Results and discussion

The essential oil yields and physicochemical properties of the A. spinosa leaves can be seen in Table 1. Examination of these results shows that the oil content varies greatly with a range of 0.03 to 2.7 % (ml per 100 g of dried material). The oil yield was particularly high using SFE (2.7 %). It was much lower using HD (0.03 %). The solubility of essential oil obtained by SFE-CO2 is much higher than that obtained by hydrodistillation.

A qualitative and quantitative comparison of the constituents present in SFE, M and in HD A.

spinosa essential oil was studied. The identified components and their percentage are given in Table 2. A total of 57 compounds, amounting 95

% of the oil, were identified. A particular corre- lations were found between the oils samples, which was confirmed by the cluster analysis, with a correlation coefficient varying between (0.38 and 0.89), (Figure 2). HD and SFE showed a high degree of correlation and formed one group (Scorr

= 0.89) being dominated by cubenol (35.7 - 42.1

%) (Figure 3), viridiflorl (2.4 - 10.8 %) and β- eudesmol (2.3 - 8.3 %). Microwave (M) oil showed a low degree of correlation (Scorr = 0.38), Table 1. Yields and physicochemical properties of the leaves Argania spinosa essential

oils extracted by hydrodistillation, microwave and Supercritical Fluid Extraction Type of extraction

Argania spinosa oils Hydrodistillation Microwave Supercritical

(HD) (M) Fluid (SFE)

Yield (%) 0.03-0.08 1.2-1.8 2.1-2.7

Specific gravity 0.820 0.846 0.973

Refractive index 1.3681 1.3711 1.4102

Optical rotation + 1.1° + 1.4° + 2.2°

Solubility in alcohol 0.55 vol. at 70 % 0.85 vol. at 70 % 0.95 vol. at 70 %

Values of the yield given obtained from 5 samples with two distillation replicates each Values of the physicochemical properties given represent the average of two replicates

Determination at 20°C

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Table 2. Chemical composition of the leaves Argania spinosa essential oils extracted by hydrodistillation (HD), microwave (M) and Supercritical Fluid Extraction (SFE)

DB 5 column %

Compounds RI HD M SFE

α-Pinene* 938 a, c 3.2 2.4 1.3

Camphene* 952 a, c 2.1 1.2 0.2

Sabinene* 976 a, c 0.4 0.1 0.3

β-Pinene* 980 a, c 2.8 2.1 1.4

Myrcene* 993 a, c 3.3 5.8 0.9

α-Phellandrene* 1006 a, c 0.1 t 0.2

α-Terpinene* 1018 a, c 0.8 0.1 0.4

para-Cymene* 1026 a, c 0.2 0.1 t

γ-Terpinene* 1062 a, c 1.0 0.3 0.1

Menth2-en-1-ol* 1068 b, c t 0.6 0.4

Camphor 1144 a, c 8.6 2.5 1.5

β-Terpineol* 1145 a,c 0.8 0.3 0.1

Terpinen-4-ol* 1178 a, c 1.1 0.4 0.1

α-Terpineol* 1189 a, c 0.8 0.2 0.4

Dodecane 1200 b,c t 0.3 0.1

Bornyl acetate 1286 a, c 9.3 0.4 0.6

α-Cubebene 1352 a, c t 0.3 0.1

α-Ylangene* 1373 a,c 0.1 0.4 0.2

α-Copaene* 1377 a, c t 0.3 0.2

β-Elemene* 1392 a, c t 0.5 0.3

β-Caryophyllene 1419 a, c 0.7 0.1 0.2

α-Cadinene 1451 a,c 0.1 0.4 0.1

α-Humulene* 1454 a,c t 0.3 0.2

γ-Muurolene* 1477 a,c t 0.4 0.1

Germacrene D 1485 a,c 0.2 14.3 6.5

β-Bisabolene* 1509 a,c 0.1 0.3 t

γ-Cadinene 1510 a,c 0.1 0.4 0.3

γ-Bisabolene 1516 a,c t 0.1 0.1

δ-Cadinene 1524 a, c t 0.2 0.3

Calamenene 1529 a,c 0.1 0.5 0.1

Caryophyllene oxide 1581 a,c 0.3 9.3 0.1

Viridiflorol 1595 a,c 2.4 - 10.8

1-epi-Cubenol 1627 a,c 2.1 21.8 0.3

γ-Eudesmol* 1630 a, c t 0.4 0.1

T-Cadinol 1641 b,c t 0.2 0.1

Cubenol 1644 a,c 35.7 6.5 42.1

β-Eudesmol 1653 a,b,c 8.3 - 2.3

α-Bisabolol* 1673 a, c 0.2 0.1 -

Cadalene 1680 a, c 0.4 0.8 0.5

Pentadecanol* 1777 b,c - 0.2 -

Octadecene 1795 a,b - 0.3 -

Octadecane 1800 a,b - 0.2 -

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table 2. (continued).

DB 5 column %

Compounds RI HD M SFE

Hexadecanol* 1879 a,b - 0.1 -

Nonadecane* 1920 a,c - 0.2 -

Phytol 1949 a.b - - 10.1

Eicosene* 1994 a,c - 0.2 -

Eicosane* 2000 a,c - 0.3 0.1

Octadecanol* 2082 a, c - - 0.3

Heinecosane* 2100 a,c - 0.1 0.4

Tricosane* 2295 a, c - 0.4 0.8

Eicosanol* 2310 a - 0.1 -

Nonadecanol* 2313 a,c - 0.2 8.1

Docosanoic acid, 2531 a,b - 0.3 1.5

Methyl ester*

Hexacosane* 2600 a - - 0.2

Heptacosane* 2695 a - - 0.4

Octacosane* 2800 a - - t

Nonacosane* 2900 a - - 1.1

Total 85.3 77.0 95.6

a = Comparison of our MS data with NBS75K library data b = Comparison of our MS data with literature data (Adams)

c = Comparison of our RI data with literature data (Laseve data base, Chicoutimi Univ., Quebec, Canada t: trace (<0.1 %); compounds are listed in order of their elution from a DB5 column

- : compound not present in oil

*: components which have not been previously described in the Argania spinosa essential oil in the literature

Figure 2. Dendrogram obtained by cluster analysis of the percentage composition of the essential oils isolated from Argania spinosa leaves based on correlation and

using unweighted pair-group method with arithmetic average (UPGMA).

Abbreviation: H.D: Hydrodistillation; S.F.E: supercritical fluid extraction; M: microwave.

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characterized by the dominance of 1-epi-cubenol (21.8 %) (Figure 3), germacrene D (14.3 %) and caryophyllene oxide ( 9.3 %).

Using HD, we have identified only 39 components amounting 85.3 %. The major constituents were cubenol (35.7 %), bornyl acetate (9.3 %), camphor (8.6 %) and β-eudesmol (8.3 %). In addition, we observed that eighteen compounds in the oils obtained by HD have not been identified in the oils obtained by M and SFE.

Using SFE, 49 compounds were identified. The main components were cubenol (42.1 %), viridiflorol (10.8 %), phytol (10.1 %), nonadecanol (8.1

%) and germacrene D (6.5 %). In addition, eight compounds were not found in the SFE and are present in the M extract or HD oils such as:

bisabolol, pentadecanol, octadecene, octadecane, hexadecanol, nonadecane, eicosene and eicosanol.

Using M, 50 compounds were identified. The main components were 1-epi-cubenol (21.8 %), germacrene D (14.3 %), caryophyllene oxide (9.3

%), cubenol (6.5 %) and myrcene (5.8 %). It is interesting to note that seven constituents were not found in the M extract and are present in the SFE such as: viridiflorol, β-eudesmol, phytol, hexacosane, heptacosane, octacosane and nonacosane.

In comparison to the only two results previously reported in the literature for the A. spinosa essential oils, there were considerable differences qualitatively and quantitatively. Tahrouche et al.

21 have studied the volatiles obtained by HD of A.

spinosa leaves collected from Agadir region, at the South of Morocco, and reported that the principal constituent was 14-methylidene-2,6,10- trimethylhexadecene (51.2 %). The authors identified only 17 constituents. From these components, the phytol (1.5 %) is the alone compound present in our oils analysed. The percentage of phytol

Figure 3. Chemical structures of main constituents in Argania spinosa essential oils leave Cubenol (CAS 21284-22-0) 1-epi-cubenol (CAS 19912-67-5)

(10.1 %) in our samples was particularly higher than the mentioned by the authors. The sixteen others compounds are not found in our oils such as: nonane (0.4 %), 3-octanone 0.9 %), decane (0.2 %), methyl benzoate (0.1 %), undecane (0.3

%), pulegone (0.7 %), 2-undecanone (0.2 %), tridecane (0.3 %), p-hydroxyphenylethanol (1.8

%), pentadecane (0.3 %), heptadecane (0.2 %), hexahydrofarnesyl acetone (0.8 %), 14-methylidene-2,6,10-trimethylhexadecene (51.2 %), octanol (8.6 %), (E)-2,6,10-trimethylhexadeca- 1,3-diene (12.3 %), (Z)-2,6,10-trimethylhexadeca-1,3-diene (17 %).

El Kabouss et al.¹⁸, studied the chemical composition of the leaf essential oil of A. spinosa obtained by HD and reported that the principal constituent was 1,10-di-epicubenol (20.5 %).

They have identified only 33 constituents, in which twenty are present in the oils analysed in the present work. The thirteen others are not present in the oils obtained in the current assay such as: cis-muurola-4-(14),5-diene (0.7 %), β- selinene (1 %), α-muurolene (0.3 %), selina- 3,7(11)-diene (5.1 %), α-calacorene (0.6 %), germacrene B (1.1 %), globulol (0.3 %), 1,10-di- epicubenol (20.5 %), T-muurolol (1 %), α- muurolol (0.6 %), α-cadinol (1.5 %), cis-14- muurol-5-en-4-one (1.7 %) and juniper camphor (1.3 %).

In addition, thirty six constituents found in our oils have not been identified by Tahrouche et al.

21 and El Kabouss et al.¹⁸. As far as we know, these thirty six components of our A. spinosa essential oil leaf have not been previously described in the A. spinosa essential oil in the literature.

Recently Harhar et al. ²⁴ studied the composition of the essential oil from the fresh and dried pulp of the fruit of A. spinosa and mentioned that camphor was the major component in both oil Abdelaziz El Amrani et al., / TEOP 18 (5) 2015 1138 - 1147 1144

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types; in addition, the fresh fruit oil had significant amounts of 1,8-cineole, endo-borneol, and 2-(4- methylcyclohex-3-enyl)-propan-2-ol., and the dried pulp oil were rich on 3,5-dimethyl-4- ethylidene-cyclohex-2-ene-1-one, 1,8-cineole, and 2-methylbutanoic acid.

Conclusion

We conclude from this study that the extraction

techniques influenced the chemical composition of Moroccan A. spinosa essential oils. They are characterized by the percentage of oxygenated components, particularly the alcohols cubenol, which was higher with SFE and HD, and 1-epi- cubenol which was higher with M. The result of this investigation will allow us to better promoting this species.

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