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SUPPLEMENTARY MATERIAL
Chemotypes of essential oil of unripe galls of Pistacia atlantica Desf. From Algeria

Ibrahim Sifia, Nadhir Gourinea, Emile M. Gaydoub and Mohamed Yousfia



aLaboratoire des Sciences Fondamentales à l’Université Amar TÉLIDJI de Laghouat, (03000), Algeria

bLaboratoire de Recherche en Systèmes Chimiques Complexes, Faculté des Sciences et Technique de Saint-Jérôme, Université Paul Cézanne, Marseille, France
Ibrahim Sifi

Email: sifi_ibrahim@yahoo.fr

Affiliation: Laboratoire des Sciences Fondamentales

Address: Laboratoire des Sciences Fondamentales à l’Université Amar TÉLIDJI de Laghouat, Route de Ghardaïa, BP37G, (03000), Algérie.


Nadhir Gourine

Email: n.gourine@mail.lagh-univ.dz

gourine.nadir@gmail.com

Affiliation: Laboratoire des Sciences Fondamentales

Address: Laboratoire des Sciences Fondamentales à l’Université Amar TÉLIDJI de Laghouat, Route de Ghardaïa, BP37G, (03000), Algérie.
Emile M. Gaydou

Email: emile.gaydou@univ-cezanne.fr

Affiliation: UMR CNRS 6263, Équipe AD2EM (Phytochimie), Institut des Sciences Moléculaires de Marseille.

Address: Université Paul Cézanne, Faculté des Sciences et Techniques de Saint-Jérôme, Avenue Escadrille Normandie Niémen, Case 461, Marseille Cedex 20, France.


Mohamed Yousfi

Email: yousfim8@gmail.com

Affiliation: Laboratoire des Sciences Fondamentales

Address: Laboratoire des Sciences Fondamentales à l’Université Amar TÉLIDJI de Laghouat, Route de Ghardaïa, BP37G, (03000), Algérie.


*Corresponding author at:

Laboratoire LSF, B.P. 37G, Université Amar TÉLIDJI, Laghouat, (03000), Algeria.

Tel.: +213 29 90 00 66

Fax: +213 29 90 00 66

E-mail address: n.gourine@mail.lagh-univ.dz

gourine.nadir@gmail.com


Abstract

The essential oils of unripe galls (from male and female plants), of a total number of 52 samples of Pistacia atlantica collected from different regions in Algeria were analysed by GC/MS and GC. The yields of the extraction of the essential oil by hydrodistillation varies from low to high values (0.08-1.89% v/w). The results of both methods of Principal Component Analysis (PCA) and Hierarchical Ascendant Classification (HAC) revealed the presence of two different chemotypes: α-pinene chemotype and α-pinene/sabinene/terpinen-4-ol chemotype.


Keywords

Pistacia atlantica; unripe galls; essential oil; chemotypes.

1. Plant description

In Greece the fruits of P. atlantica are used for tanning and as fodder for cattle and since they contain oil, it is used for soap making . The fruits of P. atlantica which smell like mastic are also occasionally chewed by local inhabitant (for mouth flavouring). From the bark of the wood, a resin is collected for laquer production and it is also used in popular medicine “as an antiseptic to wounds, etc.” . The oleoresin of P. atlantica var. mutica, known as “Turk terebinth gum,” is used to make chewing gum in Iran. This plant has also been used traditionally in the treatment of peptic ulcer and as a mouth freshener . In Algeria and Morocco, P. atlantica is important because it is the source of mastic gum, an exudates which strengthens gums, deodorizes breath and combats coughs, chills and stomach diseases . Furthermore, P. atlantica along with Ferula persica, Paronychia argentea are the three plants which are widely recommended by the herbalists and used for their hypoglycaemic activity in Jordan . In Algeria, the galls of P. atlantica are used as an embalming agent by rural inhabitant. They are also known in Arabic as ‘‘afse’’ and are edible and sold in markets.


Gall-formers are parasitic organisms that manipulate plant traits for their own benefit. Galls have been shown to protect their inhabitants from natural enemies such as predators and parasitoids by various chemical and mechanical means. Much less attention, however, has been given to the possibility of defense against microbial pathogens in the humid and nutrient-rich gall environment . Gall insects are parasitic herbivores that do not only consume plant resources, but also induce physiological and morphological changes in plant tissue. These growth transformations are the result of both the gall-inducer’s stimulus and the plant’s reactions. As a consequence, gall-inducers may damage the host plants . The galls of P. atlantica are induced by infection of a variety of galling aphids “e.g., Forda riccobonii (Stefani), Slavum wertheimae HRL, Geoica sp., etc.” .
The main previous few studies on the chemical composition of the essential oils of different parts of P. atlantica tree deal with leaves , fruits and oleoresin . In our continuing research on the essential oils of P. atlantica grown in Algeria and considering the fact that the smell and the color of the essential oils of the unripe galls found herein our investigation are quite different from those of mature galls, the aim of the present work is to study the chemical composition of these essential oils at large scale (different regions, male and female trees) and to investigate the possible occurrence of different chemotypes within these essential oils.
2. Experimental
2.1. Chemicals

Pure authentic samples myrcene, β-phellandrene, globulol and phytol were purchased from Aldrich. Linalool, terpinen-4-ol, α-terpineol, were purchased from Fluka.


2.2. Plant material

Fresh unripe galls, from both male and female trees of P. atlantica Desf., were randomly collected from the bottom of the trees branches during the period of the end of July and the beginning of August 2010. Two regions in Algeria were selected. The first region Aïn-Oussera belongs to the town of Djelfa (39 samples, among them 13 were from male trees), while the second region belongs to the town of Laghouat (13 samples). For this region of Laghouat, we have selected three locations of the plant collections: the location of south Laghouat (2 samples, male and female), the location of Kheneg (3 male samples and 4 female samples) and the location of Tilghemt (2 female, 2 male). The chosen numbers of samples collected from each region were roughly proportional to the availability of the plant in each one of these regions.

The locations of Aïn-Oussera, Kheneg, south of Laghouat and Tilghemt “Hassi R’mel” are located at 200, 400, 440 and 500km, respectively south Algiers, the capital of Algeria. The location of Aïn-Oussera (latitude 35°33' (N); longitude 02°31' (E); altitude 649m) was characterized by annual precipitation of 25mm and mean summer temperature of 37.8 °C. The locations of Laghouat and south of Laghouat (latitude 33°47' (N); longitude 02°52' (E); altitude 750m) were characterized by annual precipitation of 18mm and mean summer temperature of 41.4 °C. Finally the location of Tilghemt (latitude 32°95' (N); longitude 03°18' (E); altitude 774m) was characterized by annual precipitation of 16mm and mean summer temperature of 42.3°C. All these locations have same the kind of soil which is the clay soil. This soil is locally known as “Daya”.

For comparison purpose, mature galls (dark brown color) were also collected from the regions of Aïn-Oussera and Laghouat. In addition, other samples were purchased from local markets of medicinal plants in the town of Laghouat (Algeria).

The plant material was identified by staff at the herbarium of National Superior School of Agronomics (ex. INA), Algiers, Algeria. A voucher specimen (PAUG-52S/08/10) was deposited in the herbarium of the Fundamental Sciences Research Laboratory at Laghouat University.

2.3. Preparation of samples

The essential oils were obtained by hydrodistillation using a Clevenger apparatus for almost 2 hours, time which was necessary for the complete extraction. The obtained essential oils were dried over anhydrous sodium sulphate, and stored at +4°C until analyzed.


2.4. Gas chromatography (GC)

A CP-Varian 3800 gas chromatograph was used with a flame ionization detector (FID), and a UB-Wax fused silica capillary column (60m×0.32mm, 0.25μm film thickness). Oven temperature was programmed from 50°C to 250°C at a rate of 3°C/min and held at 250°C for 10 min. Injector and detector temperatures were set at 250°C and 260°C, respectively. Helium was the carrier gas at a flow rate of 1mL/min. Diluted samples (1:100 v/v, in n-pentane) of 1 μL were injected manually using split ratio of 80:1.


2.5. Gas chromatography–mass spectroscopy (GC/MS)

The GC/MS analyses were performed on an Agilent 6890 GC machine equipped with a capillary column DB-Wax (30 m × 0.25 mm, 0.25 μm film thickness) and coupled with a Quadrupole CMSD 5973 detector in electron ionization mode (EI 70 eV). Helium was the carrier gas, at a flow rate of 1 mL/min. Injector and MS transfer line temperatures were set at 250 and 220°C, respectively. Oven temperature was programmed from 50°C to 250°C at a rate of 3°C/min and held at 250°C for 10 min. Diluted samples (1:100 v/v, in n-pentane) of 1 μL were injected manually using split ratio of 80:1.


2.6. Compound identifications

Linear retention indices were calculated relative to linear homologous series of n-alkanes (C8–C40). The identifications of the components were based on the comparison of their mass spectra with those of Wiley and NIST (National Institute of Standards and Technology) libraries, as well as by comparison of their retention indices with those of the values of a home made database and those of components of known oils and by co-injections of some pure authentic samples.


3. Satistical Analysis

The results shows that the main compounds were not the same for the overall analyzed samples (male and female tree) and coming from the different locations i.e. the major compound is sometimes being a minor compound and vice-versa. In order to investigate the differences between the essential oil samples of the different regions of our collections, we have chosen two methods: Principal Component Analysis (PCA) and Hierarchical Ascendant Classification (HAC). The data set, composed of 52 samples and 14 variables (main compounds), were subjected to multivariate statistical analysis for chemotype determination.


3.1. Principal Component Analysis (PCA)

For the PCA method, loading factors for principal axes F1 and F2 with rotation Varimax are given in Figure 1S. The factor loading of the 14 variables on axes F1 and F2 (circle of correlations), using PCA of the chemical composition obtained by gas chromatography of the 52 essential oil samples represented in Figure 1S, shows strong correlations with some components of the essential oils. Tricyclene is strongly correlated with camphene (R=0.992), sabinene is highly correlated with both p-cymene (R=0.839) and terpinen-4-ol (R=0.868) and p-cymene is also highly correlated with terpinen-4-ol (R=0.861). On the other hand, α-pinene is reversibly highly correlated with terpinen-4-ol (R= −0.766), sabinene (R= −0.766) and p-cymene (R= −0.71). Axis F1, which represents 33.92% of the total information, is highly negatively correlated with α-pinene and in good correlation with both tricyclene and camphene, and positively highly correlated with terpinen-4-ol, sabinene and p-cymene. This axis is also in good correlation with γ-terpinene and isocineole. This axis separates the samples into two point clouds (Figure 1). The F2 Axis, which represents 18.84% of the total information, is positively and highly correlated with only one compound which is spathulenol, and negatively correlated with limonene, β-pinene, δ-3-carene and myrcene. This last axis can note separates the clouds of samples (Figure 1). The α-pinene chemotype was found to be more abundant in comparison with sabinene/terpinen-4-ol chemotype, since the number of the samples of this cluster represents 59.6% of the total samples used.


3.2. Hierarchical Ascendant Classification (HAC)

The second method used was HAC (Hierarchical Ascendant Classification). The cluster analysis was performed using the Ward’s technique. Similarly to PCA method, the result of HAC showed and confirmed the existence of the same two different clusters (I and II) within the essential oils of the individuals of P. atlantica (Fig. 2). The distance measuring the dissimilarity was very important between the two identified clusters, which mean that the two chemotypes could be easily distinguished. Taking into account the chemical content range, we can consider two groups or chemotypes, as shown in Figure 2S. These two chemotypes could be easily differentiated upon a three different components: α-pinene, sabinene and terpinen-4-ol. The table 1S gives the data for each chemotype. Finally, it was detected only one high value of unidentified component “unknown 5” with a percentage of 18.4% which was the opposite of the rest of samples where its content didn’t exceed 3.3%.


Regarding the minor compounds, there were some differences related to some components. For example, the contents of bicyclogermacrene and globulol had reached very important values in the essential oils of mature galls with maximal percentages of 7.2 and 3.6%, respectively. At the contrary in the EO’s of unripe galls, the contents of bicyclogermacrene and globulol haven’t been detected.


Figure S1: Factor loading of 14 variables on axes F1 and F2 using Principal Component Analysis of the 52 P. atlantica essential oil samples from Algeria.





A: Region of Aïn-Oussera, L: Region of Laghouat, K: Location of Kheneg, T: Location of Tihlghemt, M: male tree, F: female tree.
Figure S2: Histograms showing the compositions distribution of the main compounds in the essential oil samples of P. atlantica (two identified chemotypes).

Table S1: Mean and range variation of the two identified chemotypes of P. atlantica galls (unripe) essential oil.


Compounds

chemotype (I)a

α-pinene




chemotype (II)b

α-pinene/sabinene/terpinen-4-ol




RIc

Identification




min

max

mean

sd




min

max

mean

sd










Tricyclene

0.1

1.4

0.7

0.3




tr

0.7

0.3

0.2




1011

MS, RI

α-Pinene

12.5

81.5

55.2

15.7




14.2

32.8

23.2

5.1




1025

MS, RI

Camphene

0.5

5.8

2.5

1.3




0.3

2.0

0.9

0.4




1063

MS, RI

β-Pinene

2.3

18.0

9.0

4.3




3.4

9.4

5.8

1.95




1106

MS, RI

Sabinene

tr

1.9

0.6

0.4




8.1

28.0

21.5

4.6




1119

MS, RI

δ-3-Carene

tr

1.1

0.4

0.3




tr

0.5

0.2

0.1




1147

MS, RI

Myrcene

0.5

20.4

4.5

4.2




1.1

12.4

5.6

3.1




1165

MS, RI, AS

α-Phellandrene

tr

1.3

0.2

0.4




tr

1.1

0.2

0.3




1169

MS, RI

Isocineole

tr

0.8

0.1

0.2




tr

2.1

0.4

0.6




1183

MS, RI

Limonene

1.3

6.9

3.7

1.3




1.3

7.2

2.2

1.2




1205

MS, RI

β-Phellandrene

tr

3.1

1.3

0.81




tr

2.5

1.1

0.5




1214

MS, RI, AS

γ-Terpinene

tr

0.9

tr

0.2




tr

7.8

1.2

1.8




1247

MS, RI

p-Cymene

0.4

1.9

1.0

0.4




0.5

11.4

6.4

2.5




1274

MS, RI

α-Terpinolene

tr

4.0

0.7

1.0




tr

4.4

0.9

1.0




1296

MS, RI

Unknown 1

tr

0.8

0.3

0.2




tr

0.2

0.1

0.1




1386

MS, RI

β-Thujone

tr

0.2

0.1

0.1




tr

0.2

0.1

0.1




1450

MS, RI

Camphor

tr

0.2

tr

0.1




tr

0.1

tr

tr




1477

MS, RI

p-Menth-3-en-1-ol

tr

2.2

0.3

0.5




tr

0.6

0.1

0.2




1586

MS, RI

Bornyl acetate

0.1

3.7

1.0

0.8




0.1

0.7

0.3

0.2




1591

MS, RI

Terpinen-4-ol

0.2

3.8

0.6

0.6




8.7

20.9

13.1

3.1




1617

MS, RI, AS

Myrtenal

tr

0.4

tr

0.1




tr

0.2

0.1

0.1




1643

MS, RI

Unknown 2

tr

0.1

tr

tr




0.1

0.6

0.4

0.1




1662

MS, RI

E-Pinocarveol

tr

0.5

0.2

0.1




tr

0.3

tr

0.1




1670

MS, RI

Cryptone

0.1

1.8

0.7

0.4




tr

0.8

0.4

0.2




1694

MS, RI

Unknown 3

tr

2.7

0.4

0.6




tr

1.7

0.5

0.4




1701

MS, RI

α-Terpineol

0.7

6.7

2.7

1.6




1.2

3.3

2.2

0.6




1707

MS, RI, AS

Myrtenol

tr

0.3

tr

0.1




tr

0.2

tr

tr




1772

MS, RI

Unknown 4

tr

0.8

0.3

0.2




tr

0.6

0.2

0.1




1807

MS, RI

p-Cymen-ol

0.1

1.5

0.6

0.4




0.2

0.9

0.4

0.2




1869

MS, RI

Unknown 5

tr

18.4

1.1

3.3




tr

2.4

0.5

0.7




2038

MS, RI

Unknown 6

tr

0.2

tr

0.1




tr

0.6

0.3

0.2




2116

MS, RI

Unknown 7

tr

0.1

0.2

0.2




0.1

1.5

0.6

0.3




2130

MS, RI

Spathulenol

0.1

5.0

1.0

1.1




0.2

1.5

0.8

0.3




2143

MS, RI

Myristic acid

tr

6.0

0.8

1.2




0.2

1.0

0.5

0.2




2726

MS, RI

Palmitic acid

tr

0.9

0.1

0.2




tr

0.3

0.1

0.1




2912

MS, RI








































Monoterpenes HC

19.3

95.1

79.9

16.6




49.0

84.5

69.5

8.3










Oxygenated monoterpenes

2.2

14.4

5.5

2.8




12.0

27.0

16.8

3.7










Total monoterpenes

22.9

97.7

85.4

15.7




67.8

96.5

86.3

7.2










Oxygenated sesquiterpenes

0.1

5.0

1.0

1.1




0.2

1.5

0.84

0.3










Other compounds

0.5

20.6

3.0

3.6




0.6

5.8

2.9

1.3










Fatty acids

0.0

6.0

0.9

1.3




0.2

1.3

0.6

0.3

















































EO yield % (v/w)

0.32

1.89

0.96

0.37




0.08

0.98

0.71

0.20










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