Bacteriostatic and Bactericidal Profile of Leaves and Twigs Essential oils of Moroccan Pistacia lentiscus

Taoufik Haloui , Abdellah Farah , Mounyr Balouiri , Marwa Chraibi , Mouhcine Fadil , Kawtar Fikri Benbrahim , Aziz Belrhiti Alaoui 1 1 Laboratory of Functional Ecology and Environment, Faculty of Sciences and Technology, Sidi Mohamed Ben Abdellah University, BP 2202 Road Imouzzer. Fez, Morocco. 2 Laboratory of Aromatic Plants, Medicinal and Natural Substances, National Institute of Medicinal and Aromatic Plants, Sidi Mohamed Ben Abdellah University, P.O. Box 159. Taounate, 34025, Morocco. 3 Laboratory of Microbial Biotechnology, Faculty of Science and Technology Saïss. Sidi Mohamed Ben Abdellah University, P.O.Box 2202. Fez, Morocco.


INTRODUCTION
For centuries, medicinal plants have been used as a remedy for various diseases. Over the past few years, the use of plant-based natural antimicrobials in the treatment of bacterial infections has gained much recognition (Kasrati et al., 2014).The progressive interest of these plant's use has urged researchers to look for new methodologies (Hemaiswarya et al., 2008), and for suitable design to develop new therapeutic value products with highly effective anti-infective agents in general and antibacterial agents in particular (Turgis et al., 2012).
Pistacia lentiscus L. belong to the family of Anacardiaceae, also called pistachio mastic or mastic tree (Hmamouchi et al., 1999). Shrub up to 5 m high, the mastic is one of the most characteristic trees of the Mediterranean region (Aafi et al, 2002), which grows on all kinds of soil (Bayer et al., 2009) and it is a native species of Morocco (Aafi et al, 2002). The therapeutic properties of this species were known, long time ago, when the Egyptians used mastic for embalming (De Pooter et al., 1991). The essential oil of mastic tree has been shown to have antibacterial (Derwich et al., 2010), anti-fungal (Darua et al., 2003, insecticides (Bachrouch et al., 2010) and antioxidants effects (Barra et al., 2007). Mastic tree is also used in cosmetics, perfumes and as a flavoring in food preparations (Daferera et al., 2002). The aim of this study was to evaluate the antibacterial activity and to identify the chemical profiles of essential oils from twigs and leaves of Pistacia lentiscus L. collected in Taounate region (Morocco).

Plant material
Leaves and twigs of P. lentiscus L. were randomly collected from natural populations at the flowering stage during May 2013 in the Ifrane's forest at Taounate region in Morocco (Altitude: 475 m, 34° 35'12.5" N 4° 38'31.1" W). Pistacia lentiscus leaves were separated from stem, and the twigs were cut into small pieces to facilitate the extraction of essential oils.

Extraction of essential oil
The essential oils were extracted from P. lentiscus L. by hydrodistillation process in Clevenger-type apparatus (Clevenger, 1928). Plant material was dried at 105 °C for 4 hours to determine the moisture (Zrira et al., 1995) and the yield was expressed relatively to the dry matter. Essential oils obtained were stored in opaque glass bottles at 4 °C.

Chemical analysis of essential oil
The essential oil was analyzed using Gas chromatography (GC) coupled to mass spectrometry GC / MS (Polaris Q ion trap MS). Hence, analyses were performed on a Hewlett-Packard (HP 6890) gas chromatograph (FID), equipped with a 5% phenyl methyl silicone HP-5 capillary column (30m x 0.25 mm x film thickness 0.25 µm). The temperature was programmed from 50°C after 5 min initial hold to 200°C at 4°C/min. Chromatography carrier gas was N2 (1.8 ml/min), split mode was used (Flow: 72.1 ml/min, ratio: 1/50), temperature of injector and detector was 250 °C, final hold time was 48 min. The machine was led by a computer system type "HP Chem Station", managing its functioning and allowing to follow the evolution of chromatographic analyses. Diluted samples (1/20 in methanol) of 1µl were injected manually.

Bacterial strains
The in vitro antibacterial effect of essential oils was tested against the following bacterial strains: Pseudomonas aeruginosa ATCC 27853, Bacillus subtilis ATCC 3366, Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922. All of these strains were maintained in 20 % glycerol at -20 °C as stock.

Inoculum preparation
The direct colony suspension method was used for the inoculum preparation. Briefly, bacteria were sub-cultured in the Luria Bertani agar (LB). Plates were incubated at 37 °C for 24 h. A loop full of isolated colony was aseptically transferred into physiologic saline solution and the turbidity of the suspension was adjusted to 0.5 McFarland (Murray et al., 2007).

Antibacterial screening
The screening of antibacterial activity was performed by the agar disc-diffusion method (Murray et al., 2007). Petri dishes (90 mm in diameter) containing LB-agar were seeded using the previously prepared inoculum. The seeding was done so as to ensure a homogeneous distribution of bacteria, then excess liquid was eliminated with a Pasteur pipette and the Plates were dried for 20 minutes. The sterile filter paper discs (6 mm in diameter) were individually soaked with 10 µl of each essential oil then placed on the surface of plates seeded, which were placed at 4 °C for 2 h. After incubation for 24 h at 37 °C, inhibition diameters were measured. All the tests were performed in triplicate.

Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
The broth microdilution method was used to evaluate the minimum inhibitory concentration (MIC) according to the CLSI guidelines M7-A7 (CLSI, 2007), with slight modifications. Briefly, agar at 0.15% (w⁄v) was used as emulsifier and resazurin was used as bacterial growth indicator (Mann and Markham, 1998). Firstly, 50 µl of Mueller Hinton Broth (MHB) supplemented with bacteriological agar (0.15% w/v) were distributed from the second to the 12th well of a 96-well polypropylene microtitre plate. Essential oils dilutions were prepared in MHB supplemented with agar (0.15% w/v), 100 µl of these suspensions were added to the first test well of each microtitre line, then 50 µl of scalar dilution were transferred from the second to the 11th well. The 12 th well was considered as growth control. Then, 50 µl of a bacterial suspension was added to each well at a final concentration of approximately 10 6 CFU/ml. The final concentration of the essential oil was between 16 and 0.0015% (v⁄ v) for leaves and twigs. After incubation at 37°C for 20 h, 5 µl of resazurin was added to each well (Mann and Markham, 1998). After further incubation at 37°C for 2 h, the MIC was determined as the lowest essential oil concentration that prevented a change in resazurin color (CLSI, 2007). Experiments were conducted in triplicate.
To determine the minimum bactericidal concentration (MBC), 2 µL of each negative well, in which microbial growth was not observed, were spotted on LB plates and incubated at 37 °C for 24 h. The MBC corresponded to the lowest concentration of the essential oil at which the incubated microorganism was completely killed (Bassole et al., 2001). Each test was performed in triplicate.

Yield and chemical composition of the leaves and twigs essential oils
The hydrodistillation of Pistacia lentiscus leaves and twigs gave essential oils with yields of 0.3% and 0.5% (v/w) respectively.
Twenty six compounds, which represented 84.72% of the twigs essential oils, were identified. The major constituents of this essential oil were α-pinene (19.24%), tricyclene (8.16%), trans-β-Ocimene (6.9%), caryophyllene (6.18%), 3-carene (5.18%) and germacrene (5.17%). Similar compounds have been reported at different percentages by other authors (Amhamdi et al., 2009). However, the number of compounds identified in leaves and twigs essential oil (29 and 26) respectively, was inferior to the number of compounds identified in a previous study (Hafsé et al., 2013). This could be explained by: environmental factors such as geography, temperature and collection period, etc., which were considered to play a key role in the chemical composition of essential oils (Derwich et al., 2010).

Antibacterial activity
Results of both essential oil's antibacterial activity against S. aureus, E. coli, B. subtilis and P. aeruginosa are shown in table 2. As can be noted in this finding, both essential oils tested have shown a remarkable antibacterial effect. So, crude essential oils of the two Pistacia lentiscus parts were active against all strains examined. Indeed, the MIC values ranged from 4 to 0.015% (v/v) for leave's essential oil and from16 to 0.5 % (v/v) for twig's essential oil. Hence, leaves essential oil exhibit a higher antibacterial effect with MIC values 0.015, 0.5, 1 and 4 % fold least compared to twigs essential oil with MIC values 0.5, 4, 4 and 16% against B. subtilis, S. aureus, E. coli and P. aeruginosa respectively. Also, it can be noted that E. coli and P. aeruginosa (Gram-negative) were more resistant to the leaves and twigs essential oil compared to S. aureus and B. subtilis (Gram-positive).
Regarding the MBC values of both essential oils tested (Table 3), we found that MBC values could well be similar to their MIC values against P. aeruginosa and B. subtilis and two fold higher toward S. aureus and E. coli for twigs essential oil. In addition we noted that MBC values were 2, 4, 8 and 2 fold higher toward B. subtilis, S. aureus, E. coli and P. aeruginosa respectively than MIC for leaves essential oil.   Antimicrobial activity of the essential oils of P. lentiscus against tested bacteria has shown that Gram-negative strains were more resistant compared to the Gram-positive ones. Similar findings have been reported by other authors (Hafsé et al., 2013) who found that Gram negative strains were less sensitive to this essential oil than Gram-positive strains.
Pseudomonas aeruginosa has shown low sensitivity with MIC values of 16 and 8% for leaves and twigs essential oils respectively. Similarly, previous investigations showed that this bacterial strain was more resistant to leaves essential oils of P. lentiscus (Benhammou et al., 2008). Typically, Gram-negative bacteria are more resistant to essential oils than Gram-positive bacteria, due to the differing structures of their cell wall. Outer membrane of the Gram-negative bacteria contains primarily lipopolysaccharides molecules and forms a hydrophilic barrier conferring protection against the effects of highly hydrophobic compounds (Trombetta et al., 2005). The mechanism of action assigned at both essential oils has not been studied in detail in the past. The results of this study confirm the findings in previous reports, which state that the strength and spectrum of activity varied between Gram type of target bacteria and the investigated parts of P. lentiscus (Djenane et al., 2011).
The antibacterial activity of the essential oils of P. lentiscus could be attributed to their high content of different groups of chemical compounds known for their antibacterial effect. Nevertheless, the antibacterial activities of the essential oils are difficult to correlate to a specific compound due to their complexity (Mélanie et al., 2012). The low antibacterial activity of twigs essential oil compared to the leaves one could be explained by their high content on terpene (α-pinene, tricyclene, 3-carene, trans-β-Ocimene and D-germacrene) known for their relatively low antibacterial activity (Inouye et al., 2001). Moreover, many reports have shown that α-terpineol, present with high percentage in leaves essential oil, exhibit an inhibitory effect against E. coli (Alessandra et al., 1999). Similarly geraniols, present only in the leaves oil, exhibit a higher antibacterial effect (Antonio et al., 2007).

CONCLUSION
This work aims to evaluate the bacteriostatic and bactericidal profile of essential oils of leaves and twigs of Pistacia lentiscus L.against B. subtilis, S. aureus, E. coli and P. aeruginosa. Both studied essential oils presented remarkable antibacterial activity against tested strains. B. subtilis was the most sensitive strain in regard to the two essential oils while P. aeruginosa was the most resistant strain. The high antibacterial performance of Pistacia lentiscus essential oils from leaves and twigs should be studied in more details in order to make them a promising antibacterial agent for the control in food and pharmaceutical industries.