Chemical composition and antimicrobial activity of the essential oils from four Ruta species growing in Algeria

Chemical composition and antimicrobial activity of the essential oils from four Ruta species growing in Algeria

Food Chemistry 141 (2013) 253–258 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodch...

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Food Chemistry 141 (2013) 253–258

Contents lists available at SciVerse ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Chemical composition and antimicrobial activity of the essential oils from four Ruta species growing in Algeria Farah Haddouchi a,⇑, Tarik Mohammed Chaouche a, Yosr Zaouali b, Riadh Ksouri c, Amina Attou a, Abdelhafid Benmansour a a b c

Laboratory of Natural Products, Department of Biology, Faculty of Sciences, Abou Bekr Belkaïd University, B.P. 119, Tlemcen 13000, Algeria Laboratory of Plant Biotechnology, Department of Biology, National Institute of Applied Science and Technology (INSAT), B.P. 676, 1080 Tunis Cedex, Tunisia Laboratory of Plant Adaptation to Abiotic Stresses, Biotechnologic Center in Borj-Cedria Technopol (CBBC), B.P 901, 2050 Hammam-Lif, Tunisia

a r t i c l e

i n f o

Article history: Received 2 February 2013 Received in revised form 1 March 2013 Accepted 2 March 2013 Available online 14 March 2013 Keywords: Antibacterial activity Antifungal activity Ruta species Essential oil GC/MS analysis

a b s t r a c t Antimicrobial properties of plants essential oils have been investigated in order to suggest them as potential tools to overcome the microbial drug resistance and the increasing incidence of food borne diseases problems. The aim of this research is to study the antibacterial and antifungal effects of four traditional plants essential oils, Ruta angustifolia, Ruta chalepensis, Ruta graveolens and Ruta tuberculata, against standard bacterial and fungal strains. The chemical compounds of the oils were examined by GC/MS. Results revealed a powerful antifungal activity against filamentous fungi. Aspergillus fumigatus and Cladosporium herbarum are the most sensitive strains to these oils with MIC values less than 3.5 lg ml1 for certain oils, reaching 7.8 lg ml1 for other. GC/MS essay exhibited ketones as the most abundant constituent of these oils except for R. tuberculata essential oil which has a completely different composition, monoterpenes alcohols being the most abundant. These compositions explain their potential antifungal activity. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction The development of drug resistance as well as the appearance of side effects of certain antibiotics has led to the search of new antimicrobial agents mainly among plant extracts with the goal to discover new chemical structures which overcome the above disadvantages (Lewis & Ausubel, 2006). Thus, the food industry at present uses chemical preservatives to prevent the growth of food borne and spoiling microbes and to extend the life of foods. Mainly due to undesirable effects such as toxicity and carcinogenicity of synthetic additives, interest has considerably increased for finding naturally occurring antimicrobial compounds suitable for use in food (Feng & Zheng, 2007). With antimicrobial studies, the chemical composition should ideally be used to correlate any structure activity relationships (Van-Vuuren & Viljoen, 2007) . Essential oils are complex mixtures comprising many single compounds. Each of these constituents contributes to the beneficial or adverse effects of these oils. Therefore, the intimate knowledge of essential oil composition allows for a better and specially directed application (Dorman & Deans, 2000). Many oils have been identified as antimicrobials. This activity is variable of one to another and from one microbial strain to another (Kalemba & Kunicka, 2003). ⇑ Corresponding author. E-mail address: [email protected] (F. Haddouchi). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.03.007

Ruta chalepensis var. bracteosa is characterised by its petals fringed, matching only half the width of the petals and bracts much larger than the stem to which they are attached. Ruta graveolens is branded by its petals not fringed and it fruit rounded, while Ruta angustifolia is determined by its oval leaves in their general outline, 2–3 times divided into segments oblong and ciliate-fringed sepals (Bonnier, 1999). Ruta tuberculata is characterised by its leaves lanceolate or often elongated and small flowers with four petals yellow (Ozenda, 1991). The phytochemical studies conducted on these species characterise the presence of amino acids, saponins (Hnatyszyn, Arenas, Moreno, Rondina, & Coussio, 1974), alkaloids, flavonoids, coumarins, tannins, volatile oil, glycosides, sterols and triterpenes (Chen, Huang, Huang, Wang, & Ou, 2001). They are used in the traditional medicine of many countries for the treatment of a variety of diseases. Exciting, diaphoretic, antiseptic, antispasmodic, anthelmintic (Bonnier, 1999), emmenagogue, abortifacient and anti-inflammatory properties (Raghav, Gupta, Agrawal, Goswami, & Das, 2006), are assigned to R. chalepensis var. bracteosa, R. graveolens and R. angustifolia. R. tuberculata treats bone and joint pain, dysmenorrhea, infertility in women, anaemia and headache (Hammiche & Maiza, 2006). In this paper we report the chemical composition and the antibacterial and antifungal activities of four Ruta species essential oils belonging to the family of Rutaceae. These are R. chalepensis var. bracteosa (DC.) Boiss., R. graveolens L., R. angustifolia Pers.,

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originating around the Mediterranean and R. tuberculata Forsk. common throughout the northern Sahara. 2. Materials and methods 2.1. Plant material The aerial parts of plants were collected at flowering, in June 2011, from four different regions of Algeria. R. chalepensis var. bracteosa was collected from Ain Temouchent (35°140 5400 N, 1°140 5600 W) and R. angustifolia from Tlemcen (35°000 4700 N, 1°440 5100 W), both in the West of Algeria. R. graveolens was collected from Anaba (36°540 1500 N, 7°450 0700 E) in the East and R. tuberculata from Bechar (31°370 0000 N, 2°130 0000 W) in the south of Algeria. They were identified in the Laboratory of Natural Products, Department of Biology, University of Tlemcen, Algeria. Voucher specimens were deposited at the Herbarium of the Laboratory. The plants were dried at room temperature for two weeks. 2.2. Essential oils isolation procedure One hundred grams of aerial parts from each species were submitted to hydro-distillation for 3 h using a Clevenger apparatus. Oils were recovered directly, using a micro-pipette from above the distillate without adding any solvent, and stored in dark vials at 4 °C until analysis. Yields, expressed as 100 g of dried weight, were stated in mean ± standard deviation of three replicates. 2.3. Essential oil analysis The essential oils were analysed by gas chromatography-mass spectrometry (GC–MS), a gas chromatograph, Agilent Technologies 7890A using a HP 5975C Mass spectrometer with electron impact ionisation (70 eV). A HP-5MS capillary column (30 m  250 lm, 0.25 lm film thickness) was used. Oven temperature was programmed to rise from 60 to 220 °C at a rate of 40 °C/min; transfer line temperature was 230 °C. The carrier gas was He with a flow rate of 0.8 ml/min and a split ratio of 50:1. Scan time and mass range were 1 s and 50–550 m/z, respectively (Zaouali, Chograni, Trimech, & Boussaid, 2013). The components were identified by comparing their relative retention times and mass spectra with the data from the library of essential oil constituents, Wiley, Mass-Finder and Adams GC/ MS libraries. Determination of the percentage composition was based on peak area normalisation without using correction factors. 2.4. Sources of microbial cultures The antimicrobial activity of Ruta species essential oils was evaluated using laboratory reference strains (American Type Culture Collection ‘‘ATCC’’ for bacteria and Candida albicans, National Museum of Natural History ‘‘NMHN’’ for filamentous fungi), obtained from Laboratory of Natural Products, Department of Biology (Tlemcen University, Algeria): Gram-positive bacteria: Bacillus cereus (ATCC 10876), Enterobacter cloacea (ATCC 13047), Enterococcus faecalis (ATCC 49452), Listeria monocytogenes (ATCC15313), Staphylococcus aureus (ATCC 25923). Gram-negative bacteria: Acinetobacter baumanii (ATCC 19606), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 700603), Pseudomonas aeruginosa (ATCC 27853), Proteus mirabilis (ATCC 35659), Salmonella typhimurium (ATCC 13311), Citrobacter freundii (ATCC 8090). Fungal microorganisms: Candida albicans (ATCC 26790), Aspergillus fumigatus (MNHN 566), Aspergillus flavus (MNHN 994294), Cladosporium

herbarum (MNHN 3369), Fusarium oxysporum (MNHN 963917), Alternaria alternaria (MNHN 843390). Test strains were cultured and turbidity was adjusted to the 0.5 standard of the McFarland scale, corresponding to 1–2  108 CFU/ ml for bacteria (DO = 0.08–0.1/k = 625 nm), 1–5  106 CFU/ml for yeast C. albicans (DO = 0.12–0.15/ k = 530 nm) (NCCLS, 2001) and 106 spores/ml (68–82% transmittance/k = 530 nm) for filamentous fungi (Pfaller, Messer, Karlsson, & Bolmstrom, 1998). The cultures were diluted with Mueller–Hinton broth (MHB) for bacteria, Sabouraud dextrose broth (SDB) for C. albicans and sterile saline for fungal strains, to achieve optical densities corresponding for each test. 2.5. Antimicrobial screening Two different methods were employed for the determination of in vitro antimicrobial activity of the essential oils: an agar disc diffusion method and dilution methods (Broth micro-dilution method for bacteria and C. albicans, Agar dilution method for fungi). The inhibition zones and minimum inhibitory concentrations (MICs) of gentamycin and amphotericin B were also determined in parallel experiments in order to control the sensitivity of the test microorganisms. All tests were performed in duplicate. 2.5.1. Determination of Antimicrobial activity by the disc diffusion method The essential oils were tested for their antimicrobial activity by the disc diffusion method according to the National Committee for Clinical Laboratory Standards (NCCLS, 2001) using 100 ll of suspension of the tested microorganisms, containing 2  108 CFU/ml for bacteria, 1–5  106 CFU/ml for C. albicans and 2  105 spores/ ml for fungal strains. Mueller–Hinton agar and Sabouraud dextrose agar sterilized in a flask and cooled to 45–50 °C were distributed into sterilized Petri dishes with a diameter of 9 cm (15 ml). The filter paper discs (6 mm in diameter) were individually impregnated with 10 ll of the oil and then placed onto the agar plates which had previously been inoculated with the tested microorganisms. The Petri dishes were kept at 4 °C for 2 h. The plates were incubated at 37 °C for 24 h for bacteria and at 30 °C for 24 and 48 h for C. albicans and fungal strains, respectively. The diameters of the inhibition zones (mm) were measured including the diameter of discs. All the tests were performed in duplicate. Gentamycin (15 lg/disc) and amphotericin B (20 lg/disc) served as positive controls. 2.5.2. Determinations of the minimum inhibitory concentration (MIC) For bacteria and C. albicans, a broth micro-dilution method was used to determine the MIC according to the National Committee for Clinical Laboratory Standards (NCCLS, 2001). All tests were performed in MHB. The investigated oils were dissolved in 1% dimethylsulfoxide (DMSO) and then diluted to the highest concentration. Serial doubling dilutions of oils were prepared in a 96-well microtiter plate over the range of 0.3–163 lg ml1. Overnight broth cultures of each strain were prepared and the final concentration in each well was adjusted to 5  105 CFU/ml for bacteria and to 2.5  106 CFU/ml for C. albicans. The bacteria and C. albicans were incubated for 24 h, at 37 and at 30 °C, respectively. The MIC is defined as the lowest concentration of the essential oil at which the microorganism does not demonstrate visible growth. The microorganism growth was indicated by the turbidity. For filamentous fungi, MICs were determined by the dilution method in agar medium (Soliman & Badeaa, 2002). The tested strains were grown on potato dextrose agar (PDA) medium, on petri dishes, for 5–7 days. Each of the tested oils dissolved in 1% DMSO was used at different concentrations. Each concentration was mixed with sterilized semi-solidified PDA medium and then

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poured into sterilized Petri dishes (15 ml in each plate) over the range of 3.5–9 lg ml1. A 6 mm diameter agar disk covered with mycelium was placed on the surface of the agar. Plates were incubated for 5–7 days at 28 °C. Two replicates were used for each treatment. Gentamycin and Amphotericin B were used as reference compound. 3. Results 3.1. Essential oils isolation Air-dried herbal of R. chalepensis var. bracteosa, R. graveolens, R. angustifolia and R. tuberculata were subjected to hydrodistillation and the yields of essential oils are respectively 0.9 ± 0.04, 0.18 ± 0.01, 1.49 ± 0.36 and 0.11 ± 0.01% (w/w). 3.2. Chemical composition of the essential oil Essential oils, extracted from R. chalepensis var. bracteosa, R. graveolens, R. angustifolia and R. tuberculata, were analysed by GC/MS. Tables 1 and 2 shows the chemical composition of the essential oils together with the retention times of the compounds. A total of 16 compounds were identified from the essential oil of R. chalepensis var. bracteosa, which represented 97.65% of the oil extracted. 2-Nonanone and 2-Undecanone are the dominant ones at 32.79% and 32.58%, respectively, followed by 1-Nonene at 13.95% and a-Limonene at 5.27%. The profile of the oil components of R. graveolens was almost similar to that of R. chalepensis var. bracteosa. 10 compounds, representing 97.3% of the essential oil, were

Table 1 Essential oils composition of R. chalepensis var.bracteosa (RCb), R. graveolens (RG) and R. angustifolia (RA). Rt

5.479 6.406 6.778 7.87 9.799 9.976 10.091 11.155 11.327 11.332 12.923 14.388 16.35 16.493 18.513 18.519 19.457 20.636 22.57 27.669 31.411 31.662 39.988

Compounds

a-Pinene Sabinene 2-Octanone a-Limonene 2-Nonanone NI Nonanal 2-Octanol acetate 5,6-Diethenyl-1-methyl-cyclohexene Geyrene 2-Decanone 1-Nonene 2-Undecanone NI NI 11-Dodecen-2-one 2-Dodecanone 1-Tetradecanol methacrylate 2-Tridecanone NI NI NI 12-Methoxy-19-norpodocarpa3,5,8,11,13-pentaen-7-one

Chemical classes Ketones 2-Ketones Acyclic alkenes Monoterpene hydrocarbons Sesquiterpenes Esters Aldehydes All identified components Rt: retention time, NI: not identified,

% RCb

RA

RG

0.31 0.49 0.39 5.27 32.79 1.14 0.62 0.89 – 1.46 2.42 13.95 32.58 1.21 – 0.99 0.6 3.29 0.63 – – – 0.97

– – – 0.96 – 0.84 – – – 10.03 – 82.46 1.56 – – 1.51 – 0.67 0.83 – 1.13 –

– – – 4.26 21.62 – – – 3.24 – 1.46 4.35 55.4 – 1.03 – 1.02 2.22 1.58 – 1.67 – 2.14

71.37 69.41 13.95 06.07 01.46 04.18 00.62 97.65

95.63 95.63 – – – – 00.84 96.47

83.22 81.08 04.35 07.50 – 02.22 – 97.30

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Table 2 Essential oil composition of R. tuberculata (RT). Rt

Compounds

RT (%)

5.307 5.479 6.406 6.789 7.201 7.355 7.762 7.89 10.69 11.264 13.077 13.489 15.006 16.001 16.35 17.752 20.327 20.722 23.938 24.498 25.259

a-Thujene a-Pinene

0.99 1.79 1.53 3.05 2.22 5.45 2.9 10.9 13.14 11.22 4.12 12.31 13.62 1.25 1.59 0.98 1.87 0.79 1.14 7.32 1.84

Sabinene b-Myrcecne (R)-a-Phellandrene 3-Carene p-Cymene b-Phellandrene trans-p-Menth-2-en-1-ol cis-p-Menth-2-en-1-ol trans-p-Menth-1-en-3-ol (trans-Piperitol) cis-p-Menth-1-en-3-ol (cis-Piperitol) Piperitone Bornyl acetate 2-Undecanone a-Terpinene b-Caryophyllene c-Elemene NI Germacrene-B b-Caryophyllene epoxide

Chemical classes Ketones 2-Ketones Monoterpene hydrocarbons Sesquiterpene hydrocarbons Esters Monoterpene alcohols Oxides tricyclic All identified components

15.21 1.59 29.81 9.98 01.25 40.79 01.84 98.88

Rt: retention time, NI: not identified.

identified. The major components were 2-Undecanone and 2-Nonanone (55.4 and 21.62%) followed by 1-Nonene and a-Limonene at 4.35 and 4.26% respectively. For R. angustifolia, 6 compounds, representing 96.47% of the essential oil, were identified with the dominance of 2-Undecanone (82.46%). The 2-Decanone represents 10.03%. In contrast, 20 compounds were identified in essential oil of R. tuberculata, representing 98.88% of the volatile compounds. 54.41% of these compounds were represented by Piperitone (13.62%) and Monoterpene alcohols which are, trans-p-Menth2-en-1-ol (13.14%), cis-p-Menth-2-en-1-ol (11.22%), trans-pMenth-1-en-3-ol (4.12%) and cis-p-Menth-1-en-3-ol (12.31%). b-Phellandrene, Germacrene-B and 3-Carene represents 10.9, 7.32 and 5.45% respectively. 3.3. Antimicrobial activity The in vitro antimicrobial activity of Ruta species essential oils against the microorganisms employed and its activity potentials were qualitatively and quantitatively assessed by the presence or absence of inhibition zones, zone diameters and MIC values. 3.3.1. Antibacterial activity The disc diameters of zone of inhibition of essential oils against the bacteria tested are shown in Table 3. The results obtained indicated that the essential oils of the investigated species had low in vitro potential of antibacterial activity against all 12 bacteria tested. The highest activity was observed against S. aureus and S. typhi with the strongest inhibition zones (15 and 17 mm, respectively) recorded for R. chalepensis var. bracteosa essential oil, followed by B. cereus and A. baumanii (12 mm). R. graveolens oil exhibited weak inhibition zones on B. cereus, S. aureus and S. typhi (12 mm) while R. tuberculata oil exhibited modest antimicrobial activity against E. faecalis (14 mm). For R. angustifolia oil, no antibacterial activity was detected on all strains tested. Tested bacteria

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Table 3 In vitro antibacterial activity of essential oils.

Table 5 In vitro antifungal activity of essential oils.

Inhibition diametersa (mm)

Inhibition diametersa (mm)

Essential oils strains

RA RCb 10 ll/disc

RG

RT

Gentamycin 15 lg/disc

Essential oils strains

RA RCb 10 ll/disc

RG

RT

Amphotericin B 20 lg/disc

Enterobacter cloacea Enterococcus faecalis Acinetobacter baumanii Bacillus cereus Escherichia coli Citrobacter freundii Pseudomonas aeroginosa Klebsielle pneumaniae Lysteria monocytogenes Proteus mirabilis Staphylococcus aureus Salmonella typhi

6 8 6 10 6 7 6 6 6 6 6 6

6 9 6 12 7 6 6 6 6 7 12 12

6 14 6 12 6 6 6 6 6 6 10 8

26 9 12 16 20 27 19 17 12 14 17 16

Candida albicans Cladosporium herbarum Fusarium oxysporum Alternaria alternaria Aspergillus flavus Aspergillus fumigatus

35 18 20 25 15 20

33 25 20 25 22 15

17 34 16 20 17 17

22 37 08 27 17 28

6 10 12 12 7 6 6 6 6 10 17 15

RG: R. graveolens, RT: R. tuberculata. a Diameter disc (6 mm) included, RA: R. angustifolia, RCb: R. chalepensis var.bracteosa.

were more sensitive to Gentamycin (12–27 mm) than to the essential oils tested, except for S. aureus and S. typhi which are more susceptible to R. chalepensis var. bracteosa oils, and for E. faecalis being more susceptible to R. tuberculata and the most resistant to this antibiotic (9 mm). For R. chalepensis var. bracteosa and R. tuberculata, results were confirmed by the MIC values (Table 4). The inhibitory properties of the oils were observed within a range of concentrations from 18 to163 lg ml1. MICs of R. tuberculata oil against Enterococcus faecalis was ranged between 18 and 37 lg ml1. Both oils are less active than the antibiotic. 3.3.2. Antifungal activity Essential oils, applied at 10 ll/disc, showed consistent antifungal effects against all fungi strains tested (Table 5). The highest antifungal activity of R. angustifolia and R. graveolens oils was observed against C. albicans (35 and 33 mm). Other strains react differently with diameters between 15 and 25 mm. C. herbarum was the most sensitive microorganism to R. chalepensis var. bracteosa and R. tuberculata essential oils (35 and 34 mm, respectively). Other strains behave differently with diameters between 15 and 23 mm, except for F. oxysporum and A. alternaria. The activities of R. chalepensis var. bracteosa and R. tuberculata essential oils against C. herbarum, F. oxysporum, A. flavus and of R. angustifolia and R. graveolens essential oils against F. oxysporum, A. alternaria, A. flavus and C. albicans, are close or more important to that of Amphotericin B. F. oxysporum being the most resistant to both Amphotericin B and R. chalepensis var. bracteosa oil. The essential oils (Table 6) showed inhibitory effects on the six tested fungi, through the MICs. It could be seen that this effect is proportional to its concentration. Against C. albicans, R. graveolens essential oil has most inhibitory effect (18 lg ml1) than other three Ruta essential oils tested. Oils have a lower activity than amphotericin B. For filamentous fungi, MIC values are less than 3.5 lg ml1 for certain oils, reaching 8.7 lg ml1 for other. MICs of amphotericin B are between 1.25 and 5 lg ml1, except for F. oxysporum which Table 4 In vitro MIC (lg ml1) values of essential oils against bacteria.

Enterococcus faecalis Staphylococcus aureus Salmonella typhi

Essential Oils MICs

Antibiotic MICs

RCb

RT

Gentamycin

ND 81–163 41–81

18–37 ND ND

5–10 0.25–0.5 1.25–2.5

RCb: R. chalepensis var.bracteosa, RT: R. tuberculata ND: not detected.

15 35 08 10 16 23

a Diameter disc (6 mm) included, RA: R. angustifolia, RCb: R. chalepensis var.bracteosa, RG: R. graveolens, RT: R. tuberculata.

Table 6 In vitro MIC values (lg ml1) of essential oils against yeast and filamentous fungi. Essential oils strains

RA

RCb

RG

RT

Amphotericin B

Candida albicans Cladosporium herbarum Fusarium oxysporum Alternaria alternaria Aspergillus flavus Aspergillus fumigatus

154 4.7 7 8.2 8.2 <3.5

41 4.9 8.7 8.7 8.7 6.2–7.4

18 6.5 7.6 7.6 7.6 <3.5

74 7.8 6.7 6.7 <4.5 <4.5

2.5 2.5 20 1.25 5.0 2.5

RA: R. angustifolia, RCb: R. chalepensis var.bracteosa, RG: R. graveolens, RT: R. tuberculata.

a value of MIC is very high (20 lg ml1), even compared to the four oils tested. A. fumigatus is the most sensitive strain to these oils, followed by C. herbarum. A. flavus was more resistant to these oils, except to R. tuberculata oil. 4. Discussion The extraction yields obtained for R. angustifolia, R. graveolens and R. chalepensis var. bracteosa are comparable or similar to those reported in the literature (Dob, Dahmane, Gauriat-Desrdy, & Daligault, 2008; Merghache, Hamza, & Tabti, 2009). As reported by Salgueiro et al. (1997), climate, genotype, growth location, rainfall and harvesting regime can affect the total essential oil content of plants. Oils of the Ruta genus are generally characterised by a mixture of 2-Ketones, which can reach 84% of the oil (Verzera, Mondello, Ragusa, & Dugo, 2000). In the present work, the 2-ketone represents 95.63%, 81.08%, 69.41% and 1.59% in essential oils of R. angustifolia, R. graveolens, R. chalepensis var. bracteosa and R. tuberculata, respectively. The major compound is 2-Undecanone except for R. tuberculata, which Piperitone is the major ketone. The monoterpene alcohols represent 40.79% of the total R. tuberculata oil, followed by the monoterpene hydrocarbons (29.81%). So we can deduce that R. tuberculata essential oil, which has never been published, has a completely different composition compared to other species of Ruta. Abundance of 2-Undecanone in oils is completely in accordance with the works done on species of Ruta genus such as R. chalepensis essential oils from Algeria (Merghache et al., 2009) and Tunisia (Ben-Bnina, Hammami, Daamii-remadi, Ben-Jannet, & Mighri, 2010; Mejri, Abderrabba, & Mejri, 2010; Saidani-Tounsi et al., 2011), R. graveolens essential oils from Egypt (El-Sherbeny, Hussein, & Khalil, 2007), Iran (Soleimani, Aberoomand-Azar, SaberTehrani, & Rustaiyan, 2009), and Malaysia (Yaacob-Karim, Abdullah, Che Mazenah, & Joulain, 1989) and R. angustifolia from Algeria (Dob et al., 2008). The nature and proportions of other compounds which constitute our oils are not always the same in comparison with these same previous works. There are affected by climatic,

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seasonal and geographic conditions, harvest period, chemotypes and/or extraction procedure (Palá-Paúl et al., 1993). According Corduan and Reinhard (1972), the light has a decisive effect on the composition of the oil. A plant of Ruta growing in shade will have different properties from another pushing sunlight. Therefore, it is hard to make a complete comparison with previously issued data. Some researchers reported that there is a relationship between the chemical structures of the most abundant compounds in the tested oil, the proportions in which they are present and to interactions between them and the antimicrobial activity (Delaquis, Stanich, Girard, & Mazza, 2002; Dorman & Deans, 2000). Pure oxygenated components showed a higher activity. This is the case of alcohols and phenols which are widely reported to possess high levels of antimicrobial activity (Dorman & Deans, 2000; Lambert, Skandamis, Coote, & Nychas, 2001). Highest antimicrobial activity was also observed for the oxygenated terpenes, compared with terpene hydrocarbons (Dorman & Deans, 2000; Griffin, Wyllie, Markham, & Leach, 1999). Because their cyclic structure, monoterpene hydrocarbons are excellent antiseptic atmospheric, but not in contact method (Aromatogram). The monoterpene alcohols are anti-infectives and ketones are weakly antiseptic, but they have antifungal action (Mailhebiau, 1994). This data explain the potential activity of the compounds of essential oils, which has been confirmed and extended in the present studies. Numerous studies demonstrated that the essential oils of Ruta species are among the less potent essential oils with regard to antibacterial properties (Ben-Bnina et al., 2010; Proestos, Boziaris, Nychas, & Komaitis, 2006). In this study, the antibacterial results showed variation between different Ruta species oils. This difference is probably due to the chemical composition of the essential oils. For R. angustifolia oil, no antibacterial activity was detected. This result was probably due to the presence of 95.63% of ketones in the total oil. The modest antibacterial nature of the R. graveolens and R. chalepensis var. bracteosa essential oils studied was apparently related to the presence of monoterpene hydrocarbons. That of R. tuberculata against E. faecalis and B. cereus can be related to the presence of monoterpene alcohols and monoterpene hydrocarbons. MICs showed larger range of values (18–163 lg ml1) in comparison with inhibition zones (14–17 mm) and MICs of antibiotic (0.25–10 lg ml1). These suggest that the size of the inhibition zone does not reflect the real antibacterial effectiveness of a compound. This point was in agreement with Cimanga et al. (2002) suggestions. These values do not mean that these oils are inactive because according to Lewis and Ausubel (2006), a phytochemical molecule is considered antimicrobial if it inhibits the growth of microorganisms for MICs ranging between 100 and 1000 lg ml1. For antibiotics of microbial origin, MICs ranging from 0.01 to 10 lg ml1 are sufficient to generate an inhibitory activity. The demonstration of the antifungal properties of essential oils has broad potential significance. This activity can be attributed to the abundances of ketones in oils and of monoterpene alcohols in R. tuberculata oil. According Belghazi et al. (2002), Mentha pulegium essential oil whose major component is a ketone, (R)-Pulegone (82%), has a strong antifungal activity against Penicillium and Mucor. Further, activity of R. chalepensis essential oil screened against nine fungal species and fifteen Candida species, shows an important antifungal activity against Trichodrema viride and significant effects against C. albicans ATCC 90028 (Ben-Bnina et al., 2010).

5. Conclusion Multiple drug resistance in microbial pathogens is a continuing problem throughout the world. Some synthetic organic

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formulations have been recommended to control storage losses of food items. However, none of these represent an efficient strategy to control the mould growth. There is an established need to develop new antimicrobial agents to combat these pathogens. Our results are a contribution to a valorisation of some medicinal plants from Algeria. R. angustifolia, R. graveolens and R. chalepensis var. bracteosa essential oils, investigated in the present study, showed a large variation in their chemical composition with R. tuberculata essential oil. The first three essential oils were dominated by 2-ketones while the R. tuberculata oil contains a piperitone and some Monoterpene alcohols with similar proportions. We evaluated the antibacterial and antifungal activities of these oils. To the best of our knowledge, our results on R. tuberculata essential oil can be assessed as the first report about the antimicrobial properties in respect to the chemical composition. In view of their potential as inhibitory of fungal growth, Ruta essential oils investigated or some of their components may be recommended for formulation of plant based preservatives for enhancement of shelf life of food items during post harvest processing and of raw and processed food. However, issues of off-flavor, safety and toxicity should be addressed. Further research is needed in order to obtain information regarding the practical effectiveness of essential oil to prevent the growth of food borne and spoiling microbes under the specific application conditions. Several other biological tests will be worthwhile to search for more eventual activities of these plants. Phytochemical investigations will be planned to identify and characterise active principles, and assess toxicity by laboratory assays.

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