See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/303299458

16.food chemistry

Dataset · May 2016

CITATIONS READS

0 61

6 authors, including:

Farah Haddouchi Tarik Med Chaouche

Abou Bakr Belkaid University of Tlemcen Abou Bakr Belkaid University of Tlemcen
27 PUBLICATIONS 80 CITATIONS 29 PUBLICATIONS 70 CITATIONS

SEE PROFILE SEE PROFILE

Faten Medini
Razi Hospital
36 PUBLICATIONS 88 CITATIONS

SEE PROFILE

All in-text references underlined in blue are linked to publications on ResearchGate, Available from: Tarik Med Chaouche
letting you access and read them immediately. Retrieved on: 09 October 2016
 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
Laboratory of Natural Products, Department of Biology, Faculty of Sciences, Abou Bekr Belkaïd University, B.P. 119, Tlemcen 13000, Algeria
b
Laboratory of Plant Biotechnology, Department of Biology, National Institute of Applied Science and Technology (INSAT), B.P. 676, 1080 Tunis Cedex, Tunisia
c
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 a b s t r a c t

Article history: Antimicrobial properties of plants essential oils have been investigated in order to suggest them as poten-
Received 2 February 2013 tial tools to overcome the microbial drug resistance and the increasing incidence of food borne diseases
Received in revised form 1 March 2013 problems. The aim of this research is to study the antibacterial and antifungal effects of four traditional
Accepted 2 March 2013
plants essential oils, Ruta angustifolia, Ruta chalepensis, Ruta graveolens and Ruta tuberculata, against stan-
Available online 14 March 2013
dard 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
Keywords:
herbarum are the most sensitive strains to these oils with MIC values less than 3.5 lg ml1 for certain oils,
Antibacterial activity
Antifungal activity
reaching 7.8 lg ml1 for other. GC/MS essay exhibited ketones as the most abundant constituent of these
Ruta species oils except for R. tuberculata essential oil which has a completely different composition, monoterpenes
Essential oil alcohols being the most abundant. These compositions explain their potential antifungal activity.
GC/MS analysis Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Ruta chalepensis var. bracteosa is characterised by its petals

The development of drug resistance as well as the appearance of
side effects of certain antibiotics has led to the search of new antimi-
crobial 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 composi-
tion should ideally be used to correlate any structure activity rela-
tionships (Van-Vuuren & Viljoen, 2007) .
Essential oils are complex mixtures comprising many single
compounds. Each of these constituents contributes to the benefi-
cial or adverse effects of these oils. Therefore, the intimate knowl-
edge 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 an-
other and from one microbial strain to another (Kalemba & Kuni-
cka, 2003).
⇑ Corresponding author.
E-mail address: biofar23@yahoo.fr (F. Haddouchi).
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 lan-
ceolate or often elongated and small flowers with four petals yel-
low (Ozenda, 1991).
The phytochemical studies conducted on these species charac-
terise 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 dis-
eases. Exciting, diaphoretic, antiseptic, antispasmodic, anthelmintic
(Bonnier, 1999), emmenagogue, abortifacient and anti-inflamma-
tory 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.,

0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.03.007
254 F. Haddouchi et al. / Food Chemistry 141 (2013) 253–258
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 col-
lected 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 Prod-
ucts, Department of Biology, University of Tlemcen, Algeria. Vou-
cher 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 sub-
mitted 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 pro-
grammed 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 Cul-
ture Collection ‘‘ATCC’’ for bacteria and Candida albicans, National
Museum of Natural History ‘‘NMHN’’ for filamentous fungi), ob-
tained from Laboratory of Natural Products, Department of Biology
(Tlemcen University, Algeria): Gram-positive bacteria: Bacillus cer-
eus (ATCC 10876), Enterobacter cloacea (ATCC 13047), Enterococcus
faecalis (ATCC 49452), Listeria monocytogenes (ATCC15313), Staph-
ylococcus aureus (ATCC 25923). Gram-negative bacteria: Acineto-
bacter baumanii (ATCC 19606), Escherichia coli (ATCC 25922),
Klebsiella pneumoniae (ATCC 700603), Pseudomonas aeruginosa
(ATCC 27853), Proteus mirabilis (ATCC 35659), Salmonella typhimu-
rium (ATCC 13311), Citrobacter freundii (ATCC 8090). Fungal micro-
organisms: 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, Sab-
ouraud 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 dif-
fusion 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 paral-
lel experiments in order to control the sensitivity of the test micro-
organisms. 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 sus-
pension 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 fil-
ter 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 incu-
bated 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 per-
formed in MHB. The investigated oils were dissolved in 1% dimeth-
ylsulfoxide (DMSO) and then diluted to the highest concentration.
Serial doubling dilutions of oils were prepared in a 96-well micro-
titer plate over the range of 0.3–163 lg ml1. Overnight broth cul-
tures 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 de-
fined as the lowest concentration of the essential oil at which the
microorganism does not demonstrate visible growth. The microor-
ganism 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 pet-
ri 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
 F. Haddouchi et al. / Food Chemistry 141 (2013) 253–258 255

poured into sterilized Petri dishes (15 ml in each plate) over the Table 2
range of 3.5–9 lg ml1. A 6 mm diameter agar disk covered with Essential oil composition of R. tuberculata (RT).

mycelium was placed on the surface of the agar. Plates were incu- Rt Compounds RT (%)
bated for 5–7 days at 28 °C. Two replicates were used for each 5.307 a-Thujene 0.99
treatment. Gentamycin and Amphotericin B were used as reference 5.479 a-Pinene 1.79
compound. 6.406 Sabinene 1.53
6.789 b-Myrcecne 3.05
7.201 (R)-a-Phellandrene 2.22
3. Results 7.355 3-Carene 5.45
7.762 p-Cymene 2.9
7.89 b-Phellandrene 10.9
3.1. Essential oils isolation 10.69 trans-p-Menth-2-en-1-ol 13.14
11.264 cis-p-Menth-2-en-1-ol 11.22
Air-dried herbal of R. chalepensis var. bracteosa, R. graveolens, R. 13.077 trans-p-Menth-1-en-3-ol (trans-Piperitol) 4.12
angustifolia and R. tuberculata were subjected to hydrodistillation 13.489 cis-p-Menth-1-en-3-ol (cis-Piperitol) 12.31
15.006 Piperitone 13.62
and the yields of essential oils are respectively 0.9 ± 0.04,
16.001 Bornyl acetate 1.25
0.18 ± 0.01, 1.49 ± 0.36 and 0.11 ± 0.01% (w/w). 16.35 2-Undecanone 1.59
17.752 a-Terpinene 0.98
20.327 b-Caryophyllene 1.87
3.2. Chemical composition of the essential oil 20.722 c-Elemene 0.79
23.938 NI 1.14
Essential oils, extracted from R. chalepensis var. bracteosa, R. 24.498 Germacrene-B 7.32
graveolens, R. angustifolia and R. tuberculata, were analysed by 25.259 b-Caryophyllene epoxide 1.84
GC/MS. Tables 1 and 2 shows the chemical composition of the Chemical classes
essential oils together with the retention times of the compounds. Ketones 15.21
2-Ketones 1.59
A total of 16 compounds were identified from the essential oil of R.
Monoterpene hydrocarbons 29.81
chalepensis var. bracteosa, which represented 97.65% of the oil ex- Sesquiterpene hydrocarbons 9.98
tracted. 2-Nonanone and 2-Undecanone are the dominant ones at Esters 01.25
32.79% and 32.58%, respectively, followed by 1-Nonene at 13.95% Monoterpene alcohols 40.79
and a-Limonene at 5.27%. The profile of the oil components of R. Oxides tricyclic 01.84
All identified components 98.88
graveolens was almost similar to that of R. chalepensis var. bracte-
osa. 10 compounds, representing 97.3% of the essential oil, were Rt: retention time, NI: not identified.

identified. The major components were 2-Undecanone and

Table 1
Essential oils composition of R. chalepensis var.bracteosa (RCb), R. graveolens (RG) and
2-Nonanone (55.4 and 21.62%) followed by 1-Nonene and a-Limo-
R. angustifolia (RA). nene at 4.35 and 4.26% respectively.
For R. angustifolia, 6 compounds, representing 96.47% of the
Rt Compounds %
essential oil, were identified with the dominance of 2-Undecanone
RCb RA RG (82.46%). The 2-Decanone represents 10.03%.
5.479 a-Pinene 0.31 - – In contrast, 20 compounds were identified in essential oil of R.
6.406 Sabinene 0.49 – – tuberculata, representing 98.88% of the volatile compounds.
6.778 2-Octanone 0.39 – –
54.41% of these compounds were represented by Piperitone
7.87 a-Limonene 5.27 – 4.26
9.799 2-Nonanone 32.79 0.96 21.62 (13.62%) and Monoterpene alcohols which are, trans-p-Menth-
9.976 NI 1.14 – – 2-en-1-ol (13.14%), cis-p-Menth-2-en-1-ol (11.22%), trans-p-
10.091 Nonanal 0.62 0.84 – Menth-1-en-3-ol (4.12%) and cis-p-Menth-1-en-3-ol (12.31%).
11.155 2-Octanol acetate 0.89 – –
b-Phellandrene, Germacrene-B and 3-Carene represents 10.9,
11.327 5,6-Diethenyl-1-methyl-cyclohexene – – 3.24
11.332 Geyrene 1.46 – –
7.32 and 5.45% respectively.
12.923 2-Decanone 2.42 10.03 1.46
14.388 1-Nonene 13.95 – 4.35 3.3. Antimicrobial activity
16.35 2-Undecanone 32.58 82.46 55.4
16.493 NI 1.21 1.56 –
The in vitro antimicrobial activity of Ruta species essential oils
18.513 NI – – 1.03
18.519 11-Dodecen-2-one 0.99 – – against the microorganisms employed and its activity potentials
19.457 2-Dodecanone 0.6 1.51 1.02 were qualitatively and quantitatively assessed by the presence or
20.636 1-Tetradecanol methacrylate 3.29 – 2.22 absence of inhibition zones, zone diameters and MIC values.
22.57 2-Tridecanone 0.63 0.67 1.58
27.669 NI – 0.83 –
31.411 NI – – 1.67 3.3.1. Antibacterial activity
31.662 NI – 1.13 – The disc diameters of zone of inhibition of essential oils against
39.988 12-Methoxy-19-norpodocarpa- 0.97 – 2.14 the bacteria tested are shown in Table 3. The results obtained indi-
3,5,8,11,13-pentaen-7-one cated that the essential oils of the investigated species had low
Chemical classes in vitro potential of antibacterial activity against all 12 bacteria
Ketones 71.37 95.63 83.22 tested. The highest activity was observed against S. aureus and S.
2-Ketones 69.41 95.63 81.08
typhi with the strongest inhibition zones (15 and 17 mm, respec-
Acyclic alkenes 13.95 – 04.35
Monoterpene hydrocarbons 06.07 – 07.50 tively) recorded for R. chalepensis var. bracteosa essential oil, fol-
Sesquiterpenes 01.46 – – lowed by B. cereus and A. baumanii (12 mm). R. graveolens oil
Esters 04.18 – 02.22 exhibited weak inhibition zones on B. cereus, S. aureus and S. typhi
Aldehydes 00.62 00.84 –
(12 mm) while R. tuberculata oil exhibited modest antimicrobial
All identified components 97.65 96.47 97.30
activity against E. faecalis (14 mm). For R. angustifolia oil, no anti-
Rt: retention time, NI: not identified, bacterial activity was detected on all strains tested. Tested bacteria
256 F. Haddouchi et al. / Food Chemistry 141 (2013) 253–258

Table 3 Table 5
In vitro antibacterial activity of essential oils. In vitro antifungal activity of essential oils.

Inhibition diametersa (mm) Inhibition diametersa (mm)

Essential oils strains RA RCb RG RT Gentamycin Essential oils strains RA RCb RG RT Amphotericin B
10 ll/disc 15 lg/disc 10 ll/disc 20 lg/disc
Enterobacter cloacea 6 6 6 6 26 Candida albicans 35 15 33 17 22
Enterococcus faecalis 8 10 9 14 9 Cladosporium herbarum 18 35 25 34 37
Acinetobacter baumanii 6 12 6 6 12 Fusarium oxysporum 20 08 20 16 08
Bacillus cereus 10 12 12 12 16 Alternaria alternaria 25 10 25 20 27
Escherichia coli 6 7 7 6 20 Aspergillus flavus 15 16 22 17 17
Citrobacter freundii 7 6 6 6 27 Aspergillus fumigatus 20 23 15 17 28
Pseudomonas aeroginosa 6 6 6 6 19 a
Klebsielle pneumaniae 6 6 6 6 17 Diameter disc (6 mm) included, RA: R. angustifolia, RCb: R. chalepensis
Lysteria monocytogenes 6 6 6 6 12 var.bracteosa, RG: R. graveolens, RT: R. tuberculata.
Proteus mirabilis 6 10 7 6 14
Staphylococcus aureus 6 17 12 10 17
Salmonella typhi 6 15 12 8 16
Table 6
RG: R. graveolens, RT: R. tuberculata. In vitro MIC values (lg ml1) of essential oils against yeast and filamentous fungi.
a
Diameter disc (6 mm) included, RA: R. angustifolia, RCb: R. chalepensis
var.bracteosa. Essential oils strains RA RCb RG RT Amphotericin B
Candida albicans 154 41 18 74 2.5
were more sensitive to Gentamycin (12–27 mm) than to the essen- Cladosporium herbarum 4.7 4.9 6.5 7.8 2.5
tial oils tested, except for S. aureus and S. typhi which are more sus- Fusarium oxysporum 7 8.7 7.6 6.7 20
ceptible to R. chalepensis var. bracteosa oils, and for E. faecalis being Alternaria alternaria 8.2 8.7 7.6 6.7 1.25
more susceptible to R. tuberculata and the most resistant to this Aspergillus flavus 8.2 8.7 7.6 <4.5 5.0
Aspergillus fumigatus <3.5 6.2–7.4 <3.5 <4.5 2.5
antibiotic (9 mm).
For R. chalepensis var. bracteosa and R. tuberculata, results were RA: R. angustifolia, RCb: R. chalepensis var.bracteosa, RG: R. graveolens, RT: R.
confirmed by the MIC values (Table 4). The inhibitory properties of tuberculata.
the oils were observed within a range of concentrations from 18
to163 lg ml1. MICs of R. tuberculata oil against Enterococcus fae- a value of MIC is very high (20 lg ml1), even compared to the four
calis was ranged between 18 and 37 lg ml1. Both oils are less ac- oils tested. A. fumigatus is the most sensitive strain to these oils,
tive than the antibiotic. followed by C. herbarum.
A. flavus was more resistant to these oils, except to R. tuberculata
oil.
3.3.2. Antifungal activity
Essential oils, applied at 10 ll/disc, showed consistent anti- 4. Discussion
fungal effects against all fungi strains tested (Table 5). The highest
antifungal activity of R. angustifolia and R. graveolens oils was ob- The extraction yields obtained for R. angustifolia, R. graveolens
served against C. albicans (35 and 33 mm). Other strains react dif- and R. chalepensis var. bracteosa are comparable or similar to those
ferently with diameters between 15 and 25 mm. reported in the literature (Dob, Dahmane, Gauriat-Desrdy, & Dalig-
C. herbarum was the most sensitive microorganism to R. chalep- ault, 2008; Merghache, Hamza, & Tabti, 2009). As reported by Sal-
ensis var. bracteosa and R. tuberculata essential oils (35 and 34 mm, gueiro et al. (1997), climate, genotype, growth location, rainfall and
respectively). Other strains behave differently with diameters be- harvesting regime can affect the total essential oil content of
tween 15 and 23 mm, except for F. oxysporum and A. alternaria. plants.
The activities of R. chalepensis var. bracteosa and R. tuberculata Oils of the Ruta genus are generally characterised by a mixture
essential oils against C. herbarum, F. oxysporum, A. flavus and of R. of 2-Ketones, which can reach 84% of the oil (Verzera, Mondello,
angustifolia and R. graveolens essential oils against F. oxysporum, Ragusa, & Dugo, 2000). In the present work, the 2-ketone repre-
A. alternaria, A. flavus and C. albicans, are close or more important sents 95.63%, 81.08%, 69.41% and 1.59% in essential oils of R.
to that of Amphotericin B. F. oxysporum being the most resistant angustifolia, R. graveolens, R. chalepensis var. bracteosa and R. tuber-
to both Amphotericin B and R. chalepensis var. bracteosa oil. culata, respectively. The major compound is 2-Undecanone except
The essential oils (Table 6) showed inhibitory effects on the six for R. tuberculata, which Piperitone is the major ketone. The mono-
tested fungi, through the MICs. It could be seen that this effect is terpene alcohols represent 40.79% of the total R. tuberculata oil, fol-
proportional to its concentration. Against C. albicans, R. graveolens lowed by the monoterpene hydrocarbons (29.81%). So we can
essential oil has most inhibitory effect (18 lg ml1) than other deduce that R. tuberculata essential oil, which has never been pub-
three Ruta essential oils tested. Oils have a lower activity than lished, has a completely different composition compared to other
amphotericin B. species of Ruta.
For filamentous fungi, MIC values are less than 3.5 lg ml1 for Abundance of 2-Undecanone in oils is completely in accordance
certain oils, reaching 8.7 lg ml1 for other. MICs of amphotericin with the works done on species of Ruta genus such as R. chalepensis
B are between 1.25 and 5 lg ml1, except for F. oxysporum which essential oils from Algeria (Merghache et al., 2009) and Tunisia
Table 4 (Ben-Bnina, Hammami, Daamii-remadi, Ben-Jannet, & Mighri,
In vitro MIC (lg ml1) values of essential oils against bacteria. 2010; Mejri, Abderrabba, & Mejri, 2010; Saidani-Tounsi et al.,
Essential Oils MICs Antibiotic MICs
2011), R. graveolens essential oils from Egypt (El-Sherbeny, Hus-
sein, & Khalil, 2007), Iran (Soleimani, Aberoomand-Azar, Saber-
RCb RT Gentamycin
Tehrani, & Rustaiyan, 2009), and Malaysia (Yaacob-Karim, Abdul-
Enterococcus faecalis ND 18–37 5–10 lah, Che Mazenah, & Joulain, 1989) and R. angustifolia from Algeria
Staphylococcus aureus 81–163 ND 0.25–0.5
(Dob et al., 2008). The nature and proportions of other compounds
Salmonella typhi 41–81 ND 1.25–2.5
which constitute our oils are not always the same in comparison
RCb: R. chalepensis var.bracteosa, RT: R. tuberculata ND: not detected. with these same previous works. There are affected by climatic,
 F. Haddouchi et al. / Food Chemistry 141 (2013) 253–258
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 inter-
actions between them and the antimicrobial activity (Delaquis,
Stanich, Girard, & Mazza, 2002; Dorman & Deans, 2000). Pure oxy-
genated 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 ter-
pene hydrocarbons (Dorman & Deans, 2000; Griffin, Wyllie, Mark-
ham, & 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 poten-
tial activity of the compounds of essential oils, which has been con-
firmed 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 anti-
bacterial properties (Ben-Bnina et al., 2010; Proestos, Boziaris, Ny-
chas, & Komaitis, 2006). In this study, the antibacterial results
showed variation between different Ruta species oils. This differ-
ence 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 appar-
ently 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 com-
parison 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 com-
pound. 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 Mu-
cor. Further, activity of R. chalepensis essential oil screened against
nine fungal species and fifteen Candida species, shows an impor-
tant 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
257
formulations have been recommended to control storage losses
of food items. However, none of these represent an efficient strat-
egy 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 domi-
nated by 2-ketones while the R. tuberculata oil contains a piperito-
ne 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 antimicro-
bial 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 rec-
ommended for formulation of plant based preservatives for
enhancement of shelf life of food items during post harvest pro-
cessing and of raw and processed food. However, issues of off-fla-
vor, safety and toxicity should be addressed. Further research is
needed in order to obtain information regarding the practical effec-
tiveness 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 investiga-
tions will be planned to identify and characterise active principles,
and assess toxicity by laboratory assays.
References
Belghazi, L., Lahlou, N., Alaoui, I., Abousaouiria, T., Habti, N., Tantaoui, I. A., et al.
(2002). Extraction et analyse par chromatographie en phase gazeuse de l’huile
essentielle de la menthe pouliot. Test antifongique. Congrès de biochimie.
Casablanca. Biochimie et santé, 38–40.
Ben-Bnina, E., Hammami, S., Daamii-remadi, M., Ben-Jannet, H., & Mighri, Z. (2010).
Chemical composition and antimicrobial effects of Tunisian Ruta chalepensis L.
essential oils. Journal de la Société Chimique de Tunisie, 12, 1–9.
Bonnier, G. (1999). La Grande Flore en Couleur. (pp. 205–206). BELIN Ed, Tome 3.
Chen, C. C., Huang, Y. L., Huang, F. I., Wang, C. W., & Ou, J. C. (2001). Water soluble
glycosides from Ruta graveolens. Journal of Natural Products, 64, 990–992.
Cimanga, K., Kambu, K., Tona, L., Apers, S., Bruyne, T., Hermans, N., et al. (2002).
Correlation between chemical composition and antibacterial activity of
essential oils of some aromatic medicinal plants growing in the Democratic
Republic of Congo. Journal of Ethnopharmacology, 79, 213–220.
Corduan, G., & Reinhard, E. (1972). Synthesis of volatile oils in tissue cultures of Ruta
graveolens. Phytochemistry, 11, 917–922.
Delaquis, P. J., Stanich, K., Girard, B., & Mazza, G. (2002). Antimicrobial activity of
individual and mixed fractions of dill, cilantro, coriander and eucalyptus
essential oils. International Journal of Food Microbiology, 74, 101–109.
Dob, T., Dahmane, D., Gauriat-Desrdy, B., & Daligault, V. (2008). Volatile
constituents of the essential oil of Ruta chalepensis L. subsp. Angustifolia
(Pers.) P. Cout. Journal of Essential Oil Research, 20(30), 306–309.
Dorman, H. J. D., & Deans, S. G. (2000). Antimicrobial agents from plants:
Antibacterial activity of plant volatile oils. Journal of Applied Microbiology,
88(2), 308–316.
El-Sherbeny, S. E., Hussein, M. S., & Khalil, M. Y. (2007). Improving the production of
Ruta graveolens L. plants cultivated under different compost levels and various
sowing distance. American-Eurasian Journal of Agricultural & Environmental
Sciences, 2(3), 271–281.
Feng, W., & Zheng, X. (2007). Essential oils to control Alternaria alternate in vitro and
in vivo. Food Control, 18(9), 1126–1130.
Griffin, S. G., Wyllie, S. G., Markham, J. L., & Leach, D. N. (1999). The role of structure
and molecular properties of terpenoids in determining their antimicrobial
activity. Flavour & Fragrance, 14(5), 322–332.
Hammiche, V., & Maiza, K. (2006). Traditional medicine in Central Sahara:
Pharmacopoeia of Tassili N’ajjer. Journal of Ethnopharmacology, 105, 358–367.
Hnatyszyn, O., Arenas, P., Moreno, A. R., Rondina, R., & Coussio, J. D. (1974). Plantas
reguladoras de la fecundidad segun la medicina folklòrica. Revista de la Sociedad
del Paraguay, 14, 23–57.
Kalemba, D., & Kunicka, A. (2003). Antibacterial and antifungal properties of
essential oils. Current Medicinal Chemistry, 10, 813–829.
Lambert, R. J. W., Skandamis, P. N., Coote, P., & Nychas, G. J. E. (2001). A study of the
minimum inhibitory concentration and mode of action of oregano essential oil,
thymol and carvacrol. Journal of Applied Microbiology, 91, 453–462.
258 F. Haddouchi et al. / Food Chemistry 141 (2013) 253–258
Lewis, K., & Ausubel, F. M. (2006). Prospects for plant derived antibacterials. Nature
Biotechnology, 24, 1504–1507.
Mailhebiau, P. (1994). La nouvelle aromathérapie: caractérologie des essences et
tempéraments humains: [biochimie aromatique et influence psychosensorielle des
odeurs]. Lausanne: Jakin Ed., pp. 635.
Mejri, J., Abderrabba, M., & Mejri, M. (2010). Chemical composition of the essential
oil of Ruta chalepensis L: Influence of drying, hydro-distillation duration and
plant parts. Industrial Crops and Products, 32, 671–673.
Merghache, S., Hamza, M., & Tabti, B. (2009). Etude physicochimique de l’huile
essentielle de Ruta Chalepensis L. de Tlemcen, Algérie. Afrique Science, 05(1),
67–81.
NCCLS, National Committee for Clinical Laboratory Standards. (2001). Performance
standards for antimicrobial susceptibility testing: Eleventh informational
supplement, M100-S11, Wayne, PA, USA.
Ozenda, P. (1991). Flore et végétation du Sahara. CNRS Ed (3Eds). Paris p. 326.
Palá-Paúl, J., Pérez-Alonso, M. J., Velasco-Negueruela, A., Varadé, J., Ma Villa, A., Sanz,
J., et al. (1993). Composition and antimicrobial properties of essential oils of
four Mediterranean Lamiaceae. Journal of Ethnopharmacology, 39, 167–170.
Pfaller, M. A., Messer, S. A., Karlsson, Å., & Bolmstrom, A. (1998). Evaluation of the
Etest method for determining fluconazole susceptibilities of 402 clinical yeast
isolates by using three different agar media. Journal of Clinical Microbiology,
36(9), 2586–2589.
Proestos, C., Boziaris, I. S., Nychas, G. J. E., & Komaitis, M. (2006). Analysis of
flavonoids and phenolic acids in Greek aromatic plants: Investigation of their
antioxidant capacity and antimicrobial activity. Food Chemistry, 95, 664–671.
Raghav, S. K., Gupta, B., Agrawal, C., Goswami, K., & Das, H. R. (2006). Anti-
inflammatory effect of Ruta graveolens L. in murine macrophage cells. Journal of
Ethnopharmacology, 104, 234–239.
Saidani-Tounsi, M., Aidi-Wannes, W., Ouerghemmi, I., Msaada, K., Smaoui, A., &
Marzouk, B. (2011). Variation in essential oil and fatty acid composition in
different organs of cultivated and growing wild Ruta chalepensis L. Industrial
Crops and Products, 33, 617–623.
Salgueiro, L. R., Vila, R., Tomi, F., Figueiredo, A. C., Barroso, J. G., & Canigueral, S.
(1997). Variability of essential oils of Thymus caespititius from Portugal.
Phytochemistry, 45, 307–311.
Soleimani, M., Aberoomand-Azar, P., Saber-Tehrani, M., & Rustaiyan, A. (2009).
Volatile Composition of Ruta graveolens L. of North of Iran. World Applied
Sciences Journal, 7(1), 124–126.
Soliman, K. M., & Badeaa, R. I. (2002). Effect of oil extracted from some medicinal
plants on different mycotoxigenic fungi. Food and Chemical Toxicology, 40,
1669–1675.
Van-Vuuren, S. F., & Viljoen, A. M. (2007). Antimicrobial activity of limonene
enantiomers and 1,8-cineole alone and in combination. Flavour & Fragrance, 22,
540–544.
Verzera, V., Mondello, L., Ragusa, S., & Dugo, G. (2000). Essential oil from leaves of
typical Mediterranean plants. Note II Ruta chalepensis L.. Essenze e derivati
agrumari, 70, 207–210.
Yaacob-Karim, B., Abdullah Che Mazenah & Joulain (1989). Essential oil of Ruta
graveolens L. Kebangsaan Malaysia, Selangor, Malay. Journal of Essential Oil
Research, 1(5), 203–207.
Zaouali, Y., Chograni, H., Trimech, R., & Boussaid, M. (2013). Changes in essential oil
composition and phenolic fraction in Rosmarinus officinalis L. var. typicus Batt.
organs during growth and incidence on the antioxidant activity. Industrial Crops
and Products, 43, 412–419.