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Industrial Crops and Products

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Antioxidant and antibacterial activities of Thymus numidicus and

Salvia officinalis essential oils alone or in combination
Nabil Adrar, Naima Oukil, Fatiha Bedjou ∗
Laboratoire de Biotechnologie Végétale et d’Ethnobotanique, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, Algérie

a r t i c l e i n f o a b s t r a c t

Article history: Combinations between antibiotics and other antimicrobial substances such as plant essential oils repre-
Received 3 July 2015 sent one of the most promising advances against drug-resistant microorganisms. The aim of this study
Received in revised form 1 December 2015 was to evaluate, by the microdilution method, the antibacterial effects of different combinations of two
Accepted 7 December 2015
essential oils with their major components or antibiotics (cephalosporines) against Staphylococcus aureus,
Available online xxx
Escherichia coli, Serratia marcescens, Klebsiella pneumoniae, Pseudomonas aeruginosa and the antioxidant
effect of the same essential oils combined with thymol or DL-˛-tocopherol against DPPH free radical. Two
Keywords:
aromatic plants widely growing in north Algeria, Thymus numidicus (Poiret) and Salvia officinalis (Linné),
Antibacterial activity
Antioxidant activity
were investigated. Essential oils were extracted from these plants through hydrodistillation method.
Essential oil Extraction yields were evaluated at 1.83% for T. numidicus (Poiret) and 0.97% for S. officinalis L. Synergistic
Components interaction was observed with DL-˛-tocopherol-T. numidicus essential oil tested against DPPH free radi-
Antibiotics cal. Additional effect was noted with ciprofloxacine-T. numidicus essential oil combination tested against
FIC index S. aureus. However no significant action was observed among the other combinations used in this study
for the investigation of antibacterial or antioxidant activities.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction
Decreased efficacy and resistance of pathogens to antibiotics
has necessitated development of new alternatives. Combination
of essential oil and antibiotics showed substantial antimicrobial
effects (Janssen et al., 1987). This need to exploit natural products is
potentially ascribed both to the increasing emergence of bacterial
resistance to antibiotic therapy and to newly emerging pathogens
(Boyle, 1955; Schafer and Wink, 2009).
Among all natural products, essential oils showed pharmaco-
logical activities. They have been recognized for their antibacterial,
antifungal, antioxidant and insecticidal properties (Burt, 2004;
Giordani et al., 2008; Ayvaz et al., 2010). They are widely used
in medicine and in food preservation (Bassolé and Rodolfo-Juliani,
2012).
In foods systems higher concentrations of essential oils are
needed to have similar antimicrobial effects as those obtained in
vitro. The use of essential oils and their isolated components are
new approaches to increase their efficacy, taking advantage of their
synergistic and additive effects (Bassolé and Rodolfo-Juliani, 2012).
∗ Corresponding author.
E-mail address: fatihabedjou2015@gmail.com (F. Bedjou).
Probably their biological profiles are the result of a synergism of all
molecules present in the oil.
Better efficiency of essential oils or their major components
were observed when they are combined with synthetic agents
(Rosato et al., 2008). For this reason we associated essential
oils, major components and antibiotics in order to provide bet-
ter efficacy for combating various infections and drug resistance
microorganisms. We combined essential oils with DL-˛-tocopherol
to evaluate their effect on oxidative stress. Eighteen combinations
were tested against bacterial strains and five combinations against
DPPH free radical.
2. Material
In this study we combined essential oils of two aromatic plants:
Thymus numidicus (Poiret) and Salvia officinalis L., belonging to the
family of Lamiaceae, with major components of essential oils and
antibiotics. The antimicrobial effects of these combinations were
examined against several bacterial strains.
Samples were harvested in June 2013. Leaves and flowers of S.
officinalis were collected in the area of Tichy 15 km from Bejaia.
Leaves and flowers of T. numidicus were harvested in the region of
Toudja at 25 km from Bejaia city. Identification of these two plants
was made according to Quezel and Santa (1963).

http://dx.doi.org/10.1016/j.indcrop.2015.12.007
0926-6690/© 2015 Elsevier B.V. All rights reserved.

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Table 1
Antibiotics used for the combinations.
Antibiotics Origine
Ampicilline SIGMA® , Chine
Amoxicilline
Tetracicline
Chloramphenicol
Cefotaxime Shanghai Pharmaceutical Group (SPG), China
Ciprofloxacine Hikma Farmaceutica, Portugal
Imipeneme RANBAXY Laboratories, India
Table 2
Bacterial strains used in this study.
Bacterial strain GRAM
Staphylococcus Aureus subsp. aureus Positive
Klebsiella pneumoniae Negative
Escherichia coli Negative
Escherichia coli Negative
Serratia marcescens Negative
Pseudomonas aeruginosa Negative
Plant samples were thoroughly washed to remove all attached
material and immediately used for essential oil extraction. The used
major components of essential oils were the following:
• Thymol (lot number 70110, E CLabel C.O.O Germany)
• DL-˛-tocopherol (BASF Personal Care and Nutrition GmbH, Illertis-
sen, Germany)
Antibiotics selected for this study are shown in Table 1.
Six bacterial strains, from them, three from American type cul-
ture collection were used to test the antibacterial properties of
combination of essential oils with major components or antibi-
otics. These microbial strains were aerobic and Gram positive and
negative.
Antimicrobial activities of all combinations were investigated
against the following strains from American type collection culture:
Staphylococcus aureus subsp. aureus methicillin resistant or MRSA,
ATCC® 25923TM Klebsiella pneumoniae ATCC 1603, Escherichia coli
ATCC 25922, and three other clinical strains (Table 2).
3. Methods
3.1. Water distillation of essential oils
Leaves and flowers of S. officinalis and T. numidicus were used for
essential oil extraction. Hundred grams of each sample were mac-
erated in 500 ml of distillated water during 24 h before extraction.
Plants were then submitted to Clevenger hydrodistillation during
3 h (Clevenger, 1928). The obtained essential oils were dried over
anhydrous sodium sulphate and after filtration stored at 4 ◦ C until
evaluation (Williams and Lasunzi 1994; Auclair and Coté, 2002).
The yield of extraction was evaluated according to Williams and
Lasunzi (1994).
3.2. Preparation of antibiotics and bacterial suspension
Antibiotics used in this study were dissolved in NaCl solution
at 0.9%. To enhance oil and major components solubility, a solu-
tion of agar at 0.2% was added before their incorporation in the
checkerboard (Remmal et al.,1993; Satrani et al., 2001).
Bacterial species were cultured on Mueller Hinton agar “MHA,
scharlau S.I. Barcelona, spain”. Each bacterial suspension was com-
posed of 2 or 3 colonies of each strain taken on MHA plates and
dissolved in 5–9 ml NaCl solution at 0.9% and then adjusted to 107
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essential oils alone or in combination. Ind. Crops Prod. (2015), http://dx.doi.org/10.1016/j.indcrop.2015.12.007
or 108 CFU/ml at 625 nm, equivalent to 0.5MC Farland (Courvalin
et al., 1991).
Mi Minimum inhibitory concentration (MIC) is the lowest con-
centration which inhibits growth of microorganisms compared
with the growth in the control plate (Santos et al., 1997). The
minimum bactericidal concentration (MBC) represents the lowest
concentration at which a percentage of 99.9% of bacteria have been
killed (Mah, 2014; Gradinaru et al., 2014). Values of MIC and MBC
were determined by the agar dilution method as envisaged by CLSI
protocol M7 A7 (2006).
3.3. Dilution checkerboard method used to evaluate the different
combinations
Reference
Microdilution techniques are used to test the interactions
ATCC 25923
between essential oils and other substances like major compo-
ATCC 1603
ATCC 25922 nents or antibiotics (Gradinaru et al., 2014). All these methods
161 were developed for the detection of drug interactions, thus
338 there is no standardized method known to evaluate interaction
867
between essential oil and their components or antibiotics (Mackay
et al., 2000; Tallarida, 2001). Dilution procedures were performed
according to CLSI protocol M7A7-2006.
Serial dilutions of essential oils and its major component or
antibiotics were prepared, and different combinations of these
antibacterial were made and tested. Each well of the plate contain:
100 ␮l of Mueller Hinton broth
5 ␮l of bacterial suspension
50 ␮l of an antibacterial dilution if it was tested alone and 25 ␮l
of each sample with a binary combination.
The final volume in each well was equal to 155 ␮l. The last well
containing 100 ␮l of Mueller Hinton, 50 ␮l of agar and 5 ␮l of bac-
terial suspension was used as positive control (growth control)
(Clinical and Laboratory Standards Institute, 2006) (Table 3).
The plate was covered with a sterile plate sealer. Contents of
the plate were mixed on a plate shaker at 300 rpm for 20 s and then
incubated at 37 ◦ C for 18 H (Denis et al., 2007).
The checkerboard test requires determination of the fractional
inhibitory concentration (FIC). The FIC of a component represent
the concentration which inhibit bacterial growth when it is used
with another agent, divided by the concentration that has the same
effect when this component is used alone (Krostad and Moellering,
1986). The FIC index for the combination of two substances is the
sum of their individual FIC values. The FIC index is used for the
determination of the nature of the interaction between two sub-
stances (Burt, 2004; Goni et al., 2009; Pei et al., 2009).
In our study, the effect of combinations (Table 4) was deter-
mined according to Rosato et al. (2010) as following:
3.3.1. Isobologram
Isobologram illustrates the results of the checkerboard tests and
the fractional inhibitory concentration index (FIC index or FICI) val-
ues. The “axis” should be replaced with “x-coordinate” or “abscissa”,
in the Cartesian system both Ox and Oy are axes. The straight
line connecting the intercept points represents zero interaction
(Williamson, 2001).
3.4. DPPH radical scavenging assay
This assay was determined according to Wu and Ng (2008),
1 ml of an ethanolic solution (0.1 mmol) was mixed with 3 ml of
an essential oil dilution or thymol or DL-˛-tocopherol, each one
at different concentrations. The mixture was left in the dark at
room temperature during 30 min. The absorbance at 517 nm was
read using a Zuzi model 4111 RS spectrophotometer. DPPH ethano-
lic solution was used as blank sample. The antioxidant effect of
the tested samples, expressed as percentage of DPPH inhibition,
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Table 3
Microdilution technique used for the evaluation of combination effect on bacterial strain.

A MIC/16
B MIC/8
C MIC/4
D MIC/2
E MIC
F 2.MIC
G 4.MIC
H 4.MIC 2.MIC MIC MIC/2 MIC/4 MIC/8 MIC/16 MIC/32 MIC/64 MIC/128 MIC/256
1 2 3 4 5 6 7 8 9 10 11 12

The well H1 contains 100 ␮l of BMH, 5 ␮l of the bacterial inoculum, and 50 ␮l of 0.2% agar (growth control).
- With the exception of H1 well, each well of the line H (abscissa) contains 100 ␮l of Mueller Hinton broth, 5 ␮l of inoculum, and 50 ␮l of a dilution of the antimicrobial agent
X (most concentrated—the more diluted), starting with a concentration of 4 times the MIC (MIC of each antimicrobial agent is previously determined by the same technique).
- With the exception of H1 well, each well of the ordinate line contains 100 ␮l of Mueller Hinton broth, 5 ␮l of inoculum, and 50 ␮l of a dilution of the antimicrobial agent Y
(the more concentrated—the more diluted, starting with a concentration of 4 times the MIC).
- Each well from the rest of the microdilution plate contains identical amounts of Mueller Hinton broth, 5 ␮l of inoculum, and 25 ␮l of each antimicrobial agent (combination
of different dilutions which correspond to the two axes (combination of doses).

was calculated according to the following formula as described by Table 4

Effect of combination of essential oil with another essential oil or antibiotic or major
Sharififar et al. (2007):
component (Rosato et al., 2010).

Abs (blank) − Abs (sample) Obtained effect FIC index

I% = × 100
Abs (blank) Synergistic effect ≤0.5
Addition 0.5 < FIC index ≤ 1
Inhibition concentrations at 50% (IC50) were calculated for all the Indifferent effect FIC index = 1
Antagonistic effect >1
samples using the «Origin® Pro 9» software.
FIC index (Fractional inhibitory concentration index).
Where synergistic effect is overlapping addition and addition is overlapping indif-
3.4.1. Statistical analysis ferent effect, with values equal to 0.5 and 1.

All the tests were made in triplicate on three separate sam-

ples, and the results were expressed as an average of the three
assays. (Analysis of variance (ANOVA) was performed using XLSTAT
2009.1.02. computer software and followed by Duncan’s multiple
comparative test.
3.5. Evaluation of antioxidant activities of essential oils in
combination
Five combinations were tested against DPPH free radical, T.
numidicus essential oil/S. officinalis essential oil, thymol/S. officinalis
essential oil, DL-˛-tocopherol/S. officinalis essential oil, DL-˛-
tocopherol/ T. numidicus essential oil and DL-˛-tocopherol/thymol.
To establish if the binary mixtures tested are synergistic, antagonis-
tic, indifferent, the fractional inhibitory concentration fifty percent
index (FIC50 I) was determined according to Santiesteban-Lopez
et al. (2007).
4. Results and discussion
4.1. Extraction yield of essential oils
The extraction yield for T. numidicus estimated at 1.83% is lower
than that one of Thymus algeriensis growing in the same region and
higher than the extraction yield for the plant growing in Morocco
which was evaluated to 0.3% (Amarti et al., 2011) and T. numidicus
cultivated in the region of Morroco (1.3%) (Dob et al., 2006).
The extraction yield for S. officinalis estimated at 0.97% is lower
than that one obtained from the same specie growing in Tunisia
(1.8%), in France (2.05%), in Portugal (2.9%) and in Romania (2.3%)
(Fellah et al., 2006).
The difference in this value was attributed to several factors
like origin of species, harvesting period and extraction method
(Russo et al., 1998; Karousou et al., 2005; Pibiri, 2005; Curado, 2006;
Loziene et al., 1998).

FIC50 I = FIC50 (A) + FIC50 (B) 4.2. Antibacterial activities of essential oils

The antibacterial activities of T. numidicus and S. officinalis essen-

IC50 (of compound A in the presence of B)
FIC50 (A) =
IC50 (of compound A individually)
IC50 (of compound B in the presence of A)
FIC50 (B) =
IC50 (of compound B individually)
According to Santiesteban-Lopez et al. (2007) a combination is con-
sidered synergistic if its FIC50 I < 0.9; indifferent if 0.9 < FIC50 I < 1.1
and antagonistic if its FIC50 I > 1.1.
For each combination, the FIC50 I was expressed as the mean
of three FIC50 I obtained with three different concentrations of a
binary mixture. Therefore, FIC50 I were compared with two theo-
retical means (0.9 and 1.1) using unilateral compliance test. This
statistical test was performed using XLSTAT 2009.1.02 computer
software. In addition, isobole curves are plotted.
tial oils are shown in Table 4. MBC/MIC value was calculated for
the determination of essential oil effect on bacteria. According to
Traoré et al. (2012) essential oil exercises a bactericidal effect when
MBC/MIC ≤ 4. In our case this value is equal to 1 excepted for S.
aureus (MBC/MIC = 2). Essential oils of T. numidicus and S. offici-
nalis have a bactericidal effect on all strains used in this study.
T. numidicus essential oil exhibited higher activity, than S. offici-
nalis essential oil, against all bacterial strains tested in this study.
According other studies there is a relationship between the antibac-
terial activity of essential oils and their chemical composition.
Most of the antimicrobial activity in essential oils is attributed
to oxygenated terpenoids (e.g., alcohols and phenolic terpenes)
while some hydrocarbons also exhibit antimicrobial effect (Burt,
2004; Koroch et al., 2007). Some studies demonstrated that whole
essential oils exhibit higher antimicrobial activity than their major
components, suggesting that the minor components are critical for

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Fig. 1. Isobole curves showing the effect of combination of Thymus numidicus EO; Salvia officinalis EO and thymol with antibiotics on growth’s inhibiting of three bacterial
strains: S. aureus subsp. aureus ATCC 25923 (a–e curves); E. coli ATCC 25922 (f; g;h;i, j curves) and E. coli 161 (k, l curves).

their activity (Davidson and Parish, 1989; Mourey and Canillac,
2002). It has been reported that essential oils containing aldehyde
and phenols such as cinnamaldehyde, citral, carvacrol, eugenol or
thymol as major components exhibited the highest antibacterial
activity, followed by essential oils containing terpene alcohols (El-
Hosseiny et al., 2014). Other essential oils containing ketones or
esters, such as beta myrcene, alpha thujone or geranyl acetate
had much weaker activity (Dormans and Deans, 2000; Inouye
et al., 2001; Ait-Ouazzou et al., 2011). Essential oil of Thymus
numidicus, endemic species of East Algeria, showed a high phenolic
content. Its major component is thymol (57.20–66.31%), followed
by linalool (8.62–9.26%), gamma terpinene (6.12–9.19%) and p-
cymene (6.20–7.55%) (Giordani et al., 2008). The presence of these
components could explain the high activities found against all bac-
terial strains tested in this study, and the high antioxidant activity
of T. numidicus essential oil.
4.3. Antimicrobial activities of the different combinations
Antimicrobial activities of the different combinations are shown
in Table 6.

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Fig. 2. Isobole curves showing the effect of five binary combinations including Thymus numidicus EO; Salvia officinalis EO; thymol and DL- ␣-tocopherol in inhibiting DPPH
free radical.

Table 5
Antibacterial activities of Thymus numidicus and Salvia officinalis essential oils.

Thymus numidicus EO (mg/ml) Salvia officinalis EO (mg/ml)

MIC MBC MBC/MIC MIC MBC MBC/MIC

E. Coli ATCC 25922 0.117 0.117 1 3.749 3.749 1

E. Coli 161 0.234 0.234 1 7.497 7.497 1
S. aureus subsp. aureus ATCC 25923 0.234 0.469 2 7.497 14.995 2
S. marcescens 338 0.469 0.469 1 14.995 14.995 1
K. pneumoniae ATCC1603 0.234 0.234 1 14.995 14.995 1
P. aeruginosa 867 0.469 0.469 1 7.497 7.497 1

MIC (minimal inhibitory concentration).

MBC (minimal bactericidal concentration).
EO (essential oil).

The following figures (Fig. 1a–l) represent isobolograms show-
ing the results of the checkerboard tests and the FIC index values
cited in Table 6.
The effect of tested combinations was evaluated by MIC and
FIC index. As shown in Table 5, an additive effect is obtained
against S. aureus by using thyme essential oil/ciprofloxacine
combination. In another hand, an indifferent effect is obtained
against S. aureus subsp. aureus ATCC® 25923TM , (E. coli
(ATCC 25922)), and E. coli 161 with the following combi-
nations, cefotaxime/thymol and thyme essential oil/cefotaxime
Thymol–imipinene and Thymol–chloramphenicol, respectively. All
these results are confirmed by the isobole curves. According to

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Table 6
Effects of combinations between essential oils, antibiotics and major components of essential oils.

Bacterial Combinationa MIC0 MICC FIC FICI Effect of the compounds in combination
strain

S. aureus ATCC 25923 Thymus numidicus EO 0.234 0.234 1 1.5 Antagonistic

Imipeneme 0.252 0.126 0.5
Thymol 0.202 0.006 0.030 1.030 Antagonistic
Imipeneme 0.252 0.252 1
Thymus numidicus EO 0.234 0.117 0.5 1.00 Indifferent
Cefotaxime 0.504 0.252 0.5
Thymol 0.202 0.101 0.5 1.00 Indifferent
Cefotaxime 0.504 0.252 0.5
Thymus numidicus EO 0.234 0.059 0.25 0.75 Additive
Ciprofloxacine 0.448 0.224 0.5

E. coli ATTC 25922 Thymus numidicus EO 0.117 0.004 0.034 1.034 Antagonistic
Imipeneme 0.063 0.063 1
Thymol 0.202 0.101 0.5 1.00 Indifferent
Imipeneme 0.063 0.031 0.5
Thymus numidicus EO 0.117 0.004 0.034 1.034 Antagonistic
Cefotaxime 0.126 0.126 1
Thymol 0.202 0.006 0.030 1.030 Antagonistic
Cefotaxime 0.126 0.126 1
Salvia officinalis EO 3.749 0.117 0.031 1.031 Antagonistic
Cefotaxime 0.126 0.126 1

E. coli 161 Thymus numidicus EO 0.234 0.234 1 1.031 Antagonistic

Salvia officinalis EO 7.497 0.234 0.031
Thymus numidicus EO 0.234 0.007 0.030 1.030 Antagonistic
Chloramphenicol 2.016 2.016 1
Thymol 0.202 0.101 0.5 1.00 Indifferent
Chloramphenicol 2.016 1.008 0.5
Thymus numidicus EO Resistance against all
Cefotaxime the antibiotics
Thymus numidicus EO
Ampicilline
Thymus numidicus EO
Tetracycline
Thymus numidicus EO
Amoxicilline
Thymus numidicus EO
Ciprofloxacine

MIC0 : MIC of the compound individually.

MICC : MIC of the compound in combination.
EO: essential oil FIC (Fractional inhibitory concentration).
FICI (Fractional inhibitory concentration index).
a
MIC of antibiotics are given in ␮g/ml, MIC of essential oils and thymol are given in mg/ml.

Pereira de Sousa et al. (2012), it seems that the compounds, which
have similar structures or which act with the same mechanism of
action, exhibit an additive or indifferent effect against microorgan-
isms, when they are combined. Effectively, several studies showed
an additive effect against microorganisms when thymol and car-
vacrol, both isomers, are combined (Pereira de Sousa et al., 2012;
Fu et al., 2007; Lambert et al., 2001). The increased antimicro-
bial activity caused by the mixture of carvacrol and 1,8-cineole
could be partially explained by considering the different structure,
and therefore possibly different mechanisms of action, for each
compound (Pereira de sousa et al., 2012). Additionally, the type
of interaction (synergistic, additive and/or antagonistic) between
essential oils was shown to be dependent on the proportion of the
two components combined (Van Vuuren et al., 2009).
4.4. Antioxidant activity of the different compounds
The different compounds were tested individually against DPPH
free radical to investigate their antioxidant capacity (Table 7).
ANOVA of IC50 identified significant differences between the
four samples at 95% confidence interval. Duncan’s test subdivided
them into two groups. The first one, group “A” consists only of S.
officinalis essential oil and has a very distant IC50 of the three other
samples which are relatively homogeneous in group “B”. This can
be explained by the weak activity of S. officinalis essential oil.
As seen in Table 7, DL-˛-tocopherol exhibited the highest activ-
ity against DPPH, followed by Thymol, T. numidicus essential oil
and S. officinalis essential oil which showed the lowest activity. T.
numidicus essential oil exhibited not only a high anti bacterial activ-
ity but also a high antioxidant effect. This is probably related to
its high phenol content such as thymol which represent the major
component (57.20–66.31%) for the endemic species of East Algeria
(Giordani et al., 2008).
4.5. Antioxidant activity of the different combinations
The antioxidant capacity of compounds in combinations is
shown in Table 8.
Study by isobologram, where a diverse range of concentrations
of both components of the binary mixture that reached the IC50
value of 50% initial DPPH quenching, is evaluated (Fig. 2a–2e). This
study revealed that when DL-˛-tocopherol is mixed with T. numidi-
cus essential oil lower doses than those expected if this compound
is used alone are needed to achieve the same antioxidant effect.
The IC50 values are very low in all combinations which contain T.
numidicus essential oil. These results show the efficiency of this oil
alone or in combination.

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Table 7
Antioxidant activity of compounds individually.

Compound Concentration (␮g/ml) Inhibition (%) IC50 (␮g/ml) Group

Thymus numidicus EO 232.6 67.11 ± 3.06 156.53 ± 20.23 B

116.3 37.95 ± 4.03
58.15 23.89 ± 4.43
29.07 13.2 ± 4.11
14.53 5.78 ± 1.43

Thymol 212.5 70.59 ± 3.21 104.44 ± 6.76 B

106.25 48.84 ± 2.99
53.125 33.61 ± 2.27
26.56 18.97 ± 1.71
13.28 9.61 ± 2.00

DL-˛-tocopherol 6.87 85.77 ± 3.05 4.11 ± 0.25 B

3.43 40.77 ± 4.25
1.72 18.53 ± 2.89
0.86 9.17 ± 0.99
0.43 4.09 ± 0.42

Salvia officinalis EO 5746.25 87.73 ± 1.44 1999.28 ± 168.89 A

2871.25 64.05 ± 2.99
1435.62 38.41 ± 3.99
717.81 20.93 ± 3.45
358.9 10.52 ± 2.61

IC50 (inhibitory concentration at 50%).

Table 8
FIC50 and FIC50 Index of compounds in combination.

Combination IC50 (␮g/ml) IC50c (␮g/ml) FIC50 FIC50 I FIC50 I average

Thymus numidicus EO 156.53 100.72 0.643 0.955 0.934

Salvia officinalis EO 1999.28 621.67 0.312 ± 0.019
Thymus numidicus EO 156.53 16.42 0.105 0.916
Salvia officinalis EO 1999.28 1621.63 0.811
Thymus numidicus EO 156.53 49.79 0.318 0.933
Salvia officinalis EO 1999.28 1229.35 0.61489

Thymol 104.44 54.63 0.523 1.261 1.215

Salvia officinalis EO 1999.28 1476.45 0.738 ± 0.159
Thymol 104.44 80.23 0.768 1.039
Salvia officinalis EO 1999.28 541.99 0.271
Thymol 104.44 21.17 0.2027 1.347
Salvia officinalis EO 1999.28 2288.46 1.1446

DL-˛-tocopherol 4.11 1.62 0.394 1.07 0.992

Salvia officinalis EO 1999.28 1351.4 0.676 ± 0.082
DL-˛-tocopherol 4.11 0.52 0.126 0.999
Salvia officinalis EO 1999.28 1746.91 0.8737
DL-˛-tocopherol 4.11 2.61 0.635 0.907
Salvia officinalis EO 1999.28 544.36 0.272

DL-˛-tocopherol 4.11 1.64 0.340 0.694 0.655

Thymus numidicus EO 156.53 55.4 0.354 ± 0.063
DL-˛-tocopherol 4.11 2.1 0.512 0.739
Thymus numidicus EO 156.53 35.61 0.227
DL-˛-tocopherol 4.11 0.55 0.133 0.614
Thymus numidicus EO 156.53 75.39 0.481

DL-˛-tocopherol 4.11 2.87 0.698 1.546 1.789

Thymol 104.44 88.63 0.848 ± 0.219
DL-˛-tocopherol 4.11 5.84 1.421 1.853
Thymol 104.44 45.14 0.432
DL-˛-tocopherol 4.11 1.38 0.336 1.97
Thymol 104.44 170.68 1.634

EO = essential oil.
FIC50 (Fractional inhibitory concentration at 50%).
FIC50 I (Fractional inhibitory concentration at 50% index).

Statistical analysis confirmed synergistic interaction between
DL-˛-tocopherol and T. numidicus essential oil, antagonistic inter-
action when DL-˛-tocopherol is combined with Thymol and
indifferent effect of the combinations T. numidicus essential oil/S.
officinalis essential oil, Thymol/S. officinalis essential oil and DL-˛-
tocopherol/Salvia essential oil. These data indicate that a positive
antioxidant interaction between alpha tocopherol and T. numidi-
cus essential oil might take place. This research indicates that the
interaction of T. numidicus essential oil components with alpha
tocopherol appears to increase the reactivity of this antioxidant
substance involved in the mixture. The reason for this is unclear,
but one possible explanation would be that a heterologous activa-
tion of an oxydryle group in an antioxidant molecule by another
antioxidant takes place to enhance the formation of a hydrogen
radical which rapidly reacts with DPPH to quench it (Romano et al.,
2009).

Please cite this article in press as: Adrar, N., et al., Antioxidant and antibacterial activities of Thymus numidicus and Salvia officinalis
essential oils alone or in combination. Ind. Crops Prod. (2015), http://dx.doi.org/10.1016/j.indcrop.2015.12.007
G Model
INDCRO-8575; No. of Pages 8 ARTICLE IN PRESS
8 N. Adrar et al. / Industrial Crops and Products xxx (2015) xxx–xxx
5. Conclusion
In this study, we report for the first time antimicrobial and
antioxidant activities of essential oils combined with other compo-
nents. Analyses show that essential oils in combination with other
substances exhibit different effects (indifferent, additive, antago-
nistic and synergistic) against bacteria and DPPH free radical. This
is probably due to different mechanisms of action involved in this
case. Essential oil extracted in T. numidicus showed high antioxi-
dant and antibacterial activities. Subsequently, it will be tested with
other components against pathogenic bacteria and free radicals in
order to support their credibility as combination’s therapeutic in
the treatment of infectious diseases and to prevent effect of oxidant
radicals.
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