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Antioxidant and antibacterial activities of Thymus numidicus and Salvia officinalis essential oils alone or in combination
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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
Article history: Received 3 July 2015 Received in revised form 1 December 2015 Accepted 7 December 2015 Available online xxx Keywords: Antibacterial activity Antioxidant activity Essential oil Components Antibiotics FIC index
a b s t r a c t Combinations between antibiotics and other antimicrobial substances such as plant essential oils represent one of the most promising advances against drug-resistant microorganisms. The aim of this study was to evaluate, by the microdilution method, the antibacterial effects of different combinations of two essential oils with their major components or antibiotics (cephalosporines) against Staphylococcus aureus, 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 aromatic plants widely growing in north Algeria, Thymus numidicus (Poiret) and Salvia officinalis (Linné), were investigated. Essential oils were extracted from these plants through hydrodistillation method. Extraction yields were evaluated at 1.83% for T. numidicus (Poiret) and 0.97% for S. officinalis L. Synergistic interaction was observed with DL-˛-tocopherol-T. numidicus essential oil tested against DPPH free radical. Additional effect was noted with ciprofloxacine-T. numidicus essential oil combination tested against 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 pharmacological 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: [email protected] (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 better 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.
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
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Table 1 Antibiotics used for the combinations. Antibiotics
Origine
Ampicilline Amoxicilline Tetracicline Chloramphenicol
SIGMA® , Chine
Cefotaxime Ciprofloxacine Imipeneme
Shanghai Pharmaceutical Group (SPG), China Hikma Farmaceutica, Portugal RANBAXY Laboratories, India
or 108 CFU/ml at 625 nm, equivalent to 0.5MC Farland (Courvalin et al., 1991). Mi Minimum inhibitory concentration (MIC) is the lowest concentration 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
Table 2 Bacterial strains used in this study. Bacterial strain
GRAM
Reference
Staphylococcus Aureus subsp. aureus Klebsiella pneumoniae Escherichia coli Escherichia coli Serratia marcescens Pseudomonas aeruginosa
Positive Negative Negative Negative Negative Negative
ATCC 25923 ATCC 1603 ATCC 25922 161 338 867
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, Illertissen, Germany) Antibiotics selected for this study are shown in Table 1. Six bacterial strains, from them, three from American type culture collection were used to test the antibacterial properties of combination of essential oils with major components or antibiotics. 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 macerated 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).
Microdilution techniques are used to test the interactions between essential oils and other substances like major components or antibiotics (Gradinaru et al., 2014). All these methods were developed for the detection of drug interactions, thus there is no standardized method known to evaluate interaction 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 bacterial 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 substances (Burt, 2004; Goni et al., 2009; Pei et al., 2009). In our study, the effect of combinations (Table 4) was determined 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) values. 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).
3.2. Preparation of antibiotics and bacterial suspension
3.4. DPPH radical scavenging assay
Antibiotics used in this study were dissolved in NaCl solution at 0.9%. To enhance oil and major components solubility, a solution 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 composed 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
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 ethanolic 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 B C D E F G H
MIC/16 MIC/8 MIC/4 MIC/2 MIC 2.MIC 4.MIC 1
4.MIC 2
2.MIC 3
MIC 4
MIC/2 5
MIC/4 6
MIC/8 7
MIC/16 8
MIC/32 9
MIC/64 10
MIC/128 11
MIC/256 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 Sharififar et al. (2007): I% =
Abs (blank) − Abs (sample) × 100 Abs (blank)
Inhibition concentrations at 50% (IC50) were calculated for all the samples using the «Origin® Pro 9» software.
3.4.1. Statistical analysis All the tests were made in triplicate on three separate samples, 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.
Table 4 Effect of combination of essential oil with another essential oil or antibiotic or major component (Rosato et al., 2010). Obtained effect
FIC index
Synergistic effect Addition Indifferent effect Antagonistic effect
≤0.5 0.5 < FIC index ≤ 1 FIC index = 1 >1
FIC index (Fractional inhibitory concentration index). Where synergistic effect is overlapping addition and addition is overlapping indifferent effect, with values equal to 0.5 and 1.
4. Results and discussion 4.1. Extraction yield of essential oils
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, antagonistic, indifferent, the fractional inhibitory concentration fifty percent index (FIC50 I) was determined according to Santiesteban-Lopez et al. (2007).
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
3.5. Evaluation of antioxidant activities of essential oils in combination
IC50 (of compound A in the presence of B) FIC50 (A) = IC50 (of compound A individually)
FIC50 (B) =
IC50 (of compound B in the presence of A) IC50 (of compound B individually)
According to Santiesteban-Lopez et al. (2007) a combination is considered 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 theoretical 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.
The antibacterial activities of T. numidicus and S. officinalis essential 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. officinalis have a bactericidal effect on all strains used in this study. T. numidicus essential oil exhibited higher activity, than S. officinalis essential oil, against all bacterial strains tested in this study. According other studies there is a relationship between the antibacterial 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 (ElHosseiny 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 pcymene (6.20–7.55%) (Giordani et al., 2008). The presence of these components could explain the high activities found against all bacterial 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)
E. Coli ATCC 25922 E. Coli 161 S. aureus subsp. aureus ATCC 25923 S. marcescens 338 K. pneumoniae ATCC1603 P. aeruginosa 867
Salvia officinalis EO (mg/ml)
MIC
MBC
MBC/MIC
MIC
MBC
MBC/MIC
0.117 0.234 0.234 0.469 0.234 0.469
0.117 0.234 0.469 0.469 0.234 0.469
1 1 2 1 1 1
3.749 7.497 7.497 14.995 14.995 7.497
3.749 7.497 14.995 14.995 14.995 7.497
1 1 2 1 1 1
MIC (minimal inhibitory concentration). MBC (minimal bactericidal concentration). EO (essential oil).
The following figures (Fig. 1a–l) represent isobolograms showing 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 combinations, 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 strain
Combinationa
MIC0
MICC
FIC
FICI
Effect of the compounds in combination
S. aureus ATCC 25923
Thymus numidicus EO Imipeneme Thymol Imipeneme Thymus numidicus EO Cefotaxime Thymol Cefotaxime Thymus numidicus EO Ciprofloxacine
0.234 0.252 0.202 0.252 0.234 0.504 0.202 0.504 0.234 0.448
0.234 0.126 0.006 0.252 0.117 0.252 0.101 0.252 0.059 0.224
1 0.5 0.030 1 0.5 0.5 0.5 0.5 0.25 0.5
1.5
Antagonistic
1.030
Antagonistic
1.00
Indifferent
1.00
Indifferent
0.75
Additive
Thymus numidicus EO Imipeneme Thymol Imipeneme Thymus numidicus EO Cefotaxime Thymol Cefotaxime Salvia officinalis EO Cefotaxime
0.117 0.063 0.202 0.063 0.117 0.126 0.202 0.126 3.749 0.126
0.004 0.063 0.101 0.031 0.004 0.126 0.006 0.126 0.117 0.126
0.034 1 0.5 0.5 0.034 1 0.030 1 0.031 1
1.034
Antagonistic
1.00
Indifferent
1.034
Antagonistic
1.030
Antagonistic
1.031
Antagonistic
Thymus numidicus EO Salvia officinalis EO Thymus numidicus EO Chloramphenicol Thymol Chloramphenicol Thymus numidicus EO Cefotaxime Thymus numidicus EO Ampicilline Thymus numidicus EO Tetracycline Thymus numidicus EO Amoxicilline Thymus numidicus EO Ciprofloxacine
0.234 7.497 0.234 2.016 0.202 2.016 Resistance against all the antibiotics
0.234 0.234 0.007 2.016 0.101 1.008
1 0.031 0.030 1 0.5 0.5
1.031
Antagonistic
1.030
Antagonistic
1.00
Indifferent
E. coli ATTC 25922
E. coli 161
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 microorganisms, when they are combined. Effectively, several studies showed an additive effect against microorganisms when thymol and carvacrol, both isomers, are combined (Pereira de Sousa et al., 2012; Fu et al., 2007; Lambert et al., 2001). The increased antimicrobial 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 activity 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 activity 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. numidicus 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
232.6 116.3 58.15 29.07 14.53
67.11 37.95 23.89 13.2 5.78
± ± ± ± ±
3.06 4.03 4.43 4.11 1.43
156.53 ± 20.23
B
Thymol
212.5 106.25 53.125 26.56 13.28
70.59 48.84 33.61 18.97 9.61
± ± ± ± ±
3.21 2.99 2.27 1.71 2.00
104.44 ± 6.76
B
6.87 3.43 1.72 0.86 0.43
85.77 40.77 18.53 9.17 4.09
± ± ± ± ±
3.05 4.25 2.89 0.99 0.42
4.11 ± 0.25
B
5746.25 2871.25 1435.62 717.81 358.9
87.73 64.05 38.41 20.93 10.52
± ± ± ± ±
1.44 2.99 3.99 3.45 2.61
1999.28 ± 168.89
A
DL-˛-tocopherol
Salvia officinalis EO
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
Thymus numidicus EO Salvia officinalis EO Thymus numidicus EO Salvia officinalis EO Thymus numidicus EO Salvia officinalis EO
156.53 1999.28 156.53 1999.28 156.53 1999.28
100.72 621.67 16.42 1621.63 49.79 1229.35
0.643 0.312 0.105 0.811 0.318 0.61489
0.955
Thymol Salvia officinalis EO Thymol Salvia officinalis EO Thymol Salvia officinalis EO
104.44 1999.28 104.44 1999.28 104.44 1999.28
54.63 1476.45 80.23 541.99 21.17 2288.46
0.523 0.738 0.768 0.271 0.2027 1.1446
1.261
DL-˛-tocopherol Salvia officinalis EO DL-˛-tocopherol Salvia officinalis EO DL-˛-tocopherol Salvia officinalis EO
4.11 1999.28 4.11 1999.28 4.11 1999.28
1.62 1351.4 0.52 1746.91 2.61 544.36
0.394 0.676 0.126 0.8737 0.635 0.272
1.07
DL-˛-tocopherol Thymus numidicus EO DL-˛-tocopherol Thymus numidicus EO DL-˛-tocopherol Thymus numidicus EO
4.11 156.53 4.11 156.53 4.11 156.53
1.64 55.4 2.1 35.61 0.55 75.39
0.340 0.354 0.512 0.227 0.133 0.481
0.694
DL-˛-tocopherol Thymol DL-˛-tocopherol Thymol DL-˛-tocopherol Thymol
4.11 104.44 4.11 104.44 4.11 104.44
2.87 88.63 5.84 45.14 1.38 170.68
0.698 0.848 1.421 0.432 0.336 1.634
1.546
FIC50 I average 0.934 ± 0.019
0.916 0.933 1.215 ± 0.159
1.039 1.347 0.992 ± 0.082
0.999 0.907 0.655 ± 0.063
0.739 0.614 1.789 ± 0.219
1.853 1.97
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 interaction 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. numidicus 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 activation 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).
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5. Conclusion In this study, we report for the first time antimicrobial and antioxidant activities of essential oils combined with other components. Analyses show that essential oils in combination with other substances exhibit different effects (indifferent, additive, antagonistic 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 antioxidant 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. References Ait-Ouazzou, A., Cherrat, L., Espina, L., Loran, S., Rota, C., Pagan, R., 2011. The antimicrobial activity of hydrophobic essential oils constituents alone or combined processes of food preservation. Innov. Food Sci. 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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
Article history: Received 3 July 2015 Received in revised form 1 December 2015 Accepted 7 December 2015 Available online xxx Keywords: Antibacterial activity Antioxidant activity Essential oil Components Antibiotics FIC index
a b s t r a c t Combinations between antibiotics and other antimicrobial substances such as plant essential oils represent one of the most promising advances against drug-resistant microorganisms. The aim of this study was to evaluate, by the microdilution method, the antibacterial effects of different combinations of two essential oils with their major components or antibiotics (cephalosporines) against Staphylococcus aureus, 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 aromatic plants widely growing in north Algeria, Thymus numidicus (Poiret) and Salvia officinalis (Linné), were investigated. Essential oils were extracted from these plants through hydrodistillation method. Extraction yields were evaluated at 1.83% for T. numidicus (Poiret) and 0.97% for S. officinalis L. Synergistic interaction was observed with DL-˛-tocopherol-T. numidicus essential oil tested against DPPH free radical. Additional effect was noted with ciprofloxacine-T. numidicus essential oil combination tested against 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 pharmacological 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: [email protected] (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 better 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 Amoxicilline Tetracicline Chloramphenicol
SIGMA® , Chine
Cefotaxime Ciprofloxacine Imipeneme
Shanghai Pharmaceutical Group (SPG), China Hikma Farmaceutica, Portugal RANBAXY Laboratories, India
or 108 CFU/ml at 625 nm, equivalent to 0.5MC Farland (Courvalin et al., 1991). Mi Minimum inhibitory concentration (MIC) is the lowest concentration 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
Table 2 Bacterial strains used in this study. Bacterial strain
GRAM
Reference
Staphylococcus Aureus subsp. aureus Klebsiella pneumoniae Escherichia coli Escherichia coli Serratia marcescens Pseudomonas aeruginosa
Positive Negative Negative Negative Negative Negative
ATCC 25923 ATCC 1603 ATCC 25922 161 338 867
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, Illertissen, Germany) Antibiotics selected for this study are shown in Table 1. Six bacterial strains, from them, three from American type culture collection were used to test the antibacterial properties of combination of essential oils with major components or antibiotics. 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 macerated 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).
Microdilution techniques are used to test the interactions between essential oils and other substances like major components or antibiotics (Gradinaru et al., 2014). All these methods were developed for the detection of drug interactions, thus there is no standardized method known to evaluate interaction 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 bacterial 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 substances (Burt, 2004; Goni et al., 2009; Pei et al., 2009). In our study, the effect of combinations (Table 4) was determined 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) values. 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).
3.2. Preparation of antibiotics and bacterial suspension
3.4. DPPH radical scavenging assay
Antibiotics used in this study were dissolved in NaCl solution at 0.9%. To enhance oil and major components solubility, a solution 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 composed 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
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 ethanolic 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 B C D E F G H
MIC/16 MIC/8 MIC/4 MIC/2 MIC 2.MIC 4.MIC 1
4.MIC 2
2.MIC 3
MIC 4
MIC/2 5
MIC/4 6
MIC/8 7
MIC/16 8
MIC/32 9
MIC/64 10
MIC/128 11
MIC/256 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 Sharififar et al. (2007): I% =
Abs (blank) − Abs (sample) × 100 Abs (blank)
Inhibition concentrations at 50% (IC50) were calculated for all the samples using the «Origin® Pro 9» software.
3.4.1. Statistical analysis All the tests were made in triplicate on three separate samples, 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.
Table 4 Effect of combination of essential oil with another essential oil or antibiotic or major component (Rosato et al., 2010). Obtained effect
FIC index
Synergistic effect Addition Indifferent effect Antagonistic effect
≤0.5 0.5 < FIC index ≤ 1 FIC index = 1 >1
FIC index (Fractional inhibitory concentration index). Where synergistic effect is overlapping addition and addition is overlapping indifferent effect, with values equal to 0.5 and 1.
4. Results and discussion 4.1. Extraction yield of essential oils
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, antagonistic, indifferent, the fractional inhibitory concentration fifty percent index (FIC50 I) was determined according to Santiesteban-Lopez et al. (2007).
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
3.5. Evaluation of antioxidant activities of essential oils in combination
IC50 (of compound A in the presence of B) FIC50 (A) = IC50 (of compound A individually)
FIC50 (B) =
IC50 (of compound B in the presence of A) IC50 (of compound B individually)
According to Santiesteban-Lopez et al. (2007) a combination is considered 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 theoretical 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.
The antibacterial activities of T. numidicus and S. officinalis essential 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. officinalis have a bactericidal effect on all strains used in this study. T. numidicus essential oil exhibited higher activity, than S. officinalis essential oil, against all bacterial strains tested in this study. According other studies there is a relationship between the antibacterial 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 (ElHosseiny 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 pcymene (6.20–7.55%) (Giordani et al., 2008). The presence of these components could explain the high activities found against all bacterial 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)
E. Coli ATCC 25922 E. Coli 161 S. aureus subsp. aureus ATCC 25923 S. marcescens 338 K. pneumoniae ATCC1603 P. aeruginosa 867
Salvia officinalis EO (mg/ml)
MIC
MBC
MBC/MIC
MIC
MBC
MBC/MIC
0.117 0.234 0.234 0.469 0.234 0.469
0.117 0.234 0.469 0.469 0.234 0.469
1 1 2 1 1 1
3.749 7.497 7.497 14.995 14.995 7.497
3.749 7.497 14.995 14.995 14.995 7.497
1 1 2 1 1 1
MIC (minimal inhibitory concentration). MBC (minimal bactericidal concentration). EO (essential oil).
The following figures (Fig. 1a–l) represent isobolograms showing 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 combinations, 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 strain
Combinationa
MIC0
MICC
FIC
FICI
Effect of the compounds in combination
S. aureus ATCC 25923
Thymus numidicus EO Imipeneme Thymol Imipeneme Thymus numidicus EO Cefotaxime Thymol Cefotaxime Thymus numidicus EO Ciprofloxacine
0.234 0.252 0.202 0.252 0.234 0.504 0.202 0.504 0.234 0.448
0.234 0.126 0.006 0.252 0.117 0.252 0.101 0.252 0.059 0.224
1 0.5 0.030 1 0.5 0.5 0.5 0.5 0.25 0.5
1.5
Antagonistic
1.030
Antagonistic
1.00
Indifferent
1.00
Indifferent
0.75
Additive
Thymus numidicus EO Imipeneme Thymol Imipeneme Thymus numidicus EO Cefotaxime Thymol Cefotaxime Salvia officinalis EO Cefotaxime
0.117 0.063 0.202 0.063 0.117 0.126 0.202 0.126 3.749 0.126
0.004 0.063 0.101 0.031 0.004 0.126 0.006 0.126 0.117 0.126
0.034 1 0.5 0.5 0.034 1 0.030 1 0.031 1
1.034
Antagonistic
1.00
Indifferent
1.034
Antagonistic
1.030
Antagonistic
1.031
Antagonistic
Thymus numidicus EO Salvia officinalis EO Thymus numidicus EO Chloramphenicol Thymol Chloramphenicol Thymus numidicus EO Cefotaxime Thymus numidicus EO Ampicilline Thymus numidicus EO Tetracycline Thymus numidicus EO Amoxicilline Thymus numidicus EO Ciprofloxacine
0.234 7.497 0.234 2.016 0.202 2.016 Resistance against all the antibiotics
0.234 0.234 0.007 2.016 0.101 1.008
1 0.031 0.030 1 0.5 0.5
1.031
Antagonistic
1.030
Antagonistic
1.00
Indifferent
E. coli ATTC 25922
E. coli 161
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 microorganisms, when they are combined. Effectively, several studies showed an additive effect against microorganisms when thymol and carvacrol, both isomers, are combined (Pereira de Sousa et al., 2012; Fu et al., 2007; Lambert et al., 2001). The increased antimicrobial 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 activity 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 activity 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. numidicus 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
232.6 116.3 58.15 29.07 14.53
67.11 37.95 23.89 13.2 5.78
± ± ± ± ±
3.06 4.03 4.43 4.11 1.43
156.53 ± 20.23
B
Thymol
212.5 106.25 53.125 26.56 13.28
70.59 48.84 33.61 18.97 9.61
± ± ± ± ±
3.21 2.99 2.27 1.71 2.00
104.44 ± 6.76
B
6.87 3.43 1.72 0.86 0.43
85.77 40.77 18.53 9.17 4.09
± ± ± ± ±
3.05 4.25 2.89 0.99 0.42
4.11 ± 0.25
B
5746.25 2871.25 1435.62 717.81 358.9
87.73 64.05 38.41 20.93 10.52
± ± ± ± ±
1.44 2.99 3.99 3.45 2.61
1999.28 ± 168.89
A
DL-˛-tocopherol
Salvia officinalis EO
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
Thymus numidicus EO Salvia officinalis EO Thymus numidicus EO Salvia officinalis EO Thymus numidicus EO Salvia officinalis EO
156.53 1999.28 156.53 1999.28 156.53 1999.28
100.72 621.67 16.42 1621.63 49.79 1229.35
0.643 0.312 0.105 0.811 0.318 0.61489
0.955
Thymol Salvia officinalis EO Thymol Salvia officinalis EO Thymol Salvia officinalis EO
104.44 1999.28 104.44 1999.28 104.44 1999.28
54.63 1476.45 80.23 541.99 21.17 2288.46
0.523 0.738 0.768 0.271 0.2027 1.1446
1.261
DL-˛-tocopherol Salvia officinalis EO DL-˛-tocopherol Salvia officinalis EO DL-˛-tocopherol Salvia officinalis EO
4.11 1999.28 4.11 1999.28 4.11 1999.28
1.62 1351.4 0.52 1746.91 2.61 544.36
0.394 0.676 0.126 0.8737 0.635 0.272
1.07
DL-˛-tocopherol Thymus numidicus EO DL-˛-tocopherol Thymus numidicus EO DL-˛-tocopherol Thymus numidicus EO
4.11 156.53 4.11 156.53 4.11 156.53
1.64 55.4 2.1 35.61 0.55 75.39
0.340 0.354 0.512 0.227 0.133 0.481
0.694
DL-˛-tocopherol Thymol DL-˛-tocopherol Thymol DL-˛-tocopherol Thymol
4.11 104.44 4.11 104.44 4.11 104.44
2.87 88.63 5.84 45.14 1.38 170.68
0.698 0.848 1.421 0.432 0.336 1.634
1.546
FIC50 I average 0.934 ± 0.019
0.916 0.933 1.215 ± 0.159
1.039 1.347 0.992 ± 0.082
0.999 0.907 0.655 ± 0.063
0.739 0.614 1.789 ± 0.219
1.853 1.97
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 interaction 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. numidicus 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 activation 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).
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5. Conclusion In this study, we report for the first time antimicrobial and antioxidant activities of essential oils combined with other components. Analyses show that essential oils in combination with other substances exhibit different effects (indifferent, additive, antagonistic 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 antioxidant 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. References Ait-Ouazzou, A., Cherrat, L., Espina, L., Loran, S., Rota, C., Pagan, R., 2011. The antimicrobial activity of hydrophobic essential oils constituents alone or combined processes of food preservation. Innov. Food Sci. 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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