Protective effect of some essential oils against oxidative and nitrosative stress on Tetrahymena thermophila growth

This article was downloaded by: [University of New Mexico]

On: 14 October 2014, At: 15:35 Publisher: Taylor & Francis

Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Journal of Essential Oil Research

Publication details, including instructions for authors and subscription information:

http://www.tandfonline.com/loi/tjeo20

Protective effect of some essential oils against oxidative and nitrosative stress on Tetrahymena thermophila growth

N. Errafiy

, E. Ammar

& A. Soukri

a

Laboratory of Physiology and Molecular Genetics, Department of Biology, Faculty of Sciences Aïn Chock, University Hassan II - Aïn Chock , Casablanca , Morocco

b

Research Unit of Urban and Coastal Environments Management – LARSEN, National Engineering School of Sfax , Sfax , Tunisia

Published online: 14 Mar 2013.

To cite this article: N. Errafiy , E. Ammar & A. Soukri (2013) Protective effect of some essential oils against oxidative and nitrosative stress on Tetrahymena thermophila growth, Journal of Essential Oil Research, 25:4, 339-347, DOI:

10.1080/10412905.2013.775681

To link to this article: http://dx.doi.org/10.1080/10412905.2013.775681

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no

representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.

This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any

form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://

www.tandfonline.com/page/terms-and-conditions

Protective effect of some essential oils against oxidative and nitrosative stress on Tetrahymena thermophila growth

N. Erra fi y

*, E. Ammar

and A. Soukri

a

Laboratory of Physiology and Molecular Genetics, Department of Biology, Faculty of Sciences Aïn Chock, University Hassan II - Aïn Chock, Casablanca, Morocco;

Research Unit of Urban and Coastal Environments Management – LARSEN, National

Engineering School of Sfax, Sfax, Tunisia (Received 31 August 2012; final form 8 February 2013)

The present work evaluates the anti-oxidative and anti-nitrosative stress effect of seven essential oils supplementations on Tetrahymena thermophila growth. Hydrogen peroxide and sodium nitroprusside were used to create oxidative and nitrosative stress, respectively. Results showed that the two agents of stress modify the growth curve of the proto- zoan. The protective effect of lavender, geranium, thyme, rosemary, cypress, juniper and clove essential oils were assessed. These essential oils act differently depending on the type of stress and the plants from which they arise.

Most of them remarkably increase the cell number at the exponential phase. Moreover, the synergistic interaction between essential oils does not seem to be stress type selective, and significantly reduce both oxidative and nitrosa- tive stress especially at the exponential phase of growth.

Keywords: Essential oils; aromatic plants; anti-stress effect; Tetrahymena thermophila; synergism

Introduction

Living organisms are continuously exposed to reactive oxygen (ROS) (1) and reactive nitrogen species (RNS) (2) that are produced during metabolism or in response to external stimuli. These may arise from endogenous as well as exogenous sources (3) such as infectious agents, pollution, UV rays, cigarette smoke and radia- tions (4). Common examples of ROS and RNS are hydroxyl radical (OH

), hydrogen peroxide (H

O

), superoxide anion (O

) and nitric oxide (NO) (5).

Under physiological conditions, these are counterbal- anced by an array of defense pathway and have many physiological roles. Thereby, in situations where defenses are compromised, they can cause damage to all molecular targets; DNA, proteins and lipids (6, 7).

Oxidative and nitrosative stress has been linked to inhi- bition of cell growth (8).

To cope with the fatal cellular consequences trig- gered by ROS and/or RNS, cells have evolved multiple endogenous defense mechanisms, which are based on enzymatic (superoxide dismutase, catalase and glutathi- one peroxidase) (9, 10) as well as non-enzymatic (vitamins A, C and E, carotenoids and flavonoids) pro- cesses, which are sufficient for reversing oxidative and/

or nitrosative stress, called antioxidants. These antioxi- dants have such principal role to neutralize and to degrade toxic free radicals. Therefore, the therapy con- cept based on the use of antioxidants to reinforce the

endogenous antioxidants defenses for a more efficient protection against oxidative and/or nitrosative stress represents an important therapeutic objective with a sci- enti fi c and public interest.

The use of natural antioxidants is an area of increasing interest. Some of them can be found in veg- etables, fruits, spices and plants extracts. Indeed, plants are known to produce various antioxidant compounds to interact with ROS (11). Since ancient time, folk medicine has presented empirical evidence that aroma- therapy with essential oils has physiological effects.

Essential oils are volatile, natural and a complex mix- ture obtained from plant material (flowers, seeds, buds, leaves, twigs, bark, wood, fruits and roots) through steam or hydrodistillation. They are known to induce a wide range of biological effects through their antiseptic properties such as antibacterial (12), antiviral (13), antiparasitic (14), insecticidal (15) and their fragrance properties. They are used as analgesic, sedative, anti-in fl ammatory, spasmolytic and locally anesthetic remedies (16). Moreover, several studies highlighted the anticancer potential of essential oils (17, 18). Thus, some of them were shown to induce apoptosis in can- cer cells (17).

Despite the widespread use of essential oils by humans, little is known about their anti-stress effect.

This paper presents a preliminary study describing the protective effect of some essential oils against oxidative

*Corresponding author. Email: [email protected]

Ó2013 Taylor & Francis

Downloaded by [University of New Mexico] at 15:35 14 October 2014

and nitrosative stress on Tetrahymena thermophila growth, in order to investigate the possible use of these compounds as anti-stress agents.

Tetrahymena thermophila was chosen based on its typical eukaryotic characteristics. This unicellular eukaryote presents an advantage to be used in mecha- nistic studies due, among other reasons, to its cell size (30 50 μm), which is larger than many mammalian cells. Moreover, this ciliated protozoan possesses many core processes conserved across a wide diversity of eukaryotes (including humans), differently from other commonly used unicellular model organisms (19). Fur- thermore, T. thermophila belongs to ciliates, which are one of three major groups within a monophyletic assemblage. Thus, this group is part of the alveolates (20, 21) that include also the dinoflagellates, which are of relevance for fishery (22), and the group of unicellu- lar parasitic organisms called apicomplexans (23), which have a great medical significance.

Experimental

Microorganism and cell culture

The T. thermophila strain SB1969 (Tetrahymena Stock Center, Cornell University), kindly supplied by Profes- sor Juan Carlos Gutiérrez (Universidad Complutense de Madrid) was maintained axenically in 5 mL of prote- ose-peptone (1.5%, w/v) and yeast extract (0.25%, w/v) (PPYE) medium at room temperature (25±1°C) (24).

Cultures of T. thermophila were prepared in 500-mL Erlenmeyer flasks containing 100 mL of sterile PPYE medium inoculated with 1% (v/v) of 72-hour preculture, in the same medium, grown at 32°C without shaking.

Oxidative and nitrosative stress

The ciliated protozoan T. thermophila was cultivated over 140 hours, as previously described, in PPYE medium containing H

O

(Fluka) or sodium nitro- prusside (SNP) (Sigma) at 50% inhibitory concentra- tions (

). These concentrations have been determined from T. thermophila growth inhibitory curves of the two stress reagents (Figure 1). The

values have been then estimated using prohibit analy- sis and showed to be 0.7 and 1.8 mM for H

O

and SNP, respectively. Cell growth was monitored micro- scopically by counting cell numbers at different time intervals using a haemocytometer (Malassez cell). A control was performed in the same conditions without the stress reagents.

Essential oils

Essential oils from seven aromatic plants were used to determine their anti-oxidative and anti-nitrosative stress activities. They were kindly supplied by Professor Emna Ammar (National Engineering School of Sfax) and stored at 4°C until use. These essential oils have been extracted from lavender (Lavandula angustifolia), geranium (Pelargonium robertianum), thyme (Thymus capitalus), rosemary (Rosmarinus officinalis), cypress (Cupressus sempervireus), juniper (Juniperus phoenica) and clove (Syzygium aromaticum) (Table 1).

Determination of minimal inhibitory concentration of essential oils

The assay to determine minimum inhibitory concentration (MIC) of the essential oils studied was

0 5 10 15 20 25

0 2 4 6 8 10

Concentration (mM)

Cell number / ml (x10

) H

O

SNP

Figure 1. Effects of H

O

and sodium nitroprusside (SNP) on Tetrahymena thermophila growth. The cell number was determined after 72 hours of growth at 32°C in proteose-peptone – yeast extract (PPYE) medium containing various concentrations of the stress reagent (H

O

or SNP). Data are means of three separate experiments±SEM.

340 N. Erra fi y et al.

Downloaded by [University of New Mexico] at 15:35 14 October 2014

performed by the twofold serial dilution method (25).

The extract of each essential oil was dissolved in dimethylsulfoxide (DMSO) and then dilutions series from 10

to 10

were prepared. Five microliters of each dilution was added to 5 mL of PPYE medium in the test tube. Then, 50 μl of a calibrated inoculum (1.5 10

cells/mL) of T. thermophila was added to each tube that contained oil. A tube containing DMSO (0.1%, v/v) and without essential oils was used as control. The MIC results were taken as the lowest concentration of the essential oil that showed no turbidity after 72 hours of incubation at 32°C.

Each extract was assayed in triplicate.

Antioxidant potential of essential oils

To evaluate the antioxidant potential of the essential oils, T. thermophila was cultivated in the presence of the stress reagent at the

(0.7 mM for H

O

or 1.8 mM for SNP) in PPYE medium supplemented with the essential oil tested at a non-toxic concentration of 10

. The stress reagent and the essential oil were added simultaneously to the growth medium before inoculation. Growth curves were performed, as described previously, by counting cell numbers at dif- ferent time intervals using a haemocytometer (Malassez cell). Control experiments were carried out using only the PPYE medium inoculated with T. thermophila in Table 1. Major components of the essential oils tested against oxidative and nitrosative stress.

Plant species

Details of plant oils Common

name Main compound (area, %)

Chemical structure of the major compound

Lavandula angustifolia

Lavender Linalyl acetal (34.5), linalool (20.5), methylcyclopentane (10.2)

Pelargonium robertianum

Geranium Geranyl acetate (42.3), geraniol (36.7)

Thymus capitalus Thyme Borneol (24.1), thymol (6.7)

Rosmarinus of fi cinalis

Rosemary 1,8-Cineole (27.22), trans-beta-ocimene (10.7), camphor (10.4)

Cupressus sempervirens

Cypress δ -3-Carene (22.9), α -pinene (20)

Juniperus phoenica Juniper α -Pinene (59.1), linalol (3.3)

Syzygium aromaticum Clove Eugenol (75.3), eugenyl acetate (15.1)

Note:^⁄According to the gas chromatography–mass spectrometry (GC–MS) analysis (35).

Downloaded by [University of New Mexico] at 15:35 14 October 2014

presence of the stress reagent and without essential oil.

All tests were run in triplicate.

Synergistic effect of essential oils

Essential oils selected to evaluate the synergistic effect were those from lavender, geranium and thyme. A mixture of two essential oils was added at a non-toxic concentration (10

) to the growth medium of T. thermophila containing the stress reagent at

before inoculation. Growth curves were monitored, as described previously, by counting cell numbers at dif- ferent time intervals using a haemocytometer (Malassez cell). Control experiments were carried out using the PPYE medium inoculated with T. thermophila in pres- ence of the stress reagent. All experiments were run in triplicate.

Statistical analysis

Statistical significance was determined by Student’s t- test. Differences were considered signi fi cant if p<0.05 and highly signi fi cant if p<0.01 when compared with control.

Results

Response of Tetrahymena thermophila growth under stress conditions

H

O

and SNP (a NO-donating compound) were used to create oxidative and nitrosative stress on the proto- zoa, respectively. Both stress reagents completely inhib- ited the growth of T. thermophila at the concentrations

of 1 mM of H

O

and 10 mM of SNP (Figure 1).

Thus, the

values of H

O

(0.7 mM) and SNP (1.8 mM), estimated using probit analysis from Figure 1, were used to evaluate the effect of these stress reagents on T. thermophila growth. The typical growth curve (lag, exponential and stationary phases) was consider- ably modified by the addition of H

O

or SNP. Never- theless, a significant decrease at the lag phase of growth was observed in cultures supplemented with either stress reagents when compared with control (Figure 2). Similarly, the exponential phase of growth showed a significant decrease under both oxidative and nitrosative stress. No change at the stationary phase was observed under oxidative stress when compared with control. However, significant change at the station- ary phase was observed under nitrosative stress com- pared with control (Figure 2).

Determination of the sensitivity of Tetrahymena thermophila to essential oils

The seven natural essential oils used in this study, in their pure state, inhibited the growth of T. thermophila when added to culture medium. Thus, a series of dilu- tions were done to determine the minimal inhibitory concentrations. Therefore, the MIC was obtained at the dilution 10

. Nevertheless, from the dilution 10

, a normal growth of T. thermophila was shown.

For further studies related to anti-stress effect of essential oils, the dilution 10

was chosen. Monitoring the T. thermophila growth curve, in the presence of dif- ferent essential oils at this dilution, showed no effect on the growth or on the shape (data not shown).

0 5 10 15 20

Control H

O

-treated SNP-treated Cell number / ml (x10

)

Lag phase Exponentiel phase Stationary phase

** **

**

* **

Figure 2. Effects of H

O

and sodium nitroprusside (SNP) on Tetrahymena thermophila growth. Cell numbers were determined after 24, 72 and 140 hours of growth at 32°C corresponding to the lag, exponential and stationary phases, respectively.

Tetrahymena thermophila was grown in proteose-peptone–yeast extract (PPYE) medium containing 0.7 mM of H

O

(H

O

- treated) or 1.8 mM of SNP (SNP-treated). Tetrahymena thermophila grown in absence of stress reagents was taken as control.

Values are given as means±SEM. For comparisons, Student’s t-test was used (

p<0.05 and

p<0.01).

342 N. Erra fi y et al.

Downloaded by [University of New Mexico] at 15:35 14 October 2014

Anti-oxidative stress effect of essential oils

Essential oils tested (lavender, geranium, thyme, rose- mary, cypress, juniper and clove) showed no signi fi cant change (p<0.05) at the lag phase of growth compared with the control (H

O

-treated T. thermophila). Unlike the lag phase, significant change at the exponential phase was observed. The H

O

-treated T. thermophila cell number was significantly higher (p<0.01) when the cultures were added by cypress or juniper essential oils (Figure 3). Similarly, thyme and rosemary essential oils

increase significantly (p<0.05) the cell number at the exponential phase, whereas no significant change was observed when the essential oil of lavender or geranium or clove was added to the H

O

-treated cultures. The cell number at the stationary phase increases signifi- cantly (p<0.01) with cypress or juniper essential oils when compared with control. Also, rosemary essential oil increases significantly (p<0.05) the cell number at the stationary phase. Like the exponential phase, no signi fi cant change was observed at the stationary phase

0 5 10 15 20 25 30

Control Lavender Geranium Thyme Rosemary Cypress Juniper Clove Cell number / ml (x104)

Lag phase Exponentiel phase Stationary phase

*

**

* *

**

** **

Figure 3. Protective effect of lavender, geranium, thyme, rosemary, cypress, juniper and clove essential oils against oxidative stress created by 0.7 mM of H

O

on Tetrahymena thermophila growth. Cell numbers were determined after 24, 72 and 140 hours of growth at 32°C corresponding to the lag, exponential and stationary phases, respectively. H

O

-treated cells were considered control. Values are given as means±SEM. For comparisons, Student’s t-test was used (

p<0.05 and

p<0.01).

0 2 4 6 8 10 12 14

Control Lavender Geranium Thyme Rosemary Cypress Juniper Clove Cell number / ml (x104)

Lag phase Exponentiel phase Stationary phase

* *

*

*

Figure 4. Protective effect of lavender, geranium, thyme, rosemary, cypress, juniper and clove essential oils against nitrosative stress created by 1.8 mM of sodium nitroprusside (SNP) on Tetrahymena thermophila growth. Cell numbers were determined after 24, 72 and 140 hours of growth at 32°C corresponding to the lag, exponential and stationary phases, respectively. SNP-treated cells were considered control. Values are given as means±SEM. For comparisons, Student ’ s t-test was used (

p<0.05 and

⁄⁄

p<0.01).

Downloaded by [University of New Mexico] at 15:35 14 October 2014

with lavender, geranium or clove essential oils. How- ever, the essential oil of thyme had no significant change at the stationary phase (Figure 3).

Anti-nitrosative stress effect of essential oils

The action of natural essential oils, used in this study, on SNP-treated T. thermophila was presented in Fig- ure 4. As can be seen, no signi fi cant change (p<0.05) in cell number was observed at the lag phase of growth for different essential oils used when compared with control without essential oils. However, cypress, rose- mary, clove and juniper essential oils, when added to SNP-treated T. thermophila cultures, increase signifi- cantly (p<0.05) the cell number at the exponential

phase of growth with respect to control, whereas laven- der, geranium and thyme essential oils were not effec- tive enough as anti-nitrosative stress. The stationary phase was not much changed for the different essential oils.

Synergistic effect of essential oils

In order to study whether the mixture of essential oils can influence their ability to inhibit the action of stress reagents, essential oils of lavender, geranium and thyme were chosen. These essential oils used separately had not shown an interesting effect against the two types of stress (oxidative and nitrosative).

0 5 10 15 20 25

Control Lavender+Thyme Lavender+Geranium Thyme+Geranium Cell number / ml (x104)

Lag phase Exponentiel phase Stationary phase

**

**

**

*

0 2 4 6 8 10 12 14

Control Lavender+Thyme Lavender+Geranium Thyme+Geranium Cell number / ml (x104)

Lag phase Exponentiel phase Stationary phase

*

* *

(a)

(b)

Figure 5. Synergistic effect of three essential oils (lavender, geranium and thyme) on Tetrahymena thermophila growth. In each experiment, two essential oils were added to the treated culture medium. Cell numbers were determined after 24, 72 and 140 hours of growth at 32°C corresponding to the lag, exponential and stationary phases, respectively. (a) Synergistic effects on H

O

- treated cultures; (b) Synergistic effects on sodium nitroprusside (SNP)-treated cultures. H

O

and SNP were used at 0.7 and 1.8 mM, respectively. Values are given as means±SEM. For comparisons, Student’s t-test was used (

p<0.05 and

p<0.01).

344 N. Erra fi y et al.

Downloaded by [University of New Mexico] at 15:35 14 October 2014

+ On oxidative stress

The addition of two essential oils on H

O

-trated T.

thermophila cultures showed no significant change (p<0.05) at the lag phase, whereas at the exponential phase the cell number was significantly (p<0.01) enhanced under different essential oils mixtures. Thus, the mixture of lavender and thyme essential oils increases signi fi cantly (p<0.01) the cell number at exponential and stationary phases (Figure 5a). Never- theless, signi fi cant increase (p<0.01) in cell number was observed at the exponential phase when thyme and geranium essential oils were mixed. However, at the stationary phase, no interesting change was noted (Figure 5a). Similarly, a lavender–geranium essential oils mixture enhanced significantly (p<0.01) the cell number at the exponential phase without change at the stationary phase.

+ On nitrosative stress

Under nitrosative stress, the synergistic effect of two essential oils mixed showed an improvement at the exponential phase without change at lag and stationary phases when compared with control (SNP-treated T.

thermophila) (Figure 5b). Indeed, mixtures of laven- der–thyme, thyme–geranium and lavender–geranium essential oils increased significantly (p<0.05) the cell number at the exponential phase, whereas no signi fi cant (p<0.05) increase in cell number was observed at lag and stationary phases.

Discussion

Under normal conditions, T. thermophila has the typical growth curve with lag, exponential and stationary phases. This growth curve is disturbed under stress conditions. The results reported here indicate that H

O

and SNP markedly affect the growth curve of T. thermophila. As shown for Tetrahymena pyriformis (26), the two agents of stress (H

O

and SNP) completely inhibited the growth of T. thermophila (Figure 1). Therefore, it might be explained by the dis- ruption of the equilibrium between ROS generation and its scavenging, which is lethal to the cell. Oxidative stress caused significant decrease in cell number at the two first phases of growth (Figure 2). Indeed, a high toxicity was observed at the lag phase followed by the exponential one. However, no significant change in cell number was observed at the stationary phase. Similar results have been reported for Yarrowia lipolytica (27).

Also, Lee et al. (28) showed that H

O

at sub-lethal concentrations induced growth arrest in human lung fi broblasts. Similarly to oxidative stress, nitrosative stress leads to the diminution of cell number at lag, exponential and stationary phases (Figure 2). The toxic effect of nitrosative stress has been reported on yeasts

by Sahoo et al. (29), which had observed a signi fi cant inhibition of growth on Rhodotorula mucilginosa and Saccharomyces cerevisiae in the presence of S-nitroso- glutathione, a NO-donating compound.

To cope with the toxic effect of stress reagents, nat- ural essential oils were used. These essential oils are widely used and well known for their therapeutic vir- tues. They are used in aromatherapy, in cosmetics, for flavoring food and drink, and as medicaments like brain stimulation, anxiety-relieving sedation and antide- pressant (30). Because of their concentrated nature, uti- lization of essential oils as natural anti-stress agents requires the establishment of optimal conditions. Essen- tial oils as anti-protozoal agents have been reported (31). These results are consistent with ours, which showed inhibition of T. thermophila growth under the different essential oils used at pure state. However, the essential oils used at non-lethal dose 10

did not caused alteration on the viability or the morphology of T. thermophila cells when they were observed by microscopy.

Essential oils have been widely studied for their antimicrobial, insecticidal, antifungal, antibacterial and cytotoxic activities (32). The rationale for this work is to test the possibility of creating a protective effect by using natural essential oils that could minimize the oxi- dative and nitrosative stress on T. thermophila. The var- ious essential oils used act differently with respect to stress. Their action differs depending on the nature of the plants from which it arises and the type of stress which is subjected to the cell (oxidative or nitrosative).

Throughout this study, different essential oils show no effect on anti-oxidative or nitrosative stress at the lag phase of growth. However, interesting results at the exponential phase are reflected by the significant increase in cell number. Indeed, essential oils of cypress and juniper exhibited a high anti-H

O

stress by increasing significantly the cell number, while thyme and rosemary demonstrated a moderately protec- tive effect (Figure 3). Similar results were seen in SNP- treated T. thermophila, excepted for thyme, which showed no significant change (Figure 4). The difference observed in the reaction of T. thermophila cells to the essential oils depending upon their growth stages could be explained by their physiological states, which may affect their adaptive responses to the environmental conditions. On the other hand, it is potentially interest- ing to correlate the anti-stress behavior of the essential oils with their composition and the chemical nature of their constituents. Thus, as shown in Figures 3 and 4, the highest anti-stress effect of cypress and juniper essential oils may be attributed to their major constitu- ents, which were δ-3-carene and α-pinene, respectively.

Indeed, these compounds seem to have a structural analogy compared with the other major compounds of

Downloaded by [University of New Mexico] at 15:35 14 October 2014

the essential oils used (Table 1). These essential oils have different composition, however, and it is difficult to attribute the anti-stress effect to a compound. It was reported that essential oils present specificity in the amplitude but not in the mode of action of the biologi- cal effect (33, 34). Essential oils of lavender, geranium and clove do not protect T. thermophila cells against oxidative stress induced by H

O

, while clove essential oil demonstrates a significant effectiveness against nitrosative stress during the exponential phase. At the stationary phase, no protective effect was shown against SNP-treated cells, although cypress, juniper and rosemary essential oils enhanced the cell number signif- icantly under oxidative stress.

According to our results, none of the three essential oils of lavender, geranium and thyme could reduce the two types of stress when used separately. Thus, we attempted to evaluate the synergistic role of essential oils as an anti-stress effect. Very little information has been reported on the synergistic role of essential oils as anti-stress agents. The mixture of two essential oils added to the culture medium of stressed T. thermophila by H

O

or SNP did not affect or protect cells at the lag phase against oxidative and nitrosative stress, but, at the exponential phase of growth, the three essential oils demonstrated a highly significant increase in cell number against H

O

-treated cells, when they are mixed (Figure 5a). On the other hand, these essential oil mixtures (lavender–thyme, lavender–geranium and thyme – geranium) improved the growth of T. thermo- phila SNP-treated cells (Figure 5b). The synergistic interaction does not seem to be stress-type selective or affect both oxidative and nitrosative stress. It seems the anti-stress effect of essential oils can be attributed to the synergistic interaction of the compounds constitut- ing the oils rather than to the use of the essential oil separately.

Since essential oils of lavender, geranium, thyme, rosemary, cypress, juniper and clove are used internally in folk medicine and in aromatherapy, our results as well as others may be useful for the consideration of these essential oils as possible functional food ingredi- ents or food supplements for the prevention of stress in the future.

Acknowledgments

The authors thank Professor Juan Carlos Gutiérrez from

‘ Universidad Complutense de Madrid ’ for supplying the strain of T. thermophila SB1969. The authors are also thankful to Ms Wafaa Fatma Ben Saida for bringing the essential oils.

Funding

This work was supported by the Moroccan CNRST (URAC).

References

1. D.J. Jamieson, Oxidative stress responses of the yeast Saccharomyces cerevisiae. Yeast, 14, 1511–1527 (1998).

2. C. Nathan and M.U. Shiloh, Reactive oxygen and nitro- gen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc. Natl. Acad. Sci.

USA, 97, 8841–8848 (2000).

3. R.S. Ray, S. Mehrotra, U. Shankar, G.S. Babu, P.C. Joshi and R.K. Hans, Evaluation of UV-induced superoxide radical generation potential of some common antibiotics.

Drug Chem. Toxicol., 24, 191 – 200 (2001).

4. Ch.S. Foote, Light, oxygen and toxicity. In: Pathology of Oxygen. Edit., P. Anne, pp. 21 – 44, Academia Press, Lon- don (1982).

5. T.L. Dormandy, An approach to free radical. Lancet, 29, 1010 – 1014 (1983).

6. N. Erra fi y and A. Soukri, Puri fi cation and partial char- acterization of glyceraldehydes-3-phosphate dehydroge- nase from the ciliate Tetrahymena thermophila. Acta Biochim. Biophys. Sin., 44, 527–534 (2012).

7. M. Maes, I. Mihaylova, M. Kubera, M. Uytterhoeven, N.

Vrydags and E. Bosmans, Increased 8-hydroxy-deoxygua- nosine, a marker of oxidative damage to DNA, in major depression and myalgic encephalomyelitis/chronic fatigue syndrome. Neuro. Endocrinol. Lett., 30, 715 – 722 (2009).

8. H.E. Marshall, K. Merchant and J.S. Stamler, Nitrosation and oxidation in the regulation of gene expression. Faseb J., 14, 1889 – 1900 (2000).

9. B.N. Ames, M.K. Shigenaga and T.M. Hagen, Oxidants, antioxidants and the degenerative diseases of aging.

Proc. Natl. Acad. Sci. USA, 90, 7915 – 7922 (1993).

10. V.M. Sardesai, Role of antioxidants in health mainte- nance. Nutr. Clin. Pract., 10, 19 – 25 (1995).

11. F. Lu and L.Y. Foo, Phenolic antioxidant components of evening primrose. In: Nutrition, Lipids, Health and Dis- ease. Edits., A.S.H. Ong, E. Niki and L. Packer, pp. 86 – 95, American Oil Chemists Society Press, Champaign, IL (1995).

12. N. Canillac and A. Mourey, Antibacterial activity of the essential oil of Picea excelsa on Listeria, Staphylococcus aureus and coliform bacteria. Food Microbiol., 18, 261–268 (2001).

13. C.D. Bishop, Antiviral activity of the essential oil of Mel- aleuca alternifolia (Maiden and Betche) Cheel (tea tree) against tobacco mosaic virus. J. Essent. Oil Res., 7, 641 – 644 (1995).

14. L.M. Pessoa, S.M. Morais, C.M.L. Bevilaqua and J.H.S.

Luciano, Anthelmintic activity of essential oil of Ocimum gratissimum Linn. and eugenol against Haemonchus con- tortus. Vet. Parasitol., 109, 59 – 63 (2002).

15. I. Konstantopoulou, L. Vassilopoulou, P. Mavragani-Tsi- pidou and Z.G. Scouras, Insecticidal effects of essential oils. A study of the effects of essential oils extracted from eleven Greek aromatic plants on Drosophila auraria.

Experientia, 48, 616–619 (1992).

16. F. Bakkali, S. Averbeck, D. Averbeck and M. Idaomar, Biological effects of essential oils – A review. Food Chem. Toxicol., 46, 446–475 (2008).

17. A. Kumar, F. Malik, S. Bhushan, V.K. Sethi, A.K. Shash- i, J. Kaur, S.C. Taneja, G.N. Qazi and J. Singh, An essen- tial oil and its major constituent isointermedeol induce apoptosis by increased expression of mitochondrial cyto- chrome c and apical death receptors in human leukaemia HL-60 cells. Chem. Biol. Interact., 171, 332 – 347 (2008).

346 N. Erra fi y et al.

Downloaded by [University of New Mexico] at 15:35 14 October 2014

18. J. Soeur, L. Marrot, P. Perez, I. Iraqui, G. Kienda, M.

Dardalhon, J.R. Meunier, D. Averbeck and M. Huang, Selective cytotoxicity of Aniba rosaeodora essential oil towards epidermoid cancer cells through induction of apoptosis. Mut. Res., 718, 24 – 32 (2011).

19. J.A. Eisen, R.S. Coyne, M. Wu, D. Wu and M. Thiagara- jan, Macronuclear genome sequence of the ciliate Tetra- hymena thermophila, a model eukaryote. PLoS Biol., 4, 1620–1642 (2006).

20. T. Cavalier-Smith, Kingdom Protozoa and its 18 phyla.

Microbiology, 57, 953–993 (1993).

21. J.F. Saldarriaga, F.J.R. Taylor, T. Cavalier-Smith, S. Men- den-Deuer and P.J. Keeling, Molecular data and the evo- lutionary history of dinoflagellates. Eur. J. Protistol., 40, 85–111 (2004).

22. K. Steidinger, Some taxonomic and biologic aspects of toxic dino fl agellates. In: Algal Toxin in Seafood and Drinking Water. Edit., I.R. Falconer. Academic Press, New York (1993).

23. A.A. Gajadhar, W.C. Marquardt, R. Hall, J. Gunderson, E.V. Ariztia-Carmona and M.L. Sojin, Ribosomal RNA sequences of Sarcocystis muris, Theileria annulata, and Cryptothecodinium cohni reveal evolutionary relationship among apicomplexans, dino fl agellates, and ciliates. Mol.

Biochem. Parasitol., 45, 147 – 154 (1991).

24. R.C. Pousada, M.L. Cyme and D.H. Hayes, Character- ization of peribosomal ribonucleoprotein particles from T. pyriformis. Eur. J. Biochem., 102, 389–397 (1979).

25. J.E. Baron and S.M. Fingold, Methods for testing antimi- crobial effectiveness. In: Diagnostic Microbiology. Edit., C.V. Mosby, pp. 171–194, Bailey Scotts, Missouri (1990).

26. L. Fourrat, A. Iddar, F. Valverde, A. Serrano and A. Soukri, Effects of oxidative and nitrosative stress on Tetrahymena pyriformis glyceraldehyde-3-phosphate dehydrogenase. J.

Eukaryot. Microbiol., 54, 338 – 346 (2007).

27. E.N. Biriukova, A.G. Medentsev, A. Yu, Arinbasarova and V.K. Akimenko, Tolerance of the yeast Yarrowia lipolytica to oxidative stress. Microbiology, 75, 243 – 247 (2006).

28. H.-C. Lee, P.-H. Yin, C.-Y. Lu, C-W. Chi and Y-H. Wei, Increase of mitochondria and mitochondrial DNA in response to oxidative stress in human cells. Biochem. J., 348, 425 – 432 (2000).

29. R. Sahoo, R. Sengupta and S. Ghosh, Nitrosative stress on yeast: inhibition of glyoxalase-I and glyceraldehyde-3- phosphate dehydrogenase in the presence of GSNO.

Biochem. Biophys. Res. Commun., 302, 665–670 (2003).

30. G. Buchbauer and L. Jirovetz, Aromatherapy – use of fragrances and essential oils as medicaments. Flavour Fragr. J., 9, 217–222 (1994).

31. E-R. Mojica, C.C. Deocaris and M.A. Endriga, Essential oils as anti-protozoal agents. Philipp. J. Crop Sci., 29, 41 – 43 (2004).

32. L. Faleiro, G.M. Miguel, C.A.C. Guerrero and J.M.C.

Brito, Antimicrobial activity of essential oils of Rosmari- nus of fi cinalis L., Thymus mastichina (L) L. ssp. masti- china and Thymus albicans. In: Proceedings of the II WOCMAP Congress on Medicinal and Aromatic Plants, Part 2: Pharmacognosy, Pharmacology, Phytomedicine, Toxicology (1999).

33. F. Bakkali, S. Averbeck, D. Averbeck, A. Zhiri and M.

Idaomar, Cytotoxicity and gene induction by some essen- tial oils in the yeast Saccharomyces cerevisiae. Mutat.

Res., 585, 1–13 (2005).

34. F. Bakkali, S. Averbeck, D. Averbeck, A. Zhiri, D. Bau- doux and M. Idaomar, Antigenotoxic effects of three essential oils in diploid yeast (Saccharomyces cerevisiae) after treatments with UVC radiation, 8-MOP plus UVA and MMS. Mutat. Res., 606, 27–38 (2006).

35. F.W. Ben Saida, Les huiles essentielles et leur utilisation en usage thérapeutique. Master memory, Institut Supéri- eur de Biotechnologie Université de Monastir (2007).

Downloaded by [University of New Mexico] at 15:35 14 October 2014