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Composition chimique, activités antioxydantes et antibactériennes des huiles essentielles de Pistacia lentiscus et Rosmarinus officinalis

H. SELMI^1*

A. DHIFALLAH¹

A. BAHRI²

S. JEDIDI¹

C. ABBES¹

H. ROUISSI²

¹ Sylvo-Pastoral Institute of Tabarka, University of Jendouba

² Higher Institute of Agriculture of Mateur, University of Carthage

Abstract - Tunisia is characterized by a climate that allows the proliferation of many plants rich in active substances with multiple biological activities and can replace the use of antioxidants and synthetic antibiotics.For this reason, the essential oils of Rosmarinus officinalis and Pistacialentiscuswere extracted with the technique of steam distillation, the chemical composition of the essential oils was analyzed by gas chromatography, and the antioxidant capacity was evaluated by the DPPH test and the antibacterial activity by the well method. Our results showed a significant difference (p <0.05) in the yield of essential oils. Indeed, we recorded a yield of 0.7% for R.officinalis against 0.07% for P. lentiscus. Both species have an important ability to trap free radicals but the essential oil of Pistacialentiscus has an antioxidant capacity superior to that of Rosmarinus officinalis oil. Likewise, for the antibacterial activity, the essential oils of Pistacia lentiscus were the most active against the bacterial strains tested except the Salmonella strain was more sensitive with Rosmarinus officinalis essential oil than Pistacia lentiscus essential oil. The results of the GC analysis -DM essential oils showed a complex and highly variability of chemical and aromatic composition for each species, the main components of the essential oil of P.lentiscus were monoterpenes: α-pinene (21,86- 17.87%), limonene (16.98-12.02%), whereas for R.officinalisessential oil were 1.8-cineole (28.58-38.12%) and camphor (12.81-9.65%).To conclude, the antioxidant power and the antibacterial activity are strongly correlated with the chemical composition of the essential oils.

Keywords: Essential oils, Chemical Composition, Antioxidant activities, Antibacterial activities, Rosmarinus officinalis, Pistacialentiscus.

1. Introduction

Essential oils are used in the food industry for the manufacture of a wide variety of products, ranging from margarines to chocolate or used directly as salad and cooking oils (Trabelsi et al,2012). Secondary metabolites constitute biologically and chemically interesting group of substances extracted from the plant kingdom. Essential oil of plants shows many biological activities in addition to their use in food, flavor, perfumery, cosmetic and pharmaceutical industries as natural antioxidants (Wei et Shibamoto, 2010; Mothana et al, 2012). Essential oil has been used since ancient times for medicinal purposes and known for its anti-rheumatic, anti-inflammatory and antispasmodic properties (Benincá et al, 2011 ;Zaouali et al, 2013). It has demonstrated powerful antimutagenic, antibacterial and chemo preventiveproperties (Okoh et al,2010). The secondary metabolites grouped as essential oil impart the much needed curative properties to them (Derwich et al, 2010). Different studies made on the essential oil show influence of the area of culture, of variety and harvest season on the chemical composition(Rohloff et al, 2005 ;Flamini et al, 2007). The presence of phenolic compounds in herbs and spices, along with the essential oils, is gaining increasing attention because of their various functions, such as antioxidant activity and flavoring properties(Gardeli et al, 2008). Indeed, natural bioactive compounds like phenols and flavonoids are the important secondary metabolites in plants having intrinsic properties that affect appearance, taste, odor and oxidative stability of plant basedfood (Singh et al, 2012). Secondary metabolites from plants have important biological and pharmacological activities, such as anti-oxidative, anti-allergic, antibiotic, hypoglycemic and ant carcinogenic (Stankovic, 2011).

Essential oils and fatty acids, the leaves of Pistacia lentiscus and Rosmarinus officinal shave a high content of phenolic compounds and a good antioxidant activity. Polyphenols are important natural antioxidants play a major role in the prevention various pathological conditions. However, non-phenolic substances can be responsible for the antioxidant activity of Pistacia lentiscus and Rosmarinus officinalis. Therefore, further studies are needed to identify which phenolic compounds are responsible for the antioxidant activity of the species, and assess the way in which the phenolic substances contribute to this activity. Indeed, our study is interested in evaluating the chemical composition, the anti-bacterial and antioxidant activity of the tow essentials oilsPistacia lentiscus and Rosmarinus officinalis.

2. Materials and methods

2.1. Study area

The Study was carried out in Tabarka at the north-west of Tunisia. This covers an area of 3000 ha, belonging to the humid bioclimatic stage characterized by a very cold winter and a very hot summer. The rainfall is at the average of 1000 -1200mm. The study area is characterized by latitude of 36°55/ N. longitude 8°48/ N and an altitude of 108 m. The soils of the study series are permeable and devoid of limestone, characterized by their leaching and hydromorphy.

2.2. Plant materials

Two species of aromatic and medicinal plants (Pistacia lentiscus and Rosmarinus officinalis) were collected in February 2018, Stem and leaves were separated by hand and air dried at the Sylvo-Pastoral Resources Laboratory in Tabarka.

2.3. Essential oil extraction

The extraction of essential oils was realized by steam distillation, this method consists in placing the plant material on a grid located a few centimeters from the bottom of the extractor filled with water. The heated water produces steam, after passing through the plant material this vapor is enriched with volatile constituents. Then it is condensed under the effect of a cooling system. The floral water is collected in à glass balloon and the separated essential oil is collected in an opaque glass bottle. The extraction was carried out for four hours at a temperature of 100 ° C.

2.4. Essential oil gas chromatography analysis

Gas chromatography analyses were done with Shimadzu HRGC-2010 gas chromatograph (Shimadzu Co, Kyoto, Japan) equipped with flame ionization detector (FID), Auto-injector AOC-20i and auto-sampler AOC-20s. Apolar column Rtx-1 (30 m x 0.25 mm, 0.32 µm film thickness) was used. The oven temperature was held at 50°C for 10 min then programmed at 2°C/min to 190°C then held isothermal for 10 min. The injector and detector temperature were programmed at 230°C. The flow of the carrier gas (N2) was 1.6 ml/min and the split ration was 1:20. Injection volume for all samples was 0.5µl of diluted oils in n-pentane (LabScan Dublin, Ireland). The volatile compounds were identified by comparison of their retention indices (RI) relative to (C7–C20) n-alkenes with those of literature and/or with those of authentic compounds available. Relative percentage amounts of the identified compounds were obtained from the electronic integration of the FID peak areas.

2.5. Determination of total phenolic contents

The determination of the total polyphenols is carried out according to (Singleton et al, 1999). 500 μl of Folin–Ciocalteu (10%) and 1 ml of an aqueous solution of sodium carbonate Na2CO3(7.5%) are added to 500 μl of diluted extract. After shaking, the mixture was incubated for 1 hour at room temperature in dark. The absorbance of solution was then measured at 760 nm using a UV/Vis Jenway ¬6300 spectrophotometer (Jenway Ltd., United Kingdom). The total phenolics content was expressed as mg of gallic acid equivalents per gram of dry matter (mg GAE/g DM) through the calibration curve of gallic acid. All measurements were performed in triplicate.

2.6. Determination of total flavonoid contents

The flavonoid content was determined according to the method of(Yi et al, 2007),1 ml of the diluted aqueous extract was added to 1 ml of a methanol solution of aluminum chloride AlCl3 2%. After incubation at room temperature for 15 min, the absorbance was measured at 430 nm using a UV/Vis Jenway ¬6300 spectrophotometer (Jenway Ltd., United Kingdom). The total flavonoid content were calculated on the basis of the calibration curve of quercetin, and were expressed as mg quercetin equivalents per g dry matter (mg EQ/g DM). All samples were analyzed in three replications.

2.7. Determination of total tannins contents

Evaluation of the total content of condensed tannin was determined using a method described by(Sun et al, 1998). 50 μl of the suitably diluted aqueous sample, 3 ml of Vanillin solution (4% in methanol) and 1.5 mL of concentrated H2SO4 are mixed. The mixture was left in the dark for 15 minutes and the absorbance was measured at 500 nm using a UV/Vis Jenway ¬6300 spectrophotometer (Jenway Ltd., United Kingdom). The total tannins content was calculated on the basis of the calibration curve of catechin, and were expressed as mg catechin equivalents per g dry matter (mg CE/g DM). All samples were analyzed in three replications.

2.8. 1, 1-Diphenyl-2-picrylhydrazyl radical (DPPH) scavenging

The antioxidant capacity of Rosmarinus officinalis andPistacia lentiscus essential oils was evaluated according to the method described by(Grzegorczyk et al, 2007).The essential oils were diluted in dimethyl sulphoxide (DMSO) in order to prepare different concentrations of each essential oil (10, 20, 50, 75, 100, 150,200 and 300 µg/ml) and added to 1 mL of 0.1mM DPPH in ethanol the resulting mixture was then shaken. After 30 min in the dark at room temperature, the absorbances of the different concentrations already prepared of essential oils of each species were measured at 517nm against the corresponding blank. The radical-scavenging activities, expressed as percentage inhibition of DPPH, were calculated according to the following equation: I(%)=[(A0-A1)/A0] x 100

Where I was DPPH inhibition (%), A0 was the absorbance of the control, and A1 was the absorbance of the extract/standard.

The concentration of sample required for 50% inhibition was determined and represented as IC50 for each of test solution which is expressed as µg/ml. All measurements were performed in triplicate.

2.9. Antibacterial activity

The antibacterial activities of Rosmarinus officinalis andPistacia lentiscus against two Gram-positive bacteria strains (Listeria, Bacillus) and two Gram-negative (Escherichia coli, salmonella) were measured by means of the agar-well diffusion assay described by(Güven et al,2006). Twenty milliliters of mixture molten agar and Nutrim Broth (NB) were poured into sterile Petri dishes. A suspension (100ml) of bacteria was spread on the plates of nutrient agar, and then the plates must be dried aseptically at room temperature during 2 hours. After that, 6 mm wells were bored using a sterile cork borer. 60µl of essential oil were placed into the wells, then the Petri dishes were incubated at + 4°C for 3 to 4 hours in order to allow the diffusion of the essentials oils present into the wells. Finally, they were incubated at 37°C for 48h and the antibacterial activity was evaluated by measuring zone of inhibition. The tests were carried out in triplicate.

2.10. Statistical analysis

All data were subjected to statistical analysis by the variance according to the GLM procedure of the software(SAS , 1989)and compared by Duncan multiple rank tests. The model used was: Yij = μ + Ai + Eijk.

Where; Yij: dependent variable. µ: overall of Y; Ai: effect of the ith essential oil; Eijk: residual error.

3. Results and discussion

3.1. Volatile oil yield

The yields of essential oilsfrom the leaves of Pistacia lentiscus and Rosmarinus officinalis are shown in Figure 1. Theyvaryfrom 0.07% for Pistacia lentiscus to 0.7% for Rosmarinusofficinalis. This corroborates the results of(Dob et al, 2006) and that obtainedby (Amhamdi et al,2009), so they are high compared to whatwasfound by (Bouali et al, 2017). This variationin yield of essential oils may be due to the characteristics of each plant, the type and drying time before extraction (Bencheikh et al,2015).

According to our results, the observed yield of essential oils increases significantly from Pistacia lentiscus(Figure 1). In fact, the highest level is observed for the Rosmarinus officinalis with a mean value of 0.7%. However,variation in oil yield can be attributed to some factors like conditions of plant growth, environmental and region. The oil yield during plant growth is particularly susceptible to environmental conditions such as light, nutrient availability, day length and daily temperature (Skoula et al, 2000 ;Msaada et al, 2009).

3.2. Total Polyphenol, flavonoid and Tannin content

Total phenolic values of Pistacia lentiscus and Rosmarinus officinalis leaves are given in Table 1. The total phenolic content varied widely and ranged from 66.72±4.40 to 161.18±6.11mg GAE/g DM. High levels (161.18±6,11mg GAE/g DM) were found in extracts of Pistacia lentiscus. Low levels (66.72±4.40mg GAE/g DM) were found in extracts of Rosmarinus officinalis. The differences in total phenolic content between Species were statistically significant (P <0.05).

The total tannin content ofPistacia lentiscusand Rosmarinus officinalis leaves was shown in Table1. The total tannin content (mg/g) in aqueous extracts, expressed in catechin equivalent (CE), varied between 36.01±4.51 and52.17±5.16 mg EC/g DM. The highest tannin concentration was registered inPistacia lentiscus leaves extract (52.17±5.16 mg EC/g DM).In fact, as seen from Table1, tannin contents varied significantly (P<0.05) between the species.

However, there is no significant difference between the two species for total flavonoids contentsas shown in table 1 the concentration of Pistacia lentiscus and Rosmarinus officinalis in flavonoidsare respectively15.55±0.5 mg QE/g DM and 15.97±0.63mg QE/g DM.

The variations in the distribution of the total phenolic can be partially due to genotypic factors that control accumulation of these compounds in the plant, origins of plant and conditions for plant growth(Hashempour et al, 2010 ;Schmidt et al, 2010).The meteorological conditions, season and post-harvest conditions have been recently reported as additional source of variance in the total flavonoids content(Dziri et al, 2012). Moreover, other studies suggested that the biotic conditions (organ and physiological stage) and abiotic stresses (edaphic factors, salinity) can play an important role in the production and accumulation of phenolic compounds(Msaada et al, 2009 ;Andarwulan et al, 2010). Phenols are very important plant constituents because of their scavenging ability on free radicals due to their hydroxyl groups. Therefore, the phenolic content of plants may contribute directly to their antioxidant action and it is likely that the activity of the extracts is due to these compounds(Wei et Shibamoto, 2010; Besombes, 2008). Flavonoids are class of secondary plant metabolites found in leguminous, fruits, flowers and leaves having several biological activities (Harborne et Williams, 2000;Karioti et al, 2010).

3.3. Antioxidant activity

Based on the IC₅₀ values ​​of the essential oils of 2 species (Pistacialentiscus, and Rosmarinus officinalis) expressed in Table 2, the DPPH radical scavenging activity of these oils measured by the DPPH test shows a significant difference between species (p <0.0057). Indeed, the essential oils of Pistacialentiscus have an antioxidant activity more important than that expressed by the essential oil of Rosmarinus officinalis which is characterized by the highest IC₅₀ (74.29 μg / ml). In fact, the IC₅₀ is inversely related to the antioxidant capacity of a compound because it expresses the amount of antioxidant required to decrease the free radical concentration by 50%, the lower the IC₅₀ value, the lower the activity antioxidant of a compound is great.The antioxidant capacity of the two essentials oils remain inferior to ascorbic acid used as reference antioxidant (IC₅₀ = 61.3 μg / ml). Our results are close to those found by (Lardry et Haberkorn, 2007; Flamini et al, 2007).

A number of methods are available for the determination of free radical scavenging activity but the assay employing the DPPH has received the maximum attention owing to its ease of use and its convenience(Rout et al, 2011).

This variation in antioxidant activity can be related to the nature and proportion of the active compounds present in the different oils studied(Bouyahya et al,2017). In fact, various phytochemical components, especially polyphenols, are known to be responsible for the free radical scavenging and antioxidant activities of plants(Atoui et al, 2005 ;Asadujjaman et al, 2013).

3.4. Antibacterial activity

The results recorded in Table 3 show that the essential oils of both species have antibacterial activity which varies significantly against the 4 bacterial strains tested. This activity depends on the essential oil (P <0.05) and does not depend on the strain nature (P> 0.05). Indeed, Pistacialentiscusessential oil appears the most active against the bacteria tested with zones of inhibition which varies according to the strains from 16.3 mm to 20mm and Rosmarinus officinalisessential oilwas less active whose zones of inhibition varies between 6mm and 12mm.

As it isnotedin table 3 thethree Bacillus, Listeria and Escherichia coli strains have an extremesensitivity to Pistacia lentiscus essential oil (17.33±0.57mm;14.33±1.52mm and 20mm) and alowersensitivity toRosmarinusofficinalis essential oil (13.33±3.78mm; 9±1.73mm and 10.33±2.51mm). However, the Salmonella strain expresses sensitivity more important with essential oils of Rosmarinusofficinalis (21.1±1mm) than the essential oil of Pistacia lentiscus (16.33±1.52mm)

R = rosemary
L = lentisc
(+) = positive test:antibiotic (Gentamicin)
(-) = negative test: ethanol

3.5. Chemical composition of essential oils

The analysis of the essential oilidentified 45 terpene compounds accounting for approximately 52.94 to 90.12% of the total chemical composition in P. lentiscusoils, with 49 compounds accounting for 63.81 to 81.92% in R. officinalis (Table 4). The proportion and nature of the major compounds varyfrom one species to another.

The results of the P. lentiscus EO analysisshowedthat the main components of leafHEweremonoterpenes: α-pinene (21.86-17.87%), limonene (16.98-12, 02%), β-pinene (7.01-3.35%), terpinen-4-ol (7.02-3.98%) and p-cymene (4.11-3.31%).We also note the presence, to a lesserextent, of germacrene D (1.21-3.11%), α-terpinene (2.68-1.91%), α-cadinol (2.10-0.67 %), α-terpineol (2.22-1.16%), α-phellandrene (1.56-1.28%), camphene (2.23-1.01%) and sabinene (1.56-1 , 13%), γ-terpinene (2.95-0.14%), trans-caryophyllene (2.15-0.12%) and α-thujene (1.33-0.09%). alsopresent but at low proportions.

The results of the R. officinalis HE test show that the major constituents of thisoilare: 1,8-cineole (28,58-38,12%), camphor (12,81-9,65%) ), terminalol (10.87-6.08%), α-pinene (10.01-7.14%), α-terpineol (5.03-2.60%) and β-pinene (4.53- 1.60%). Other compounds are presentwithsignificantlevelsnamely camphene (3.21-1.81%), terpinen-4-ol (3.12-1.37%), β-myrcene (2. 16-2.02%) and β-caryophyllene (2.98-0.18%) (Table 3). The results of the GC-MS analysis of leaf essential oils, twospeciesstudiedshowed a complex and highly variable chemical and aromatic composition for eachspecies. The variability of the essential oilsobserved can beexplained by the influence of variousfactors. In fact, it has been shown for the majority of plant speciesthatsecondarymetabolisms are stronglyinfluenced by plant physiology, the harvestperiod. Thesetwofactorsinduce qualitative and quantitative changes in the chemical composition(Msaada et al, 2009 ; Hosni et al,2011 ; Jemâa, 2014).The composition of essential oils cans beinfluencednimbly by otherfactors (genetics, plant environment, geographical origin, age of the plant, extraction method). Genetic heritagerelated to the species, subspecies, type of clone, plant parts used (Besombes, 2008 ;Burt, 2004 ;Neffati et al, 2009 ;Smitha et al, 2005)

The environment of the plant;related to geographic sources, climatic and meteorological conditions, nature of the soil, harvest time during the day, sunshine, harvesting seasons, neighboring plant populations may influence the chemical composition of the plant(Besombes, 2008 ;Lamendin et al, 2004 ;Lardry et Haberkorn, 2007 ;Delamare et al, 2007 ; Neffati et al, 2009 ; Smitha et al, 2005).In fact, seasonal variations in the distribution betweenhydrocarbonmonoterpenes and oxygenatedmonoterpenes for the essential oils of lentiscus (Pistacia lentiscus L.) have been observed by(Gardeli et al, 2008). Otherstudies have highlighted the influence of the geographical origin of the raw material(Bakkali et al, 2008 ; Oliveira et al, 2013). This is particularly the case of rosemary, whosebiochemical specificities and propertiesvaryaccording to itsoriginwhether from North Africa, Corsica or mainland France(Lamendin et al, 2004).Theage of the plant; the degree of maturity of the plant concerned also affects the composition of the essential oils(Besombes, 2008 ;Burt, 2004). Thus (Neffati et al, 2009) reported that the essential oils of the young aerial parts of Pituranthoschloranthus have higherlevels of hydrocarbonmonoterpenes, whereas the oils of the adultaerial parts are rich in oxygenatedmonoterpenes. The influence of environmentalfactors in the chemical composition of essential oils has also been reported in A. absinthium(Bailen et al, 2013).Extraction methods, drying techniques or storage of rawmaterials affect the chemical composition of essential oils(Besombes, 2008 ; Burt, 2004 ; Smitha et al, 2005).

4. Conclusion

The present work has shown that the essential oils the two species Pistacialentiscus and Rosmarinus officinalis collected from northwestern of Tunisia are doubted of considerable antioxidant and antibacterial activities, hence the possibility of using them as antioxidants and natural antibiotics. This biological activity is strongly linked to the chemical composition of the essential oils and to the concentration of these two aromatic and medicinal plants on secondary metabolites.

Research on these two essential oils should be continued to better estimate other potential of these essential oils such as anti-inflammatory, antidiabetic and antifungal activity, even use them as supplements in the field of animal feeding.

Reference

A. Bouyahya., Y. Bakri., A. Et-Touys., A. Talbaoui., A. Khouchlaa., El Yahyaoui., El A. Idrissi., J.Abriniand N. Dakka.,(2017). In vitro Screening of Antibacterial and Antioxidant Activities of Essential Oils from Four Moroccan Medicinal Plants. MicrobiologyResearch Journal International. 18(4), 1-10.

A. Ghasemzadeh., H.Z. Jaafarand A.Rahmat.,(2010).Antioxidant activities, total phenolics and flavonoids content in two varieties of Malaysia young ginger (Zingiberofficinale Roscoe). Molecules .15(6), 4324-4333.

A. Hashempour., R.F.Ghazvini., D.Bakhshi., M.Ghasemnezhad., M.SharaftiandH. Ahmadian.,(2010). Ascorbic Acid, Anthocyanins, and Phenolics Contents and Antioxidant Activity of Ber, Azarole, Raspberry, and Cornelian Cherry Fruit Genotypes Growing in Iran.Horticulture, Environment, and Biotechnology. 51(2), 83-88.

A. Karioti., A.R.Biliaand H.Skaltsa.,(2010). Quercus ilex L.: A rich source of polyacylated flavonoid glucosides. Food chemistry .123(1), 131-142.

A. Neffati., I. Bouhlel., M.B. Sghaier., J. Boubaker., I.Limem., S.Kilani., I.Skandrani., W. Bhouri., J. Le Dauphin., D. Barillier., R.Mosrati., L.Chekir-Ghediraand R.Mosrati.,(2009).Antigenotoxic and antioxidant activities of Pituranthoschloranthus essential oils. Environmental toxicology and pharmacology. 27(2), 187-194.

A. Weiand T.Shibamoto.,(2010). Medicinal activities of essential oils: role in disease prevention. In Bioactive foods in promoting health,59-70.

A.P.L. Delamare., I.T.Moschen-Pistorello., L.Artico., L. Atti-Serafiniand S. Echeverrigaray.,(2007). Antibacterial activity of the essential oils of Salvia officinalis L. and Salvia triloba L. cultivated in South Brazil. Food chemistry. 100(2), 603-608.

Atoui, A. K., Mansouri ,A., Boskou, G.andKefalas, P.(2005).Tea and herbal infusions: their antioxidant activity and phenolic profile. Food chemistry. 89(1), 27-36.

B. Sun., J.M.Richardo-da-Siliviaand I.Spanger.,(1998). Critical factors of vanillin assay for catechins and proanthocyanidins.J.Agric.Food Chem.46,4267–4274.

3. Besombes.,(2008).Contribution à l'étude des phénomènesd'extraction hydro-thermo-mécaniqued'herbesaromatiques: applications généralisées (Doctoral dissertation, La Rochelle).

19. Burt.,(2004). Essential oils: their antibacterial properties and potential applications in foods:review. International Journal of Food Microbiology. 94, 223-253.

C. Gardeli., P.Vassiliki., M. Athanasios., T.Kibourisand M.Komaitis.,(2008). Essential oil composition of Pistacialentiscus L. and Myrtuscommunis L.: Evaluation of antioxidant capacity of methanolic extracts. Food chemistry. 107(3), 1120-1130.

E. Derwich., A.Manar., Z.Benzianeand A.Boukir.,(2010). GC/MS analysis and in vitro antibacterial activity of the essential oil isolated from leaf of Pistacialentiscus growing in Morocoo. World Applied Sciences Journal.8(10)?1267-1276.

F. Bakkali., S.Averbeck., D.Averbeckand M.Idaomar.,(2008). Biological effects of essential oils–a review. Food and chemical toxicology.46(2),446-475.

G. Flamini., P.L. Cioni., I. Morelliand A. Bader.,(2007). Essential oils of the aerial parts of three Salvia species from Jordan: Salvia lanigera, S. spinosa and S. syriaca. Food chemistry. 100(2),732-735.

G.L. Oliveira., D.D.L. Moreira., A.D.R. Mendes., E.F.Guimarães., L.S.Figueiredo., M.A.C. Kaplanand E.R. Martins.,(2013). Growth study and essential oil analysis of Piper aduncum from two sites of Cerrado biome of Minas Gerais State, Brazil. RevistaBrasileira de Farmacognosia. 23(5), 743-753.

H. Amhamdi., F.Aouinti.,J.PWatheletand A. Elbachiri.,(2009).chemical composition of the essentiol oil of PistacialentiscusL.from Eastern Morocco.UniversityMohamedI .Morocco.3(2), 90-95.

H. Lamendin., G.Toscanoand P.Requirand.,(2004).Phytothérapie et aromathérapiebuccodentaires. EMC-Dentisterie. 1(2),179-192.

H. Trabelsi., O.A.Cherif., F.Sakouhi., P. Villeneuve., J. Renaud., N.Barouh., S. Boukhchinaand P. Mayer.,(2012).Total lipid content, fattyacids and 4-desmethylsterols accumulation in developing fruit of Pistacialentiscus L. growing wild in Tunisia. Food chemistry.131(2), 434-440.

I. Grzegorczyk., A.Matkowskiand H.Wysokinska.,(2007). Antioxidant activity of extracts from in vitro cultures of Salvia officinalis L. Food Chemistry. 104,536–541.

J. Rohloff., S.Dragland., R. Mordaland T.H. Iversen.,(2005). Effect of harvest time and drying method on biomass production, essential oil yield, and quality of peppermint (Mentha× piperita L.). Journal of agricultural and food chemistry. 53(10), 4143-4148.

J.B. Harborneand C.A. Williams.,(2000).Advances in flavonoid research since 1992. Phytochemistry. 55(6), 481-504.

J.M. Lardryand Haberkorn.,(2007). Les huiles essentielles: principes d’utilisationKinésithérapie, la Revue.7(61),18-23.

J.P Benincá., J.B.Dalmarco., M.G.Pizzolattiand T.S.Fröde.,(2011).Analysis of the anti-inflammatory properties of Rosmarinus officinalis L. in mice. Food Chemistry.124(2), 468-475.

K. Güven., E.Yüceland F.Cetintaş.,(2006). Antimicrobial Activities of Fruits of Crataegus.and Pyrus. Species, Pharmaceutical Biology.44(2) ,79-83.

K. Hosni., M. Jemli., S. Dziri., Y.M’rabet., A.Ennigrou., A.Sghaier., H. Casabianca., E. Vulliet., N.Brahimand H. Sebei.,(2011). Changes in phytochemical, antimicrobial and free radical scavenging activities of the Peruvian pepper tree (Schinusmolle L.) as influenced by fruit maturation. Industrial Crops and Products. 34(3), 1622-1628.

K. Msaada., M. BenTaarit., KHosni., H.Hammamiand B.Marzouk.,(2009).Regional and maturational effects on essential oils yields and composition of coriander (Coriandrum sativum L.) fruits.ScientiaHorticulturae .122(1),116-124.

L. Bouali., A.Khadhri., S.Dallali., A.Albouchi., H.Sebei., S. BoukhchinaandE. Masso.,(2017).Chemical Composition and Antioxidant Activity of Essential Oils from Three Varieties of Caryaillinoinensis (Wangenh.) C. Koch Grown in TunisiaJournal of essential oil bearing plants. 20(6),1472-1481.

M. Asadujjaman., A. Hossainand U. Kumar-Karmakar.,(2013). Assessment of DPPH free radical scavenging activity of some medicinal plants. PharmacologyOnLine. 1,161-165.

M. Bailen., L.F Julio., C.E Diaz., J. Sanz., R.A Martínez-Díaz., R. Cabrera., J. Burilloand Gonzalez-Coloma.,(2013). Chemical composition and biological effects of essential oils from Artemisia absinthium L. cultivated under different environmental conditions. Industrial crops and products. 49,102-107.

M. Skoula., J.E. Abbesand K.B.Johnson.,(2000).Genetic variation of volatiles and rosmarinic acid in populations of Salvia fruticosa mill growing in Crete. BiochemicalSystematics and Ecology. 28(6),551-561.

M. Tosun., S.Ercisli., I.M.Sengu., H. Ozer., T.Polatand E.Ozturk.,(2009).Antioxidant Properties and Total Phenolic Content of Eight Salvia Species from Turkey.Biological Research. 42(2),175-181.

M.J.B. Jemâa.,(2014). Essential oil as a source of bioactive constituents for the control of insect pests of economic importance in Tunisia. Med Aromatic Plants. 3, 1-7.

M.S. Stankovic.,(2011).Total phenolic content, flavonoid concentration and antioxidant activity of Marrubiumperegrinum L. extracts. Kragujevac J Sci, 33,63-72.

Mothana, R. A., Alsaid,M. S., Hasoon, S. S., Al-Mosaiyb, N. M., Al-Rehaily, A. J. and Al-Yahya, M. A.(2012). Antimicrobial and antioxidant activities and gas chromatography mass spectrometry (GC/MS) analysis of the essential oils of Ajuga bracteosa Wall. ex Benth. and Lavandula dentata L. growing wild in Yemen. Journal of Medicinal Plants Research, 6(15):3066-3071.

N. Andarwulan., R. Batari., D.A. Sandrasari., B. Bollingand H.Wijaya .,(2010).Flavonoid content and antioxidant activity of vegetables from Indonesia.Food Chemistry. 121(4),1231-1235.

O.O. Okoh., A.P.Sadimenkoand A.J.Afolayan.,(2010). Comparative evaluation of the antibacterial activities of the essential oils of Rosmarinus officinalis L. obtained by hydrodistillation and solvent free microwave extraction methods. Food chemistry. 120(1),308-312.

O.P. Rout., R. Acharya and S.K.Mishra.,(2011).In-Vitro Antioxidant potentials in leaves of Coleus aromaticusBenth and rhizomes of Zingiber zerumbet (L.) SM. Journal of Applied Pharmaceutical Science.1(8), 194.

R. Singh., P.K. Verma and G. Singh.,(2012). Journal of Intercultural Ethnopharmacology. Journal of Intercultural Ethnopharmacology. 1(2), 101-104.

R.L. Smitha., S.M.Cohenb., J. Doullc., V.J.Ferond., J.L.Goodmane., L.J.Marnettf.,P.S. Portogheseg., W.J. Waddellh., B.M.Wagneri., R.L.Hallj., N.A.Higleyk., C. Lucas-GavinlandT.B. Adams.,(2005). A procedure for the safety evaluation of natural flavorcomplexes used as ingredients in food: essential oils. Food and Chemical Toxicology.43, 345-363.

S. Dziri., L. Hassen., S.Fatnassi., Y. Mrabet., H. Casabianca., B.Hanchiand K. Hosni.,(2012).phenolic constituents, antioxidant and antimicrobial activities of rosy garlic (Allium rosium var. odaratissimum ). Journal of functional foods. 4(2), 423-432.

S.E. Bencheikh., M.B.Goudjil., S.Zighmiand S.Ladjel., (2015).Effet du séchage sur le rendement des huiles essentielles de la plante Teucriumpoliumssp. AurasianumLabiatae.Annales des Sciences et Technologie.7(1).

S.Schmidt., M.Zietz., M.Schreiner., S.Rohn., L.W. Krohand A.Krumbein.,(2010).Genotypic and climatic influences on the concentration and composition of flavonoids in kale (Brassica oleracea var. sabellica).Food Chemistry. 119 (4),1293-1299.

SAS User’s Guide.( 1989). Version 6.10 for Windows, SAS Inst. Inc., Cary, NC.

T. Dob., D. DahmaneandC. chelghum.,(2006).Chemical composition of the essential oil of Pistacialentiscus L. from Algeria.journal of essential oil research. 18(3),335-338.

V.L. Singleton., R.Orthoferand R.M.Lamuela-Raventos.,(1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin–Ciocalteau reagent. Method. Enzymol.299,152–178.

Y. Zaouali., H. Chograni., R.Trimechand M.Boussaid., (2013).Changes in essential oilcomposition and phenolic fraction in Rosmarinus officinalis L. var. typicusBatt.organsduringgrowth and incidence on the antioxidant activity. Ind. Crops Prod.43, 412–419.

Z.B. Yi., Y. Yu., Y.Z. Liangand B.Zeng.,(2007). In vitro antioxidant and antimicrobial activities of the extract of PericarpiumCitriReticulatae of a new Citrus cultivar and its main flavonoids, LWT-Food Science and Technology.4,1000-1016.