IJFS#1864_bozza Ital. J. Food Sci., vol. 32, 2020 - 983 PAPER CHEMICAL CHARACTERIZATION AND BIOACTIVE POTENTIAL OF ESSENTIAL OIL ISOLATED FROM RHANTERIUM SUAVEOLENS DESF. SPECIES GROWING IN TUNISIAN ARID ZONE M. HITANA*1, H. NAJAA1, S. FATTOUCH2, T. GHAZOUANI2, C. BEN SASSI2, C. DUPAS-FARRUGIA3, N. OULAHAL3 and M. NEFFATI1 1Laboratoire d’écologie pastorale, Institut des régions arides, Km 22.5, route du Djorf, 4119 Médenine, Tunisia 2Laboratory of Protein Engineering and Bioactive Molecules (LIP-MB), National Institute of Applied Sciences and Technology, University of Crathage Tunis, 1080, Tunisia 3Université de Lyon, Université Claude Bernard Lyon 1, BioDyMIA (Bioingénierie et Dynamique Microbienne aux Interfaces Alimentaires), Equipe Mixte d'Accueil. Université Lyon 1, ISARA Lyon n°3733 *Corresponding author: mhitana@yahoo.com ABSTRACT The purpose of this work is to assess the antioxidant and the antibacterial activities of essential oil from flowers of Rhanterium suaveolens Desf. and to investigate its chemical composition. The GC-MS analysis revealed the identification of a total of thirty-one compounds representing 98.4% of the total oil. Spathulenol (18.3%), carvacrol (12.1%), linalool (9.4%), α-terpineol (7.10%), α-terpinolene (6.3%) and pinocarvone (5.6%) were identified as major constituents. The tested oil exhibited weak activities in both DPPH and ABTS radical scavenging assays and ferric reducing power test. However, it showed a good lipid peroxidation activity using the β-carotene/linoleic acid assay with an IC50 value of 26.20±1.01 µg/mL. In addition, the highest antibacterial effect was recorded against Staphylococcus aureus ((MIC=37.5 μg/mL). These findings show that essential oil of R. suaveolens flowers can be used as a promising source of natural food and drug preservatives. Keywords: antioxidant, antibacterial, essential oil, Rhanterium suaveolens, chemical composition Ital. J. Food Sci., vol. 32, 2020 - 984 1. INTRODUCTION Food preservatives are usually used to extend the shelf life of food products and to limit their deterioration caused by oxidation and growth of foodborne pathogens (RUSSELL and GOULD, 2003). As harmful effects caused by the extensive use of chemical preservatives and the increase of microbial resistance to a wide number of antibacterial drugs (SHAN et al., 2007), the search of new bioactive substances with interesting biological activities is required. In this purpose, several studies have been carried out for the prospection of new products derived from plants and their potential use as ingredients in food and pharmaceutical industries (BEN SALAH et al., 2019). Essential oils are natural products known for their multi-propose applications (DE MARTINO et al., 2015). They have shown a big interest as agents with several healthy- promoting activities such as antibacterial, antioxidant, anti-carcinogenic and antimutagenic properties (GUTIERREZ et al., 2009). Therefore, their investigation proves to be a relevant choice in order to limit the use of chemical or synthetic preservatives and minimize their toxic effect (CAILLET and LACROIX, 2007). The use of essential oils can improve food safety and protect our body against bacteria causing food poisoning (ULTEE et al., 2000). In fact, many studies have demonstrated the potential use of essential oils as natural antimicrobial agents in cheese-making industry (KHORSHIDIANA et al., 2018). As an alternative of specific applications, the essential oils can also be prepared in a large number of formulations, which can be used in food preservation. Recently, GIRARDI et al. (2018) reported that the application of microencapsulated Peumus Boldus essential oil was useful to prevent peanut deterioration caused by food spoilage microorganisms. This biological potential is mainly attributed to the presence of several constituents such as oxygenated derivatives and terpenoids (ABERRANE et al., 2019; BIDA et al., 2019). Tunisian flora is characterized by a wide variety of aromatic and medicinal species producing several bioactive substances with multiple interests (SALEM et al., 2018). However, only few of these species have been investigated for their antioxidant and antibacterial potential. For example, Rhanterium suaveolens Desf. from the Asteraceae family is an endemic species from North Africa growing in Algerian Sahara (QUEZEL and SANTA, 1963) and arid zone of Tunisia (CHAIEB and BOUKHRIS, 1998). Three species of the genus Rhanterium; namely, R. epapposum Oliver, R. adpressum Coss. & Durieu and R. suaveolens Desf. have been reported in literature. R. suaveolens commonly known as “Arfadj” is a forage plant, grazed on by sheep and camel in the desert. It is used by the local population in the production of cheese and in folk medicine as an antidiuretic (HAMIA et al., 2013). To the best of our knowledge, only few studies have been conducted on the phytochemistry of the R. suaveolens essential oil (RSEO) and information on its biological activities, particularly, antioxidant potential, are still scarce in literature. Therefore, the main purpose of this study was to investigate the chemical profile of essential oil collected from the flowers of R. suaveolens growing in arid zone of Tunisia and to evaluate its antioxidant and antibacterial activities. Ital. J. Food Sci., vol. 32, 2020 - 985 2. MATERIALS AND METHODS 2.1. Plant material Flowers of R. suaveolens were collected during the flowering period in April 2014 from a single population of this species growing in Gorthab from the Tataouine region situated in the South East of Tunisia. Taxonomic identification of the plant material was confirmed by a local botanist at the Institute of Arid Zone Research in Medenine (Tunisia). A voucher specimen (IRABS1865) was prepared and deposited in the Herbarium of the Laboratory of Pastoral Ecology. The collected plant material was cleaned and then air-dried at room temperature for eight to ten days. .The dried flowers were ground to powders and stored in air-tight glass. 2.2. Essential oil extraction Air dried flowers (100 g) of R. suaveolens were subjected to hydrodistillation in a Clevenger-type apparatus for 3h. The obtained oil was dried over anhydrous sodium sulfate (Na2SO4) to remove water traces and stored in amber glass vials at 4°C. The oil yield (%) was expressed as volume of essential oil vs. dry weight basis (v/w). 2.3. Gas Chromatography/Mass spectrometry (GC-MS) analysis The GC-MS analysis of the essential oil was carried out using an Agilent 6890N Network GC system combined with Agilent 5975 B Inert MSD detector (quadrupole) with electron impact ionization (70 eV). AHP-5MS (5% phenyl methyl siloxane) column (30 m×0.25 mm i.d, film thickness 0.25 mm). The analysis was performed using helium (purity ˃ 99.99 vol.%) as a carrier gaz at a flow rate of 1.0 mL.min-1. The column temperature was programmed to rise from 50 to 280°C at a rate of 7 °C/min. Injector and detector temperatures were maintained at 220 and 240°C, respectively. Essential oil (1 µL) was injected in a split mode ratio of 1:10. Scan time and mass range were 2.2 s and 50–550 m/z, respectively. 2.4. Identification of the essential oil constituents Identification of the R. suaveolens essential oil (RSEO) components was based on their linear retention indices (RIs) and comparison of their mass spectra with those of the computer library (Wiley 275 library and NIST98 database/ChemStation data system) provided by the instrument software and MS literature data (JOULAIN et al., 2001; ADAMS, 2001). RIs were calculated using n-alkane series (C6–C22) analysed under the same GC–MS conditions as for the samples. 2.3. Antioxidant assays 2.3.1 Scavenging effect on DPPH (2,2-diphenyl-l-1-picrylhydrazil) radical The DPPH assay was estimated as described by DHAOUADI et al. (2014), with slight modifications. Different concentrations of the RSEO were prepared in pure methanol, then 50 µL of each of them were added to 950 µL of a 40 µmol/L (v/v) DPPH methanolic solution in methanol. After vigorous shaking, the resulting mixtures were left in the dark Ital. J. Food Sci., vol. 32, 2020 - 986 at room temperature for 30 min. The absorbance of the resulting solutions was measured at 517 nm. And the radical scavenging ability of RSEO was measured as shown below: 𝐷𝑃𝑃𝐻 𝑠𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 𝑒𝑓𝑓𝑒𝑐𝑡 % = 𝐴𝑜 − 𝐴𝑡 𝐴𝑜 ×100 Where 𝐴0 is the absorption of the control sample after 30 min and 𝐴t is the sample absorption after 30 min. The antioxidant activity was expressed as IC50 value (mg/mL). 2.3.2 Scavenging effect on ABTS (2,20 azinobis-3-ethylbenzthiazoline-6- sulphonic acid) radical cation The ABTS+ assay was performed according to a slight modified version of the method described by TUBEROSO et al. (2007). The radical cation was produced by mixing the ABTS+ solution (7 mmol/L) with potassium persulfate aqueous solution (2.45 mmol/L). The ABTS+ solution was kept in the dark at room temperature for 12-16 h, then, was diluted with phosphate buffer to the absorbance of 0.7±0.02 at 734 nm. Different concentrations of RSEO were prepared in methanol. To 50 µL of each test concentration, 950 µL of diluted ABTS solution were added. The resulting mixtures were allowed to incubate in the dark for 10 min at room temperature. The absorbance of the mixtures was recorded at 734 nm. The antioxidant activity was calculated as follows: 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 % = 1 − A − B C ×100 where, A is the absorbance of the mixture containing the sample, B is the absorbance of the blank reagent and C is the absorbance of the blank sample.The concentration providing 50% of inhibition (IC50) was calculated using a calibration curve in the linear range by plotting the extract concentration. 2.3.3 Reducing power assay The reducing power of the RSEO was assessed following the method described by SINGH et al. (2012). One mL of phosphate buffer (0.2 M ‘w/v’, pH 6.6) and 1 mL of potassium ferricyanide [K3Fe (CN)6], 1% ‘w/v’ was mixed with 1 mL of different concentrations of RSEO (10, 20, 30, 40 and 50 mg/mL). The obtained mixtures were incubated at 50°C for 20 min. Then 1 ml of trichloroacetic acid (TCA) (10% ‘w/v’) was added. The resulting mixtures were revolved at 3000 rpm for 10 min. The supernatant was recovered and mixed with 1.5 mL of distilled water and 150 µL of FeCl3 (0.1% ‘w/v’). The absorbance was measured at 700 nm and the butylated hydroxyanisole (BHA) was used as standard. The result was expressed as IC50 (mg/mL). 2.3.4 Lipid peroxidation activity The lipid peroxidation activity of RSEO was carried out by β-carotene/linoleic method according to DAPKEVICIUS et al. (1998), which is based on the inhibition of the products resulting from the oxidation of linoleic acid. A stock solution of β-carotene/linoleic acid was prepared by mixing 200 mg of Tween 40, 0.5 mg of β-carotene, 25 µL of linoleic acid and 1 mL of chloroform. After chloroform evaporation, under low pressure at 40°C, 100 Ital. J. Food Sci., vol. 32, 2020 - 987 mL of oxygenated distilled water were added to the mixture with vigorous shaking. An aliquot of the resulting solution (2.5 mL) was dispersed to test tubes and 0.5 mL of prepared sample with different concentrations (5-40 µg/mL) in methanol and water were added. The obtained emulsion was incubated for 2 h at 50°C. Two controls were prepared, one with the standard BHA (positive control) and the other without BHA or extract (blank). The absorbance of each sample was immediately measured at 490 nm after 30 min, 60 min, 90 min and 120 min. The bleaching rate R of β-carotene was determined according to the following equation: 𝑅 = ln(AB) T Were ln=natural log, A=absorbance at time 0, B=absorbance at time T (30 min, 60 min, 90 min and 120 min). Antioxidant activity was calculated in terms of inhibition percentage using the following equation: 𝐴𝑛𝑡𝑖𝑜𝑥𝑖𝑑𝑎𝑛𝑡 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (%) = (𝑅𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝑅𝑠𝑎𝑚𝑝𝑙𝑒)×100 𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 Results were expressed as IC50 value (µg/mL). 2.4. In vitro evaluation of antibacterial activity 2.4.1 Tested bacterial strains and growth conditions The antibacterial activity of RSEO was tested against a range of bacterial strains collected from American Type Culture Collection (ATCC, Rockville). Gram-positive: Staphylococcus aureus (ATCC 25923), Listeria monocytogenes (ATCC 19115) and Bacillus cereus (ATCC 14579) and Gram-negative: Eescherichia coli (ATCC 35218), Salmonella Typhimurium (NRLB 4420) and Pseudomonas aeruginosa (ATCC 27853). All bacterial strains were cultured at 37°C for 24h in Mueller-Hinon agar (MHA). The cultures were started by adjusting the bacterial suspension in broth to 0.5 Mc Farland turbidity. Then the bacterial suspension was diluted using 10 fold serial dilution method in order to obtain an inoculum of 108 colony-forming units (CFU) per plates (DHAOUADI et al., 2015). 2.4.2 Disk diffusion method The in vitro antibacterial activity of RSEO was estimated using the disk diffusion method described by DHAOUADI et al. (2015) with slight modifications. 10 µL of RSEO were placed onto sterilized paper disc (6 mm ∅), and placed onto the inoculated agar surface. The petri dishes were placed at 4 °C for 1 h and then incubated at 37 °C for 24 h. After incubation, the diameters of the resulting inhibition zones were determined. Tests were performed in triplicate. Gentamicin (10 µg per disk) was used as positive control and sterile water as negative control. Ital. J. Food Sci., vol. 32, 2020 - 988 2.4.3 Microdilution method The antibacterial activity of RSEO was also assessed by the determination of minimum inhibitory and bactericidal concentrations (MIC and MBC) using broth microdilution method. The minimal inhibition concentration (MIC) was determined as described by GULLUCE et al. (2007) with slight modifications. RSEO sample previously dissolved in 10% dimethylsulfoxide (DMSO) was first diluted to the highest concentration (3 mg/mL) to be tested. The 96 well plates were prepared by dispensing into each well 95 µL of the nutrient broth and 5 µL of the inoculum. An aliquot from the stock solution of RSEO (100 µL) was added into the first well. Then, 100 µL from the serial dilutions were transferred into eleven consecutive wells. The last well containing 195 µL of nutrient broth without RSEO and 5 µL of the inoculum on each strip were used as the negative control. After that, the plates were incubated at 37°C for 24 h. All samples were screened two times against each microorganism.The MIC is defined as the lowest concentration of the sample that did not allow any visible growth of the tested bacterial strain (BEN SALAH et al., 2019). To the determination of the MBC value an aliquot (25 µl) was spreaded onto MHA plates and then incubated for 12–16 h at 37°C. The determination of surviving bacterial strains allowed the estimation of the MBC at 99.9 % of bacterial death (FATTOUCH et al., 2007). 2.5. Statistical analysis All experiments were repeated in triplicate and the results were reported as mean values and standard deviation (mean±SD). Significance differences between the results were performed by analysis of variance (ANOVA) using Tukey’s multiple comparison tests at a level of significance set at P<0.05. Data analysis was performed using Minitab 18 Statistical Software (Minitab Inc., U.S.A.). 3. RESULTS AND DISCUSSION 3.1. Essential oil composition The volatile oil extracted from R. suaveolens flowers has yellow color with an agreeable intense smell. Its extraction yield was about 0.23%±0.02 (volume/dry weight), which was similar to that reported by BEN SALAH et al. (2019) (0.22%), and was slightly, higher than that obtained from the aerial parts of Algerian R. suaveolens (0.14%) (CHEMSA et al., 2016). As depicted in Table 1, thirty-one components have been identified in the RSEO which represent 98.4% of the total composition. This oil contains a complex mixture dominated by oxygenated monoterpenes (46%) followed by oxygenated sesquiterpenes (23.6%) and monoterpenes hydrocarbons (17.5%). The major components of the RSEO were identified as spathulenol (18.3%), carvacrol (12.1%), linalool (09.4%), α-terpineol (7.10%), α- terpinolene (6.3%) and pinocarvone (5.6%). Compared to previous studies, our findings differ from those reported by BEN SALAH et al. (2019), with α-pinene (25.84 %), β-pinene (17.57 %), 1-octen-3-ol (16.23 %), camphene (12.28 %), limonene (8.03 %) and β-myrcene (5.13 %) as major compounds. Also, the composition of the Algerian R. suaveolens essential oil showed a significant difference in the chemical composition (CHEMSA et al., 2016). Ital. J. Food Sci., vol. 32, 2020 - 989 Table 1. Chemical composition of the essential oil from the flowers of R. suaveolens analysed by GC-MS. aCompounds are listed in order of their elution from a HP-5MS column. b Experimental linear retention index on a HP-5MS capillary column using the homologous series of n-alkanes. c Linear retention index from literature. dPeak area of the essential oil components. eCompounds were identified based on their RI on HP- 5MS capillary column and GC-MS data. Values are given as mean± S.D. (n=3). *Values with different letters with in the same column indicate significant difference (p<0.05). No. Compoundsa RIexp b RIlit c % Area Identification methods 1 α-thujene 926 924 0.5±0.02 RI, MS 2 α-pipene 936 939 2.5±0.03 RI, MS 3 Camphene 955 956 1.8±0.01 RI, MS 4 β-pinene 981 979 1.9±0.01 RI, MS 5 β-myrcene 990 993 0.5±0.02 RI, MS 6 α-terpinene 1016 1017 2.4±0.04 RI, MS 7 Limonene 1030 1029 1.2±0.01 RI, MS 8 γ-Terpinene 1060 1059 0.4±0.01 RI, MS 9 α-terpinolene* 1088 1089 6.3±0.10d RI, MS 10 Linalool* 1095 1098 9.4±0.08c RI, MS 11 Trans-sabinol* 1140 1142 4.1±0.01e RI, MS 12 p-menth-4(8)-ene 1157 1160 1.6±0.02 RI, MS 13 Pinocarvone* 1160 1164 5.6±0.21de RI, MS 14 α-terpineol* 1190 1192 7.10±0.02d RI, MS 15 Trans-carveol 1220 1217 1.5±0.01 RI, MS 16 Carvone 1242 1250 0.5±0.01 RI, MS 17 Geraniol 1255 1252 1.5±0.03 RI, MS 18 α-Thujenol 1287 1290 2.6±0.01 RI, MS 19 Carvacrol* 1298 1299 12.1±0.21b RI, MS 20 Trans-caryophyllene 1415 1419 0.6±0.1 RI, MS 21 Aromadendrene 1437 1437 3.3±0.1 RI, MS 22 Alloaromadendrene 1463 1458 0.7±0.2 RI, MS 23 Eremophilene 1511 1512 2.4±0.1 RI, MS 24 ð-cadinene 1522 1523 0.1±0.2 RI, MS 25 α-calacorene 1541 1546 0.4±0.1 RI, MS 26 Spathulenol* 1577 1577 18.3±0.1a RI, MS 27 Caryophyllene oxide* 1581 1582 4.8±0.2de RI, MS 28 α-cadinol 1676 1652 0.5±0.01 RI, MS 29 Myristic acid 1762 1767 0.6±0.1 RI, MS 30 Palmitic acid methyl ester 1908 1909 0.3±0.1 RI, MS 31 Palmitic acid 1970 1968 2.9±0.02 RI, MS Monoterpene hydrocarbons 17.5 Oxygenated monoterpenes 46.0 Sesquiterpene hydrocarbons 7.50 Oxygenated sesquiterpenes 23.60 Others (%) 3.80 Total identified (%) 98.4 Ital. J. Food Sci., vol. 32, 2020 - 990 The presence of spathulenol, linalool and carvacrol mentioned in this work as major constituents, had never been, already, reported for the R. suaveolens species. The differences observed between our findings and those previsouly reported by BEN SALAH et al. (2019) and CHEMSA et al. (2016) can be attributed to the environmental, agronomic, age and geoclimatic factors (season, location, fertility regime, soil type and climate) as well as the experimental extraction conditions (BOUKHATEM et al., 2014; SINGH et al., 2012). 3.2. Antioxidant activity As depicted in Figs. 1, 2 and 3, the inhibition of the DPPH and ABTS radicals, the reducing power and the inhibition of lipid peroxidation activities of the RSEO, respectively, are dose dependent. Figure 1. DPPH•− and ABTS•+ free radical-scavenging properties of the essential oil of the R suaveolens flowers. Data were presented as means±SD (n=3). Figure 2. Reducing power of the essential oil of the R. suaveolens flowers. Ital. J. Food Sci., vol. 32, 2020 - 991 Figure 3. Antioxidant activity (%) of essential oil of the R. suaveolens flowers measured by β-carotene–linoleic acid method. Values expressed are means±S.D. (n=3) As can be seen in Table 2 the IC50 values obtained for both DPPH and ABTS assays (11.48±0.11 mg/mL, 18.58±0.39 mg/mL, respectively) are significantly (p<0.05) higher than those observed for the tested standard BHA (0.041±0.002 mg/mL, 0.032±0.004 mg/mL, respectively). In other words, the activity of the selected essential oil is lower than that of the BHA standard. Our results are in agreement with the findings of the RSEO isolated from Algeria, which exhibited a weak DPPH scavenging activity (CHEMSA et al., 2016). As depicted in Table 2, the ferric reducing power of the RSEO (IC50=58.95±1.21 mg/mL) was significantly (p<0.05) lower than that of the BHA standard (IC50=0.052±0.001 mg/mL). Table 2. Antioxidative capacities of the essential oil of the Rhanterium suaveolens flowers. IC50 DPPH (mg/mL) ABTS (mg/mL) Reducing power (mg/mL) Β-carotene/linoleic acid (µg/mL) Essential oil 11.48±0.11b 18.58±0.39b 58.95±1.21b 26.15±1.01b BHA 0.041±0.002a 0.032±0.004a 0.052±0.001a 5.95±0.82a BHA standard was used as a reference. All the values are means±SD (Standard Deviation) of three parallel measurements. Different letters in the same column indicate a significant difference (p<0.05). The inhibition of the lipid peroxidation activity of the tested essential oil was carried out using the β-carotene bleaching test. As shown in Table 2, the activity of the RSEO was lower (IC50=26.15±1.01 µg/mL) than that of the synthetic standard BHA (IC50=5.95±0.82 µg/mL). Also, this activity is less important than that previously reported for the essential oil of the Algerian R. suaveolens aerial parts with an IC50 of 17.97±5.40 µg/mL (CHEMSA et al., 2016). Compared to the radical scavenging effects and the reducing power, the RSEO is more active on the inhibition of the lipid peroxidation. This can be, probably, due to the Ital. J. Food Sci., vol. 32, 2020 - 992 high specificity of the test to lypophilic molecules (HARKAT-MADOURI et al., 2015). It has been reported that the lipid peroxidation activity may be due to the richness of the tested oil on conjugated sesquiterpenoids. Indeed, these compounds can scavenge the singlet oxygen and consequently, protect the β-carotene color against bleaching, indirectly (CHEMSA et al., 2016). The weak antioxidant activity observed for the RSEO can be related to its chemical composition as well as the abundance of ineffective compounds such as the monohydroxylated compounds which are unable to chelate ferrous ions (HARKAT- MADOURI et al., 2015; AIDI WANNES et al., 2010; DZAMI et al., 2013). The low antioxidant potential of the tested oil can, also, be attributed to the degradation of bioactive compounds during their extraction. Indeed, during hydrodistillation process, plant material is usually extracted in boiling water for a long period which could cause thermal decomposition of the thermolabile target molecules inducing, therefore, a decrease in the antioxidant capacity of the extract (BAGHERI et al., 2014). 3.3. Antibacterial activity The antibacterial activity was evaluated against six foodborne pathogens (3 Gram-positive and 3 Gram-negative), using the dilution and disk diffusion methods. As shown in Table 3, the RSEO was sensitive to all tested bacteria and exhibited a variable antibacterial activity dependent on the tested strains. S. aureus was the most susceptible bacteria with the largest inhibition zone (IZ=18.25±0.35 mm) followed by L. monocytogene (IZ =17.37±0.53 mm) and B. cereus (16.0±0.0 mm). However, the highest resistance to the RSEO was observed for the S. typhimurium with the lowest (p<0.05) inhibition zone (IZ=12.25±0.35 mm). Results showed that the tested essential oil was slightly more active against Gram-positive than Gram-negative bacteria. This can be explained by the complexity of their double membrane containing cell envelope, which can limit the diffusion of hydrophobic compounds through its lipopolysaccharide covering. Generally, the bacteriostatic and/or bactericide action of the plant extracts is attributed to their ability to disrupt cell membrane structures, disturb their permeability barrier and, consequently, to cause the chemiosmotic control loss (BAGAMBOULA et al., 2004). As depicted in Table 3, the MIC and MBC values of the RSEO ranged from 75 to 300 µg/mL for the tested bacterial strains. The highest antibacterial activity was observed against S. aureus with the lowest (p<0.05) MIC and MBC values (37.5 µg/mL and 75 µg/mL, respectively). There are a few reports on the antibacterial activity of the R. suaveolens essential oil for comparison. Recently, BEN SALAH et al. (2019), developed the antibacterial activity of Tunisian RSEO against a broad spectrum of bacterial strains. Our findings showed discrepancies between their published data. Larger inhibition zones values were reported against E. coli and P. aeruginosa (19 mm, 23 mm, respectively). The corresponding MICs were found 230µg/mL for E.coli and 46 µg/mL for P. aeruginosa (BEN SALAH et al., 2019). In addition, a moderate antibiofilm potential has been reported against six Gram positive bacteria for the essential oil collected from the aerial parts of Algerian R. suaveolens (CHEMSA et al., 2016). The differences between our findings and those prevesiouly reported by other authors, may result from different chemical compositions and percentage content of active constituents in the tested essential oils. Factors such as the choice of bacterial strains and their sensitivity, the experimental conditions and the choice of methods used for in vitro antibacterial activity could also be related to the variation in the experimental results (SIDDIQUE et al., 2017). Ital. J. Food Sci., vol. 32, 2020 - 993 Table 3. Antibacterial activity of essential oil of the R. suaveolens flowers using disc diffusion method and determination of MIC and MBC values. Bacterial strains IZ (mm±SD) Gentamicine MIC (µg/mL) MBC (µg/mL) Gram positive Bacteria S. aureus (ATCC 25923) 18.25±0.35a 34.50±0.71a 37.5 75 L. monocytogenes (ATCC19115) 17.37±0.53a 31.00±0.00b 75 150 B. cereus (ATCC14579) 16.00±0.00ab 24.00±0.00d 150 300 Gram negative Bacteria E. coli (ATCC35218) 15.62±0.17b 26.00±0.00c 75 150 S. typhimurium (NRLB4420) 12.25±0.35d 21.50±0.71e 150 300 P. aeruginosa (ATCC27853) 14.00±0.00c 32.50±0.71b 150 150 IZ: The diameter of the inhibition zones (mm), including the well diameter (6 mm), are given as mean±SD (n=3). Gentamicine: is used as positive control for bacteria. Different letters in the same column indicate a significant difference (p<0.05). The appreciable antibacterial potential of the RSEO against some bacterial strains could be related to the presence of a high amount of phytochemicals such as monoterpenes and oxygenated monoterpenes (AGGARWAL et al., 2002). Effectively, many studies have proved the presence of a relationship between the chemical composition of the major components of the essential oils and the antibacterial activity (BEL-HADJ et al., 2017). The major compounds identified in the RSEO such as spathulenol, carvacrol, linalool, α- terpineol, α-terpinonene and pinocarvone have not been tested for their antibacterial activity in the present study. However, some reports have approved their antibacterial properties. Indeed, a number of researchers have shown that carvacrol and linalool are well-known substances with pronounced antimicrobial activity against several pathogenic bacteria (BOZIN et al., 2006). Likewise, it was found that spathulenol and linalool exhibited moderate to strong activities against several microorganisms (MAGIATIS et al., 2002). In addition, it has been revealed that interactions between the constituents of some essential oils may contribute to different effects such as additive, synergistic, or antagonistic (DELAQUIS et al., 2002). A study conducted on the release of the cellular materials test, showed that α-terpineol/linalool combination treatments have shown a strong effect on the release of cell constituents both from Gram-negative and Gram- positive bacteria (ZENGIN and BAYSAL. 2014). 4. CONCLUSIONS This paper reports the chemical composition and the in vitro antioxidant and antibacterial properties of the essential oil collected from the R. suaveolens flowers. The GC–MS analysis revealed the identification of 31 constituents. Spathulenol, linalool and carvacrol identified as major compounds, were reported for the first time in the essential oil of this species. 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