Evaluation of Chemical Composition and Biological Activities of Essential Oil and Methanolic Extract of Origanum vulgare L. ssp. glandulosum (Desf.) Ietswaart from Algeria

The objective of this study was to evaluate the chemical composition of O. vulgare L. ssp. glandulosum (Desf.) Ietswaart essential oil (EO), to determine total phenolic constituent in methanolic extract and investigate the antibacterial and antioxidant activities of essential oil and methanolic extract. For antibacterial activity, susceptibility tests were expressed as inhibition zone by the disc diffusion method and minimal inhibitory concentration (MIC) by broth microdillution method. Antioxidant activity was evaluated using DPPH scavaging capacity assay. GC and GC/MS analyses of the oil resulted in the identification of 43 compounds, representing 98.55% of the oil; para-cymene (25.615%), thymol (23.129%), carvacrol (20.321%) were the main components. The methanolic extract showed total phenolic contents of 526.75 μgGAE/mg. EO was particularly found to possess strong antibacterial capacity while methanolic extract remained inactive. In antioxidant activity, methanolic extract exhibited very high scavenging ability on DPPH radicals with IC50= 25.59 μg/ml whereas EO presented an IC50= 461.62μg/ml. INTRODUCTION The essential oils and various extracts of plants have attracted considerable academic attention as well as industrial interest due to their various properties, particularly, the antimicrobial and antioxidant activities. The plant oils and extracts have formed the basis of many applications because of the resistance that pathogenic microorganisms have built against antibiotics and the increasing popular concern about the safety of food and the potential toxicities of the synthetic antioxidants on health. There are several food plants (spices in particular) with a significant volatile organic compounds content (e.g Levisticum officinale, Origanum vulgare, O. majorana, Cinnamomum ssp., Zingiber ssp., Citrus ssp., Elettaria cardamomum, Foeniculum vulgare, Salvia officinalis, Syzygium aromaticum, Pimpinella anisum and Ocinum ssp., to name a few examples). Essential oil composition may vary considerably between plant species and varieties, and, within the same variety, from different geographical origin influenced by soil and environmental conditions. Essential oils are usually characterized by two or three major components at quite high concentrations (up to 80%) compared to other components present only in trace amounts. The essential oils and some of their components can often be used in different forms for human consumption. It was observed that potato starch or sunflower oil concentrations of above 5%, reduced the efficacy of oregano and thyme essential oils on Listeria monocytogenes. Their use in foods as preservatives is limited because of flavor considerations. Hence, for the study of biodiversified application, a complete investigation including the chemical composition, efficacy as antibacterial and/or antioxidant agents of an essential oil and/or extract was considered important. Origanum vulgare L. (oregano), without focusing on specific subspecies, is the most widespread and known species of Lamiaceae family. It is an important aromatic plant widely used in many countries for seasoning foods. Origanum vulgare ssp. glandulosum (Desf.) Ietswaart, synonymous O. glandulosum Desf., is an endemic spontaneous plant, growing in North Africa (Algeria and Tunisia). In Algeria, O. glandulosum Desf. is an aromatic shrub called ‘‘zaâter” which is mostly used as a medicinal plant against whooping cough, cough, fever and bronchitis. Carvacrol and thymol, the two main phenols that constitute about 78–82% of the essential oil of oregano, are principally responsible of its antibacterial and antioxidant activities . In addition, other minor constituents such as γ-terpinene and ρ-cymene, two monoterpenes hydrocarbons that constitute about 5 and 7% of the total oil, respectively, also contribute to these activities . In recent years, the essential oil of Origanum species have intensively been investigated for their chemical composition and potential use as natural antimicrobial and antioxidant agents all over the world: from France, Austria, Southern Italy, China and Pakistan, Bosnia, Bulgaria, Lithuania, Tunisia and different Mediterranean populations. As far as our literature survey could ascertain chemical composition and antibacterial activities of O. vulgare from Bouhaddouda et al. / Evaluation of Chemical... IJPPR, Volume 8, Issue 1 : January 2016 Page 105 east of Algeria have been previously published. Amrouni et al. have studied antibacterial activity on nosocomial bacteria : Pseudomonas aeruginosa, and reference strains: Staphylococcus aureus and Escherichia coli, but no information is available on the action on other microorganisms, antioxidative nature of this plant, and its potential use as natural foods preservative. The chemical composition of Algerian O. glandulosum Desf. essential oil have been studied 24, 25, 26, 27, 28 and all reports have identified thymol and carvacrol as the main components. Antimicrobial and antioxidant activity of the essential oils have been proved 25, . However, there is no report on the biological activities of methanolic extract of O. glandulosum Desf. Therefore, due to high utilization of the endemic O. glandulosum Desf. by local population to treat many diseases we were interested in this plant. For this reason, the aims of the present study was: to analyze the chemical composition of a hydrodistilled essential oil of O. glandulosum Desf collected from Guelma city in east Algeria by a GC/FID and GC/MS system in order to determine the essential oil chemotype and compare it with oregano essential oil from other localities of Algeria and world region; to determine total phenolic constituent in methanol extract and investigate the antimicrobial and antioxidant activities of both essential oil and methanol extracts from O. glandulosum Desf. MATERIALS AND METHODS Plant material O. glandulosum Desf. plants at flowering stage were collected from Nechmaya region of Guelma city (northeastern part of Algeria) in June 2012. The aerial parts (leaves and flowers) were dried in the shade at room temperature. Preparation of the extracts Isolation of the essential oil The air-dried and ground flowering parts of the plants were submitted for 2 hours to water-distillation, using a Clevenger-type apparatus. The essential oil was collected, dried over anhydrous sodium sulphate and stored at 4°C until tested and analyzed. Yield based on dried weight of the sample was calculated. Preparation of the methanol extracts (MeOH) The air-dried and finely ground samples were extracted with methanol (99.7%) by using a Soxhlet apparatus for about 6 hours. The methanolic extracts were filtered using Whatman filter paper (No. 1) and then concentrated in vacuo at 40° C using a rotary evaporator. The residues obtained were stored in a freezer until further tests. GC and GC-MS analysis GC/FID The GC-FID analysis was performed on a HewlettPackard 6890 gas chromatograph equipped with a DB5 MS column (30 m X 0.25 mm, 0.25 μm, Agilent Technologies, USA) and fitted to FID (Flame Ionisation Detector). The injector and detector were operated at 280 and 300°C, respectively. Oven temperature program was 5 min isothermal at 50°C then raised to 300°C at a heating rate of 5°C/minute, and finally isothermally held for 5 minutes. As a carrier gas, Hydrogen at 1.0ml/min was used. 1μL of the essential oil diluted in hexane (1/30) was injected in a split mode in the ratio of 1: 60. The percentage of composition of the essential oil was calculated by electronic integration of FID peak areas. GC/MS The analyses of the volatile constituents were run on a Hewlett-Packard GC-MS system: Gas Chromatograph Model 7890 coupled to a 5975 Mass Selective Detector; and equipped with a DB5 MS column (20 m X 0.18 mm, 0.18 μm, Agilent Technologies, USA). The injector and detector were operated at 280 and 300°C, respectively. Oven temperature was programmed (50°C for 3.2 minutes, then 50 to 300°C at 8°C/minute and subsequently, held isothermally for 5 minutes). As a carrier gas, Helium with a flow rate of 1.0ml/minute was used. 1μL of the essential oil diluted in hexane (1/30) was injected in a split mode in the ratio of 1:250. The MS working in electron impact mode at 70 eV; and anionization energy of 1800 V; ion source temperature, 230°C; mass spectra data were acquired in the scan mode in m/z range 33-550. The identification of components was based on comparison of retention time of each component (Rt) and their mass spectra with those of Wiley 275 mass spectra and NIST (National Institute of Standards and Technology) libraries and those described by Adams. Also, a homemade MS library with the spectra corresponding to pure substances and components of known essential oils was used. Antimicrobial activity Microbial strains The essential oil and extracts were individually tested against a panel of microorganisms, including: five Laboratory reference strains obtained from the American Type Culture Collection: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Klebsiella pneumoniae ATCC 700603, Pseudomonas aeruginosa ATCC 27853; nine clinical isolates: Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Serratia marcescens, Proteus vulgaris, Proteus mirabilis, Salmonella thyphimurium, Acinetobacter baumanii, Pseudomonas aeruginosa and one foodborne pathogen Klebsiella oxytoca. Antimicrobial screening Disc diffusion method The dried plant extracts were dissolved in the same solvent (methanol) to a final concentration of 30 mg/ml. The agar disc diffusion method was employed for the determination of antimicrobial activities of the essential oil and methanolic extract according to Vuddhakul et al.with slight modification. For the experiments, a loopful of the bacterial working stocks were enriched on a tube containing 9 ml of Mueller-Hinton broth, then incubated at 37°C for 18–24 hours. The overnight cultures were used for the antibacterial activity of the essential oil and extract used in this study and the optical density was adjusted at 0.5McFarland turbidity standards. The inocula of the respective bacteria were streaked onto MHI agar plates Bouhaddouda et al. / Evaluation of Chemical... IJPPR, Volume 8, Issue 1 : January 2016 Page 106 using a sterile swab. After inoculum absorption by agar, sterile filter discs (diameter 6 mm, Whatman paper N°1) were impregnated with 5μl of essential oil or the 30 mg/ml extracts (150 μg/disc) and placed on the inoculated agar, using forceps dipped in ethanol and flamed, as described previously by Gulluce et al.. Standard disc of Gentamycin (10 μg/disc) and blank discs (impregnated with methanol) were used as positive and negative controls, respectively. All Petri dishes were sealed with sterile laboratory parafilm to avoid eventual evaporation of the essential oils. And kept at 4°C for 2 hours, and then incubated at 37°C for 24 hours. After the incubation period, the mean diameter of inhibition halo where test microorganism did not grow (clearly visible inhibition zone) was measured in millimeters and recorded as the mean standard deviation (SD). All tests were performed in triplicate Determination of Minimum Inhibitory Concentrations (MIC) and Minimum Bactericidal Concentrations (MBC) The minimal inhibition concentration (MIC) values were studied for the bacterial strains which were sensitive to the essential oil and/or extracts in disc diffusion assay. The inocula of the bacterial strains were prepared from 12 hours broth cultures and suspensions were adjusted to 0.5 McFarland standard turbidity. Essential oil, which was dissolved in 10% dimethylsulfoxide (DMSO), was first diluted to the highest concentration (50 mg/ml) to be tested, and then serial two-fold dilutions were made in a concentration range from 0.048 to 50 mg/ml in 5 ml sterile test tubes containing nutrient broth. MIC values of O. vulgare essential oil against bacterial strains were determined based on a microwell dilution method. The 96-well plates were prepared by dispensing into each well 95 μl of nutrient broth and 5 μl of the inoculum. A 100 μl aliquot from the stock solutions of O. vulgare essential oil initially prepared at the concentration of 50 mg/ml was added into the first wells. Then, 100 μl from the serial dilutions were transferred into ten consecutive wells. The last well containing 195μl of nutrient broth without essential oil and 5 μl of the inoculums on each strip was used as negative control. The final volume in each well was 200 μl. The plates was covered with a sterile laboratory parafilm and then incubated at 37°C for 18–24 hours. The bacterial growth was indicated by the presence of a white “pellet” on the well bottom. As an indicator of microorganism growth, 10 μl of 2 mg/ml nitroblue tetrazolium (NBT) dissolved in water were added to the wells and incubated at 37 °C for 30 min. The nitroblue tetrazolium (NBT) salt acts as an electron acceptor and the yellow colored NBT is reduced to a purple-blue formazan product by biologically active organisms (Viable bacteria). The MIC was defined as the lowest concentration essential oil to inhibit the growth of the microorganisms. The MBC values were interpreted as the highest dilution (lowest concentration) of the sample, which showed clear fluid with no development of turbidity and without visible growth. All tests were performed in duplicate. Antioxidant activity Determination of total phenolic contents Total phenolic constituents of the methanolic extracts of O. glandulosum Desf. were determined by the literature methods involving the Folin–Ciocalteu reagent and gallic acid as standard. 0.1 ml of extract solution, containing 1000 μg extract, was taken in a volumetric flask, 46 ml distilled water and 1 ml Folin-Ciocalteu reagent were added, and flask was shaken thoroughly. After 3 minutes, 3 ml of a solution of 2% Na2CO3 were added and the mixture was allowed to stand for 2 hours with intermittent shaking. Absorbance was measured at 760 nm. The same procedure was repeated for all standard gallic acid solutions. The concentration of total phenolic compounds in the methanolic extract was determined as μg of gallic acid/mg dry plant material by using the regression equation that was obtained from the calibration curve of the gallic acid standard. All tests were carried out in triplicate, and gallic acid equivalent values were reported as X ± SD of triplicates. DPPH radicals scavenging capacity assay The ability of the plant essential oil and extract to scavenge diphenylpicrylhydrazyl (DPPH) free radicals was assessed using the method described by Takao et al., along with antioxidant activity index (AAI). The stock solution of the plant essential oil and extract was prepared in methanol to achieve the concentration of 2000 μg/ml. Further, two-fold dilutions were made to obtain concentrations from 1000 μg/ml to 7.81 μg/ml. Diluted solutions of extract (2 ml each) were mixed with 2 ml of DPPH methanolic solution (80 μg/ml). After 30 minutes in darkness at room temperature, the absorbance was read in a spectrophotometer at 517 nm. The control samples consisted of 2 ml of methanol added to 2 ml of DPPH solution. Ascorbic acid was used as a positive control. The experiment was performed in triplicate. Scavenging activity is expressed as the inhibition percentage calculated using the following equation: Scavenging activity (%) = 100 × [(A control A sample) / A control] Where A control is the absorbance of the control and A sample is the absorbance of the extract. The IC50 value is the effective concentration at which 50% of DPPH radicals were scavenged. It was obtained from the graph of scavenging activity (%) versus concentration of samples. Low IC50 value indicates strong ability of the extract to act as DPPH scavenger. The antioxidant activity was expressed as the antioxidant activity index (AAI), calculated using the following equation: AAI = final concentration of DPPH (μg/ml) / IC50 (μg//ml) The estimation of AAI was: if AAI < 0.5 → poor antioxidant activity; AAI > 0.5-1 → moderate antioxidant activity; AAI > 1-2 → strong antioxidant activity and AAI > 2 →very strong antioxidant activity. DPPH assay on TLC This procedure was applied for extracts and the essential oil of O. glandulosum Desf. following Tepe et al.. Five microlitre of a 1:10 dilution of the extracts in methanol were applied to the TLC plate and methanol – ethyl acetate (1:1) mixture was used as developer. The plate was sprayed with a 0.2% DPPH reagent in methanol and left at Bouhaddouda et al. / Evaluation of Chemical... IJPPR, Volume 8, Issue 1 : January 2016 Page 107 room temperature for 30 minutes. Yellow spots formed from bleaching of purple colour of DPPH reagent, were evaluated as positive antioxidant activity. RESULTS AND DISCUSSION Chemical composition of the essential oil Yield of the essential oil obtained by hydrodistillation from the aerial part of O. vulgare was 2.52 (w/w). As shown in Table 1, GC/MS analysis resulted in the identification of forty three compounds representing 98.546% of the oil. The major constituents of the oil were para-cymene (25.615%), thymol (23.129%) and carvacrol (20.321%). Gamma-terpinene (16.612%) and alpha-terpinene (1.787%) were also present at significant concentrations. Amrouni et al. reported a different composition for this essential oil which showed a carvacrol chemotype with 33.85% carvacrol, 23.64% thymol and 20.85% paracymene. Table 1: Chemical composition of O. glandulosum Desf. essential oil. RT Compounds % FID KI 1 5,78 Alpha-Thujène 0,895 924 2 5,92 Alpha-Pinène 0,716 932 3 6,25 Camphène 0,12 946 4 6,71 Sabinène 0,018 696 5 6,8 Béta-Pinène 0,154 974 6 6,87 Octène-3-ol 0,52 974 7 6,97 Octanone-3 0,161 979 8 7,04 Myrcène 1,494 988 9 7,35 Alpha-Phellandrène 0,188 1002 10 7,38 Delta-3-Carène 0,072 1008 11 7,55 Alpha-Terpinène 1,787 1014 12 7,72 Para-Cyméne 25,615 1020 13 7,77 Limonène 0,403 1024 14 7,8 Béta-Phéllandrène 0,207 1025 15 7,83 Eucalyptol 0,05 1031 16 7,89 (Z)-Béta-Ocimène 0,074 1032 17 8,08 (E)-Béta-Ocimène 0,06 1044 18 8,31 Gamma-Terpinène 16,612 1054 19 8,5 Cis-Hydrate de Sabinène 0,197 1065 20 8,74 Terpinolène 0,065 1086 21 8,83 Para-Cyménène 0,066 1089 22 8,99 Linalol 0,87 1095 23 9,05 Trans-Hydrate de Sabinène 0,12 1098 24 9,78 Camphre 0,012 1141 25 10,17 Bornéol 0,214 1165 26 10,3 Terpinène-4-ol 0,311 1174 27 10,39 Para-Cymène-8-ol 0,216 1179 28 10,53 Alpha-Terpinéol 0,386 1186 29 10,99 Thymol méthyl-Ether 0,051 1232 30 11,12 Carvacrol méthyl-Ether 0,18 1241 31 11,71 Isomère Thymol MW 150 0,163 1258 32 11,87 Thymol 23,129 1289 33 11,99 Carvacrol 20,321 1298 34 13,58 Béta-Caryophyllène 0,848 1417 35 13,75 Thymohydroquinone 0,487 1553 36 14,02 Alpha-Humulène 0,047 1452 37 14,62 Béta-Bisabolène 0,283 1505 38 14,7 Gamma-Cadinène 0,018 1513 39 14,75 Delta-Cadinène 0,025 1522 40 14,81 Béta-Sesquiphellandrène 0,539 1521 41 14,99 (E)-Alpha-Bisabolène 0,422 1529 42 15,46 Spathulénol 0,02 1577 43 15,54 Oxyde de Caryophyllène 0,41 1582 Total 98,546 Notes : a Retention time (as minutes). b Compounds listed in order of their elution c Kovats Index Bouhaddouda et al. / Evaluation of Chemical... IJPPR, Volume 8, Issue 1 : January 2016 Page 108 Except Egyptian Origanum vulgare essential oil with an important concentration of Pulegone, most essential oils from southern Mediterranean presented four main components with different percentages : carvacrol, thymol, ρ-cymene and γ-terpinene 23, . Algerian O. glandulosum Desf. From Jijel, Constantine, Setif 25,26 and Tlemcen presented a low ρ-cymene content compared to our essential oil (7.5%, 6.6%, 14.6%, 7.9% and 17,1% respectively). And all showed a thymol and/or carvacrol chemotype. However it is important to note that the compositions of these essential oils obtained by hydrodistillation may vary from one region to another. This variability concerns particularly carvacrol for which food manufacturers have a particular interest. To the best of our knowledge, there are many reports on the chemical composition of the essential oil isolated from different O. vulgare subspecies from different regions. Most of them indicate the presence of two main chemotypes of this essential oil, one contains as major components the phenols thymol and/or carvacrol 40 and other consists mainly monoterpene alcohols . It is known that O. vulgare species presents great variability in its essential oil composition due to the existence of different subspecies, but also to a numerous of parameters where mainly are the local climatic, seasonal, geological and geographical factors. However, the chemical composition of the studied essential oil differ completely with those previously reported on the literature and displayed a different specific oil chemical profile with para-cymene, thymol and carvacrol as dominant components. Antimicrobial activity The antimicrobial activities of O. glandulosum Desf. essential oil and methanolic extract against microorganisms were studied in the present work and their potency was qualitatively and quantitatively assessed by the presence or absence of inhibition zones, zone diameter, and MIC values. The results are given in Table 2. According to the results given in Table 2, O. glandulosum Desf. essential oil displayed a great in vitro potential of antimicrobial activitiy, however the methanolic extract showed no antimicrobial activity. In disc diffusion assay and Micro-well dilution assay, Gram negative strains were extremely sensitive to the 5 μl of essential oil with IZ between 43.66 ± 1.44 (mm) and 59 ± 2.64 (mm), also MIC values ranged from 0.78 (mg/ml) to 1.56 (mg/ml) ; Gram positif strains have also been extremely sensitive: Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC25923 showed a IZ at 51.83 ± 2.46 and 51.83 ± 2.56 respectively, MIC/MIB values were at 0.78 mg/ml. Staphylococcus aureus have been the most sensitive strain with IZ = 63.33 ± 3.05. Table 2: Antimicrobial activities of Origanum vulgare L. essential oil and methanolic extract against the bacterial strains