Evaluation of Chemical Composition and Biological Activities of Essential Oil and Methanolic Extract of Origanum vulgare L ssp glandulosum Desf Ietswaart from
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 mgGAE/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 mg/ml whereas EO presented an IC50= 461.62mg/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 'zaater" 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 g terpinene and r 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 4degC 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 40deg 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 mm, Agilent Technologies, USA) and fitted to FID (Flame Ionisation Detector) The injector and detector were operated at 280 and 300degC, respectively. Oven temperature program was 5 min isothermal at 50degC then raised to 300degC at a heating rate of 5degC/minute, and finally isothermally held for 5 minutes. As a carrier gas, Hydrogen at 1.0ml/min was used. 1mL 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 mm, Agilent Technologies, USA) The injector and detector were operated at 280 and 300degC, respectively. Oven temperature was programmed (50degC for 3.2 minutes, then 50 to 300degC at 8degC/minute and subsequently, held isothermally for 5 minutes) As a carrier gas, Helium with a flow rate of 1.0ml/minute was used. 1mL 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, 230degC; 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 37degC for 18 24 hours. The