J. Biol. Today's World. 2016 Aug; 5 (8): 150-156 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ Journal of Biology and Today's World Journal home page: http://journals.lexispublisher.com/jbtw Received: 25 March 2016 • Accepted: 01 September 2016 Research doi:10.15412/J.JBTW.01050803 Phytochemical and Antibacterial Properties of Echinophora Orientalis Essential Oil against Staphylococcus aureus in Soup Effat Farzanehnia1, Peyman Ghajarbeygi1, Razzagh Mahmoudi1*, Karim Mardani2 1 Department of Food Hygiene and Safety, School of Public Health, Qazvin University of Medical Sciences, Qazvin, Iran 2 Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran *Correspondence should be addressed to Razzagh Mahmoudi, Department of Food Hygiene and Safety, School of Public Health, Qazvin University of Medical Sciences, Qazvin, Iran; Tel: +989127868571; Fax: +989127868571; Email: [email protected]. ABSTRACT Using of chemical preservatives in food may have harmful effects on human health and reduce food safety; Natural preservatives can be used to improve food safety. Echinophora Orientalis is one of the medicinal herbs that traditionally has been used as natural preservative. The objective of the present investigation was to determine the chemical composition and antimicrobial effect of E. orientalis essential oil (EO) against Staphylococcus aureus in a food model. In order to preparing E. orientalis EO, the aerial parts of the plant were collected from Binalud mountain in Nishapur, East of Iran. The EO was extracted using Clevenger and its chemical composition was determined by Gas chromatography–mass spectrometry. Assessment of antibacterial activity of the EO was performed by inoculating the amount of 103 cfu/ml S. aureus into a certain amount of soup samples. Different concentrations of the EO (6.25, 12.5, 25 μg ml -1) added into the soup samples. The antimicrobial activity of different concentrations of the EO on S. aureus was examined in the commercial barley soup kept under fridge condition in 1, 2, 3, 4, 5 days after S. aureus inoculation. In total 43 components were identified in E. orientalis EO by GC-MS analysis, comprising 99.05% of the volatile oil, of which γ-decalactone (21.15%), β- cis-Ocimene (15.27%), Linalool L (8.82%), Spathulenol (7.74 %), Eugenol methyl ether (6.61%) were the major components. The EO showed strong antimicrobial activity against tested bacteria, so that no bacterial growth was observed in concentrations of 12.5 μg ml -1 and 25 μg ml -1 five days after bacterial inoculation, but bacterial growth was observed at concentrations of 6.25 μg ml -1. Average growth of bacteria in concentrations of 6.25, within five days counting were respectively 34 and 35 respectively 62.33±4.07, 42.66±3.02, 16±0.81, 1.33±0.65, 0 Cfu/ml (p<0.05). Evaluation of the sensory properties showed that concentration of 6.25 μg ml -1 of the EO was the most acceptable concentration. It was concluded that E. orientalis EO is a strong preservative and a flavoring agent in foods. Key words: Echinophora Orientalis, Essential oil, GC-MS, Staphylococcus aureus, Soup. Copyright © 2016 Effat Farzanehnia et al. This is an open access paper distributed under the Creative Commons Attribution License. Journal of Biology and Today's World is published by Lexis Publisher; Journal p-ISSN 2476-5376; Journal e-ISSN 2322-3308. 1. INTRODUCTION Two species including E. sibthorpiana and E.orientalis are he occurrence of adverse effects of chemical also growing in Anatolia, Turkmenistan, Armenia, Russia, antimicrobial preservatives and increasing the Syria, the Balkans, Crete, Cyprus and Afghanistan (4, 5). E. Tbacterial resistance to antibiotics and other orientalis is a common species in Iran. It is known by local antimicrobial agents has prompted researchers to carry out names of Khosharize, Tigh Touragh,Tigh Masti, extensive studies in order to reducing chemical Koshander, Kouzang, Tanghez or Khousharouze and preservitives and replace them by the natural chemical Kharmoshk. E. orientalis; this plant is an aromatic compounds, especially medicinal plants (1-3). The perennial herbaceous plant, tough and prickly, singular and Echinophora plant of the family Apiaceae (umbelliferae) bottom-branched stems, branches stitched with thick, includes 10 species that has been distributed from the sturdy and thick, tangled tentacles to name a few highly Mediterranean area to Iran and Afghanistan. Among ten branched with a height of 30 to 100 cm, white flowers species, four of them are found in Iran including E. integrated, since the flowering period is from June to July orientalis, E. sibthorpiana, E. cinerea and E. platyloba. (3, 6). The Echinophora EO contains alkaloid compounds and flavonoids. Flavonoids exist abundantly in various 150 J. Biol. Today's World. 2016 Aug; 5 (8): 150-156 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ vegetables, fruits, and medicinal plants. The plant and its manufacturers to supply their products, labeled "natural" or oil can be used as an antiseptic, antibacterial, antioxidant, "clean", which indicates the presence of natural antifungal and an ability to inhibit human platelet components in their products. Manufacturing of these aggregation and are also used in folk medicine to heal products has difficulties and obstacles, including high wounds, carminative and digestive properties (2, 7-9). levels of using, create undesirable color, flavor stability Aerial parts of the plant are used commonly as flavor and reduce the productivity which are due to the low levels compounds in dairy products and also used in the of natural anti-bacterial materials in them (13, 14). preparation “halva” a Turkish sweet, the sweat of these According to importance of natural preservatives and the plants create a flavor in home environment and is used as role of medicinal plants that are native to Iran, the present an anti-freeze compound (10, 11). Staphylococcal food study was conducted to assess the antibacterial properties poisoning (SFP) is one of the most common food-borne of the EO of E. orientalis against S.aureus in a soup as a diseases and caused by the ingestion of staphylococcal food model. enterotoxins (SEs) that is produced in food by enterotoxigenic strains of S.aureus. Food poisoning caused 2. MATERIALS AND METHODS by S.aureus is one of the most common disease and in 2.1. Plant most countries is primarily in terms of toxicity (12). The aerial parts of E. orientalis were collected during Preservatives are added to food, drugs, paints, flowering stage (10th June to 15th August 2015) from environmental samples for reducing the level of Binalud mountains in Nishapur in the Khorasan Razavi, deterioration and increasing their shelf life. Despite these east of Iran and identified by the Herbarium of University advantages, they are harmful and toxic, so, this encourages of Tabriz, Iran (Figure 1). researchers to find appropriate approaches for reducing the use of chemical preservatives and replacing them with natural preservatives. Natural preservatives allow food Figure 1. E. orientalis (Collected from Binalud Mountains in Nishapur in Iran) Preparation of the EO: According to the method confirmation of the results, EO was also analyzed by GC- recommended by the European Pharmacopeia, in order to MS (Agilent 6890 gas chromatograph equipped with an extracting E. orientalis EO; dried aerial parts (100 grams) Agilent 5973 mass-selective detector; Agilent, U.K.) and was milled and distilled using a Clevenger apparatus for 3 the same capillary column and analytical conditions as hours (2),(4). The obtained EO was dewatered using above. The MS was run in electron ionization mode with sodium thiosulphate and then kept at 4°C until it was used ionization energy of 70 eV (2). in the experiment. GC-MS Analysis of the EO: Chemical composition of the extracted EO was measured and 2.2. Antimicrobial activity assay in soup quantified using GC and GC-MS (15). The chromatograph 2.2.1. Bacterial strains (Agilent 6890; Agilent Technologies, Kansas) was The antimicrobial activity of extracted EO was examined equipped with an HP-5MS capillary column (30×0.25 mm against S. aureus ATCC 6538. Lyophilized culture of the ID ×0.25 mm film thickness) and the data were taken organism was obtained from Iranian Research under the following conditions: initial temperature 5°C, Organization for Science and Technology, Tehran, Iran. temperature ramp 5C/min, 240C/min to 300C (holding for Preparation of bacterial suspensions: Concentrations of 3 min) and injector temperature at 290C. The carrier gas bacteria were determined using spectrophotometer was helium and the split ratio was 0.8 mL/ min. For (Pharmacia LKB-Nova Spacell, Cambridge, UK). 151 J. Biol. Today's World. 2016 Aug; 5 (8): 150-156 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ Suspensions of vegetative form of the bacteria were considered significant when P<0.05. prepared by culturing in BHI broth at 37°C for 24 h. The optical densities (OD) of bacteria cultures were measured 3. RESULTS AND DISCUSSION 3 at 600 nm to obtain a bacterial concentration of 10 Cfu/ml In the present study, the antibacterial property