Research Article Received: 26 March 2009 Revised: 14 August 2009 Accepted: 19 August 2009 Published online in Wiley Interscience: 16 November 2009 (www.interscience.wiley.com) DOI 10.1002/jsfa.3788 The influence of harvest time on essential oil composition, phenolic constituents and antioxidant properties of Turkish oregano (Origanum onites L.) Gulcan Ozkan,a∗ Hasan Baydarb and Sabri Erbasb Abstract BACKGROUND: The aim of this research was to determine essential oil composition, phenolic constituents and antioxidant properties of Turkish oregano (Origanum onites L.) leaves harvested during the months of June to September. RESULT: The maximum essential oil yield in the leaves appeared in the middle of July. The main components of oregano oil were carvacrol, thymol, γ -terpinene, p-cymene, α-terpinene and α-pinene. Carvacrol was highest in the July harvest. The maximum extract yield was found in September. Oil distilled from early-season (June) harvested leaves had the highest antioxidant ability, expressed as low concentration providing 50% inhibition of free radical scavenging activity and high levels of reducing/antioxidant capacity. Twelve phenolic compounds of oregano extract were identified and the main components were found to be rosmarinic acid and acecetin. The maximum rosmarinic acid and acecetin were found in harvests of July and June, respectively. Total phenolic contents, free radical scavenging activities and reducing/antioxidant capacities were found to be highest in the July harvest. DISCUSSION: All yields, chemical compositions, free radical scavenging activities and reducing/antioxidant capacities of extracts and essential oils of Turkish oregano changed importantly depending on vegetative periods of growing season. c 2009 Society of Chemical Industry Keywords: Origanum onites; essential oil; phenolics; antioxidant properties; seasonal variation INTRODUCTION in variable amounts depending on different times of day and Turkey is regarded as an important gene center for the family vegetative period. It was reported that 1.9–3.0% of essential oil Lamiaceae (Labiatae). The genus Origanum is represented in was obtained from above-ground parts through steam distillation Turkey by 22 species or 32 taxa, 21 being endemic to Turkey.1 The of O. onites.12,13 The major essential oil constituent of Turkish rateofendemismamongtheTurkishOriganumspeciesisover60%, oregano is carvacrol.14 Oregano essential oils and extracts include and this high rate is suggestive that the gene centre of Origanum phenolic substances such as carvacrol and rosmarinic acid, which 15–18 is Turkey.1–4 Turkey is the leading country in the oregano trade were reported their antioxidant and antibacterial activities. in the world and exportation has reached over 8000 tonnes and Although limited reports were found on variations of essential 13,19 US $15 million in recent years.5 However, the overharvesting of oil composition during the vegetative cycle, studies on the oregano from nature has resulted in the threat of extinction of influence of harvest time on phenolic constituents and antioxidant Origanum species in recent years.6 For conservation of natural properties were lacking. sources and production with high standard and quality, oregano This research aimed to determine the effect of harvest time cultivation has been started and has expanded rapidly in recent on the yield, chemical composition and antioxidant properties years. Oregano is used mainly in the production of essential oils, of extracts and essential oils of oregano (Origanum onites L.) spices, herbal teas, folk drugs and beverages in Turkey. harvested during vegetative periods of growing season. In the essential oil plants, the content of the essential oil is mostly variable and many factors such as drying conditions, distillation techniques, geographic and climatic factors such as duration of ∗ Correspondence to: Gulcan Ozkan, Department of Food Engineering, daylight, temperature, water stress and plant growth phase play Suleyman¨ Demirel University, 32260 Isparta, Turkey. an important role in yield and composition of essential oils.7–11 E-mail: [email protected] Productivity and ontogenetic variability of O. onites have been a Department of Food Engineering, Suleyman¨ Demirel University, 32260 Isparta, studied and it has been noted that an ontogenetic variability Turkey 12 exists in the amount of essential oil yield and composition. The 205 same report reveals that secondary metabolites in the plant exist b Department of Field Crops, Suleyman¨ Demirel University, 32260 Isparta, Turkey J Sci Food Agric 2010; 90: 205–209 www.soci.org c 2009 Society of Chemical Industry www.soci.org G Ozkan, H Baydar, S Erbas MATERIALS AND METHODS Table 1. Solvent gradient conditions with linear gradient Plant material Oregano (Origanum onites L.) seeds used in this study were Final time 3 20 28 35 45 60 62 70 75 80 obtained from the Aegean Agricultural Research Institute (Izmir, Turkey). The seeds were sown in a greenhouse, and seedlings were A% 95 75 72 70 65 63 55 50 20 0 developed in small perforated plastic tubes, and then transplanted B% 52528303537455080100 into an experimental field in rows spaced 50 × 25 cm in plots A (solvent): acetic acid–water (2 : 98 v/v); B (solvent): methanol. in a randomized block design with three replications, at the Experimental Station of Suleyman Demirel University in Isparta in ◦ the Mediterranean region of Turkey (latitude 37 45 N, longitude was used: solvent A consisted of acetic acid–water (2 : 98, v/v); ◦ 2 30 33 E, altitude 997 m). Each plot in the blocks was 12.5 m in size solvent B was methanol and the gradient program used is given and consisted of five rows. There was no fertilizer application, and in Table 1. The data were integrated and analyzed using the the plants were watered with a drip irrigation system during the Shimadzu Class-VP Chromatography Laboratory Automated drought seasons. The oregano plants were harvested four times in Software System (Tokyo, Japan). Extract samples, standard approximately the middle of the months from June to September. solutions and mobile phases were filtered using a 0.45 µmpore Plants were cut at a height of 10 cm above soil level and dried in a size membrane filter (Vivascience AG, Hannover, Germany). The shaded area. amount of phenolic compounds in the extract was calculated as g kg−1 herb using external calibration curves, constructed for Isolation of essential oil each pure phenolic standard. All determinations were carried out Dried and powdered herb material (200 g each) was distilled in triplicate and the results were averaged. for 3 h using Clevenger-type apparatus. The essential oils were dried over anhydrous sodium sulfate and, after filtration, stored Determination of total phenolics, free radical scavenging ◦ at −20 Cuntiluse. activity and reducing/antioxidant capacity Total phenolics were determined according to the method 21 Preparation of extract adapted by the Folin–Ciocalteu colorimetric method. Estima- Dried and powdered herb material (15 g) was extracted with tions were carried out in triplicate and calculated from a calibration curve obtained with gallic acid, and total phenolics were expressed a 100 mL mixture of methanol–acetone–water–acetic acid − ◦ 1 (55:40:4.5:0.5) for 2h at 40 C using an ultrasonicated water as gallic acid equivalent (mg GAE g extract). Free radical scavenging activity was measured by the DPPH bath. The extracts were filtered and the solvent mixtures 22 were concentrated using a rotary evaporator (Rotavator, BUCHI¨ method of Lee et al. and calculated according to the following ◦ = × Labortechnik AG, Flawil, Switzerland; T < 40 C) under vacuum formula: free radical scavenging activity 100 (absorbance of and lyophilizers (VirTis 2K, Gradiner, NY, USA; T =−60) to obtain control sample – absorbance of sample/absorbance of control ◦ crude extracts. The residue was stored at −20 Cuntiluse. sample). Extract concentration providing 50% inhibition (IC50)was calculated from the plot of inhibition percentage against extract concentration. Analysis of essential oil components Thereducing/antioxidantcapacityoftheextractandessentialoil Analyses of the essential oil components were performed on was evaluated by the formation of phosphomolybdenum complex a gas chromatograph–mas spectrometer/quadropole detector, according to the method of Prieto et al.23 The antioxidant activity using a Shimadzu QP 5050 system (Kyoto, Japan), fitted with a was expressed relative to that of ascorbic acid. All determinations polyethylene glycol+2nitroterephthalate (FFAP) (50 m × 0.32 mm were carried out in triplicate and the results were averaged. (i.d.), film thickness: 0.25 µm) capillary column. Detector and ◦ injector temperatures were set at 240 C. The temperature ◦ ◦ Statistical analysis program for the FFAP column was from 60 C (1 min) to 220 Cata ◦ ◦ Results of the research were tested for statistical significance rate of 5 Cmin−1 and than held at 220 C for 35 min. Helium by Duncan’s multiple range test. Differences were considered was used as a carrier gas at a flow 14 psi. (Split 1 : 20) and statistically significant at the P < 0.05 level. The analysis was injection volume of each sample was 5 µL. The identification of the performed in triplicate. components was based on comparison of their mass spectra with those of Wiley and Nist, Tutore Libraries. The ionization energy was set at 70 eV. RESULTS AND DISCUSSION In this study, the essential oil and extract yield of Turkish Analysis of phenolic constituents oregano varied from 25.00 to 32.00 g kg−1 and from 129.10 to The procedure for quantitation of the phenolic compounds has 201.70 g kg−1, respectively, according to month (Table 2). While previously been described by Capanio et al.20 Reversed phase the lowest value was obtained in June, at the pre-flowering stage, high-performance liquid chromatography (RP-HPLC) was used. the highest essential oil yield was obtained in July, at the end of Detection and quantification was carried out with a SCL-10 Avp the flowering stage and beginning of the fruit-set stage. Similar System controller (Shimadzu, Japan), SIL-10AD vp Autosampler, results were reported for O.