Effect of Different Salinity Levels on Essential Oil Content and Composition of Dracocephalum moldavica.

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1 AGRICULTURAL COMMUNICATIONS, 2014, 2(2): Effect of Different Salinity Levels on Essential Oil Content and Composition of Dracocephalum moldavica. SHIMA ALAEI 1, MORTEZA KHOSH-KHUI 2, SOHEIL KOBRAEE 1 AND BITA ZAJI 1 1Department of Agronomy and Plant Breeding, College of Agriculture, Kermanshah Branch, Islamic Azad University, Kermanshah, Iran. 2 Department of Horticultural Science, College of Agriculture, Shiraz University, Shiraz, Iran. *Corresponding Author: shima1354al@yahoo.com. (Accepted: 12 Feb. 2014) ABSTRACT In order to study the effect of salinity on quantity and quality of essential oil of moldavian balm, a pot experiment was carried out in a completely randomized design with 5 treatments and 4 replications in controlled environment of green house. The treatments were different levels of salinity including 0, 10, 25, 40 and 50 mm applied by NaCl. Clevenger method was used to extract the essential oils from the foliage of the treated plants. Results indicated that by increment in salinity levels, essential oil content increased but did not have a significant effect. The highest essential oil content (0.32%) was extracted from 50 mm of salinity treatment. Essential oil components were analyzed and identified by GC and GC/MS machines. The results indicated that in different levels of salinity stress, different components were produced by Dracocephalum moldavica. The major constituents of D. moldavica oil were identified as: geranyl acetate, geraniol, geranial, neral, methyl citronellate and neryl acetate. The highest amount of the main essential oil components were geranyl acetate (44.5%) in 10 mm NaCl and geraniol in control treatments. For both geranial and neral, the highest amount was observed in 25 mm NaCl treatment. Keywords: Dragonhead, NaCl, oil composition, essence, geranyl acetate. Abbreviations: GC: Gas Chromatography; GC-MS: Gas chromatography mass spectrometry. INTRODUCTION Dragonhead (Dracocephalum moldavicum) from Lamiaceae family is one of the major medicinal and aromatic plants that its essential oil is used extensively in medicinal, food, cosmetic and health industrials. Dracocephalum moldavica is an aromatic and medicinal plant which grows in high and mountainous parts of Iran. It is a considerable medicinal plant due to its high amount of essential oil. Its essential oil have antiseptic and antibacterial properties and therefore it is used for stomach-ache and bloat (Omidbaigi, 2000). The aerial parts of the plant contain essential oil that has medicinal properties and are tranquillizing and appetizing. The essential oil of dragonhead has also antioxidant activity (Povilaityte, 2000). Salinity stress is major factors limiting plant productivity in semi-arid and arid regions around the world. It causes a decline in primary metabolites which results a shortage of precursors necessitated for the synthesis of secondary compounds (Soliman, 1992). With regard to development of salinity lands and the shortage of agricultural land, abiotic stress related studies have a great importance in agricultural studies and to make use of salinity tolerant plant. Since dragonhead is an important plant in medicine, the investigations in to the cultivation aspects of this plant are essential. Water and soil salinity is critical bioenvironmental problems which limits plant growth and yield in many crops, in vast saline areas of Iran (Alaei, 2003; Khorsandi, 2010). Previous studies have shown that salinity stress decreases herbal yield and amount of essential oil in various medicinal plants such as Anise Hyssop (Agastache foeniculum) from lamiaceae family (Khorsandi, 2010). In Dracocephalum moldavica previous study has shown that as the amount of irrigation water decreases, growth and yield of dragonhead also decreases (Alaei, 2013). Besides that, for composition of essential oils and volatile compounds, previous studies have shown differences between oil compositions of dragonhead at different harvest times (Yousefzadeh, 2011). Davazdahemami (2008) has also showed that essential oil quality of moldavian

2 ALAEI ET AL. balm had significant differences during spring and summer. As the soil water content decreased, growth, yield and essential oil yield of moldavian balm decreased but there was no significant for essential oil content (hassani, 2006). Due to the importance of this medicinal crop and the impact of salinity stress, this study focuses on the impact of salinity stress on essential oil composition of Dracocephalum moldavica. As there is no report on the oil components of Dracocephalum moldavica cultivated in saline conditions and this literature is this first report in this respect, we hope it will provide valuable information for the possible cultivation of this plants in saline lands. MATERIALS AND METHODS Plant Material: Dragonhead (Dracocephalum moldavicum L.) plants were obtained from seed plantation. The used seeds in this investigation were obtained from Medicinal and Aromatic Plants Department of Corvinus University, Budapest, Hungary. Similarsized pots were filled with 3750 g sand and soil mixture. After germination, weak seedlings were omitted during 2-3 times. Four plants per pot were preserved finally. During germination to full flowering stage (about 4 months), pots were irrigated every day based on their field capacity (F.C) that was determined by pressure plate set. Irrigation treatments were completed during germination until stage of 6-8 leaf. Treatments: A pot experiment in randomized complete block design with 5 salinity treatments each in 4 replications was carried out in green house. Salinity treatments used after rehabilitation of plants based on different levels of salinity including 0, 10, 25, 40 and 50 mm Nacl. The pots were irrigated according to their weight in 5 days interval. Sample Taking and Extraction: In order to evaluate the composition of essential oils, when plants were at full flowering stage, large foliar mounds with fresh flower stalks arising from the center of each plant were harvested with a sharp knife about 5 cm above the ground surface. Each completely harvested sample was placed in a separate paper bag and then air dried under room condition. Typically 7-10 days were required to complete the drying process. Arid- dried aerial parts were subjected to steam distillation for 1 hour in an all glass apparatus to produce the oil in 0.5% w/w yield. Clevenger method used to extract the essential oil from the plant foliage. The essential oil obtained by hydrodistillation from the herb of plants and then analyzed by using GC (Gas Chromatography) and GC/MS (Gas chromatography mass spectrometry) for identification and quantification of the components. Sample Analysis: GC analysis was performed using an Agilent 6890 gas chromatograph equipped with a HP-5 MS column (30 m x 0.25 mm, film thickness 0.25 micrometer). Oven temperature was held at 0 C for 5 minute and then programmed to 240 C at a rate of 3 C per min; holding at the final temperature 300 C for 3 min. Injector and detector temperature were 290 C, helium was used as carrier gas with a linear velocity of 0.8 ml per min. GC-MS analysis was performed using a Agilent 5973 equipped with a ionization energy 70 ev; ionization temperature were 220 C. Identification of Components: The components of the oil were identified by comparison of their mass spectra with those of a computer library or with authentic compounds and confirmed by comparison of their retention indices either with those of authentic compounds or with data published in the literature (Adams, 2001). Mass spectra from the literature were also compared (Davies, 1990). RESULTS AND DISCUSSION Essential Oil Content: Results indicate that with salinity level increment, essential oil content of Dracocephalum moldavicum increases significantly. The highest and lowest essential oil content 0.32% and 0.27% were extracted from 50 mm and 10 mm of NaCl salinity, respectively (Fig. 1). In this experiment the essential oil content of dragonhead varied and depended on environmental factors. Safikhani et al. (2007) showed that effect of drought stress on essence yield at a two year period was significant. In their study, the result s analysis of variation for a two year period showed that effect of year on essential oil percent was significant. Treatments mean comparison of plant population and different drought stress levels showed that highest essential oil percent was seen in Esfahan seed treated with 60% of field capacity drought. In a study Hassani (2006) found that there is not a significant difference between drought stress treatments on essential oil content of Moldavian balm. This was while he observed that water stress had a significant effect on essential oil yield. The highest amounts of essential oil content (0.35 mlg -1 dry weight) were observed in 70% of field capacity water stress. Previous results show that essential oil content varies between 0.8 to 3 percent (Omidbaigi, 2005; Shatar and Altantseg, 2000). Omidbaigi et al. (2010) showed that the highest oil content (3.2%) and the lowest amount (2.3%) was obtained from the herb of plants which were sown on 20 th April and 5 th March, respectively. In another study, 43

3 AGRICULTURAL COMMUNICATIONS. Mafakheri (2012) showed that the highest essential oil content (0.74%) was obtained at the 30% level of the vermicompost treatment. Yousefzadeh et al. (2011) explained that maximum and minimum essential oil percentage was observed in full flowering (0.53) and yellowmaturity stage (0.07) respectively. For Agastache foeniculum, Khorsandi et al. results showed that salt stress had a significant effects on yield and amount of essential oil per pot. Similar to our findings, Khorsandi et al. (2010) found that in different levels of salinity, biomass yield and amount of essential oil in each plant and composition of essential oil changed and decreased by stress increment. Fig. 1. Effect of salinity stress on essential oil content in Dracocephalum moldavica. Essential Oil Components: The results indicated that thirty eight and twenty one components were identified from Dracocephalum moldavica in control and 50mM salinity treatments, respectively. The major constituents of the oil of D. moldavica were found as geranyl acetate, geraniol, geranial, neral, neryl acetate, methyl citronellate and phytol in all of the treatments (Table 1). This results is in accordance to other previous studies such as Li and Ding (2001), Hawthorne et al. (1993), Yousefzadeh (2011), Mafakheri (2012), Alaei and Mahna (2013), Omidbaigi et al. (2010) and Davazdahemami et al. (2008). The highest of the main components were geranyl acetate and geraniol. By salinity levels increment, the amount of geranyl acetate decreased. Among different treatments, geranyl acetate percentage in the 10 mm salinity was higher than that in others. Geranial and neryl acetate were not observed and produced in control treatment. This was while control treatment had the highest amount of geraniol (27.3%). With salinity level increment, geraniol decreased whenever geranial increased. The highest amount of neral and geranial were observed in dragonhead plants treated with 25mM salinity level. In control treatment, methyl citronellate was produced in the amount of 12.92%. This was while by salinity treatment the mentioned was not produced in salinity treatments. The results indicated that increasing of salinity levels increases the amount of neryl acetate and phytol. In table 1, GC mass analysis of essential oil content in Dracocephalum moldavica plants treated with different salinity levels are provided. Number of identified components of essential oil in plants of each level of salinity were different. The identified compounds in oil of dragonhead plants were 21, 27, 28, 29 and 38 in salinity treatments of 0, 10, 25, 40 and 50 mm, respectively. As seen in Table 1, increment in salinity level, increased major components percentage in dragonhead. Sum of six major components neral, geraniol, geranial, phytol, neryl acetate and geranyl acetate in essence were 75.89%, 87.01%, 85.12%, 83.08% and 80.32% in salinity levels of 0, 10, 25, 40 and 50 mm, respectively. The major constituents of the oil of D. moldavica extracted by hydro distillation were found to be geranyl acetate, geranial, neryl acetate, geraniol, neral and nerol (Li, 2001). Hawthorne et al. (1993) identified geranyl acetate (65.8%), carvacrol (14.9%) and thymol (7%) as the major components of the oil of D. moldavica. But Shatar and Altantseg (2000) introduced linalool (67%) and carvone (5.9%) as the main components of the oil of D. moldavica. Chopra (1986) found that dragon head contains citral in its essential oil. But in this research citral was not identified in NaCl treated nor the control plants. Davazdahemami (2008) showed that sum of five major components neral, geraniol, geranial, neryl acetate and geranyl acetate in essence were 92% and 64% in spring and summer sowing date and maximum change was seen in geranyl acetate from 35.3% in spring to 14% in summer. Omidbaigi et al. (2010) also found that the essential oil content and its constituents of Dragonhead were significantly affected by sowing dates. The results of GC and GC/MS analysis showed that the essential oil constituents were affected by sowing dates. Eighteen components were identified from the oil of plants were sown on 5 th of June but nine to twelve components were analyzed from the essential oils of plants which were sown from 5 th of March to 20 th of May. The highest amount of geranyl acetate (50.10%), geranial (25.27%), neral (19.34%) and geraniol (28.80%), were obtained from the plants sown on 5 th of May, 5 th June and 20 th of March, respectively (Omidbaigi, 2010). Alaei (2013) showed that thirty six and twenty one components were identified from Dracocephalum moldavica in field and greenhouse 44

4 ALAEI ET AL. conditions, respectively. The major constituents of the oil of D. moldavica were found as geranyl acetate (46.72%), geraniol (15.87%), geranial (8.36%), neral (5.8%), cedroxyde (3.39%), neryl acetate (2.57%) and hinesol (2.39%) (Totally, 88.49%) in field condition and geranyl acetate (39%), geraniol (27.30%), methyl citronellate (12.92%) and neral (9.32%) (Totally 88.54%) in greenhouse condition. Geranyl acetat percentage in the field was higher than that in the greenhouse but neral and geraniol decreased in field condition. Table 1. Constituents of the essential oil of Dracocephalum moldavica grown in different salinity treatments. Component Treatment RI Control 10 mm NaCl 25 mm NaCl 40 mm NaCl 50 mm NaCl 6-Methyl-5-Hepten-2-one Linalool oxide Linalool Isophorone (4-Keto) Verbenol (trans-) Karahanaenone Chrysanthenol(CIS-) Nerol Neral Geraniol Methyl citronellate Geranial Methyl geranate Neryl acetate Alpha-copaene Geranyl acetate Beta- elemene Trans-Caryophyllene Germacrene-D Selinene (BETA) E,E-Alpha-Farnesene Spathulenol Caryophyllene oxide Veridiflorol Tetradecanal Farnesol (E,E) Methyl tetradecanoate Vetivenol (Bicyclo) Dodecanoic acid,butylester Juniper camphor acetate Pipitzol (o-methyl,beta) Isobornyl isobutyrate(8-isobotyryloxy-) Totarene Phytol Rimuene (Tetrahydro-) Nootkatin Manoyl oxide Ethyl hexadecanoate Eicosene(N) Ethylene brassylate Manool Nezukol Incensole Ethyl octadecanoate Octadecanol acetate Triacontane Means in each row with the same letter are not significantly different based on Duncan s new multiple range test at p < According to this research results, salinity treatment significantly increases essential oil content in Dracocephalum moldavica. By culturing this plant at 50mM level of salinity, the number of components and sum of six major components namely, neral, geraniol, geranial, phytol, neryl acetate and geranyl acetate in essence increases. Therefore our result indicates the advantage of saline stress for dragonhead. ACKNOWLEDGEMENT This research was financially supported by Islamic Azad University, Kermanshah branch. 45

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