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Morphological, essential oil and biochemical variation of Dracocephalum moldavica L. populations
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
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Morphological, essential oil and biochemical variation of Dracocephalum moldavica L. populations ⁎
Saeed Yousefzadeha, , Faezeh Daryaia, Ali Mokhtassi-Bidgolib, Saeid Hazratic, Tumach Yousefzadehd, Khosro Mohammadie a
Department of Agriculture, Payame Noor University, Tehran, Iran Department of Agronomy, Faculty of Agriculture, Tarbiat Modares University, PO Box 14115-336, Tehran, Iran c Department of Agronomy College of Agriculture, Azarbaijan Shahid Madani University, Tabriz, Iran d Payame Noor University, Tehran, Iran e Department of Agronomy, College of Agriculture, Sanandaj Branch, Islamic Azad University, Sanandaj, Iran b
A R T I C LE I N FO
A B S T R A C T
Keywords: Dragonhead Medicinal plants Secondary metabolites Geranial
To determine the quantitative and qualitative characteristics of five dragonhead populations at five habitats (Salmas, Uremia, Khoy, Maragheh and Tabriz), an experiment was conducted as completely randomized design with three replications in 2013. The results showed that the tallest plant height (92 cm) and the highest contents of nitrogen (2.3%), phosphorus (0.22%), chlorophyll (1.9 mg g−1 FW), carotenoids (0.68 mg g−1 FW) and essential oil (0.61%) were obtained from Salmas population. Number of secondary branches varied from 5.7 to 22.7, while number of flowering branches ranged from 4.6 to 14.0. The major components of the essential oil were geraniol, geranial, nerol and geranyl acetate. The highest and the lowest values of neral + geraniol + geranial were observed in Tabriz and Maragheh populations, respectively. The levels of neral, geranial and geraniol were greater for Salmas population followed by Tabriz population. The present study increased knowledge about the morphological and phytochemical properties of dragonhead populations and can be useful for domestication, breeding, industrial and medicinal uses.
1. Introduction Moldavian balm or dragonhead (Dracocephalum moldavica L.) is an annual, herbaceous aromatic plant belong to Lamiaceae family which reaches 80 cm in height. In Iran, it is mainly distributed in the western parts of Azerbaijan province, and in the Albourz Mountains (Dastmalchi et al., 2007). Dragonhead are greatly used in pharmaceuticals, cosmetics and food industries. In folk medicine, extracts and essential oil from dragonhead are used painkiller, toothache, anti-inflammatory, anticonvulsive and sedative properties (Racz et al., 1975). In Iran, particularly in the East and West Azerbaijan provinces, dragonhead’s distilled aqueous extracts are used as a special beverage, food ingredient and as a tea. The essential oil content and composition of Moldavian balm was different owing to the plant origin (Hussein et al., 2006). The essential oil content of Moldavian balm ranged from 0.20 to 0.74% in different reports (Racz et al., 1975; Hornok et al., 1990; Shatar and Altanstetseg, 2000). Yousefzadeh et al. (2011) stated that the highest and lowest essential oil contents for both landrace and modern cultivars were gained at the full flowering (0.53%) and yellow-maturity
⁎
(0.07%) stages, respectively. They reported that the major essential oil components of Moldavian balm were geranial, geraniol and geranyl acetate. Other researchers have identified geraniol, geranyl acetate, carvacrol, linalool, carvone, citral and thymol as the dominant components of D. moldavica (Shatar and Altanstetseg, 2000; Nikitina et al., 2008; Fallah et al., 2018). Essential oil of medicinal plants are affected by many factors such as genetic and evolution, environmental factors such as climate and soil conditions, geographic variations, physiological factors, political/social conditions and harvest time Aghaei et al., 2013). Due to the impact of environmental factors, the accessions of medicinal plants growing in various geographical conditions might be different in their chemical composition, which might lead to changes in the pharmaceutical properties and biological activities (Heywood, 2002; Aghaei et al., 2013). There is high correlation between geographical origin of plants and their active ingredients. Ibrahim et al. (2014) reported that morphological traits of Lemon verbena including plant height, number of lateral branch, leaf area, total dry mater and essential oil content were higher in Qalyupia than Giza location in Egypt. In other research
Corresponding author. E-mail address: [email protected] (S. Yousefzadeh).
https://doi.org/10.1016/j.jarmap.2018.06.005 Received 15 March 2018; Received in revised form 10 June 2018; Accepted 20 June 2018 2214-7861/ © 2018 Elsevier GmbH. All rights reserved.
Please cite this article as: Yousefzadeh, S., Journal of Applied Research on Medicinal and Aromatic Plants (2018), https://doi.org/10.1016/j.jarmap.2018.06.005
Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx–xxx
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2.4. Total flavonoid and anthocyanins
essential oil content of anise (Pimpinella anisum L.) was significantly increased in moderate year compared to the dry and hot year in Serbia (Acimovic et al., 2014). Photosynthetic pigments including chlorophyll, carotenoids, anthocyanin and flavonoid can affect by environment conditions (Zucchi and Necchi, 2001). According to Zucchi and Necchi (2001), the physical factors such as light intensity, photoperiod and temperature can alter the pigment contents. Nitrate was positively correlated with chlorophyll amount in a study (Széles et al., 2012). In addition, the fertility of soil for supply nutrients for plants is a very important agent in plant growth and development. Findings have showed that application of nitrogen in medicinal and aromatic plants improved photosynthesis pigments and essential oil yield (Ozguven et al., 2006; Sifola and Barbieri, 2006). Therefore, plants with different genetic characteristics grown in natural habitats with different climatic conditions can use as potent sources for new chemotypes, which is of particular importance for cosmetic, perfume, pharmacogical and food industries. Knowledge of metabolite diversity among plants collected from different natural habitats allows to determine the optimum growing conditions for plants domestication and breeding. The aim of this study was to evaluate variations of environments, some morphological traits, photosynthesis pigments, element contents and composition of essential oil of dragonhead from East and West Azarbaijan provinces in Iran.
Total flavonoid content was determined by aluminum chloride (AlCl3) using quercetin as a standard (Ordonez et al., 2006). The plant extract (400 μl) was added to 0.3 ml distilled water followed by 5% NaNO2 (0.03 ml). After 5 min rest at 25 °C, AlCl3 (0.03 ml, 10%) was added. After further 5 min rest, the reaction mixture was treated with 0.2 ml of 1 mM NaOH. Finally, the reaction mixture was diluted to 1 ml with water and the absorbance was measured at 510 nm. For the determination of anthocyanin concentration, 0.2 g fresh leave were taken and extracted in 15 ml glass centrifuge tubes containing 10 ml of acidified methanol (methanol: HCl, 99: 1, v: v) and kept overnight in the dark. The samples were brought up to volume, and the absorbance value at 550 nm was determined spectrophotometrically (Hosu et al., 2014). 2.5. Extraction of essential oils An all-glass Clevenger- type apparatus was used to conduct 2.5 h of hydro-distillation of dried aerial parts (50 g) of D. moldavica L., which were collected at the full flowering stage. This method for the extraction of essential oils is recommended by the European Pharmacopoeia (1983). The essential oils were dried over anhydrous sodium sulphate (Merck Chemical Co., Germany) and stored in tightly closed dark vials at 2 °C before analysis.
2. Materials and methods 2.6. Gas chromatography conditions 2.1. Experimental design, plant and soil material A Thermo-UFM Gas-Chromatograph (Model 9 A) with Hp-5 column as 10 m × 0.1 mm with 0.4 mm film thickness. The initial temperature of oven was 60 °C for 3 min which is increased to 280 at a rate of 80 °C/ min. The injector and flame-ionization temperatures were held at 285 °C and helium gas was used as the carrier gas with the flow rate of 32 cm per second. Samples of 1.0 μL were injected manually with a split ratio of 1:20. The percentages of compounds were calculated by using the area normalization method, without consideration response factors.
This experiment was conducted as completely randomized design with three replications in 2013. Treatments were five dragonhead populations including Salmas, Uremia, Khoy, Maragheh and Tabriz and a cultivar called Szk-1, as a control treatment. Only cv. Szk-1 seed was planted on 5 May 2013 at the Research Field of Payam Noor University (PNU) in Marand. Plants of populations were collected at the flowering stage in August 2013 from East and West Azarbaijan provinces in Iran (Fig. 1). Populations were called based on where they were collected. In each location, morphological characteristics such as plant height, number of lateral branch and number of flowering branches were recorded by using 25 plants randomly cut at ground level. Several physiological traits such as contents of nitrogen, phosphorus, potassium, chlorophyll, carotenoid, anthocyanin and flavonoid were measured in aerial parts of ten subsamples of 25 plants in the laboratory. The fifteen remaining plants were air dried in shade to extract essential oil. For all locations, composite soil samples were collected at a depth of 0–30 cm. The air-dried samples were used to determine pH, electrical conductivity (EC), saturation percentage, total organic carbon (Walkley and Black method) and total nitrogen (Kjeldahl method) (Tandon, 1995). The soil physical and chemical properties and the climatological details of studied sites are shown in Tables 1 and 2.
2.7. Gas chromatography–mass spectroscopy (GC–MS) conditions GC–MS analysis was done via a Varian 3400 GC–MS system equipped with a DB-5 fused silica column (30 m × 0.25 mm i.d., film thickness 0.25 μm), and then the oven temperature programmed to rise from 50 to 240 °C at a rate of 4 °C/min, the temperature of the transfer line was set at 260 °C. Helium gas was used as the carrier gas with the flow rate of 31.5 cm per second, with a split ratio of 1:60, an ionization energy of 70 eV, scan time 1 s and mass range analyzed was 40–300 amu (Salehi and Hazrati, 2017). 2.8. Identification of volatile components Constituents were determined by comprising of mass spectra with those held in a computer library or obtained using authentic compounds. The identities of the components were confirmed by comparing their retention indices, either with those of authentic compounds or with data in the literature (Adams, 1995), and the retention indices were calculated for all volatile constituents using a homologous series of n-alkanes.
2.2. Nutrient assay The total nitrogen, available phosphorus, and available potassium were measured based on the Kjeldahl method, Olsen method, and ammonium acetate extraction protocol (Tandon, 1995).
2.3. Chlorophyll content
2.9. Statistical analysis
Chlorophyll from the leaf samples was extracted in 80% acetone solution, following the method of Arnon (1949). Extracts were filtrated and total Chl content was measured spectrophotometrically at 645 and 663 nm, respectively. The Chl content was expressed as mg g−1 fresh weight.
All extractions and determinations were conducted in triplicate. The data were subjected to one way analysis of variance (ANOVA) using SAS statistical software. The data were analyzed using completely randomized design (CRD), and means comparison were employed by using least significant difference (LSD) at P < 0.05. For all essential oil 2
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Fig. 1. Geographical distribution of the studied population of dragonhead.
Uremia populations, respectively (Table 3). The height of Szk-1 (63.33 cm) was approximately 45% lower compared with Salmas (91.66 cm) population (Table 3). Perhaps Szk-1 cultivar genetically had a lower plant height compared with other studied populations except Uremia. In this regard, Yousefzadeh et al. (2013) showed that native populations were taller than Szk-1 cultivated in two locations of Iran. The reason for greater height of Salmas population maybe due to more nitrogen absorbed by the plants. According to Table 1, total nitrogen in the soil of Salmas was more than ten times than other locations. Nitrogen is one of the most important growth factors in vegetative growth and can increase plant height. Former studies have reported that application of nitrogen increased plant height in several of medicinal plants such as Artemisia (Artemisia annua L.) and dragonhead (D. moldavica L.) (Sotiropoulou and Karamanos, 2010; Yousefzadeh et al., 2013). In addition, plant height in Salmas region enhanced due to higher precipitation and soil organic matters which caused more availability of moisture and nutrients (Tables 1 and 2).
Table 1 Soil physical and chemical properties. Population
Soil texture
Organic Carbon (%)
Phosphorus (%)
Nitrogen (%)
Salmas Uremia
loam sandy clay loam silt loam sandy loam clay loam clay loam
1.29 0.93
4.76 4.40
0.10 0.08
0.87 0.93 0.87 1.02
4.36 5.06 4.45 3.93
0.09 0.08 0.07 0.09
Khoy Cultivar Szk-1 Maragheh Tabriz
composition data except for geranial and geraniol, geranyl acetate and neral acetate, the square root transformation were performed. 3. Result and discussion As Tables 3 and 4 showed, there were significant differences between the populations in plant height, number of secondary branches, contents of nitrogen, phosphorus, chlorophyll (a, b and total), anthocyanin, total flavonoids and number of flowering branches. According to Table 5 the population had significant differences in terms of essential oil, citronella, neral acetate at 5% probability level and βpinene, trans-β-ocimene, cis-rose oxide, cis-chrysanthenol, neral, geranial and geranial acetate at 1% probability level.
Number of secondary branches was the highest in Szk-1 compare to other studied population. Uremia population had the least secondary branches (Table 3). It seems, Szk-1 have a high potential in production of secondary branches in the plant. According to Yousefzadeh et al. (2013), Szk-1 had more number of secondary branches compared to landraces.
3.1. Plant height
3.3. Number of flowering branches
3.2. Number of secondary branches
Szk-1, Khoy and Maragheh populations were high in number of
The highest and lowest plant height were observed in Salmas and 3
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Table 2 Monthly meteorological data during the growing season in different regions. Sampling location (above sea levels)
Salmas m
Urmia 1328 m Khoy 1193
Marand 1334 M
Maragheh 1472 M
Tabriz 1458 M
3.9
Monthly temperatures average (°C) 10.8
Maximum relative humidity (%) 47.7
Minimum relative humidity (%) 30.4
Relative humidity average (%) 52.5
19.7 26.1 31.1 30.2 18.4 20.1 26.4 31.0 30.5 19.8 21.1 27.5 32.7 31.6 11.7 13.7 19.7
7.5 11.7 16.5 16.9 4.1 6.9 10.9 15.0 15.1 5.8 8.2 13.0 17.6 17.5 7.0 9.4 14.6
13.6 18.9 23.8 23.5 11.2 13.5 18.6 23.0 22.8 12.8 14.6 20.3 25.2 24.5 16.3 18.0 24.8
81.5 75.3 66.4 64.6 73.6 80.5 75.5 67.5 71.0 75.0 81.0 75.4 65.5 68.8 32.0 37.0 31.0
38.9 29.7 24.8 26.1 29.0 36.4 29.6 26.0 28.8 28.5 37.4 33.4 26.5 28.9 61.0 68.0 62.0
60.2 52.5 45.6 45.4 51.6 58.4 52.5 46.7 49.9 51.8 59.2 54.4 46.0 48.9 46.5 52.5 46.5
5.1 2.4 21.8 20.8 1.8 0
24.6 24.1 12.5 15.1 21.9 26.7
19.1 18.6 6.1 8.8 17.9 19.8
30.0 29.6 18.9 21.4 29.0 33.6
26.0 28.0 26.0 28.0 18.0 17.0
50.0 54.0 70.0 71.0 54.0 45.0
28.0 41.0 48.0 49.0 36.0 31.0
375.2 252.9 251.3 327.7
0 38.1 34.9 14.7
26.8 12.3 14.9 21.5
20.2 6.0 9.1 14.7
33.5 18.5 20.6 28.3
19.0 25.0 30.0 21.0
49.0 71.0 74.0 67.0
34.0 48.0 52.0 44.0
379.7 356.6
4.7 0
26.0 25.9
19.3 19.1
32.7 32.7
18.0 20.0
54.0 56.0
36.0 38.0
Total solar radation (Hours) 249.9
Total precipitation (mm)
Maximum temperature average (°C)
Minimum temperature average (°C)
March
Solar radation average (Hours 8.1
35.1
17.7
April May June July March April May June July March April May June July March April May
8.0 10.1 12.3 11.4 8.6 8.6 10.9 12.7 11.8 7.9 7.5 9.4 12.0 11.0 7.9 7.7 10.4
247.7 312.8 282.2 352.7 267.7 266.0 327.5 392.5 366.9 245.5 233.2 291.7 373.3 341.1 244.3 238.1 322.9
49.0 42.2 46.0 1.3 28.2 35.8 31.9 0 0.1 26.6 27.1 35.5 2.5 3.0 42.2 36.5 52.1
June July March April May June
12.3 11.4 8.5 8.6 11.4 12.7
381.5 353.0 263.4 267.7 354.3 392.3
July March April May
12.1 8.2 8.1 10.6
June July
12.2 11.5
Months
flowering branches and Uremia population produced the minimum number of flowering branches in plant. The number of flowering branches was three times higher in Szk-1 in comparison with Uremia population (Table 3). This result is in conformity with Yousefzadeh et al. (2013). It might be because of greater genetic potential of Szk-1 which helped the plants to produce more branches. Consequently, Szk-1 and Khoy populations due to more secondary branches produced more flowering branches, too. In contrast, Uremia population produced the least secondary and flowering branches. Less flowering branch may be attributed to average monthly temperature and minimum temperature, which were the lowest in Uremia region than other regions (Table 2). Nasrabadi et al. (2007) reported that early sowing date in comparison with late sowing date caused slower plant growth due to the low temperature and intensity of sunlight during the day in the initial of
Longitude
Latitude
44° 45′ 53″ E
38° 11′ 41″ N
45° 4′ 21″ E
37° 33′ 19″ N
45° 46′ 30″ E
38° 25′ 58″ N
46° 14′ 15″ E
37° 23′ 21″ N
46° 18′ 0″ E
38° 4′ 0″ N
growing season.
3.4. Plant nitrogen, phosphorus and potassium The greatest content of nitrogen and phosphorus in plants leaf were observed in Salmas population which was twice more than the nitrogen derived from plant leaf in Khoy and Maragheh population, respectively (Table 3). Obviously, due to the high content of nitrogen of residue in the soil of Salmas location, nitrogen uptake was higher compared to other regions. Nemeth et al. (2007) showed that plant nitrogen content increased with increased doses of nitrogen. Other finding reported that the positive correlation (r = 0.84) was observed between SPAD index and total leaf N in barley (Izsaki and Nemeth, 2007). It seems that organic matter with high nitrogen and phosphorus in Salmas location
Table 3 Some traits of dragonhead in different regions. Treatment Population
Plant height (cm)
Number of secondary branches Per plant
Number of flowering branches
Nitrogen
Phosphorus %
Potassium
Salmas Uremia Khoy Cultivar Szk-1 Maragheh Tabriz Significance
91.66a 53.66c 79.66ab 63.33bc 81ab 72bc
12a 5.66d 20.66a 22.66a 14.33bc 18ab
9ab 4.66b 13.33a 14a 10a 9ab
2.31a 1.16b 1.12b 1.32b 1.3b 1.3b
0.22a 0.19ab 0.18ab 0.17ab 0.12c 0.16bc
**
**
*
**
*
1.86 2.15 2.37 2.12 1.88 2.88 ns
Means followed by the same letter in each column are not significantly different at 5% of probability level. * Indicate significance at P level of 0.05. ** Indicate significance at P level of 0.01. 4
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Table 4 Photosynthetic pigments of dragonhead in different regions. Treatment Population
Chlorophyll a
Chlorophyll b (mg g−1 FW)
Total chlorophyll
Total carotenoid
Anthocyanin (mmolg−1FW)
Total flavonoid
Salmas Uremia Khoy Cultivar Szk-1 Maragheh Significance
1.32a 0.59c 1.24ab 1.3a 0.54c
0.57a 0.3c 0.5a 0.51a 0.29c
1.89a 0.89c 1.8ab 1.88a 0.84c
5.15ab 4.14b 6.46a 6.22a 4.12b
247.53ab 180.5b 328.2a 335.25a 148.03b
**
*
**
0.68 0.31 0.49 0.46 0.29 ns
**
*
Means followed by the same letter in each column are not significantly different at 5% of probability level. * Indicate significance at P level of 0.05. ** Indicate significance at P level of 0.01.
concentration in plant tissues is closely correlated with the N supplying capacity of the soil. It seems that, due to high level of nitrogen in Salmas, it is reasonable to assume the increase of chlorophyll amount in leaf of plants is happen. In addition, with increasing nitrogen absorption by plant root, nitrogen content would improve in plant tissue. Nitrogen is one of the essential components in the structure of chlorophyll, amino acids, proteins, nucleic acids and enzymes. Nitrogen deficiency accelerates leaf senescence and production of free radical molecules and causes decrease in the production of macromolecules such as proteins and chlorophyll (Ravier et al., 2017). In this regard, Sotiropoulou and Karamanos (2010) reported that application of 120 kg N ha−1 increased significantly the concentration of chlorophyll a and total in marjoram (Origanum majorana). Similar finding reported by Mathivanan et al. (2013) in (Arachis hypogaea L.).
enhanced nitrogen and phosphorus content in plant leaf (Table 1). Previous studies have shown that the use of organic fertilizers such as compost and manure increased organic matter and nutrients specially nitrogen in the soil and plant tissues (Hendawy, 2008), and also increases the solubility of insoluble phosphorus and turn into available phosphorus in the soil (Cooperland, 2002) Soil organic matter indirectly optimizes usable phosphorus in soil by maintaining soil within a specified pH range (Malakouti and Homaei, 2004). There was no significant difference was observed on potassium content of all populations (Table 3). There is no deficiency of the amount of potassium in the soils of Iran, especially in arid and semi-arid regions. In this regard, Samadi (2006) reported that K values varied from 207 to 538 mg kg−1 in five locations of the West Azerbaijan province in Iran. Also, exchangeable K in this study ranged from 160 to 441 mg kg−1 while Meyer and Wood (1985) stated that critical K for medium and heavy soil textures were 120 and 240 mg kg−1, respectively.
3.6. Carotenoid The highest and the lowest level of carotenoids were gained from Salmas and Maragheh populations, respectively. The same as chlorophyll, the carotenoids were high in leaves of the Salmas population (Table 4). The contents of chlorophyll and carotenoids were high in the harvested plants from Salmas location. According to Kopsell et al. (2007), with increasing nitrogen application carotenoid content increased. In this study, application of 150 mg L−1 nitrogen compare to other rates of nitrogen (6, 13, 26 and 52 mg L−1) produced the
3.5. Chlorophyll Salmas population and Szk-1 produced the highest and populations of Urmia and Maragheh produced the least contents of chlorophyll a, b and total. chlorophyll a, b and total contents of Salmas population were 2.4, 2 and 2.3 times higher compared with those of Maragheh population, respectively (Table 4). Izsaki and Nemeth (2007) reported that N Table 5 Essential oil compounds of dragonhead in studied populations. Studied populations Compound
Monoterpen ydrocarbone β -pinene (E)- β -ocimene Oxygenated monoterpen Linalool cis rose oxide Citronellal cis chrysanthenol Neral Geraniol Geranial E-dimethony citral Neryl acetate Geranyl acetate Sesquiterpen hydrocarbone E-caryophyllene Neral+geraniol+geranial Total Essential oil (%)
Tabriz
Maragheh
Salmas
Khoy (%)
Uremia
Cultivar Szk-1
Retention Index
Significance
1.6a 0.8b
0.1d 1.2a
0.6 bc 0.8b
0.3cd 1.4a
0.7b 0.7b
0.3cd 0.7b
976 1048
**
0.7 0.1b 10.3a 2.1a 20.4a 7.9ab 28.9a 0 1.3c 23.6e
0.2 0.3dc 0.4b 0.7d 10.4c 6.4ab 17.2c 0.1 1.7abc 57.3a
0.3 0.7a 0.7b 1.6b 17.7ab 5.9b 26.4c 0 2.1a 39.3c
0.3 0.5abc 0.6b 1.3c 15.4b 6.4ab 27.3a 0 1.6bc 34d
0.2 0.6ab 0.9ab 1.6bc 15.3b 6.6ab 21.8b 0 1.2ab 41.6c
0.7 0.4bc 0.5b 0.8d 11.5c 8.7a 19.3bc 0 2ab 50.6b
1100 1108 1153 1164 1238 1253 1267 1340 1360 1380
0 57.3a 97.8 0.400 b
0.1 33.9e 96 0.402 b
0 49.9b 95.9 0.611 a
0 49.01bc 89 0.523 ab
0.1 43.8de 92.1 0.422 b
0.1 39.5de 95.7 0.440 b
1425
Means followed by the same letter in each column are not significantly different at 5% of probability level. * Indicate significance at P level of 0.05. ** Indicate significance at P level of 0.01. 5
**
ns ** * ** ** * **
ns * **
ns **
**
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growth and essential oil yield. Increase in essential oil was demonstrated due to the N fertilizer consumption which plays an important role in the development and sharing of new cells contain essential oils, essence channels, excretory ducts and glandular trichomes (Alsafar and Al-Hassan, 2009). Organic matter in the soil is a source of nutrients which have slow decomposition and release that make growth successfully. They have ability to increase water retention and cation exchange capacity of sandy soils and improve soil structure and stability and permeability of clay soils (Chang et al., 1991). More organic matter in Salmas location could have a positive effect on increasing essential oil. The findings of Hendawy (2008) showed that the use of organic fertilizers significantly increased the Plantago arenaria mucilage. In another study, Hussein et al. (2006) reported that the use of 39.6 tons per hectare of compost significantly increased essential oil content of dragonhead. It seems that, more precipitation and having a loamy soil at Salmas provided more accessibility to moisture for vegetation and caused an increase in photosynthesis and plant growth and subsequently increase in essential oil content. Additionally, less solar radiation in Salmas site make decrease in evapotranspiration, which is effective in increasing the amount of essential oil.
maximum lutein and beta carotene in Kale plant. High nitrogen level in the soil and plant caused increase in carotenoids content in Salmas population. 3.7. Anthocyanin Khoy population and Szk-1 produced the highest content of anthocyanin and the lowest one was obtained from Uremia and Maragheh populations (Table 4). The anthocyanin variation between the populations can be attributed to environmental and edaphological factors. Anthocyanin was increased by reducing the amount of nitrogen in the soil. Szk-1 produced the high anthocyanin which may be due to genetic differences in this cultivar compared with other populations except Khoy. The finding showed that application of high levels of nitrogen and low temperature decreased anthocyanin content in Clematis pitcher plant (Kawa-Miszczak et al., 2009). According to Del amor et al. (2008), utilization of urea in sweet pepper decline total anthocyanin compare to control treatment. On the other hand, this difference could be related to the differences in some climate parameters. The influence of climate in anthocyanins content was also observed in red apricots by Bureau et al. (2009) who demonstrate the relationship between anthocyanin content and air temperature. The highest anthocyanin of population Khoy might also be attributed to the climatic conditions of the sampling area, as the plant was grown at the lowest altitude (1193 m) with the highest maximal mean annual temperature (32.7 °C) to other populations studied
3.10. Composition of essential oils Table 5 showed in general, 13 compounds were identified from Szk1 grown in Marand and populations collected from differen t regions. Both GC and GC–MS analyses revealed that the major constituents of the oil were geraniol, geranial, and geranyl acetate (Table 5). These components represent 87.3% of the oils that were extracted from plants of the Tabriz population, and 96% of the oils extracted from plants of the Maragheh population. This result is in conformity with other researchers findings (Yousefzadeh et al., 2013). Holm et al. (1988) reported that the main components of essential oil of dragonhead were oxygenated acyclic monoterpenes contains geraniol, geranial, neral, nerol and geranyl acetate. Sonboli et al. (2008) found that neral, geranial, geranyl acetate and geraniol were 32.1, 21.6, 19.9 and 17.6% of the main components of essential oil, respectively. Different results were reported by other researchers. In this regard, Shuge et al. (2010) reported that citral (32.55%), beta citral (23.53%) and geranial ester (21.32%), were the most dominant components of essential oil of dragonhead plant. According to the Fallah et al. (2018), geranyl acetate, neral, linalool acetate and geraniol were the most important components of the dragonhead essential oil. However, according to the Hussein et al. (2006), the main components of dragonhead essential oil were linalool and geranial. It seems that a number of considerations, including geographic origin, ecological factors, genetic differences, and agro-technical approaches, could influence the composition of essential oil extracts from medicinal and aromatic plants. Results showed that the highest (35.3%) and the lowest (23.6%) amount of geranyl acetate were gained in Maragheh and Tabriz populations, respectively (Table 5). The geranyl acetate in Maragheh population was 143% higher compared to the Tabriz population. The Szk-1 followed by Maragheh population produced the highest geranyl acetate (50.6%). Nevertheless, population of Tabriz had the greatest (28.9%) geranial while Maragheh population had the lowest (17.2%) amount of geranial. In addition, Szk-1 had the lowest amount of geranial after Maragheh population. The geraniol in cultivar Szk-1 (8.7%) was higher compared to the all populations. The least amount of geraniol was observed in Salmas population. The highest and lowest amounts of neral were identified in population of Tabriz (20.4%) and Maragheh (10.4%), respectively. Modern cultivar (Szk-1) produced the lowest amount of neral after population of Maragheh. There was a positive correlation between the amount of neral with geranial (r = 0.92**) (Table 6). There were also a negative correlation between neral and geranyl acetate (r=−0.93**) and geranial with geranyl acetate (r=−0.95**) (Table 6). In this regard, Ganjewala and Luthra (2009) for lemon grass (Cymbopogon flexuosus) observed a
3.8. Flavonoid Szk-1 produced the highest content of flavonoid, while the plants of Maragheh had the least content of flavonoid. The flavonoid in Szk-1 plants was 125% greater than those of Maragheh population (Table 4). Flavonoids are secondary metabolites which play the role of plant defendant against ultraviolet radiation, pathogen and protect from herbivores. Flavonoids have antioxidant effect and regulate enzyme activity. They are involved in activities related to texture, variety, environmental stresses such as ultraviolet radiation, drought, soil conditions, tillage and fertilizers, pests and diseases (Mierziak et al., 2014). Increase in nitrogen causes an increase in protein and soluble amino acids synthesis which decrease the synthesis of flavonoids (Dahui et al., 2010). According to Dahui et al. (2010), application of high level of nitrogen decrease flavonoid concentration to 18–35% in Chrysanthemum morifolium. It seems that high nitrogen in the soil of Salmas location may be the reason of lower flavonoids content. According to the results, Szk-1 had the ability to accumulate the more flavonoids compared with other populations which may be originated from genetically factors. 3.9. Essential oil concentration The highest essential oil content was recorded in Salmas population compared with other populations except Khoy (Table 3). Essential oil obtained from Salmas population was 53 percent higher compared with Tabriz population (Table 5). Probably higher nitrogen and organic matter in the soil of Salmas location were the factors which are effective in increasing the content of essential oil of Salmas population. Studies have showed that high application of nitrogen (300 kg ha−1) compared with the non-application of nitrogen caused an increase in the essential oil of basil (Ocimum basilicum) (Ozguven et al., 2006; Sifola and Barbieri, 2006). According to the survey of Singh and Sharma (2001), application of 200 kg N ha−1 compared to 0 and 100 kg N ha−1 significantly increased the essential oil yield of lemon grass (Cymbopogon martinii). Essential oil is consisted of terpenoid which nitrogen is necessary element in its structure. Nitrogen application causes increase in photosynthesis, chlorophyll content, rubisco activity, biomass and leaf 6
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Table 6 The correlation coefficient between the main components of essential oils in studied populations. Traits Neral Geraniol Geranial Geranyl acetate *
Geranyl acetate
Geranial
Geraniol
1 −0.95**
1 −0.12 −0.05
Resource for Urban and Rural Gardeners, Small Farmers, Turf Grass Managers and Large-Scale Producers. Center for Integrated Agricultural Systems. University of Wisconsin- Madison. Dahui, L., Wei, L., Duanwei, Z., Mingjian, G., Wenbing, Z., Tewu, Y., 2010. Nitrogen effects on total flavonoids, chlorogenic acid, and antioxidant activity of the medicinal plant Chrysanthemum morifolium. Journal of Plant Nutrotion 173, 268–274. Dastmalchi, K., Dorman, H.G., Kosar, M., Hiltunen, R., 2007. Chemical composition and in vitro antioxidant evaluation of a water soluble Moldavian balm (Dracocephalum moldavica L.) extract. Food Science and Technology 40, 239–248. Del Amor, F.M., Cuadra-Crespo, P., Varo, P., G´omezb, M.C., 2008. Influence of foliar urea on the antioxidant response and fruit color of sweet pepper under limited N supply. Journal of the Science of Food and Agriculture 89, 504–510. European Pharmacopoeia (Vol. 1) 1983. Maissoneuve, SA: Sainte Ruffine. p. 1–8. 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Effect of vermicompost on the growth and nutrient status in groundnut (Arachishypogaea. L). Asian Journal of Plant Sciences 3, 15–22. Meyer, J.H., Wood, R.A., 1985. Potassium nutrition of sugarcane in South African sugar industry Pretoria. Proc. of the Potassium Symposium P, 205–213. Mierziak, J., Kostyn, K., Kulma, A., 2014. Flavonoids as important molecules of plant interactions withthe environment. Molecules 19, 16240–16265. Nasrabadi, B., Omid Baygi, R., Sfidkon, F., 2007. Effect of sowing time on biological growth yield and essential oil content in dragonhead (Dracocephalum moldavica L.). Iranian Journal of Medicinal and Aromatic Plants 23, 307–314 (In persian). Nemeth, T., Mathe-Gaspar, G., Radimszky, L., Gyiri, Z., 2007. Effect of nitrogen fertilizer on the nitrogen, sulphur and carbon contents of Canola (Brassica napus L.) grown on a calcareous chernozem soil. Cereal Research Communications 35, 837–840. Nikitina, A.S., Popova, O.I., Ushakova, L.S., Chumakova, V.V., Ivanova, L.I., 2008. Studies of the essential oil of dracocephalum moldavica cultivated in the stavropol region. Pharmaceutical Chemistry Journal 42, 35–39. Ordonez, A.A.L., Gomez, J.D., Vattuone, M.A., Isla, M.I., 2006. Antioxidant activities of Sechium edule (Jacq.) Swartz extracts”. Food Chemistry 97, 452–458. Ozguven, M., Ayanoglu, F., Ozel, A., 2006. Effects of nitrogen rates and cutting times on the essential oil yield and components of Origanum syriacum L. var. bevanii. Journal of Agronomy 5, 101–105. Racz, G., Laza, A., Coicin, E., 1975. Gyógynövények (Medicinal Plants). Geres Kiadó, Bukarest. Ravier, C., Meynar, J.M., Cohan, J.P., Gate, P., Jeuffroy, M.H., 2017. Early nitrogen deficiencies favor high yield, grain protein content and N use efficiency in wheat. European Journal of Agronomy 89, 16–24. Salehi, A., Hazrati, S., 2017. How essential oil content and composition fluctuate in german chamomile flowers during the day? Journal of Essential Oil Bearing Plants 20 (3), 622–631. Samadi, A., 2006. Potassium exchange isotherms as a plant availability index in selected calcareous soils of Western Azarbaijan Province, Iran. Turkish Journal of Agriculture 30, 213–222. Shatar, S., Altanstetseg, S., 2000. Essential oil composition of some plants cultivated in Monogolian climate. Journal of Essential Oil Research 12, 745–750. Shuge, T., Xiaoying, Z., Fan, Z., Dongqing, A., Tao, Y., 2010. Essential oil composition of
Neral 1
1
0.92** −0.93**
and**: significance at the P value of 0.01 and 0.05, respectively.
negative correlation between geranial and geranyl acetate in which geranial decreased by increasing the amount of geranyl acetate. This reaction was due to geranyl acetate conversion into geraniol during the plant leave growth. The maximum and minimum neral + geraniol + geraniol were obtained in Tabriz (57.3%) and Maragheh populations (33.9%). In addition, Salmas population followed by Tabriz population was high in neral + geraniol + geraniol (Table 5). 4. Conclusion In summary, D. moldavica with several chemotypes has valuable potential for domestication and breeding programs as an initial source for commercial propagation and cultivation. Our results demonstrated that the tallest height, the greatest contents of nitrogen, phosphorus, chlorophyll, carotenoids and essential oil of plants were obtained from Salmas population. Cultivar Szk-1 produced the largest number of branches, flowering branches and total flavonoid. The best qualitative traits, especially essential oil content was obtained from plants collected from Salmas. Salmas population followed by Tabriz population was high in the total amount of neral, geranial and geraniol. Since essential oil gradients have different usage in industrial aims, therefore, the obtained chemotypes can utilize after primary studies on their constancies. Acknowledgements The current paper is extracted from research project entitled “Effect of climate condition and location on quantitative and qualitative traits and contents and composition of essential oil in dragonhead (Dracocephalum moldavica L.)” which was financial supported by Research Grant from Payame Noor University, Tehran, Iran. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jarmap.2018.06.005. References Acimovic, M.G., Korac, J., Jacimovic, G., Oljaca, S., Djukanovic, L., Vuga-Janjatov, V., 2014. Influence of ecological conditions on seeds traits and essential oil Contents in Anise (Pimpinella anisum L.). Notulae Botanicae Horti Agrobotanici Cluj-Napoca 42, 232–238. Adams, R.P., 1995. Identification of Essential Oil Components by Gas chromatography/ Mass Spectroscopy. Carol Stream. Allured Publishing Corp., USA. Aghaei, Y., Mirjalili, M.H., Nazeri, V., 2013. Chemical diversity among the essential oils of wild populations of Stachys lavandulifolia Vahl (Lamiaceae) from Iran. Chemistry & Biodiversity 10, 262–273. Alsafar, M.S., Al-Hassan, Y.M., 2009. Effect of nitrogen and phosphorus fertilizers on growth and oil yield of indigenous mint (Mentha longifolia L.). Biotechnology 8, 380–384. Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts. Polyphennoloxidase in Beta vulgaris. Plant Physiology 24, 1–150. Bureau, S., Renard, C.M.G.C., Reich, M., Ginies, C., Audergon, J.M., 2009. Changes in anthocyanin concentration in red apricot fruit during ripening. LWT Food Science and Technology 42, 372–377. Chang, C., Sommer Feldt, T.G., Entz, T., 1991. Barley performance under heavy application of cattle feedlot manure. Agronomy Journal 85, 1013–1018. Cooperland, L., 2002. Building Soil Organic Matter With Organic Amendments. A
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Water Management 107, 133–144. Tandon, H.L.S., 1995. Methods of Analysis of Soils, Plants, Waters and Fertilisers. Fertilisers Development and Consultation Organisation, New Delhi, India. Yousefzadeh, S., Modarres-Sanavi, S.A.M., Sefidkon, F., Asgharzadeh, A., Ghalavand, A., 2011. Effect of harvesting time on essential oil content and composition of dragonhead (Dracocephalum moldavica L.). Iranian Journal of Medicinal and Aromatic Plants 26, 561–573 (In persian). Yousefzadeh, S., Modarres-Sanavy, A.M., Sefidkon, F., Asgarzadeh, A., Ghalavand, A., Sadat-Asilan, K., 2013. Effects of Azocompost and urea on the herbage yield and contents and compositions of essential oils from two genotypes of dragonhead (Dracocephalum moldavica L.) in two regions of Iran. Food Chemistry 138, 1407–1413. Zucchi, M.R., Necchi, O., 2001. Effects of temperature, irradiance and photoperiod on growth and pigment content in some freshwater red algae in culture. Phycological Research 49, 103–114.
the Dracocephalum moldavica L. from Xinjiang in China. Pharmaceutical Research 1, 172–174. Sifola, M., Barbieri, G., 2006. Growth, yield and essential oil content of three cultivars of basil grown under different levels of nitrogen in the field. Scientia Horticulturae 108, 408–413. Singh, M., Sharma, S., 2001. Influence of irrigation and nitrogen on herbage and oil yield of palmarosa (Cymbopogon martinii) under semi-arid tropical conditions. European Journal of Agronomy 14, 157–159. Sonboli, A., Mojarrad, M., Gholipour, A., Ebrahimi, S.N., Arman, M., 2008. Biological activity and composition of the essential oil of Dracocephalum moldavica L. grown in Iran. Natural Product Communications 3, 1547–1550. Sotiropoulou, D.E., Karamanos, A.J., 2010. Field studies of nitrogen application on growth and yield of Greek oregano (Origanum vulgare ssp. hirtum (Link) Ietswaart). Industrial Crops and Products 32, 450–457. Széles, A.V., Megyes, A., Nagy, J., 2012. Irrigation and nitrogen effects on the leaf chlorophyll content and grain yield of maize in different crop years. Agricultural
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Morphological, essential oil and biochemical variation of Dracocephalum moldavica L. populations ⁎
Saeed Yousefzadeha, , Faezeh Daryaia, Ali Mokhtassi-Bidgolib, Saeid Hazratic, Tumach Yousefzadehd, Khosro Mohammadie a
Department of Agriculture, Payame Noor University, Tehran, Iran Department of Agronomy, Faculty of Agriculture, Tarbiat Modares University, PO Box 14115-336, Tehran, Iran c Department of Agronomy College of Agriculture, Azarbaijan Shahid Madani University, Tabriz, Iran d Payame Noor University, Tehran, Iran e Department of Agronomy, College of Agriculture, Sanandaj Branch, Islamic Azad University, Sanandaj, Iran b
A R T I C LE I N FO
A B S T R A C T
Keywords: Dragonhead Medicinal plants Secondary metabolites Geranial
To determine the quantitative and qualitative characteristics of five dragonhead populations at five habitats (Salmas, Uremia, Khoy, Maragheh and Tabriz), an experiment was conducted as completely randomized design with three replications in 2013. The results showed that the tallest plant height (92 cm) and the highest contents of nitrogen (2.3%), phosphorus (0.22%), chlorophyll (1.9 mg g−1 FW), carotenoids (0.68 mg g−1 FW) and essential oil (0.61%) were obtained from Salmas population. Number of secondary branches varied from 5.7 to 22.7, while number of flowering branches ranged from 4.6 to 14.0. The major components of the essential oil were geraniol, geranial, nerol and geranyl acetate. The highest and the lowest values of neral + geraniol + geranial were observed in Tabriz and Maragheh populations, respectively. The levels of neral, geranial and geraniol were greater for Salmas population followed by Tabriz population. The present study increased knowledge about the morphological and phytochemical properties of dragonhead populations and can be useful for domestication, breeding, industrial and medicinal uses.
1. Introduction Moldavian balm or dragonhead (Dracocephalum moldavica L.) is an annual, herbaceous aromatic plant belong to Lamiaceae family which reaches 80 cm in height. In Iran, it is mainly distributed in the western parts of Azerbaijan province, and in the Albourz Mountains (Dastmalchi et al., 2007). Dragonhead are greatly used in pharmaceuticals, cosmetics and food industries. In folk medicine, extracts and essential oil from dragonhead are used painkiller, toothache, anti-inflammatory, anticonvulsive and sedative properties (Racz et al., 1975). In Iran, particularly in the East and West Azerbaijan provinces, dragonhead’s distilled aqueous extracts are used as a special beverage, food ingredient and as a tea. The essential oil content and composition of Moldavian balm was different owing to the plant origin (Hussein et al., 2006). The essential oil content of Moldavian balm ranged from 0.20 to 0.74% in different reports (Racz et al., 1975; Hornok et al., 1990; Shatar and Altanstetseg, 2000). Yousefzadeh et al. (2011) stated that the highest and lowest essential oil contents for both landrace and modern cultivars were gained at the full flowering (0.53%) and yellow-maturity
⁎
(0.07%) stages, respectively. They reported that the major essential oil components of Moldavian balm were geranial, geraniol and geranyl acetate. Other researchers have identified geraniol, geranyl acetate, carvacrol, linalool, carvone, citral and thymol as the dominant components of D. moldavica (Shatar and Altanstetseg, 2000; Nikitina et al., 2008; Fallah et al., 2018). Essential oil of medicinal plants are affected by many factors such as genetic and evolution, environmental factors such as climate and soil conditions, geographic variations, physiological factors, political/social conditions and harvest time Aghaei et al., 2013). Due to the impact of environmental factors, the accessions of medicinal plants growing in various geographical conditions might be different in their chemical composition, which might lead to changes in the pharmaceutical properties and biological activities (Heywood, 2002; Aghaei et al., 2013). There is high correlation between geographical origin of plants and their active ingredients. Ibrahim et al. (2014) reported that morphological traits of Lemon verbena including plant height, number of lateral branch, leaf area, total dry mater and essential oil content were higher in Qalyupia than Giza location in Egypt. In other research
Corresponding author. E-mail address: [email protected] (S. Yousefzadeh).
https://doi.org/10.1016/j.jarmap.2018.06.005 Received 15 March 2018; Received in revised form 10 June 2018; Accepted 20 June 2018 2214-7861/ © 2018 Elsevier GmbH. All rights reserved.
Please cite this article as: Yousefzadeh, S., Journal of Applied Research on Medicinal and Aromatic Plants (2018), https://doi.org/10.1016/j.jarmap.2018.06.005
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2.4. Total flavonoid and anthocyanins
essential oil content of anise (Pimpinella anisum L.) was significantly increased in moderate year compared to the dry and hot year in Serbia (Acimovic et al., 2014). Photosynthetic pigments including chlorophyll, carotenoids, anthocyanin and flavonoid can affect by environment conditions (Zucchi and Necchi, 2001). According to Zucchi and Necchi (2001), the physical factors such as light intensity, photoperiod and temperature can alter the pigment contents. Nitrate was positively correlated with chlorophyll amount in a study (Széles et al., 2012). In addition, the fertility of soil for supply nutrients for plants is a very important agent in plant growth and development. Findings have showed that application of nitrogen in medicinal and aromatic plants improved photosynthesis pigments and essential oil yield (Ozguven et al., 2006; Sifola and Barbieri, 2006). Therefore, plants with different genetic characteristics grown in natural habitats with different climatic conditions can use as potent sources for new chemotypes, which is of particular importance for cosmetic, perfume, pharmacogical and food industries. Knowledge of metabolite diversity among plants collected from different natural habitats allows to determine the optimum growing conditions for plants domestication and breeding. The aim of this study was to evaluate variations of environments, some morphological traits, photosynthesis pigments, element contents and composition of essential oil of dragonhead from East and West Azarbaijan provinces in Iran.
Total flavonoid content was determined by aluminum chloride (AlCl3) using quercetin as a standard (Ordonez et al., 2006). The plant extract (400 μl) was added to 0.3 ml distilled water followed by 5% NaNO2 (0.03 ml). After 5 min rest at 25 °C, AlCl3 (0.03 ml, 10%) was added. After further 5 min rest, the reaction mixture was treated with 0.2 ml of 1 mM NaOH. Finally, the reaction mixture was diluted to 1 ml with water and the absorbance was measured at 510 nm. For the determination of anthocyanin concentration, 0.2 g fresh leave were taken and extracted in 15 ml glass centrifuge tubes containing 10 ml of acidified methanol (methanol: HCl, 99: 1, v: v) and kept overnight in the dark. The samples were brought up to volume, and the absorbance value at 550 nm was determined spectrophotometrically (Hosu et al., 2014). 2.5. Extraction of essential oils An all-glass Clevenger- type apparatus was used to conduct 2.5 h of hydro-distillation of dried aerial parts (50 g) of D. moldavica L., which were collected at the full flowering stage. This method for the extraction of essential oils is recommended by the European Pharmacopoeia (1983). The essential oils were dried over anhydrous sodium sulphate (Merck Chemical Co., Germany) and stored in tightly closed dark vials at 2 °C before analysis.
2. Materials and methods 2.6. Gas chromatography conditions 2.1. Experimental design, plant and soil material A Thermo-UFM Gas-Chromatograph (Model 9 A) with Hp-5 column as 10 m × 0.1 mm with 0.4 mm film thickness. The initial temperature of oven was 60 °C for 3 min which is increased to 280 at a rate of 80 °C/ min. The injector and flame-ionization temperatures were held at 285 °C and helium gas was used as the carrier gas with the flow rate of 32 cm per second. Samples of 1.0 μL were injected manually with a split ratio of 1:20. The percentages of compounds were calculated by using the area normalization method, without consideration response factors.
This experiment was conducted as completely randomized design with three replications in 2013. Treatments were five dragonhead populations including Salmas, Uremia, Khoy, Maragheh and Tabriz and a cultivar called Szk-1, as a control treatment. Only cv. Szk-1 seed was planted on 5 May 2013 at the Research Field of Payam Noor University (PNU) in Marand. Plants of populations were collected at the flowering stage in August 2013 from East and West Azarbaijan provinces in Iran (Fig. 1). Populations were called based on where they were collected. In each location, morphological characteristics such as plant height, number of lateral branch and number of flowering branches were recorded by using 25 plants randomly cut at ground level. Several physiological traits such as contents of nitrogen, phosphorus, potassium, chlorophyll, carotenoid, anthocyanin and flavonoid were measured in aerial parts of ten subsamples of 25 plants in the laboratory. The fifteen remaining plants were air dried in shade to extract essential oil. For all locations, composite soil samples were collected at a depth of 0–30 cm. The air-dried samples were used to determine pH, electrical conductivity (EC), saturation percentage, total organic carbon (Walkley and Black method) and total nitrogen (Kjeldahl method) (Tandon, 1995). The soil physical and chemical properties and the climatological details of studied sites are shown in Tables 1 and 2.
2.7. Gas chromatography–mass spectroscopy (GC–MS) conditions GC–MS analysis was done via a Varian 3400 GC–MS system equipped with a DB-5 fused silica column (30 m × 0.25 mm i.d., film thickness 0.25 μm), and then the oven temperature programmed to rise from 50 to 240 °C at a rate of 4 °C/min, the temperature of the transfer line was set at 260 °C. Helium gas was used as the carrier gas with the flow rate of 31.5 cm per second, with a split ratio of 1:60, an ionization energy of 70 eV, scan time 1 s and mass range analyzed was 40–300 amu (Salehi and Hazrati, 2017). 2.8. Identification of volatile components Constituents were determined by comprising of mass spectra with those held in a computer library or obtained using authentic compounds. The identities of the components were confirmed by comparing their retention indices, either with those of authentic compounds or with data in the literature (Adams, 1995), and the retention indices were calculated for all volatile constituents using a homologous series of n-alkanes.
2.2. Nutrient assay The total nitrogen, available phosphorus, and available potassium were measured based on the Kjeldahl method, Olsen method, and ammonium acetate extraction protocol (Tandon, 1995).
2.3. Chlorophyll content
2.9. Statistical analysis
Chlorophyll from the leaf samples was extracted in 80% acetone solution, following the method of Arnon (1949). Extracts were filtrated and total Chl content was measured spectrophotometrically at 645 and 663 nm, respectively. The Chl content was expressed as mg g−1 fresh weight.
All extractions and determinations were conducted in triplicate. The data were subjected to one way analysis of variance (ANOVA) using SAS statistical software. The data were analyzed using completely randomized design (CRD), and means comparison were employed by using least significant difference (LSD) at P < 0.05. For all essential oil 2
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Fig. 1. Geographical distribution of the studied population of dragonhead.
Uremia populations, respectively (Table 3). The height of Szk-1 (63.33 cm) was approximately 45% lower compared with Salmas (91.66 cm) population (Table 3). Perhaps Szk-1 cultivar genetically had a lower plant height compared with other studied populations except Uremia. In this regard, Yousefzadeh et al. (2013) showed that native populations were taller than Szk-1 cultivated in two locations of Iran. The reason for greater height of Salmas population maybe due to more nitrogen absorbed by the plants. According to Table 1, total nitrogen in the soil of Salmas was more than ten times than other locations. Nitrogen is one of the most important growth factors in vegetative growth and can increase plant height. Former studies have reported that application of nitrogen increased plant height in several of medicinal plants such as Artemisia (Artemisia annua L.) and dragonhead (D. moldavica L.) (Sotiropoulou and Karamanos, 2010; Yousefzadeh et al., 2013). In addition, plant height in Salmas region enhanced due to higher precipitation and soil organic matters which caused more availability of moisture and nutrients (Tables 1 and 2).
Table 1 Soil physical and chemical properties. Population
Soil texture
Organic Carbon (%)
Phosphorus (%)
Nitrogen (%)
Salmas Uremia
loam sandy clay loam silt loam sandy loam clay loam clay loam
1.29 0.93
4.76 4.40
0.10 0.08
0.87 0.93 0.87 1.02
4.36 5.06 4.45 3.93
0.09 0.08 0.07 0.09
Khoy Cultivar Szk-1 Maragheh Tabriz
composition data except for geranial and geraniol, geranyl acetate and neral acetate, the square root transformation were performed. 3. Result and discussion As Tables 3 and 4 showed, there were significant differences between the populations in plant height, number of secondary branches, contents of nitrogen, phosphorus, chlorophyll (a, b and total), anthocyanin, total flavonoids and number of flowering branches. According to Table 5 the population had significant differences in terms of essential oil, citronella, neral acetate at 5% probability level and βpinene, trans-β-ocimene, cis-rose oxide, cis-chrysanthenol, neral, geranial and geranial acetate at 1% probability level.
Number of secondary branches was the highest in Szk-1 compare to other studied population. Uremia population had the least secondary branches (Table 3). It seems, Szk-1 have a high potential in production of secondary branches in the plant. According to Yousefzadeh et al. (2013), Szk-1 had more number of secondary branches compared to landraces.
3.1. Plant height
3.3. Number of flowering branches
3.2. Number of secondary branches
Szk-1, Khoy and Maragheh populations were high in number of
The highest and lowest plant height were observed in Salmas and 3
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Table 2 Monthly meteorological data during the growing season in different regions. Sampling location (above sea levels)
Salmas m
Urmia 1328 m Khoy 1193
Marand 1334 M
Maragheh 1472 M
Tabriz 1458 M
3.9
Monthly temperatures average (°C) 10.8
Maximum relative humidity (%) 47.7
Minimum relative humidity (%) 30.4
Relative humidity average (%) 52.5
19.7 26.1 31.1 30.2 18.4 20.1 26.4 31.0 30.5 19.8 21.1 27.5 32.7 31.6 11.7 13.7 19.7
7.5 11.7 16.5 16.9 4.1 6.9 10.9 15.0 15.1 5.8 8.2 13.0 17.6 17.5 7.0 9.4 14.6
13.6 18.9 23.8 23.5 11.2 13.5 18.6 23.0 22.8 12.8 14.6 20.3 25.2 24.5 16.3 18.0 24.8
81.5 75.3 66.4 64.6 73.6 80.5 75.5 67.5 71.0 75.0 81.0 75.4 65.5 68.8 32.0 37.0 31.0
38.9 29.7 24.8 26.1 29.0 36.4 29.6 26.0 28.8 28.5 37.4 33.4 26.5 28.9 61.0 68.0 62.0
60.2 52.5 45.6 45.4 51.6 58.4 52.5 46.7 49.9 51.8 59.2 54.4 46.0 48.9 46.5 52.5 46.5
5.1 2.4 21.8 20.8 1.8 0
24.6 24.1 12.5 15.1 21.9 26.7
19.1 18.6 6.1 8.8 17.9 19.8
30.0 29.6 18.9 21.4 29.0 33.6
26.0 28.0 26.0 28.0 18.0 17.0
50.0 54.0 70.0 71.0 54.0 45.0
28.0 41.0 48.0 49.0 36.0 31.0
375.2 252.9 251.3 327.7
0 38.1 34.9 14.7
26.8 12.3 14.9 21.5
20.2 6.0 9.1 14.7
33.5 18.5 20.6 28.3
19.0 25.0 30.0 21.0
49.0 71.0 74.0 67.0
34.0 48.0 52.0 44.0
379.7 356.6
4.7 0
26.0 25.9
19.3 19.1
32.7 32.7
18.0 20.0
54.0 56.0
36.0 38.0
Total solar radation (Hours) 249.9
Total precipitation (mm)
Maximum temperature average (°C)
Minimum temperature average (°C)
March
Solar radation average (Hours 8.1
35.1
17.7
April May June July March April May June July March April May June July March April May
8.0 10.1 12.3 11.4 8.6 8.6 10.9 12.7 11.8 7.9 7.5 9.4 12.0 11.0 7.9 7.7 10.4
247.7 312.8 282.2 352.7 267.7 266.0 327.5 392.5 366.9 245.5 233.2 291.7 373.3 341.1 244.3 238.1 322.9
49.0 42.2 46.0 1.3 28.2 35.8 31.9 0 0.1 26.6 27.1 35.5 2.5 3.0 42.2 36.5 52.1
June July March April May June
12.3 11.4 8.5 8.6 11.4 12.7
381.5 353.0 263.4 267.7 354.3 392.3
July March April May
12.1 8.2 8.1 10.6
June July
12.2 11.5
Months
flowering branches and Uremia population produced the minimum number of flowering branches in plant. The number of flowering branches was three times higher in Szk-1 in comparison with Uremia population (Table 3). This result is in conformity with Yousefzadeh et al. (2013). It might be because of greater genetic potential of Szk-1 which helped the plants to produce more branches. Consequently, Szk-1 and Khoy populations due to more secondary branches produced more flowering branches, too. In contrast, Uremia population produced the least secondary and flowering branches. Less flowering branch may be attributed to average monthly temperature and minimum temperature, which were the lowest in Uremia region than other regions (Table 2). Nasrabadi et al. (2007) reported that early sowing date in comparison with late sowing date caused slower plant growth due to the low temperature and intensity of sunlight during the day in the initial of
Longitude
Latitude
44° 45′ 53″ E
38° 11′ 41″ N
45° 4′ 21″ E
37° 33′ 19″ N
45° 46′ 30″ E
38° 25′ 58″ N
46° 14′ 15″ E
37° 23′ 21″ N
46° 18′ 0″ E
38° 4′ 0″ N
growing season.
3.4. Plant nitrogen, phosphorus and potassium The greatest content of nitrogen and phosphorus in plants leaf were observed in Salmas population which was twice more than the nitrogen derived from plant leaf in Khoy and Maragheh population, respectively (Table 3). Obviously, due to the high content of nitrogen of residue in the soil of Salmas location, nitrogen uptake was higher compared to other regions. Nemeth et al. (2007) showed that plant nitrogen content increased with increased doses of nitrogen. Other finding reported that the positive correlation (r = 0.84) was observed between SPAD index and total leaf N in barley (Izsaki and Nemeth, 2007). It seems that organic matter with high nitrogen and phosphorus in Salmas location
Table 3 Some traits of dragonhead in different regions. Treatment Population
Plant height (cm)
Number of secondary branches Per plant
Number of flowering branches
Nitrogen
Phosphorus %
Potassium
Salmas Uremia Khoy Cultivar Szk-1 Maragheh Tabriz Significance
91.66a 53.66c 79.66ab 63.33bc 81ab 72bc
12a 5.66d 20.66a 22.66a 14.33bc 18ab
9ab 4.66b 13.33a 14a 10a 9ab
2.31a 1.16b 1.12b 1.32b 1.3b 1.3b
0.22a 0.19ab 0.18ab 0.17ab 0.12c 0.16bc
**
**
*
**
*
1.86 2.15 2.37 2.12 1.88 2.88 ns
Means followed by the same letter in each column are not significantly different at 5% of probability level. * Indicate significance at P level of 0.05. ** Indicate significance at P level of 0.01. 4
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Table 4 Photosynthetic pigments of dragonhead in different regions. Treatment Population
Chlorophyll a
Chlorophyll b (mg g−1 FW)
Total chlorophyll
Total carotenoid
Anthocyanin (mmolg−1FW)
Total flavonoid
Salmas Uremia Khoy Cultivar Szk-1 Maragheh Significance
1.32a 0.59c 1.24ab 1.3a 0.54c
0.57a 0.3c 0.5a 0.51a 0.29c
1.89a 0.89c 1.8ab 1.88a 0.84c
5.15ab 4.14b 6.46a 6.22a 4.12b
247.53ab 180.5b 328.2a 335.25a 148.03b
**
*
**
0.68 0.31 0.49 0.46 0.29 ns
**
*
Means followed by the same letter in each column are not significantly different at 5% of probability level. * Indicate significance at P level of 0.05. ** Indicate significance at P level of 0.01.
concentration in plant tissues is closely correlated with the N supplying capacity of the soil. It seems that, due to high level of nitrogen in Salmas, it is reasonable to assume the increase of chlorophyll amount in leaf of plants is happen. In addition, with increasing nitrogen absorption by plant root, nitrogen content would improve in plant tissue. Nitrogen is one of the essential components in the structure of chlorophyll, amino acids, proteins, nucleic acids and enzymes. Nitrogen deficiency accelerates leaf senescence and production of free radical molecules and causes decrease in the production of macromolecules such as proteins and chlorophyll (Ravier et al., 2017). In this regard, Sotiropoulou and Karamanos (2010) reported that application of 120 kg N ha−1 increased significantly the concentration of chlorophyll a and total in marjoram (Origanum majorana). Similar finding reported by Mathivanan et al. (2013) in (Arachis hypogaea L.).
enhanced nitrogen and phosphorus content in plant leaf (Table 1). Previous studies have shown that the use of organic fertilizers such as compost and manure increased organic matter and nutrients specially nitrogen in the soil and plant tissues (Hendawy, 2008), and also increases the solubility of insoluble phosphorus and turn into available phosphorus in the soil (Cooperland, 2002) Soil organic matter indirectly optimizes usable phosphorus in soil by maintaining soil within a specified pH range (Malakouti and Homaei, 2004). There was no significant difference was observed on potassium content of all populations (Table 3). There is no deficiency of the amount of potassium in the soils of Iran, especially in arid and semi-arid regions. In this regard, Samadi (2006) reported that K values varied from 207 to 538 mg kg−1 in five locations of the West Azerbaijan province in Iran. Also, exchangeable K in this study ranged from 160 to 441 mg kg−1 while Meyer and Wood (1985) stated that critical K for medium and heavy soil textures were 120 and 240 mg kg−1, respectively.
3.6. Carotenoid The highest and the lowest level of carotenoids were gained from Salmas and Maragheh populations, respectively. The same as chlorophyll, the carotenoids were high in leaves of the Salmas population (Table 4). The contents of chlorophyll and carotenoids were high in the harvested plants from Salmas location. According to Kopsell et al. (2007), with increasing nitrogen application carotenoid content increased. In this study, application of 150 mg L−1 nitrogen compare to other rates of nitrogen (6, 13, 26 and 52 mg L−1) produced the
3.5. Chlorophyll Salmas population and Szk-1 produced the highest and populations of Urmia and Maragheh produced the least contents of chlorophyll a, b and total. chlorophyll a, b and total contents of Salmas population were 2.4, 2 and 2.3 times higher compared with those of Maragheh population, respectively (Table 4). Izsaki and Nemeth (2007) reported that N Table 5 Essential oil compounds of dragonhead in studied populations. Studied populations Compound
Monoterpen ydrocarbone β -pinene (E)- β -ocimene Oxygenated monoterpen Linalool cis rose oxide Citronellal cis chrysanthenol Neral Geraniol Geranial E-dimethony citral Neryl acetate Geranyl acetate Sesquiterpen hydrocarbone E-caryophyllene Neral+geraniol+geranial Total Essential oil (%)
Tabriz
Maragheh
Salmas
Khoy (%)
Uremia
Cultivar Szk-1
Retention Index
Significance
1.6a 0.8b
0.1d 1.2a
0.6 bc 0.8b
0.3cd 1.4a
0.7b 0.7b
0.3cd 0.7b
976 1048
**
0.7 0.1b 10.3a 2.1a 20.4a 7.9ab 28.9a 0 1.3c 23.6e
0.2 0.3dc 0.4b 0.7d 10.4c 6.4ab 17.2c 0.1 1.7abc 57.3a
0.3 0.7a 0.7b 1.6b 17.7ab 5.9b 26.4c 0 2.1a 39.3c
0.3 0.5abc 0.6b 1.3c 15.4b 6.4ab 27.3a 0 1.6bc 34d
0.2 0.6ab 0.9ab 1.6bc 15.3b 6.6ab 21.8b 0 1.2ab 41.6c
0.7 0.4bc 0.5b 0.8d 11.5c 8.7a 19.3bc 0 2ab 50.6b
1100 1108 1153 1164 1238 1253 1267 1340 1360 1380
0 57.3a 97.8 0.400 b
0.1 33.9e 96 0.402 b
0 49.9b 95.9 0.611 a
0 49.01bc 89 0.523 ab
0.1 43.8de 92.1 0.422 b
0.1 39.5de 95.7 0.440 b
1425
Means followed by the same letter in each column are not significantly different at 5% of probability level. * Indicate significance at P level of 0.05. ** Indicate significance at P level of 0.01. 5
**
ns ** * ** ** * **
ns * **
ns **
**
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growth and essential oil yield. Increase in essential oil was demonstrated due to the N fertilizer consumption which plays an important role in the development and sharing of new cells contain essential oils, essence channels, excretory ducts and glandular trichomes (Alsafar and Al-Hassan, 2009). Organic matter in the soil is a source of nutrients which have slow decomposition and release that make growth successfully. They have ability to increase water retention and cation exchange capacity of sandy soils and improve soil structure and stability and permeability of clay soils (Chang et al., 1991). More organic matter in Salmas location could have a positive effect on increasing essential oil. The findings of Hendawy (2008) showed that the use of organic fertilizers significantly increased the Plantago arenaria mucilage. In another study, Hussein et al. (2006) reported that the use of 39.6 tons per hectare of compost significantly increased essential oil content of dragonhead. It seems that, more precipitation and having a loamy soil at Salmas provided more accessibility to moisture for vegetation and caused an increase in photosynthesis and plant growth and subsequently increase in essential oil content. Additionally, less solar radiation in Salmas site make decrease in evapotranspiration, which is effective in increasing the amount of essential oil.
maximum lutein and beta carotene in Kale plant. High nitrogen level in the soil and plant caused increase in carotenoids content in Salmas population. 3.7. Anthocyanin Khoy population and Szk-1 produced the highest content of anthocyanin and the lowest one was obtained from Uremia and Maragheh populations (Table 4). The anthocyanin variation between the populations can be attributed to environmental and edaphological factors. Anthocyanin was increased by reducing the amount of nitrogen in the soil. Szk-1 produced the high anthocyanin which may be due to genetic differences in this cultivar compared with other populations except Khoy. The finding showed that application of high levels of nitrogen and low temperature decreased anthocyanin content in Clematis pitcher plant (Kawa-Miszczak et al., 2009). According to Del amor et al. (2008), utilization of urea in sweet pepper decline total anthocyanin compare to control treatment. On the other hand, this difference could be related to the differences in some climate parameters. The influence of climate in anthocyanins content was also observed in red apricots by Bureau et al. (2009) who demonstrate the relationship between anthocyanin content and air temperature. The highest anthocyanin of population Khoy might also be attributed to the climatic conditions of the sampling area, as the plant was grown at the lowest altitude (1193 m) with the highest maximal mean annual temperature (32.7 °C) to other populations studied
3.10. Composition of essential oils Table 5 showed in general, 13 compounds were identified from Szk1 grown in Marand and populations collected from differen t regions. Both GC and GC–MS analyses revealed that the major constituents of the oil were geraniol, geranial, and geranyl acetate (Table 5). These components represent 87.3% of the oils that were extracted from plants of the Tabriz population, and 96% of the oils extracted from plants of the Maragheh population. This result is in conformity with other researchers findings (Yousefzadeh et al., 2013). Holm et al. (1988) reported that the main components of essential oil of dragonhead were oxygenated acyclic monoterpenes contains geraniol, geranial, neral, nerol and geranyl acetate. Sonboli et al. (2008) found that neral, geranial, geranyl acetate and geraniol were 32.1, 21.6, 19.9 and 17.6% of the main components of essential oil, respectively. Different results were reported by other researchers. In this regard, Shuge et al. (2010) reported that citral (32.55%), beta citral (23.53%) and geranial ester (21.32%), were the most dominant components of essential oil of dragonhead plant. According to the Fallah et al. (2018), geranyl acetate, neral, linalool acetate and geraniol were the most important components of the dragonhead essential oil. However, according to the Hussein et al. (2006), the main components of dragonhead essential oil were linalool and geranial. It seems that a number of considerations, including geographic origin, ecological factors, genetic differences, and agro-technical approaches, could influence the composition of essential oil extracts from medicinal and aromatic plants. Results showed that the highest (35.3%) and the lowest (23.6%) amount of geranyl acetate were gained in Maragheh and Tabriz populations, respectively (Table 5). The geranyl acetate in Maragheh population was 143% higher compared to the Tabriz population. The Szk-1 followed by Maragheh population produced the highest geranyl acetate (50.6%). Nevertheless, population of Tabriz had the greatest (28.9%) geranial while Maragheh population had the lowest (17.2%) amount of geranial. In addition, Szk-1 had the lowest amount of geranial after Maragheh population. The geraniol in cultivar Szk-1 (8.7%) was higher compared to the all populations. The least amount of geraniol was observed in Salmas population. The highest and lowest amounts of neral were identified in population of Tabriz (20.4%) and Maragheh (10.4%), respectively. Modern cultivar (Szk-1) produced the lowest amount of neral after population of Maragheh. There was a positive correlation between the amount of neral with geranial (r = 0.92**) (Table 6). There were also a negative correlation between neral and geranyl acetate (r=−0.93**) and geranial with geranyl acetate (r=−0.95**) (Table 6). In this regard, Ganjewala and Luthra (2009) for lemon grass (Cymbopogon flexuosus) observed a
3.8. Flavonoid Szk-1 produced the highest content of flavonoid, while the plants of Maragheh had the least content of flavonoid. The flavonoid in Szk-1 plants was 125% greater than those of Maragheh population (Table 4). Flavonoids are secondary metabolites which play the role of plant defendant against ultraviolet radiation, pathogen and protect from herbivores. Flavonoids have antioxidant effect and regulate enzyme activity. They are involved in activities related to texture, variety, environmental stresses such as ultraviolet radiation, drought, soil conditions, tillage and fertilizers, pests and diseases (Mierziak et al., 2014). Increase in nitrogen causes an increase in protein and soluble amino acids synthesis which decrease the synthesis of flavonoids (Dahui et al., 2010). According to Dahui et al. (2010), application of high level of nitrogen decrease flavonoid concentration to 18–35% in Chrysanthemum morifolium. It seems that high nitrogen in the soil of Salmas location may be the reason of lower flavonoids content. According to the results, Szk-1 had the ability to accumulate the more flavonoids compared with other populations which may be originated from genetically factors. 3.9. Essential oil concentration The highest essential oil content was recorded in Salmas population compared with other populations except Khoy (Table 3). Essential oil obtained from Salmas population was 53 percent higher compared with Tabriz population (Table 5). Probably higher nitrogen and organic matter in the soil of Salmas location were the factors which are effective in increasing the content of essential oil of Salmas population. Studies have showed that high application of nitrogen (300 kg ha−1) compared with the non-application of nitrogen caused an increase in the essential oil of basil (Ocimum basilicum) (Ozguven et al., 2006; Sifola and Barbieri, 2006). According to the survey of Singh and Sharma (2001), application of 200 kg N ha−1 compared to 0 and 100 kg N ha−1 significantly increased the essential oil yield of lemon grass (Cymbopogon martinii). Essential oil is consisted of terpenoid which nitrogen is necessary element in its structure. Nitrogen application causes increase in photosynthesis, chlorophyll content, rubisco activity, biomass and leaf 6
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Table 6 The correlation coefficient between the main components of essential oils in studied populations. Traits Neral Geraniol Geranial Geranyl acetate *
Geranyl acetate
Geranial
Geraniol
1 −0.95**
1 −0.12 −0.05
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How essential oil content and composition fluctuate in german chamomile flowers during the day? Journal of Essential Oil Bearing Plants 20 (3), 622–631. Samadi, A., 2006. Potassium exchange isotherms as a plant availability index in selected calcareous soils of Western Azarbaijan Province, Iran. Turkish Journal of Agriculture 30, 213–222. Shatar, S., Altanstetseg, S., 2000. Essential oil composition of some plants cultivated in Monogolian climate. Journal of Essential Oil Research 12, 745–750. Shuge, T., Xiaoying, Z., Fan, Z., Dongqing, A., Tao, Y., 2010. Essential oil composition of
Neral 1
1
0.92** −0.93**
and**: significance at the P value of 0.01 and 0.05, respectively.
negative correlation between geranial and geranyl acetate in which geranial decreased by increasing the amount of geranyl acetate. This reaction was due to geranyl acetate conversion into geraniol during the plant leave growth. The maximum and minimum neral + geraniol + geraniol were obtained in Tabriz (57.3%) and Maragheh populations (33.9%). In addition, Salmas population followed by Tabriz population was high in neral + geraniol + geraniol (Table 5). 4. Conclusion In summary, D. moldavica with several chemotypes has valuable potential for domestication and breeding programs as an initial source for commercial propagation and cultivation. Our results demonstrated that the tallest height, the greatest contents of nitrogen, phosphorus, chlorophyll, carotenoids and essential oil of plants were obtained from Salmas population. Cultivar Szk-1 produced the largest number of branches, flowering branches and total flavonoid. The best qualitative traits, especially essential oil content was obtained from plants collected from Salmas. Salmas population followed by Tabriz population was high in the total amount of neral, geranial and geraniol. Since essential oil gradients have different usage in industrial aims, therefore, the obtained chemotypes can utilize after primary studies on their constancies. Acknowledgements The current paper is extracted from research project entitled “Effect of climate condition and location on quantitative and qualitative traits and contents and composition of essential oil in dragonhead (Dracocephalum moldavica L.)” which was financial supported by Research Grant from Payame Noor University, Tehran, Iran. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jarmap.2018.06.005. References Acimovic, M.G., Korac, J., Jacimovic, G., Oljaca, S., Djukanovic, L., Vuga-Janjatov, V., 2014. Influence of ecological conditions on seeds traits and essential oil Contents in Anise (Pimpinella anisum L.). Notulae Botanicae Horti Agrobotanici Cluj-Napoca 42, 232–238. Adams, R.P., 1995. Identification of Essential Oil Components by Gas chromatography/ Mass Spectroscopy. Carol Stream. Allured Publishing Corp., USA. Aghaei, Y., Mirjalili, M.H., Nazeri, V., 2013. Chemical diversity among the essential oils of wild populations of Stachys lavandulifolia Vahl (Lamiaceae) from Iran. Chemistry & Biodiversity 10, 262–273. Alsafar, M.S., Al-Hassan, Y.M., 2009. Effect of nitrogen and phosphorus fertilizers on growth and oil yield of indigenous mint (Mentha longifolia L.). Biotechnology 8, 380–384. Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts. Polyphennoloxidase in Beta vulgaris. Plant Physiology 24, 1–150. Bureau, S., Renard, C.M.G.C., Reich, M., Ginies, C., Audergon, J.M., 2009. Changes in anthocyanin concentration in red apricot fruit during ripening. LWT Food Science and Technology 42, 372–377. Chang, C., Sommer Feldt, T.G., Entz, T., 1991. Barley performance under heavy application of cattle feedlot manure. Agronomy Journal 85, 1013–1018. Cooperland, L., 2002. Building Soil Organic Matter With Organic Amendments. A
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