Chemical constituents of essential oil of Dracocephalum moldavica L . and Dracocephalum kotschyi Boiss . from Iran

Dracocephalum moldavica L. and Dracocephalum kotschyi Boiss. are aromatic plants belonging to Lamiaceae family. The aim of this study was to identify the chemical components of D. kotschyi and D. moldavica from Iran. The aerial parts of D. kotschyi were collected from (Kamu Mountain) Isfahan province and the aerial parts of D. moldavica were collected from Sari (Mazandaran province) North of Iran, during 2014. The essential oil was extracted by a Clevenger approach and analyzed using GC/MS. In total, 32 and 24 compounds were identified in the essential oil from the aerial parts D. kotschyi and D. moldavica, respectively. The results obtained in our study indicated that the major components in the oil D. kotschyi were limonene (23.56 %), carvacrol (14.65 %), γterpinene (12.99 %), α -pinene (12.62 %), 2-methyl-1-octen-3yne (9.73 %), camphene (4.66 %), myrcene (3.65 %) and α terpinene (3.12 %). The major constituents of the oil D. moldavica were geranyl acetate (36.62 %), geraniol (24.31 %), neral (16.25 %) and geranial (11.21 %). D. kotschyi is one of the important sources of limonene and D. moldavica is one of the important sources of geranyl acetate.


INTRODUCTION
Dracocephalum moldavica L. and Dracocephalum kotschyi Boiss.are aromatic plants belonging to Lamiaceae family.D. moldavica known as Moldavian balm or Moldavian dragonhead is an annual herb and it is native to central Asia and is naturalized in eastern and central Europe.There is 45 species of herbaceous and shrub's dragonhead in the world and there is 8 species of annual and perennial fragrant herb in Iran, from which three are endemic to Iran (Rechinger, 1986;Mozaffarian, 2008).D. kotschyi is an herbaceous plant, endemic in Iran and known as Badrandjboie-Dennaie and Zarrin-Giah.Its oil has been used in folk medicine as an antispasmodic agent.Aerial parts of D. kotschyi plants are sources of valuable flavonoids and essential oils (Fattahi et al., 2013).Chromosome number of 2n = 2x = 10 and 2n = 2x = 20 are for D. moldavica and D. kotschyi, respectively (Salehi et al., 2014).Dracocephalum is used in folk medicine as painkiller and for treatments of kidney complaints, against toothache and colds as well as antirheumatism, antitumor (Chachoyan and Oganesyan, 1996), antimutagens, antioxidant, antiseptic and stimulant properties (Kakasy et al., 2006;Dastmalchi et al., 2007).Recent findings indicated that some of the medicinal plant characteristics can be affected by genetic and ecological factors, including precipitation, temperature and plant competition.Since essential oils are the product of a predominantly biological process further studies are needed to evaluate if the reported characteristics of each population are maintained at the level of individual plants and along the breeding and selection program when grown under climatic conditions (Ghasemi Pirbalouti and Mohammadi, 2013).Maham et al., (2013) reported the major components of D. moldavica essential oil collected from Maragheh of East Azerbaijan province as follows: citral (31.14 %), 3,7-dimethyl -2,6 octadienal (21.43 %), cis-geraniol (17.08 %), neral (9.63 %) and neryl acetate (4.03 %).The major constituents of the essential oil of D. moldavica collected from North Iran were limonene (19.8 %), α-pinene (14.4 %), methyl geranate (8.5 %), geranyl acetate (7.9 %), carvacrol (7.8 %) and geranial (5.4 %) (Morteza-Semnani et al., 2007).In studies Saeidnia et al., (2014) reported the folowing main components of the oil of D. kotschyi collected from Iran: geranial (37.2 %), limonene-10-al (28.5 %), limonene (20.1 %) and 1,1-dimethoxy decane (14.5 %).Javidnia et al. (2005) reported the main components of the oil of D. kotschyi as α-pinene, caryophyllene oxide, terpinen-4-ol and germacrene.Golshani et al., (2004), and also Yaghmai and Tafazzoli (1988) reported citral, myrcene, β-caryophyllene and terpinyl acetate as the main constituents of D. kotschyi from northeast mountains.The aim of this study was to identify of the chemical components of Dracocephalum moldavica L and Dracocephalum kotschyi Boiss from Iran.

Plant material
The aerial parts of the plant samples of Dracocephalum kotschyi Boiss were collected from Kamu Mountain, Isfahan province.Kamu is a city in Qamsar district, Kashan County, Isfahan province, in center Iran (33 • , 36´ N and 51 • , 14´ E) and the aerial parts of the plant samples of Dracocephalum moldavica L. were collected from Sari (Mazandaran province), North of Iran (36 • , 39´ N, and 53 • , 4´ E) , during 2014.The samples of the plants were identified by regional floras and authors with floristic and taxonomic references, and voucher specimens were deposited at the Herbarium of Agriculture Researches Islamic Azad University, Isfahan (Khorasgan), Iran.

Essential oil extraction
The fresh aerial part of D. kotschyi and D. moldavica were dried inside for six days at room temperature (25 ± 5 °C), and ground to fine powder using Moulinex food processor.The essential oil was extracted from 50 g of ground tissue in 1 L of water contained in a 2 L flask and heated by heating jacket at 100 °C for 3 h in a Clevengertype apparatus, according to producers outlined British Pharmacopoeia.The collected essential oil was dried over anhydrous sodium sulphate and stored at 4 °C until analyzed.

GC/MS analysis
Compositions of the essential oils were determined by GC-MS.The GC/MS analysis was carried out with an Agilent 5975 GC-MSD system.HP-5MS column (30 m x 0.25 mm, 0.25 μm film thickness) was used with helium as carrier gas with flow rate of 1.0 mL/min.The oven temperature was kept 20 °C at 50 °C for 4 min and programmed to 280 °C at a rate of 5 °C /min, and kept 20 °C constant at 280 °C for 5 min, at split mode.The injector temperature was at 20 °C at 280 °C.Transfer 20 line temperatures 280 °C.MS were taken at 70 eV.Mass range was from m/z 35 to 450.Identification of the essential oil components was accomplished based on comparison of retention times with those of authentic standards and by comparison of their mass spectral fragmentation patterns (Adams 2007).
Structural identification of a number of monoterpene synthases has indicated that they all have similar properties (like molecular mass, a divalent metal ion and neutral pH optimum requirements).Interestingly, a terpene synthase is able to form multiple products (Rajaonarivony et al., 1992;Bohlmann et al., 1998), the pinene synthases (from sage and grand fir) can catalyze the production of both α-and β-pinene (Bohlmann et al., 1997).
The biosynthesis of secondary metabolites, although controlled genetically, is strongly affected by the environmental influences of a particular growing region, and also by the agronomic conditions, harvesting time and the type of processing.In addition, for maximum oil production, long days and high light intensities are required during the maturation period (Thompson, 2003;Golparvar et al., 2015).
In the study of Abd-El-Baky and El-Baroty ( 2007) on the dragonhead they found that 44 combination of essential oils was obtained which consist 97.18 % of essential oil and 90 % of them was combined with oxygenated monoterpenes and consisted less than one percent of the weight of the plant which include compounds such as: gerannyl acetate, neryl acetate, geranial, geraniol, neral, nerol, linalool.(Kakasy et al., 2006).

CONCLUSION
In conclusion, the results obtained in our study indicated that the major components of oil of D. kotschyi were limonene, carvacrol, γ-terpinene and α -pinene.The major components of oil of D. moldavica were geranyl acetate, geraniol, neral and geranial.A comparison of our results with different reports ibndicates that differences in the volatile oil composition of the plants could be attributed to genetic (genus, species, and ecotype), chemotype, distinct environmental and climatic conditions, seasonal sampling periods, geographic origins, plant populations, vegetative plant phases, and extraction and quantification methods.

ACKNOWLEDGMENTS
This research project has been supported by Islamic Azad University, Isfahan (Khorasgan) branch, Isfahan, Iran.This support is highly appreciated.

Figure 2 :
Figure 2: The chromatograms found in essential oils of D. moldavica.

Table 1 :
Chemical compositions of essential oils of Dracocephalum moldavica L. and Dracocephalum kotschyi Boiss Terpene synthases have been cloned from different species and also the phylogenetic distances among them have been well documented.D. moldavica contains 0.06-0.92% essential oil, with the maximal level during flowering.Its lemon-like scented essential oil consists mainly of oxygenated acyclic monoterpenes, geraniol, geranyl acetate, geranial, neral and nerylacetate