Chemical composition of Chenopodium botrys L. (Chenopodiaceae) essential oil

Chemical composition of essential oil isolated from aerial parts of Chenopodium botrys L. (Chenopodiaceae) collected from five different locations in the Republic of Macedonia was analysed by GC/FID/MS. Seventy five compounds were identified representing 90.0291.24% of the oil. The analysis has shown that the oils were rich in sesquiterpenе components (83.18-87.54%) comprising elemol acetat (9.88%-21.98%), seline-11-en-4α-ol (9.81%-13.5%), selina-3,11-dien-6α-ol (6.42%-9.71%) and elemol (5.57%-9.49%) as major oxygen containing sesquiterpenes, followed by lower content of α-eudesmol acetat (3.24%-4.11%), α-chenopodiol (2.42%-5.43%), botrydiol (1.87-5.73%) and α-chenopodiol-6-acetat (1.9%-4.73%).


Introduction
Chenopodiaceae is a large family consisting of approximately 102 genera and 1400 species (Kokanova-Nedialkova et al., 2009). The typical genus Chenopodium comprises numerous species of perennial and annual plants known as goosefoots, which occur anywhere in the world. According to Fuentes-Bazan et al. (2012) the species of Chenopodium could grow as herbaceous plant or as shrubs and small trees and they are mainly non-aromatic but could be fetid. Only few species produce essential oil (glandular goosefoots) usually with characteristic chemical composition. Thus, for South America (Argentina) C. ambrosioides L., C. multifidum L., C. pumilio R. Br., and few other species were claimed as aromatic (Bonzani et al., 2003). In Europe, the most interesting is C. botrys L. (syn. Dyspha-nia botrys (L.) Mosyakin and Clemants, known as Jerusalem Oak Goosefoot also called Feathered Geranium, native to the Mediterranean region. As native or introduced plant it also grows in the territory of Asia, India, Himalayas, Northern Europe, Turkey, Cyprus, Africa, Australia and North and South America (Seidemann, 2005). Chenopodium species have been used in folk medicine worldwide for treatment of different ailments. It is well known that C. album improves the appetite and act as anthelmintic, laxative, diuretic and tonic. In South America C. ambrosioides is used against intestinal parasites from immemorial time. The plant is also known as carminative, diaphoretic and emmenagogue and as a remedy aginst cough, pulmonary obstruction and amenorrhea (Yadav et al., 2007). In Europe, C. botrys has been used for treatment of catarrh and humoral asthma and is known as a good substitute for C. ambrosioides (Yadav et al., 2007).
In the flora of the Republic of Macedonia (RM), 15 species of the genus Chenopodium occur including C. botrys, C. multifidum and C. ambrosioides as aromatic plants and 12 other species without glands (non-aromatic) such as C. bonus-henricus, C. hybridum, C. glaucum, C. murale, C. album etc. (Miceveski, 1995). Dried herbal parts of C. botrys are used from local people for preparing infusions or liquid extracts with diuretic, antispasmodic, carminative and antidiarrheal properties (Maksimovic et al., 2005). The herb of the plant has not been issue of chemical characterisation so far. Therefore, the aim of this study is determination of the chemical composition of the essential oil of wild samples of C. botrys collected from different localities of the Republic of Macedonia.

Plant material
The aerial flowering parts of C. botrys were collected in the period from July to September in 2013 at five localities in RM: Kozuf, Pretor, Strumica, Zletovo and Radovish. Botanical identification of the species was performed at the Department of Pharmaceutical Botany, Faculty of Pharmacy, University Ss. Cyril and Methodius, Skopje, RM. Voucher specimens (CB-K-1/13; CB-P-1/13, CB-S-1/13, CB-Z-1/13 and CB-R-1/13 for samples from Kozuf, Pretor, Strumica, Zletovo and Radovish, respectively) were deposited at the herbarium of the Faculty of Pharmacy, University Ss. Cyril and Methodius, Skopje, RM.
Collected plant material was air-dried and preserved in paper bags until analysis was performed, when it was minced and homogenised appropriately.

Isolation of the essential oil
The essential oil was isolated with steam distillation using all-glass Clevenger type apparatus according to the European pharmacopoeia method (Ph. Eur. 7; Method 2.8.12.). Briefly, 20 g of dry plant material was measured and transferred in a balloon of Clevenger distillation apparatus, in which 500 ml of distilled water and 0.5 ml of xylene were added and distillized for 2 hours. The obtained oil in xylene was treated with anhydrous sodium sulfate in order to be purified and dried.

GC/FID/MS analysis of essential oil
Essential oil samples were analysed on Agilent 7890А Gas Chromatography system equipped with FID detector and Agilent 5975C Mass Quadrupole detector as well as capillary flow technology which enables simultaneous analysis of the samples on both detectors. For that purpose, HP-5ms capillary column (30 m x 0.25 mm, film thickness 0.25 µm) was used. Operating conditions were as follows: the oven was heated to 60 °C with a gradual increasement in temperature of 3°C/min to 240°C, which was maintained 1 min and then at rate of 10 °C/min the temperature was rised to 280 °C and held 1 min; helium as carrier gas at a flow rate of 1ml/min; injector temperature 220 °C and that of the FID 270 °C. 1µl of each sample was injected at split ratio 1:1. The mass spectrometry conditions were: ionization voltage, 70 eV, ion source temperature 230 °C, transfer line temperature 280 °C and mass range from 50 -500 Da. The MS was operated in scan mode. For GC/FID/ MS analysis, the essential oil was dissolved in xylene to obtain 1 µl/ml oil solution.
Identification of the components present in essential oils was made by comparison of obtained mass spectra with those from Nist, Wiley and Adams mass spectra libraries, using AMDIS (Automated Mass Spectral Deconvolution and Identification System) as well as by literature and estimated Kovat′s (retention) indices that were determined using mixture of homologous series of normal alkanes from C 9 to C 25 in hexane, under the same above mentioned conditions.
The percentage ratio of essential oils components was computed by the normalisation method of the GC/FID peak areas without any correction factors.

Results and discussion
The results of the analysis of the chemical composition of essential oils isolated from samples of C. botrys collected from five different locations in RM are presented in Table 1. Using GC/FID/MS, 75 components were identified representing 90.02 to 91.24% of the total oil content.
Evaluation of the biological activity of the essential oil of C. botrys was most likely related to the high content of ОS with significant antimicrobial activity (Maksimovic et al., 2005;Mahboubi et al., 2011;Tzakou et al., 2007). These findings rationalize further investigations for determination of any antimicrobial activity of essential oil of C. botrys from RM. Positive results of this activity could be used in assessment of the antimicrobial potential of this raw material in order to use it in the pharmaceutical and cosmetic industry as well as in food production.

Conclusion
GC/FID/MS analysis of the essential oil composition of Chenopodium botrys collected at five different locations in the Republic of Macedonia showed sesquiterpene profile of the oil with major components belonging to the frac-tion of oxygen-containing sesquiterpenes. The most abundant components were elemol acetate, saline-11-en-4α-ol, selina-3, 11-diene-6α-ol and elemol, followed by lower content α-eudesmol acetate, α-henopodiol, botrydiol and α-chenopodiol-6-acetate.