Chemical composition of berry essential oils from Juniperus communis L . ( Cupressaceae ) growing wild in Republic of Macedonia and assessment of the chemical composition in accordance to European Pharmacopoeia

Common juniper, Juniperus communis L. (Cupressaceae) is widely spread throughout the territory of Republic of Macedonia (Micevski, 1998). The berries of this plant are extensively utilized in production of blended teas and other herbal medicinal products, in food industry, as a spice, in production of alcoholic beverages, etc. The cone-berries (Juniperi pseudofructus) are used traditionally to cure cystitis, digestive disorders, in therapy of chronicle arthritis and other indications. They contain essential oil with characteristic conifer-like aroma and bitterly taste. The pharmaceutical and the medicinal use of the juniper Chemical composition of berry essential oils from Juniperus communis L. (Cupressaceae) growing wild in Republic of Macedonia and assessment of the chemical composition in accordance to European Pharmacopoeia


Common
juniper, Juniperus communis L. (Cupressaceae) is widely spread throughout the territory of Republic of Macedonia (Micevski, 1998).The berries of this plant are extensively utilized in production of blended teas and other herbal medicinal products, in food industry, as a spice, in production of alcoholic beverages, etc.The cone-berries (Juniperi pseudofructus) are used traditionally to cure cystitis, digestive disorders, in therapy of chronicle arthritis and other indications.They contain essential oil with characteristic conifer-like aroma and bitterly taste.The pharmaceutical and the medicinal use of the juniper oil are of multi-purpose: diuretic, antiseptic, digestive, stomachic, antireuma thic, etc. (Yarnell, 2002).Antimicrobial (antibacterial and antifungal) activity of the oil was studied in vitro and many data on this issue point out on a strong antimicrobial activity that could be of interest in medicine and other fields (Filipowitz et al., 2003;Stassi et al., 1995;Emami et al., 2007;Pepeljnjak et al., 2005;Asili et al., 2008;Kumar et al., 2009;Miceli et al., 2009;Glisic et al., 2007;Öztürk et al., 2010;Consentino et al., 2003).Juniper oils are used in production of medicinal products with diuretic and antiseptic activity than in food industry, production of alcohol beverages, cosmetic and perfume production, etc.
The yield and the composition of the essential oils of the juniper berries depends on the geographical origin of Maced.pharm.bull., 57 (1, 2) 43 -51 (2011) Floresha Sela, Marija Karapandzova, Gjoshe Stefkov, Svetlana Kulevanova the plant, the maturity of the berries, the age of the plant, the meteorological condition (temperature, insolation, etc.) and other environmental factors (Orav, 2010;Chatzapoulou andKatsiotis 1993, 1995).Therefore the essential oil composition can vary significantly and that is the reason for very wide-ranging the values in European Pharmacopoeia monograph's requirements for characteristic constituents of the juniper oil (Ph.Eur. 7).
The juniper berries and the juniper essential oil are natural drugs that are exported from Republic of Macedonia, for years behind.Until now, there was no detailed study on the chemical composition of Macedonian juniper oil, which led us to the goal of the present research -determination of the chemical composition and possible chemical variability of the juniper essential oils from wild growing plants in the Republic of Macedonia and assessment of the essential oil chemical composition according to the European Pharmaco poeia.

Distillation of the essential oils
The essential oils were isolated by steam-distillation in a Clevenger-type apparatus using the method from European Pharmacopoeia (Ph.Eur. 7).The oils were dried over anhydrous sodium sulfate and stored in vials under refrigeration prior to analysis.
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.Identification of the components present in essential oils was made by comparing mass spectra of components in essential oils with those from Nist, Wiley and Adams mass spectra libraries, by AMDIS (Automated Mass Spectral Deconvolution and Identification System) and by comparing 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 normalization method of the GC/FID peak areas and average values were taken into further consideration (n=3).

Chemical composition of the essential oils
By the means of GC/FID/MS method, all 15 juniper oil samples were analyzed and 74 different components were indentified, representing 91.08 -99.83% of the entire oils (Table 1).Monoterpene components were presented in larger amounts in all samples of the oils than sesquiterpenes (Fig. 1.).Beside mono and sesquiterpenes, small amounts of few non-terpene components were identified, such as undecanone-2 and tricycle ne (Table 1).
Terpene hydrocarbons represented the most significant part of the oil rather than oxygen containing terpenes (Fig.
Evaluation of the chemical composition and the content of selected components in the investigated essential oils showed that twelve Macedonian samples comply with Ph.Eur. 7 requirements (α-pinene 20-50%; myrcene 1-35.5%,sabinene < 20%, limonene 2-12%; β-pinene 1-12%; trans (E) caryophyllene < 7%; terpinen-4-ol 0.5-10%; bornyl acetate < 2% and α-phellandrene < 1%).However, three oil samples obtained from the berries from Karaorman (JC6), Kicevo (1) (JC7) and Debar (JC9) contained insufficient amount of α-pinene (18.62%, 18.32% and 15.59%, respectively) nevertheless the amounts of other components were satisfactory.Eventually prepared mixture of equal parts of all fifteen investigated juniper oils, mix oil, should have average percentages of the components that comply with the Ph.Eur. 7 requirements for chemical composition quality (Table 2).This could be a solution for solving problems that appeared on chemical composition of the juniper essential oil from berries originated from different areas of collection.However, for complete assessment of the quality of the Macedonian juniper oils requires further analysis on several physical and chemical characteristics of the oils, set under the section tests as well as other features of the oil mentioned in the Juniper oil monograph.
Differences and the variability in the oil composition of juniper essential oils were reported for the oils originated from different regions in Europe and in America.The main constituents of the oils are pinenes, mostly α-pinene which could be present in a wide range, from 27% in the Greek samples (Chatzopoulou andKatsiotis, 1993), over 28.6-38.2% in Montenegro (Damjanovic et al., 2006),  Макед.фарм.билт., 57 (1, 2) 43 -51 ( 2011) and up to 46,63% in the Iranian samples (Rezvani, 2010).
Other important components such as sabinene, germacrene D, myrcene, β-pinene and limonene were found in higher amounts in juniper oil (Chatzopoulou and Katsiotis, 1993;Orav et al., 2010;Pepeljnjak et al., 2005).Terpine-4-ol is an important constituent of juniper oil, usually considered to be responsible for the diuretic effect of the oil.Its content in juniper oil can be variable and it was attained in small amount of 1.37% in Greek samples and 2.86% in samples from Iran (Chatzopoulou and Katsiotis, 1993;Rezvani, 2010).

Table 1 .
Chemical composition of the essential oils obtained by hydrodistillation from berries of Macedonian Juniperus communis a

Table 2 .
Variations in the main components composition of the essential oils obtained from berries of Macedonian Juniperus communis (+) -fulfilled monograph's requirements, (−) -not fulfilled monograph's requirements