CHEMICAL COMPOSITION OF ESSENTIAL OIL OF COMMON JUNIPER (JUNIPERUS COMMUNIS L.) BRANCHES FROM ESTONIA

For the treatment of the urinary system and kidney diseases, common juniper (Juniperus communis L.) fruits are widely used. This raw material is included in the European Pharmacopoeia (Ph.Eur.) and is one of the most popular kinds of official medicinal plant material with diuretic and uroantiseptic activity. However, the main biomass of bushes consists of green branches, which also contain a significant amount of essential oil that can be used in pharmaceutical practice. The branches become waste during bush cutting. So, it is advisable to investigate the chemical composition of essential oil isolated from common juniper branches from different regions of Estonia to prove the possibility of using this essential oil and branches in pharmaceutical practice. 
Aim. Therefore, the aim of the research was to determine the chemical composition of essential oil from common juniper (J. communis L.) branches from Estonia. 
Materials and Methods. The branches of juniper shrubs were collected in the summer months from 27 different habitats in Estonia. The essential oil was isolated from fresh juniper branches by the modified distillation method described in the European Pharmacopoeia monograph of Juniperi pseudo-fructus. GC/MS analysis was carried out using an Agilent 5975 Series MSDMSD, Agilent7890A GC (Agilent Technologies, Inc.) with two detectors (MSMS and FID) on a fused silica capillary column (30 m x 0.25 mm) with a bonded stationary phase: poly(5 %-diphenyl-95 %-dimethyl)siloxane (DB-5). The carrier gas was helium with a split ratio of 1:30, and the flow rate of 1.3 mL/min was applied. The temperature program was from 50°–240˚C at 2˚C/min and the injector temperature was 300˚C. The MS detector was operated in the EIEI mode of 70 eV and at a scan rate of 2 scans/s with a mass acquisition range of 20–400 u. 
Research results. The average amount of juniper essential oil in branches extracted during distillation using the Ph. Eur. method was 0.23±0.04 ml. 103 substances were identified in 27 different samples of juniper branches and quantified by the GC/MS method. The dominant components of Estonian common juniper essential oil are α-pinene (37.5-69.3 %), pinene, sabinene, β-myrcene and β-phellandrene. The juniper essential oils from Estonian raw materials were compared with Serbian, Iran, Portuguese, French and Greek ones. It was established that the common juniper growing in Estonia belongs to the α-pinene chemotype. 
Conclusions. Common juniper growing in Estonia belongs to the α-pinene chemotype. 103 substances were identified, and their assay was established in 27 different samples of juniper branches. The dominant components of Estonian common juniper essential oil are α-pinene (37.5-69.3 %), so it could be used as a source of a-pinene for the pharmaceutical industry. 
As the essential oils of common juniper branches didn’t meet all the requirements of the European Pharmacopoeia for juniper berries oil, so separate regulatory documentation must be developed for the essential oil from the branches


Introduction
Diseases of the urinary system and kidneys occupy a leading place among illnesses all over the world [1].For their treatment, common juniper (Juniperus communis L.) fruits are widely used.This raw material is included in the European Pharmacopoeia (Ph.Eur.) [2] and is one of the most popular kinds of official medicinal plant material with diuretic and uroantiseptic activity [3].However, the main biomass of bushes consists of green branches, which also contain a significant amount of essential oil that can be used in pharmaceutical practice.
among the different plant species [4,6].Its population is spread globally, being the only Juniperus species found in both hemispheres, with reports of this plant in Arctic regions of Asia and North America.In Europe, the largest population is found in some parts of the Alps, Scandinavia, Poland, northwest European lowlands, and Mediterranean mountain regions [4,7].The wide geographical distribution is the principal reason for the remarkable variation in the morphological characteristics and secondary metabolites' chemical composition [5].Therefore, it is advisable to conduct comparative studies of raw materials and products of their processing from different regions of the world.
The aim of the research was to determine the chemical composition of essential oil of common juniper (J.communis L.) branches from different natural growth sites in Estonia and to determine the chemotype of the local juniper.

Planning (methodology) of the research
In Fig. 1, a graphical representation of the research planning process is shown.

Materials and methods
The branches of juniper shrubs were collected in the summer months from 27 different possible habitats in Estonia.Most samples were collected in the south and the north of Estonia (Table 1).
The tops of branches (length 20 cm) without berries were cut off from the juniper shrub and were stored fresh in well-closed bags in a freezer at -18 °C.The voucher specimen is deposited in the herbarium of the Institute of Pharmacy, University of Tartu (1 Nooruse Str, Tartu, Estonia).Directly before the distillation of essential oil, the plant material was cut using scissors to fragments about 1 cm, containing needles and woody parts.The essential oils were distilled not later than four months after collecting the plant material.
Isolation of essential oil.The essential oil was isolated from fresh juniper branches by the modified distillation method described in the Ph.Eur.monograph of Juniperi galbulus [2,38] using 30 g of materials, a 500 mL round-bottomed flask, 300 ml water as the distillation liquid and 0.5 mL of xylene in the graduated tube was added to take up the essential oil.The distillation time was 1.5 h at a rate of 3 -4 ml/min.To improve consecutive chromatographic analyses, hexane was used instead of xylene.The percentage yields obtained were measured by v/w.Only one batch from each sample was analyzed, as it is stated by Ph.Eur.The oils were kept before analysis in well-closed containers at room temperature in the absence of light.

. Planning of the research
Step 1 • Review of literary primary sources about morphological and anatomical features, chemical composition and medical use of common juniper raw material Step 2 • Preparation of raw material samples (Estonia) Step 3 • Obtaining essential oil from the raw materials Step 4 • Сarrying out research in essential oils by GC-MS method Step 5 • Analysis of the obtained results 70 eV and at a scan rate of 2 scans/s with a mass acquisition range of 20-400 u.The method has also been used for detailed analysis of the composition of essential oils of several other plants [46][47][48][49][50][51].Determination of the quantitative content of essential oil components was settled in percentages by the method of internal normalization [2].
The GCGC analysis of α-pinene and sabinene in essential oils (samples 18-27) was performed using Agilent GCGC 7890a chromatograph with software Agilent Open Lab CDS Chem Station and with FID on two fused silica capillary columns with stationary phases DB-5 and HP-Innowax (both 30m × 0.25mm, Agilent).Carrier gas hydrogen with a split ratio 1:150 and a flow rate of 30 mL/min was applied.The temperature program was from 50-250 °C at 2.92 °C/min, and the injector temperature was 250 °C.The identification of the oil components was accomplished by comparing their retention indices (RIRI) on two columns with the RIRI values [53,54].Determination of the quantitative content of essential oil components was settled in percentages by the method of internal normalization [2].
Statistical Analysis.Statistical properties of random variables with n-dimensional normal distribution are given by their correlation matrices, which can be calculated from the original matrices.Statistical assessment of all data are reported as mean ± SEMSEM and were analyzed using STATISTICA 6 software [2].

1. Yield and content of essential oil
The essential oil was isolated from fresh juniper branches in amounts of 0.03-0.56% by the modified distillation method described in the monograph Juniper galbulus Ph.Eur. 10 [2, 54] (Table 2).103 substances were identified, and their assay was established in the 17 samples by the GC/MS method.The contents of the components of the essential oil of juniper branches from the concentration of 0.1 % and more are given in Table 2.The data of the contents of all 103 components can be requested from the corresponding author if interested.Additionally, the content of α-pinene and sabinene in samples 18-27 is shown in Table 3.
The dominant substances in the juniper branches were α-pinene, sabinene, β-myrcene and β-phellandrene.The main component of the essential oil was α-pinene 37.5-69.3%, the amount of which is higher compared to Ph. Eur.requirements.Consequently, it is possible to state that the common juniper growing in Estonia belongs to the α-pinene chemotype.
After processing the data, it became clear that not a single sample of the essential oil from branches met all Ph.Eur.requirements.The majority of samples (67 % or 11 essential oils out of 17) contain α-pinene in a concentration greater than 50 %.The amounts of sabinene, α-pinene, β-myrcene and β-caryophyllene in the essential oils were within limits required by Ph.Eur.The content of α-phellandrene is elevated, and no essential oil with an α-phellandrene content of less than 1 % has been analyzed.Samples 13 and 14 contain more bornyl acetate than specified.Also, the content of terpinene-4-ol in most samples (67 % or 11 essential oils out of 17) was a bit lower than needed by the pharmacopoeia.The peaks of limonene and β-phellandrene were not separated, and these substances were determined together.The total concentration of β-phellandrene and limonene was relatively high (8.8-17.4%).In this way, the essential oil obtained from juniper branches does not meet the requirements of Ph.Eur.monography "Juniper aetheroleum" distilled from the juniper fruits, so separate regu-latory documentation must be developed for the essential oil from the branches.

Conclusions
Common juniper growing in Estonia belongs to the α-pinene chemotype.The average amount of juniper essential oil in branches extracted during distillation using the Ph.Eur.method is 0.23±0.04ml.103 substances were identified, and their assay was established.The dominant components of Estonian common juniper essential oil are α-pinene (37.5-69.3%), pinene, sabinene, β-myrcene and β-phellandrene.
No sample of the essential oil of common juniper branches met all the requirements of the European Pharmacopoeia for juniper berries oil, so separate regulatory documentation must be developed for the essential oil from the branches.
The prospects for further research.The obtained results could be used for the development of an analytical method for quality control of the essential oil from juniper branches.Further phytochemical and pharmacological studies of the juniper branches and their essential oil will show the prospect of creating new pharmaceutics.

Conflicts of interest
The authors declare that they have no conflict of interest in relation to this research, whether financial, personal, authorship or otherwise, that could affect the research and its results presented in this article.

Funding
The research was funded by the Ministry of Health Care of Ukraine at the expense of the State Budget in framework # 2301020 "Scientific and scientifictechnical activity in the field of health protection" on the topic "Modern approaches to the creation of new medicines for a correction of metabolic syndrome".

Fig. 1
Fig. 1.Planning of the research

Table 1
The studied juniper samples from different habitats in Estonia

Table 2
The yield of essential oil and dominant substances of the juniper essential oils from Estonia*