Chemical characterization and radical scavenging activity of leaves of Juniperus foetidisima , J . excelsa and J . communis from Macedonian flora

Chemical characterization of three Juniperus species: J. foetidisima (JF), J. excelsa (JE) and J. communis (JC) from Macedonian flora enclosed determination of yield and essential oil composition of the oils obtained by hydro-distillation of dried leaves and determination of the content of total phenols and total flavonoids in dried plant material. GC/FID/MS analysis showed mainly monoterpene profile of the JC oil and combined monoterpene/sesquiterpene profile of JF and JE oils. Sesquiterpene cedrol was found as an important constituent of the JF and JE, thus the JF oil was characterized by three main components (a-pinene, limonene and cedrol, in amount up to 67.63%, 27.11% and 33.91%, respectively) and JE oil by four components (a-pinene, sabinene, cis-thujone and cedrol, in amount up to 33.83%, 29.49%, 26.20% and 24.44%, respectively). The JC oil was free of cedrol, but contained relatively large sesquiterpene fraction (sesquiterpene hydrocarbons and oxygen containing sesquiterpenes in amounts up to 28.64% and 13.57%, respectively). The JC oil was characterized by three monoterpene components (a-pinene, sabinene and terpinen-4-ol, presented up to 28.68%, 16.27% and 12.16%, respectively). The content of total phenols determined by Folin-Ciocalteu method ranged from 96.18-122.91 mg GAE/g dw (water extraction) while the content of total flavonoids ranged from 2.05-11.91 mg CE/g dw (ethanol extraction). Both water and ethanol extracts possessed radical scavenging activity against DPPH radical. Water extracts were more powerful with % of inhibition of DPPH ranging up to 64.52%, 67.40% and 78.23% for water extract (10 mg/ml) of JF, JE and JC, respectively. Obtained results showed correlation with the content of total phenols.


Introduction
Juniperus is one of the major genera of Cupressaceae family consisting of approximately 70 species variable in size and shape, from tall trees to columnar or low spreading shrubs.The plants are evergreen with leave-like or scalelike leaves.Many of Juniperus species are known and used as medicinal or commercially valuable plants.The common juniper, Juniperus communis L. (Cupressaceae), is an evergreen shrub or small coniferous tree, wide spread through the cool temperate Northern Hemisphere.Juniperus excelsa Bieb. is a large shrub or tree, spread mainly throughout the eastern Mediterranean starting from northeastern Greece and southern Bulgaria across Turkey to the Middle-East countries (Syria and Lebanon) and the Caucasus Mtn.It occurs in Iran, Pakistan and Oman as well (Khan et al., 2012).Juniperus foetidissima Willd. is a medium-size tree, spread mainly throughout the southeastern Europe and southwestern Asia, starting from southeastern Albania and northern Greece, across Turkey, Syria and Lebanon to the northern Iran and southwestern Turkmenistan.It often occurs together with J. excelsa Bieb., but it could be distinguished by its thicker shoots and green leaves (Marcysiak et al., 2007).
The above-ground parts, especially leaves and berries of Juniperus species are rich in essential oil that has characteristic aromatic flavour and bitter taste.Due to its diuretic and gastrointestinal properties, common juniper (J.communis) is used as medicinal plant for centuries.Besides berry essential oil, other juniper essential oil can be obtained from leaves, wood and seeds of the plant, usually by hydrodistillation (Orav et al., 2010a;Chatzopoulou and Katsiotis, 1993;Kumar et al., 2007).The oils are used in the pharmaceutical and cosmetic industries, for food and beverages, as well as for the production of perfumes.J. excelsa is a medicinal plant that has been used in folk medicine to treat dysmenorrhe, cough, bronchitis and colds, jaundice and tuberculosis and to induce menses and expel fetus (Emami et al., 2011).It is known as a remedy for diarrhea, abdominal spasm, asthma, fever, gonorrhea, headache and leucorrhoea (Khan et al., 2012).Among limited biological and pharmacological properties studied in vitro such as cytotoxic (Topcu et al., 2005) and antispasmodic activity (Atas et al., 2012), the most investigated were the antioxidant (Atas et al., 2012;Emami et al., 2007;Moein et al., 2010;Emami et al., 2011a) and antimicrobial activity (Ehsani et al., 2012;Atas et al., 2012;Asili et al., 2008;Unlu et al., 2008).However, there are few investigations regarding the biological activities of J. foetidissima also, with reports on antifungal (Balaban et al., 2007), antimicrobial (Asili et al., 2010), cytotoxic (Sadaeghi-Aliabdi et al., 2009), anti cholinesterase (Ozturk et al., 2010), fumigant (Tayoub et al., 2012), anti-inflamatory (Orhan et al., 2012;Lesjak et al., 2013) and antioxidant effects (Lesjak et al., 2013;Emami et al., 2007;Emami et al., 2011b).These activities are probably result to the complex chemical pattern of terpene and other components.
On the other hand, there is lack of information for the chemical composition and content of other secondary metabolites derived from the leaves of Juniperus species.Modnicki and Labedzka (2009) reported 2.40-3.43% of total phenol contents (TPC) estimated as pyrogallol in leaves of J. communis.Aditionally, Moein at al. (2014) found a relationship between antioxidant potentials and phenolic compounds in fruits of J. excelsa, pointing out that most active fraction posses the highest reducing power (IC 50 61.4µg/ml) and highest phenolic content (82.9 mg/g) (Moein et al., 2014).Further, the leave extracts of some other Juniperus species such as J. oxycedrus exhibited very high contents of polyphenols (133.08 mgGAE/g dw) and flavonoids (61.52 mg CE/g dw) (Chaouche et al., 2014).
Juniperus species occur in Macedonian flora as an important floristic elements.J. communis is widely spread shrub throughout the whole territory of Republic of Macedonia (RM) (Micevski, 1998).The berries of this plant are extensively utilized in production of blended teas and other herbal medicinal products, in food industry and as a spice in production of alcoholic beverages.For years, the juniper berries and the juniper essential oil are exported from RM.Additionally, the juniper leaves are used in folk medicine for various purposes.J. excelsa medicinal properties are also known by people of RM as pain reliever, for curing cold, asthma, edema or skin diseases.J. foetidissima grows in southern parts of the country, but could be found in the valleys of the River Crn Drim and the River Treska in western and Karadzica Mtn. in central RM (Micevski, 1998).Shoots decoction of the plant is known in folk medicine for curing coughs and common cold.Up to now the chemistry of the leaves of these three Juniperus species from Macedonian flora is scientifically unknown and only one report on chemical composition, antioxidant and anti-inflammatory effects of Macedonian J. foetidissima was published (Lesjak et al., 2013).Therefore the aim of the present study was chemical characterization of the Chemical characterization and radical scavenging activity of leaves of Juniperus foetidisima, J. excelsa and J. communis... leaves as well as assessment of radical scavenging activity of leaves extracts from J. communis, J. excesla and J. foetidissima from RM.

Plant material
Plant samples were collected in late autumn in 2010 and 2011.The terminal twigs of J. communis (JC) were collected from tree different localities in RM (Shara Mtn., Galicica Mtn. and Bistra Mtn.) (6 samples), terminal twigs with leaves of J. excelsa (JE) from two different localities (Velestovo and Dojran) (4 samples) and terminal plant twigs of J. foetidissima (JF) from four different localities in RM (Valandovo, Udovo, Veles and Velestovo) (8 samples).After air-drying in shadow the leaves were separated from stems, packed in paper bags and kept at dark and cool place until analysis.

Essential oil isolation
Essential oil isolation from plant leaves was made by steam distillation in special all-glass Clevenger type apparatus.For that purpose, 50 g of minced and dried leaves was distilled for 4 hours.After isolation, anhydrous sodium sulfate was added to remove residual water from the oil.Essential oil yield was expressed as %, calculated on dried weight (dw).

Extraction procedure
In order to determine the content of total flavonoids and total phenolic compounds, samples containing 1.0 g of powder dried plant material were processed.Two types of extracts were prepared, with 96% ethanol and water.The extraction procedure for sample preparation was performed with 10 ml of extractive solvent for 30 min in ultrasonic bath (50/60 Hz, 720 W).The extracts were filtered and the volume was made up to 10 ml.The obtained ethanol and water extracts were used for evaluation of radical scavenging activity as well.

GC/FID/MS analysis of essential oil
Essential oil samples were analyzed 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 mm) was used.Operating conditions were as follows: oven temperature at 60 °C (5 min), 1 °C/min to 80 °C (2 min) and 5 °C/min to 280 °C (5 min); helium as carrier gas at a flow rate of 1ml/min; injector temperature 260 °C and that of the FID 270 °C.1ml 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 ml/ml oil solution.
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 without any correction factors.

Determination of total phenolic content
The total phenolic content (TPC) of the leaves was determined with the Folin-Ciocalteu reagent according to a procedure described by Singleton et al. (1999) with slight modifications.To 1.0 ml of test sample (leaves extract), 0.5 ml (1:10 v/v diluted with distilled water) Folin-Ciocalteau reagent was added and stirred for 5 min at room temperature.After 5 min, 0.4 ml of 7.5% of sodium carbonate was added and made up to 10 ml with distilled water.These mixtures were incubated at room temperature in the dark for 2 hours.After incubation, absorbance of blue color was measured at 765 nm using a UV-Vis spectrophotometer (Agilent 8453 UV-Vis spectrophotometer, Agilent Technologies, USA).The total phenolic content was determined as mg of gallic acid equivalents per gram of dried weight of plant material (mg GAE/g dw) using an equation obtained from standard gallic acid calibration graph.

Determination of total flavonoid content
The total flavonoid content (TFC) was determined using the aluminum chloride assay described by Talari et al. (2012) with slight modification.To an aliquot of the test sample (1.0 ml of extract), 4.0 ml of distilled water and 0.3 ml of 5.0 % sodium nitrite were added and allowed to stand for 5 min.Later, 0.3 ml of 10.0 % aluminum chloride was added and the mixture was incubated for 6 min.2.0 ml of 1 M sodium hydroxide was added and the volume was made up to 10.0 ml with distilled water.After incubation of 15 min, the mixture turned to pink and the absorbance was measured at 510 nm using a UV-Vis spectrophotometer (Agilent 8453 UV-Vis spectrophotometer, Agilent Technologies, USA).The TFC was expressed in mg of catechin equivalents per gram of dried weight of plant material (mg CE/g dw) using an equation obtained from standard (+)-catechin calibration graph.

Free radical scavenging activity -DPPH assay
The scavenging activity of DPPH free radical of leaves extracts was done according to the method reported by Gyamfi et al. (1999) with minor modifications.200 ml of different concentrations of tested samples (100, 50, 20 and 10 mg/ml for ethanol extracts and 10, 5, 2 and 1 mg/ml for water extracts) were placed in a cuvette and 4 ml of 100 mm methanolic solution of DPPH was added.Mixtures were shaken vigorously for 1 min and left to stand 10 min in the dark at ambient temperature.The reduction of the DPPH free radical was measured by reading the absorbance at 517 nm.Methanol was used as control.
The percentage of inhibition was calculated from the absorbance of the control (A c ) and the sample (A s ) using the following equation:

Essential oil yield and chemical composition
The essential oil yield of Juniperus leaves was found in variable amounts in all investigated species, ranging from 0.50-1.38%, 0.16-3.30% and 0.75-0.90% dw for JF, JE and JC, respectively (Table 1).GC/FID/MS analysis showed presence of four main classes of components in all investigated samples of oils: monoterpene hydrocarbons (MH), oxygen-containing monoterpenes (OM), sesquiterpene hydrocarbons (SH) and oxygen-containing sesquiterpenes (OS).In some samples diterpenes (D) were also determined in very small amounts as well as some non-terpene components (NT) (Table 1).The monoterpene hydrocarbons were the most abundant fraction in all investigated oils ranging from 39.97% to 53.39%, 40.96% to 42.80% and 38.22% to 83.21%, in the essential oils of JC, JE and JF, respectively.The second dominated fraction was variable for different oils, thus for JC it was sesquiterpene hydrocarbons (12.27-28.64%),while for other two species it was oxygen containing sesquiterpenes, presenting a broad range from 10.10% to 39.71% and from 0.67% to 25.28%, for essential oil of JF and JE, respectively (Table 1).
The two main components in JC essential oil were α-pinene (21.27-28.68%)and sabinene (2.29-16.27%).Additionally, limonene was presented up to 6.95% and terpinene-4-ol up to12.16%.The monoterpene α-pinene was extremely high in some samples of JE and JF (up to 33.83% and up to 67.61%, for JE and JF, respectively).Additionally, the percentage of limonene was high, ranging up to 6.14% and 27.11% in samples of JE and JF, respective-ly.Some samples of JE contained high percentage of transsabinyl acetate (up to 10.38%).In the same time, these oils were the only one containing thujones (cis-thujone up to 26.20% and trans-thujone up to 12.86%).
Considering leaves essential oils of J. excelsa, many authors reported higher amounts of α-pinene and cedrol.In Chemical characterization and radical scavenging activity of leaves of Juniperus foetidisima, J. excelsa and J. communis...  (Adams, 2007); RIE -Retention index experimentally determined with reference to a homologous series of n-alkanes on HP-5ms column (AMDIS); (-) -not found, tr -traces < 0.02 some earlier papers Adams reported that the oil from wild growing J. excelsa from Greece contained cedrol (28.1%), α-pinene (22.5%) and limonene (22.7%) as predominant constituents (Adams, 1990).Also, Turkish authors found α-pinene (29.7%) and cedrol (25.3%) (Topcu et al., 2005) as dominant for this species.Moreover, Iranian researchers confirmed these two components as predominant in the leaf essential oil from wild growing J. excelsa from Iran (α-pinene 32.34% and cedrol 13.06%) (Emami et al., 2011a).Recently, Adams et al., revealed presence of mod-erate geographical variations in the volatile leaves oil of J. excelsa, comparing the samples from Greece, Bulgaria, Turkey and Cyprus (Adams et al., 2013) where cedrol was found to be the most abundant constituent of the oils, ranging from 11.3 to 35.8%.These data are in correlation with our findings for some samples of J. excelsa, but some other samples from Republic of Macedonia possess essential oil with deferent oil composition characterized with larger percentages of sabinene and thujone (cis + trans) (29.49% and 39.06%, respectively).Chemical characterization and radical scavenging activity of leaves of Juniperus foetidisima, J. excelsa and J. communis...

The contents of total phenols and total flavonoids and DPPH radical scavenging activity
The obtained values for total phenols content determined with the Folin-Ciocalteu reagent (TPC-FC) as well as total flavonoid content determined spectrophotometrycally using AlCl 3 as chelating agent (TFC-AlCl 3 ) are presented in Table 2.In all investigated samples of Juniperus leaves the content of TPC-FC was almost twice higher in water extracts (96.18-122.91 mg GAE/g dw) compared to ethanol extracts (47.72-64.42mg GAE/g dw).On the contrary, the contents of TFC-AlCl 3 were slightly higher in ethanol extracts (2.05-11.91mg CE/g dw) compared to water extracts (1.95-10.25 mg CE/g dw).Leaves extract of JF and JE, both water and ethanol contained much higher content of TFC-AlCl 3 than JC extracts.
Leaves extracts of investigated Juniperus species (JF, JE and JC), both water and ethanol extracts, showed radical scavenging capacity expressed as % of inhibition of the DPPH radical that had broad range depending on the extract concentration.In both cases more concentrated extracts have shown better results and water extracts have demonstrated stronger scavenging activity (Table 3).Thus, the highest radical scavenging activity against DPPH radi-  Tavares et al. (2009) evaluated the possibility of application of Juniperus leaves from species naturally occurring in Portugal (J.phoenicea subsp.phoenicea, J. turbinata, J. oxycedrus subsp.oxycedrus, J. oxycedrus subsp.badia and J. navicularis) against some diseases in which oxidative reactions play a crucial role.They found that all species exhibited minimum polyphenol and flavonoid contents in March/April and July and therefore a reduced antioxidant activity while the maximum concentrations of these compounds were detected in November/December, when they demonstrated higher antioxidant capacity (Taveras et al., 2009).
The antioxidant activity of leaves and fruits of 11 different conifer taxons growing wild in Iran were evaluated by Emami et al. (2007).Methanol extract of leaves and fruits were prepared and antioxidant activity of each extracts was measured using two different tests (the ferric thiocyanate method and thiobarbituric acid test, TBA).Results indicated that the methanol extracts of leaves, of male and female, and fruits of all these species possessed antioxidant activity when tested with both methods.The antioxidant capacity was then compared with those of a-tocopherol (a natural antioxidant) and butylated hydroxytoluene (BHT, a synthetic antioxidant).Methanol extract of Juniperus species, especially J. excelsa, J. excelsa ssp.polycarpos, J. oblonga and J. foetidisima, demonstrated antioxidant activity comparable to the BHT and even higher than a-tocopherol (Emami et al., 2007).

Conclusion
Chemical characterization of three Juniperus species, J. foetidisima (JF), J. excelsa (JE) and J. communis (JC) from Macedonian flora enclosed determination of yield and essential oil composition of the oils obtained by hydrodistillation of dried leaves and determination of the content of total phenols and total flavonoids in dried plant ma-terial.With GC/FID/MS analysis the essential oil profile of all investigated species was characterized dominantly with monoterpene hydrocarbon fraction.Sesquiterpene cedrol was found as an important constituent of the JF and JE oils, however the essential oil from JF was additionally characterized by two other main components (a-pinene and limonene), while the JE oil by three components (a-pinene, sabinene and cis-thujone).The essential oil of JC was free of cedrol, despite the fact that the fraction of sesquiterpene components of this oil was relatively high, but it consisted of many components presented in lower percentages and this oil was characterized mainly by three monoterpene components (a-pinene, sabinene and terpinen-4-ol).
The content of total phenols determined by Folin-Ciocalteu method ranged from 96.18-122.91 mg GAE/g dw (water extraction) while the content of total flavonoids ranged from 2.05-11.91mg CE/g dw (ethanol extraction).Both water and ethanol extracts possessed radical scavenging activity against DPPH radical but water extract were more powerful with % of inhibition of DPPH ranging up to 64.52%, 67.40% and 78.23% for water extract (10 mg/ ml) of JF, JE and JC, respectively.Obtained results showed correlation with the content of total phenols as the water extracts contained higher amounts of total phenols and exhibited better antioxidant activity.
The leaves of Juniperus species from Macedonian flora (J.foetidisima, J. excelsa and J. communis) can be taken in further consideration as a plant source for isolation of essential oil as well for extraction of phenolic compounds with promising antioxidant activity.Further research is needed to evaluate the chemical and biological potential of this raw plant material.

Table 3 .
Radical scavenging activity against DPPH radical expressed as % of inhibition for leaves extracts of Juniperus foetidissima (JF), Juniperus excelsa (JE) and Juniperus communis (JC)Sample% of inhibition of DPPH radical cal showed water extract of JF, JE and JC leaves in concentration 10 mg/ml, with % of inhibition of DPPH ranging up to 64.52%, 67.40% and 78.23%, respectively.