1 Comparative Antioxidant Properties of Juniperus communis L. and Juniperus virginiana L. Extracts Obtained by Refluxing and Sonication Methods… [610257]

1 Comparative Antioxidant Properties of Juniperus communis L. and Juniperus
virginiana L. Extracts Obtained by Refluxing and Sonication Methods

Carmen Manuela PLEȘA *, Aurel ARDELEAN , Mihai -Cosmin PASCARIU, Alfa Xenia
LUPEA
“Vasile Goldiș” Western University of Arad, Faculty of Pharmacy , 86 Liviu Rebreanu, RO-
310414, Arad, Romania , tel./fax: +[anonimizat], +[anonimizat]
* Corresponding author: [anonimizat]

The aim of this study was to evaluate the antioxidant activity of ethyl acetate and
tetrahydrofuran extracts of Juniperus berries, branches and needles , by using the 2,2-
diphenyl -1-picrylhydrazyl (DPPH) assay . Two Juniperus species , Juniperus co mmunis and
Juniperus virginiana , from different regions were used . The extracts obtain ed by sonication
and refluxing ex traction were studied by gas chromatography – mass spectrometry , which
revealed that the Romanian juniper extracts are largely comprised of monoterpene
hydrocarbons , such as α-pinene, β-pinene, β -phellandrene, sabinene , β-cadinene, τ -cadinene,
β-caryophyllene, β -cubebene, β -elemene and germacrene D . The antioxidant activity was
highe st in ethyl acetate Juniperus communis needles extract from Lipova (Romania ) and in
ethyl acetate Juniperus virginiana branches extract from Macea Botanical Garden
(Romania ). The m ean rate s (vm) of DPPH consumption were higher for the tetrahydrofuran
Juniperus communis branches extract from Lipova and ethyl acetate Juniperus virginiana
branches extract than in tetrahydrofuran Juniperus communis berries extract from Lipova .

Keywords : Juniperus communis, Juniperus virginiana, sonication extraction, refluxing
extraction, antioxidant activity

2
It is commonly accepted that free radicals play an important role in the pathogenesis of many
disease s. Antioxidants, on the other hand, can cont rol the degradation of biomolecules caused
by the free radicals. Thus, considerable efforts have been made towards locating naturally
occurring antioxidants for use in food or medicines , in order to replac e the synthetic
antioxidants [1]. The side effects of artificial antioxidants are a strong reason for consider ing
their replacement with natural equivalents . Essential oils are one source of natural
antioxidants , with a great potential for application in pharmaceutical products [2]. Juniperus
species , for example, contain such essential oils and are used for treatment of hyperglycemia,
tuberculosis, bronchitis, pneumonia, ulcers, intestinal worms and other [2].
For centuries, juniper berries have been used in folk medicine for the treatment of opportunisti c
infections, as a spice for meat, and as flavor in the preparation of gin and raki [3]. Juniperus
communis oil is of interest to perfumery, cosmetic and pharmaceutical industries because of its
aromatic and diuretic (based on its terpinen -4-ol content ) properties [4 ,5].
Juniperus L. (Cupressaceae family ) is a genus of evergreen shrubs or trees and the second
most diverse conifer , with 67 species in the world [6]. Juniperus communis L. (section
Juniperus ) grows in scrubs, pastures and cliffs, from sea leve l to high mountain regions,
throughout Europe, Asia and North America [7,8] . Juniperus communis is an evergreen
dioecious shrub or tree, with fleshy female cones , in which the cone scales are fused
resembling “berries” of dark blue – black color [8,9].
The m ain Juniperus compounds of this study are β-phellandrene, α-pinene, sabinene and
germacrene D . The hydrodistillation method was previously applied to needles [4, 8-10] and
berries [ 5-7,9,11, 12] of Juniperus communis ; supercritical CO 2 extraction was also us ed for
berries [13]. A number of studies have shown that monoterpenes, contained in juniper essential
oil, enhance , through their antioxidant activity, the oxidative stress resistance of living organisms.

3 Their antiradical activity affects the levels of th e most important enzymes responsible for the
neutralization of ROS: SOD, CATs, peroxidases, and glutathione transferase [3].
The purpose of this work was to evaluate the antioxidant activity of different extracts of
Juniperus communis and Juniperus virgini ana.

Experimental part
Materials and methods
The plant material was collected from wild growing Juniperus communis shrubby trees from
Lipova (Arad County , Romania ) and from Albac (Alba County , Romania ). Juniperus
virginiana was collected from Macea Bota nical Garden (Arad County , Romania ). Three kind
of sample s were selected , i.e. black mature berries , needles and branch es, which were all
dried at room temperature.
Ethyl acetate and tetrahydrofuran, both from Chimopar Bucharest, were used as solvents for
refluxing extractions . The former was also used as solvent for sonication extractions . All
solvents used were of puriss or p.a. grade.
All filtered extracts were dried on anhydrous sodium sulfate (Fluka Chemie AG).

Extraction methods
Refluxing extraction
The plant material was dried and stored at room temperature. Dried berries, needles, and
branches (2 g in each case) were grounded, treated with 15 mL solvent ( tetrahydrofuran or
ethyl acetate) and refluxed for 30 min. After cooling, the extract was filter ed, dried over
anhydrous Na 2SO 4 and stored at -4 °C in glass containers, until the gas chromatography –
mass spectrometry (GC -MS) analysis was performed .

4 Sonication extraction
In order to obtain the volatile compounds from the three anatomical parts (berri es, needles,
and branches) of the two Juniperus species, 1 g of dried and grounded plant material was
placed in a vial with 6 mL solvent (ethyl acetate). The vial was covered and then placed in
the sonication water bath (HK2200, 100 W, 50 kHz) for 10 minut es [14]. After sonication,
the extract was filtered, dried over anhydrous Na 2SO 4 and stored at -4 °C until the GC-MS
analysis was performed .

GC-MS analysis
The extracts obtained by refluxing and sonication were analyzed by GC-MS in order to
identify the ma in components. A Hewlett Packard HP 6890 Series gas chromatograph
coupled with a Hewlett Packard 5973 mass selective detector (GC -MS) system was used. A
HP-5 MS capillary column was used for the GC system. The temperature program was set
from 50 to 250 °C, with a rate of 6 °C/min , using helium as carrier gas. The relative
percentage concentration s of the volatile compounds f or the two species of juniper were
computed from the GC peak areas. The identification of the main compounds was performed
by matching the experimental mass spectra with those from the NIST/EPA/NIH Mass
Spectral Library 2.0.

2,2-Diphenyl -1-picrylhydrazyl radical -scavenging
A DPPH solution of approximately 1 mM was prepared in ethanol (96 %) and diluted to
obtain a standard curve for DPPH. The absorbance was recorded at 517 nm (Lambda 25 UV-
VIS Spectrophotometer) using ethanol as blank. The mean rate ( vm) of DPPH consumption
was calculated for all extracts as the ratio between the decrease in concentration ( Δc, μM) and
the time interv al (Δt, s), according to equation 1 [15].

5
tcvm (1)
The antioxidant activity was calculated according to equation 2.

100 %
015 0
 
tt t
AA AA
(2)
where A(t=0) is the absorbance for the positive control (DPPH solution, without the sample)
and A(t=15) is the absorbance after 15 minutes.

Results and d iscussion s

Composition of Juniperus extracts
The amount of the extracted compounds is expressed as a percentage of the obtained peak
area, compared with the total area of all peaks (t able 1). Of the large number of compounds
found in all extracts (over 100) , only the most important (26 compounds ) were discussed .

Table 1
The main components obtained by refluxing and sonication extraction from Juniperus
communis and Juniperus virginiana needles, b erries and branches (% of total area)

The GC -MS analyses showed that α-pinene is present in all extracts. α-Pinene is present in
highe r amount ( 26.70%) in J. communis branches from Lipova ethyl acetate extract obtained
by sonication , by compar ison with the supercritical CO 2 extraction (α -pinene 7 .11 %) [13 ].
Also, germacrene D is present in a higher amount (14.42 %) in J. communis branches from
Lipova ethyl acetate extract prepared by sonication , along with sabinene (10 .22%) from the
Juniperus virginiana needles extract in tetrahydrofuran from Macea Botanical Garden , when
compar ed with commercial essential oil from Bulgaria (sabinene 5.8%) [3].

6 To c onclud e, the extracts are mainly comprised of monoterpene s (limonen e, τ-terpinen e,
terpinolen e, cis -β-terpineol, β-phellandren e, α-pinen e, β-pinen e) and sesquiterpene s (β-
cadinen e, τ-cadinen e, β-caryophyllene, germacren e D, α-humulen e).

Antioxidant activity
The DPPH assay test was used in this study in order to determine the ability of juniper extract
to act as hydrogen atom donors. All nine extracts reduce the DPPH radical from a deep violet
color to colorless when neutralized , as can be seen in f ig. 1. By monitoring the reaction at 517
nm, a decrease in absorbance for extracts E 1, E4, E5 and E 7 in the f irst three minutes can be
observed (see t able 2) . For bra nches, this decrease was slower than for needles and berries.

Table 2
The abbreviation for antioxidant activity extracts

Fig. 1. Reduction of DPPH by juniper extracts

The antioxidant activity was calculated by using the absorbance values for the positive
control and for the sample after 15 minutes (equation 2, table 3). The obtained a ntioxidant
activity value s were between 33.31% (E 5) and 89.98% (E 3). Juniperus communis needles
extract in et hyl ace tate from Lipova had the highest antioxidant activity ( fig. 2); also, for
Juniperus communis branches extract in ethyl acetate from the Macea Botanical Garden , the
value was very close (89.69%, fig. 3).

Fig. 2. Antioxidant activity of Juniperus communis needles extract in ethyl acetate from
Lipova

7 Fig. 3. Antioxidant activity of Juniperus communis branches extract in ethyl acetate from
Macea Botanical Garden

Juniperus communis branches extract in tetrahydrofuran from Lipova had the highest mean
rate ( vm), calcul ated with e quation 1, (0.0212 µM/s) , followed by the Juniperus communis
branches extract in et hyl acetate from Macea Botanical Garden (0.0122 µM/s) . Juniperus
virginiana berries extract in et hyl acetate from Macea Botanical Garden had the lowest
values (0.026 µM/s) , see table 3 .

Table 3
Antioxidant activity and mean rate for juniper extracts

Conclusion s
Nine juniper extracts , obtained using ethyl acetate and tetrahydrofuran as solvents , were used
for antioxidant activity determinations . Sonication e xtracts sh owed the higher contents of α –
pinene and limonene (5.05 %, compara ble with the essential oil from literature [5]) in
Juniperus communis branches extract in et hyl acetate from Albac (Romania ). By refluxing
extraction , the sabinene from Juniperus communis needles extract in et hyl acetate from
Lipova (Romania ) showed the highest percent. The highest antioxidant activity was attributed
to the Juniperus communis needles extract in et hyl acetate from Lipova , which was follow by
the Juniperus communis branches extr act in et hyl acetate from Macea Botanical Garden. The
mean rate of Juniperus communis branches extract in tetrahydrofuran from Lipova and
Juniperus virginiana branches extract in et hyl acetate from Macea Botanical Garden was
higher than in the Juniperus vi rginiana berries extract in et hyl acetate from Macea Botanical
Garden .

8
Acknowledgements
This work was partially supported by the strategic grant POSDRU 6/1.5/S/13 (2008)
from the Ministry of Labor, Family and Social Protection, Romania, co -financed by the
European Social Fund – Investing in People . “Vasile Goldiș” Western University of Arad is
gratefully acknowledged for providing the Juniperus virginiana samples .

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-2 0 2 4 6 8 10 12 14 160,00,20,40,60,81,0Abs, 517nm
Time (min) E1
E2
E3
E4
E5
E6
E7
E8
E9

Fig. 1. Reduction of DPPH by juniper extracts

10
0,0 2 4 6 8 10 12 14 16 18 20,00,0890,150,200,250,300,350,400,450,500,550,600,650,700,750,800,850,901
minA
Fig. 2. Antioxidant activity of Juniperus communis needles extract in ethyl acetate from
Lipova

0,0 2 4 6 8 10 12 14 16 18 20,00,0640,100,150,200,250,300,350,400,450,500,550,600,625
minA

Fig. 3. Antioxidant activity of Juniperus communis branches extract in ethyl acetate from
Macea Botanical Garden

11
Table 1
The main components obtained by refluxing and sonication extraction from Juniperus
communis and Juniperus virginiana needles, berries and branches (% of tota l area)

No. Compounds % of total area
Refluxing extraction Sonication
E1
[16] E3
[16] E4 E5 E6 E7
[16] E8 E9 E2
1. α-Pinene 19.73 13.81 9.36 0.66 2.57 14.36 1.51 1.74 26.70
2. β-Pinene 10.10 0.47 0.85 0.28 4.27 0.85 0.33 2.59 2.47
3. Bisabolene epoxide 0.16 0.10 0.13 0.03 0.11 0.19 – – –
4. 3-Carene 0.61 – 0.15 – 0.05 – 0.08 – –
5. β-Cadinene 0.65 0.47 – 0.51 – 0.37 0.43 0.11 2.72
6. τ-Cadinene 0.20 0.18 – 0.32 – 0.16 0.26 0.12 1.13
7. τ-Cadinol 0.08 – – – – – – 2.50
8. Camfene 0.17 0.08 0.08 – – – 0.03 0.03 –
9. Cariophylene 1.83 0.44 0.10 1.23 1.92 3.10 1.18 1.20 1.88
10. Carveol 0.06 0.01 0.36 0.09 0.04 0.09 – 0.03 0.03
11. Cedrene – 0.12 – 0.02 – – 0.03 – 0.14
12. Copaene 0.55 0.04 – – – – – – 0.11
13. β-Cubebene – 2.14 0.42 – 1.75 – 0.07 1.12 –
14. β-Elemene 2.04 0.88 – 0.13 0.15 0.10 0.22 0.18 3.85
15. τ-Elemene 4.43 1.89 0.20 0.14 0.25 – 0.20 0.09 4.25
16. β-Phellandrene 10.51 8.12 0.26 7.98 46.64 10.51 0.11 26.95 4.71
17. Germacrene D 3.42 3.49 – 3.05 6.07 2.09 2.76 3.26 14.42
18. α-Humulene 1.59 0.27 0.03 0.13 0.18 0.35 0.12 0.10 1.49
19. Limonene 4.75 2.08 1.37 0.54 1.21 0.66 0.60 0.83 5.05
20. Linalool 0.07 – – 0.06 0.06 0.73 0.06 0.03 –
21. Ocimene – 0.02 – 0.06 0.09 0.33 – – 0.72
22. Sabinene – 12,07 7,70 – – – 10,22 – 0.46
23. Spatulenol 0.56 – 0.07 0.09 – – – – 0.48
24. cis-β-Terpineol 0.76 0.05 0.69 0.17 0.42 0.24 0.45 0.62 0.11
25. τ-Terpinene 0.28 – 0.23 0.42 0.09 – 0.19 1.02 0.36
26. Terpinolene 0.76 0.32 0.39 0.24 0.45 0.25 0.13 0.32 0.33

12
Table 2
The abbreviation for antioxidant activity extracts

Abbreviation Extracts for DPPH assay
E1 Juniperus communis berries extract in ethyl acetate from Albac
E2 Juniperus communis branches extract in ethyl acetate from Albac
E3 Juniperus communis needles extr act in ethyl acetate from Lipova
E4 Juniperus communis branches extract in tetrahydrofuran from Lipova
E5 Juniperus virginiana needles extract in ethyl acetate from Macea Botanical
Garden
E6 Juniperus virginiana berries extract in ethyl acetate from Macea Botanical
Garden
E7 Juniperus virginiana branches extract in ethyl acetate from Macea Botanical
Garden
E8 Juniperus virginiana needles extract in tetrahydrofuran from Macea
Botanical Garden
E9 Juniperus virginiana berries extract in tetrahydr ofuran from Macea Botanical
Garden

Table 3
Antioxidant activity and mean rate for juniper extracts
Extracts A%
(DPPH) Mean rate, vm
(µM/s)
E1 85.82 0.0050
E2 77.54 0.0043
E3 89.98 0.0065
E4 74.01 0.0212
E5 33.31 0.0032
E6 38.93 0.0026
E7 89.69 0.0122
E8 60.28 0.0049
E9 78.35 0.0034