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Chemical compounds from Phoenician juniper berries (Juniperus phoenicea)

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Chemical compounds from Phoenician

juniper berries (Juniperus phoenicea)
a c a b a
Nizar Nasri , Nizar Tlili , Walid Elfalleh , Emna Cherif , Ali
b c a
Ferchichi , Abdelhamid Khaldi & Saida Triki
a
Laboratoire de Biochimie, Département de Biologie, Faculté des
Sciences de Tunis, Université Tunis El-Manar 2092, Tunisia
b
IRA, Laboratoire d’Arido-culture & cultures oasiennes, Médenine
BP. 4119, Tunisia
c
INRGREF, Unité de Recherche Gestion et Valorisation des
Ressources Forestières, BP: 10 Ariana 2080, Tunisia

Available online: 27 Jun 2011

To cite this article: Nizar Nasri, Nizar Tlili, Walid Elfalleh, Emna Cherif, Ali Ferchichi, Abdelhamid
Khaldi & Saida Triki (2011): Chemical compounds from Phoenician juniper berries (Juniperus
phoenicea), Natural Product Research, 25:18, 1733-1742

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 Natural Product Research
Vol. 25, No. 18, October 2011, 1733–1742

Chemical compounds from Phoenician juniper berries

(Juniperus phoenicea)
Nizar Nasriac*, Nizar Tlilia, Walid Elfallehb, Emna Cherifa, Ali Ferchichib,
Abdelhamid Khaldic and Saida Trikia
a
Laboratoire de Biochimie, De´partement de Biologie, Faculte´ des Sciences de Tunis,
Universite´ Tunis El-Manar 2092, Tunisia; bIRA, Laboratoire d’Arido-culture &
cultures oasiennes, Me´denine BP. 4119, Tunisia; cINRGREF, Unite´ de Recherche Gestion
et Valorisation des Ressources Forestie`res, BP: 10 Ariana 2080, Tunisia
Downloaded by [Dr Nizar Nasri] at 03:12 10 December 2011

(Received 5 February 2010; final version received 29 July 2010)

Natural chemical compounds are a widely researched topic worldwide

because of their potential activity against cerebrovascular diseases.
Chemicals from Juniperus phoenicea berries are reported in this study.
Lipids (11%) from seeds are mainly unsaturated (86%). Minerals are also
quantified like Na (63.8 mg per 100 g DW) or K (373.9 mg per 100 g DW).
Total reduced sugars are ca 192.6 mg g1 DW. Polyphenols and flavonoids
from berries are highly present with an average of 1764  174.3 mg gallic
acid per 100 g DW and 890  47.6 mg rutin per 100 g DW, respectively.
Mean free radical scavenging activities, determined by DPPH and ABTS,
are 1337  126.2 mM TEAC per 100 g DW and 1105.7  95.9 mM TEAC
per 100 g DW, respectively. All findings improve the possible presence of
biologically active fractions in phytocomplex that could be used as such
and/or extracted for the formulation of supplements and/or ingredients for
the pharmaceutical industry.
Keywords: Juniperus phoenicea L.; berries; fatty acids; minerals; antioxi-
dant capacities

1. Introduction
In recent years, due to a rapid increase in the world population, there has been a
progressive increase in the demand for vegetable sources for domestic purposes.
Plants containing natural compounds are of particular interest. The use of natural
components for reducing cardio, cerebrovascular diseases and cancer mortality has
gained considerable attention (Hertog, Feskens, & Kromhout, 1997).
Polyunsaturated fatty acids are minor lipid phytochemicals. They are structurally
distinguished by the presence of repeating methylene units. These units produce an
extremely Fexible chain rapidly reorienting through conformational states and
constitute an inFuential group of molecules that promote health (Vermerris &
Nicholson, 2006). In addition to the common fatty acids, some gymnosperms
(like Pinaceae or Cupressuceae) contain some unusual delta-5 oleEnic fatty acids

*Corresponding author. Email: nizar.nasri@fst.rnu.tn

ISSN 1478–6419 print/ISSN 1478–6427 online

ß 2011 Taylor & Francis
http://dx.doi.org/10.1080/14786419.2010.523827
http://www.tandfonline.com
 1734 N. Nasri et al.

(Takagi & Itabashi, 1982). These fatty acids could have powerful nutraceutical
usefulness. They may be of dietetic, physiological or biochemical relevance (Wolff &
Bayard, 1995).
Polyphenols and flavonoids are natural antioxidants. These antioxidants have
been reported to play an important role against several diseases (Tapiero, Tew,
Nguyen, & Mathe, 2002). The beneficial effects derived from phenolic compounds
have been attributed to their antioxidant activity by scavenging free radicals
(Heim, Tagliaferro, & Bobilya, 2002). The oldest medical application of phenolic
compounds is the use of phenol as an antiseptic (Vermerris & Nicholson, 2006).
Recently, it has been well documented that the presence of the aromatic ring results
in the effective absorbance of the UV radiation from the sun and thus prevents
sunburns or cancer (Bravo, 1998; Miller, Wheals, & Beresford, 2001). Adding to
medical applications, polyphenols, including the flavonoids and tannins, are also
recognised to be an integral part of the human and animal diet metabolites
Downloaded by [Dr Nizar Nasri] at 03:12 10 December 2011

(Kawamura et al., 2003).

Phoenician juniper (database for North African medicinal and aromatic plants:
Juniperus phoenicea L. Sp. Pl. 1040, 1753; http://www.uicnmed.org/nabp/database/
NA_Plants.htm) is found throughout the Mediterranean Region, from Morocco and
Portugal to Turkey and Egypt. J. phoenicea is also on Madeira, on Canary Islands
and on the mountains of western Saudi Arabia near the Red Sea (Adams, 2004). In
Tunisia (ca 10,000 ha), J. phoenicea occurs on hills in the north up to the arid
mountain regions in the south, where it is popularly known as ‘ar-ar’. It is an
evergreen plant usually growing as a shrub or a tree. As a tree, it can reach the height
of 10 m. J. phoenicea berries, across about 1 cm, ripening in the second year, are
composed of 6–8 scales, with 3–9 seeds (Vidakovic, 1991).
Tunisian people recommend J. phoenicea berries and/or leaves to calm the crises
of all types of cough. Berries are also used as a hypoglycaemic or as antiseptic and
diuretic (Bellakhdar, 1997).
To our knowledge, a comprehensive study dealing with the chemical compounds
(fatty acids, storage proteins, minerals, polyphenols, etc.) from J. phoenicea berries
has not yet been reported. The objective of this study is to describe the chemicals of
samples from J. phoenicea.

2. Results and discussion

2.1. Lipids and fatty acid contents of J. phoenicea seeds
Oil content of J. phoenicea seeds ranges from 7.0  0.4% (Kesra population, coded
KS) to 18.2  0.07 (Om Jedour population, coded OK) on a dry weight basis, with an
average of 11.8  5.8%. Seed moisture contents are about 11% on a dry weight basis
(Table 1).
The extracted oils are mainly composed of unsaturated fatty acids
(ca 86%). Linolenic (18 : 2D9/12) is the major fatty acid in all populations
(33.0  1.7%) followed by linoleic, 18 : 3D9/12/15 (28.3  1.9%) and oleic acid
18 : 1D9 (12.8  0.3; Table 2). Palmitic (16 : 0) and stearic (18 : 0) fatty acids are
present at relatively high amounts, averaging 7.8  0.6% and 4.4  0.2%,
respectively.
 Natural Product Research 1735

Table 1. Oil, protein and reduced sugar contents of Phoenician juniper berries.

Population

EK OK KS Means  SD

Moisture content (%) 13.4 9.6 10.7 11.2  1.9

Total lipids (%) 10.2  1.4 18.2  0.07 07.0  0.4 11.8  5.8
Total proteins
Kjeldahl assay (%) 6.0  0.9 9.4  0.33 9.67  0.4 8.4  2.0
Bradford assay 75.9  1.7 34.7  0.36 19.6  0.9 43.4  10.6 (4.3%)
(mg g1 DW)
Total reduced sugars 228.1  18.6 168.8  25.4 180.8  13.8 192.6  31.3
(mg g1 DW)

Notes: EK, El-Ala (Kairouan) population; OK, Om Jedour (Kasserine) population; KS,
Kesra (Siliana) population.
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Table 2. Tunisian J. phoenicia fatty acid compositiona (in percent of total fatty acids) from
seeds.

Populations

Fatty acids Retention time (min) EK OK KS Mean  SD (n ¼ 3)

14 : 0 2.23 0.5 0.4 0.5 0.5  0.1

16 : 0 3.16 7.8 7.2 8.3 7.8  0.6
16 : 1 cis 3.46 0.4 0.2 0.0 0.2  0.2
16 : 1 trans 3.56 0.7 0.2 0.5 0.5  0.3
17 : 0 3.79 0.2 0.1 0.0 0.1  0.1
18 : 0 4.18 4.2 4.6 4.4 4.4  0.2
18 : 1D9 4.39 12.5 12.8 13.2 12.8  0.3
18 : 2D9/12 4.80 31.4 34.8 32.7 33.0  1.7
18 : 3D9/12/15 5.46 29.4 26.1 29.5 28.3  1.9
20 : 0 5.78 0.1 0.2 0.1 0.2  0.0
20 : 1D11 6.34 0.3 0.8 0.0 0.3  0.4
20 : 2D5/11 6.65 0.2 0.0 0.1  0.1
20 : 2D11/14 7.08 1.8 1.8 1.0 1.5  0.4
20 : 3D5/11/14 7.41 3.1 3.8 3.0 3.3  0.4
20 : 3D11/14/17 8.24 0.3 0.3 0.0 0.2  0.2
20 : 4D5/11/14/17 8.65 6.4 6.1 6.3 6.3  0.2
24 : 0 14.07 1.4 0.8 0.7 1.0  0.4
 cis-D5 fatty acids 9.5 10.0 9.3 9.6  0.4
 saturated fatty acids 12.9 13.3 14.0 13.4  0.6

Notes: EK, El-Ala (Kairouan) population; OK, Om Jedour (Kasserine) population; KS,
Kesra (Siliana) population. aAll values are not significantly higher than the mean at p ¼ 0.05
and tr, traces.

Unusual D5-olefinic acids, (or D5-unsaturated polymethylene-interrupted fatty

acids) are also detected. Juniperonic (20 : 4D5/11/14/17) and sciadonic (20 : 3D5/11/
14) acids are present at 6.3  0.2% and 3.3  0.4%, respectively. Few quantities of
20 : 2D5/11 (50.1%) were also detected. The role of these D5-olefinic acids remains
 1736 N. Nasri et al.

unknown but it has been hypothesised that it might be related to a cold

acclimatisation or a result of genetic factors (Wolff & Bayard, 1995).
The various elongation products of oleic (C18 : 1), linoleic (C18 : 2) and linolenic
(C18 : 3) acids, namely gondoic acid 20 : 1D11 (0.3  0.4%), bishomo-linoleic acid
20 : 2D11/14 (1.5  0.4%), bishomo- -linolenic acid 20 : 3D11/14/17 (0.2  0.2%)
were also identified.
Contents of myristic (14 : 0), cis-palmitoleic (16 : 1 cis), trans-palmitoleic
(16 : 1trans), heptadecanoic (17 : 0), arachidic (20 : 0) and lignoceric (24 : 0) fatty
acids are, respectively, 0.5  0.1%, 0.2  0.2%, 0.5  0.3%, 0.1  0.1%, 0.2  0.0%
and 1.0  0.4%.
Juniperus phoenicea fatty acid profile gives considerable dietetic value to this oil.
Jones, Zhong, Enomoto, Schemmer, and Thurman (1998) reported that juniperonic
fatty acid can minimise hepatic reperfusion injury by reducing cell death in the
pericentral regions of the liver lobule by 75%. The same authors confirm that trypan
Downloaded by [Dr Nizar Nasri] at 03:12 10 December 2011

blue distribution time, an indicator of the hepatic microcirculation, was reduced by

approximately 25% with fish oil and over 50% by juniper berry oil diets, particularly
due to extensive incorporation of the fatty acid 20 : 3D5/11/14 into phospholipids in
liver tissue, especially phosphatidylinositol.

2.2. Protein and reduced sugar contents

Juniperus phoenicea seed protein contents (by Kjeldahl, 1883) ranged from
6.0  0.9% (El-Ala population, coded EK) to 9.6  0.4% (KS population) with an
average of 8.4  2.0% and between 75.9  1.7 mg g1 DW (EK population) to
19.6  0.9 mg g1 DW (KS population), averaging 43.4 mg g1 DW (4.3%) using
Bradford (1976) assay (Table 1).
To compare, we see that the seeds of J. phoenicea store a quantity of protein
comparable to Graminacea, such as rice (7.5–9%), wheat (7–18%) and corn
(7–12%). But it is still less than leguminous plants such as colza (15–30%), lentils
(23–32%) and lupain (30–50%; Dulau & Thebaudin, 1998) or Pinus pinea
(gymnosperm; Nasri & Triki, 2007).
The total reduced sugar contents ranged from 168.8  25.4 (OK population) to
228.1  8.6 (EK population), with an average of 192.6  31.3 as mg glucose per
1 g DW (Table 1).
Storage chemicals from seeds of non-conventional plants should be explored.
Proteins, for example, play a significant role in human health. In developing
countries, where the average protein intake is less than required, it is essential to find
new sources of edible protein and other nutrients to overcome the population
problem.

2.3. Mineral contents of J. phoenicea seeds

Quantified minerals in J. phoenicea berries are shown in Table 3. The mean average
of Na is 63.8 mg per 100 g DW; OK population has the highest value (68.1 mg per
100 g DW). Ca and Mg average 94.6 mg per 100 g DW and 65.2 mg per 100 g DW,
respectively. Cu, Zn, Fe and Mn minerals are present in few amounts
 Natural Product Research 1737

Table 3. Mineral contents (mg per 100 g DW) of Phoenician juniper berries.

Mineral contents

Populations Na K Ca Mg Cu Zn Fe Mn

EK 60.2 153.6 82.8 37.0 0.9 0.7 1.3 0.2

OK 68.1 502.6 97.2 47.9 0.7 0.8 2.2 0.4
KS 63.0 465.7 103.6 110.7 1.5 1.8 1.6 0.4
Mean 63.8 373.9 94.6 65.2 1.1 1.1 1.7 0.3

Notes: EK, El-Ala (Kairouan) population; OK, Om Jedour (Kasserine) population; KS,
Kesra (Siliana) population.

Table 4. Total polyphenol and flavonoid contents of juniper berry extracts.

Populations
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Total polyphenol and

flavonoid contents KS EK OK Mean  SD (n ¼ 3)

Total polyphenols as mg 1963.0  108.0 1638.0  52.0 1691.0  106.0 1764.0  174.3
gallic acid per 100 g DW
Total flavonoids as mg 919.0  7.0 835.0  25.0 916.0  73.3 890.0  47.6
rutin per 100 g DW

Notes: EK, El-Ala (Kairouan) population; OK, Om Jedour (Kasserine) population; KS,
Kesra (Siliana) population.

1.1 mg per 100 g DW, 1.1 mg per 100 g DW, 1.7 mg per 100 g DW and 0.3 mg per
100 g DW, respectively.
Juniperus phoenicea berries contained high levels of potassium (373.9 mg per
100 g DW) slightly more than those found in pomegranates (211 mg per 100 g DW;
Elfalleh et al., 2009); K contents are 153.6 mg per 100 g DW, 502.6 mg per 100 g DW
and 465.7 mg per 100 g DW for EK, OK and KS populations, respectively. Contents
of minerals obtained in the studied populations remain lesser than some commonly
edible fruits (Bowen & Watkins, 1997).

2.4. Total polyphenol and flavonoid contents of juniper berry extracts

Polyphenol and flavonoid contents are shown in Table 4. Total polyphenols ranged
from 1638.0  52.0 (EK population) to 1963.0  108.0 (KS population), as mg gallic
acid per 100 g dry weight (mg gallic acid per 100 g DW). Total Favonoids varied from
835.0  25.0 (EK population) to 919.0  7.0 (KS population), as mg rutin per 100 g
of dry weight basis (mg rutin per 100 g DW).

2.5. DPPH and ABTSþ antioxidant capacity and reducing power of

juniper berry extracts
The antioxidant capacity of juniper berry extracts was evaluated with the DPPH and
ABTS tests (Table 5). The free radical scavenging activity determined by DPPH
varied from 1193.4  128.2 (OK population) to 1430.6  108.2 (KS population) mM
 1738 N. Nasri et al.

Table 5. DPPH and ABTSþ antioxidant capacity and reducing power of juniper berry
extracts.

Populations
DPPH and ABTSþ
antioxidant capacity KS EK OK Mean  SD (n ¼ 3)

DPPH mM 1430.6  108.2 1387.0  157.7 1193.4  128.2 1337  126.2

TEAC per 100 g DW
ABTSþ mM 1184.5  127.4 1133.9  215.6 998.9  108.5 1105.7  95.9
TEAC per 100 g DW

Notes: EK, El-Ala (Kairouan) population; OK, Om Jedour (Kasserine) population; KS,
Kesra (Siliana) population.
Downloaded by [Dr Nizar Nasri] at 03:12 10 December 2011

trolox equivalent antioxidant capacity per 100 g dry weight (TEAC per 100 g DW)
with an average of 1337.0  126.2 mM TEAC per 100 g DW and the values
determined by ABTS ranged from 998.9  108.5 mM TEAC per 100 g DW (OK
population) to 1184.5  127.4 mM TEAC per 100 g DW (KS population), with an
average of 1105.7  95.9 mM TEAC per 100 g DW.
Because of the multiple reaction characteristics and mechanisms involved in the
estimation of the total antioxidants, no single method could accurately reFect all the
antioxidants in a mixed system due to the complex nature of phytochemicals
(Chu et al., 2000). In this experiment, the ABTS method was used to confirm the
results from the DPPH method assay since both methods are based on a similar
antioxidant mechanism and the extracts used in both tests were methanol-soluble.
All juniper extracts are comparable without significant differences. The richness of
juniper berries with phenolic compounds confers to this species a pharmaceutical
importance. Indeed, phenolic compounds exhibit a wide range of physiological
properties, such as anti-allergenic, anti-atherogenic, anti-inflammatory,
anti-microbial, antioxidant, anti-thrombotic, cardioprotective and vasodilator
effects (Benavente-Garcı́a & Castillo, 2008; Hayouni, Abdrabba, Bouix, & Hamdi,
2007). Moreover, flavonoids have been shown to act as scavengers of various
oxidising species, that is super oxide anion (O
2 ), hydroxyl radical or peroxy radicals
(Harborne & Williams, 2000). Samoylenko et al. (2008) reported that ethanol
extracts of J. phoenicia berries showed anti-malarial and anti-leishmanial activities.
The same authors were able to demonstrate that anti-bacterial activities of these
berry extracts are due to the presence of terpenoids, especially diterpenes totarol and
ferruginol. Despite the fact that it is not clear which components of the total
polyphenols or flavonoids compounds are responsible for the total observed
antioxidant capacity, the antioxidant capacity of juniper berry extracts is confirmed.
Total polyphenols and flavonoids from Phoenician juniper berries are highly
present with an average of 1764.0  174.3 mg gallic acid per 100 g DW and
890.0  47.6 mg rutin per 100 g DW, respectively. Free radical scavenging activity,
determined by DPPH and ABTS, are 1337.0  126.2 mM TEAC per 100 g DW and
1105.7  95.9 mM TEAC per 100 g DW, respectively. Nevertheless, because of the
use of these extracts as pharmaceutical resources, extracts should not be introduced
unless their safety has been suitably confirmed. Some examples illustrate the need to
explore adequately the composition of some functional foods: pomegranate fruits
 Natural Product Research 1739

were familiarly reported to have higher antioxidant activities (Li et al., 1996) and
great pharmaceutical usefulness. However, Sanchez-Lamar et al. (2008) reported
that some galenic preparations of pomegranate were toxic because of their alkaloid
contents. There were also some reports of immunological disturbance after
consumption of this species (Igea et al., 1992).

3. Experimental
3.1. Plant material
Juniperus phoenicea berries come from three natural populations of Tunisia: El-Ala
(Kairouan): latitude 35 370 N, longitude 09 330 E, altitude 467 m; Om Jedour
(Kasserine): latitude 35 350 N, longitude 08 560 E, altitude 819 m; Kesra (Siliana):
latitude 35 480 N, longitude 09 210 E, altitude 740 m. The plant was identified by
Dr A. Khaldi from I.N.R.G.R.E.F-Tunisia and voucher specimens (VS1-JP2010,
Downloaded by [Dr Nizar Nasri] at 03:12 10 December 2011

VS2-JP2010 and VS3-JP2010) were deposited at the Herbarium of I.N.R.G.R.E.F.

Also, samples from all analysed populations were deposited in the ‘Faculté des
Sciences de Tunis, Laboratoire de Biochimie’ (VS populations JP-EK2010,
JP-OK2010 and JP-KS2010).
Berries were collected from trees randomly sampled. For each tree, several cones
were selected for antioxidant properties and mineral analysis. Seeds selected for
storage chemical analysis (fatty acids and storage proteins) were separated manually
from berries.

3.2. Oil, protein, mineral and reduced sugar extractions from seeds
Seeds were grounded in a mortar and extracted with Soxhlet apparatus as described
previously in Nasri, Khaldi, Fady, and Triki (2005).
Fatty acid composition was determined by gas chromatography as fatty acid
methyl esters (FAMEs). FAMEs were prepared by vigorous shaking of a solution of
J. phoenicea oil sample in n-hexane (0.2 g in 3 mL) with 0.4 mL methanolic potassium
hydroxide solution (2 N). Chromatographic analysis was performed as described
previously in Nasri et al. (2005). Fatty acids were identified by comparing retention
times with those of standard compounds.
Protein content was determined by Kjeldahl (1883) and Bradford (1976) assays.
Using the Bradford method, soluble proteins were extracted referring to Nasri and
Triki (2007).
In order to establish mineral contents, plant material was totally dried at 70 C.
One gram (1 g) of dry powdered sample was placed in a porcelain capsule and kept in
a muffle furnace at 550 C. After cooling, the sample was added to 5 mL deionised
water and 1 mL of hydrochloric acid and subjected to boiling. The capsule content
was filtered. The filtrate was adjusted by deionised water to 100 mL. The mineral
constituents (Ca, Na, K, Mg, Cu, Zn, Fe and Mn) from J. phoenicea seeds were
analysed separately, using an atomic absorption photometer (Shimadzu A 6800,
Kyoto, Japan). All measurements were performed in triplicate. Each sample was
converted into mg per 100 g DW.
The procedure of reduced sugar analysis is as follows: 1 g of seeds of J. phoenicea
is added in a test tube to a picric acid solution (5 g of picric acid and 27.5 g of
 1740 N. Nasri et al.

anhydrous sodium carbonate are dissolved in 1 L; Dubois, Gilles, Hamilton, Rebers,

& Smith, 1956, modified). The solution is well mixed, and then the test tube is dipped
into the boiling water for 15 min, and the colour produced is compared with standard
colour solution using glucose solution (0–1 mg) prepared under the same condition
as cited below.

3.3. Total polyphenol contents, flavonoid contents and antioxidant capacities of

J. phoenicea berries
The most efficient solvents for polyphenols extractions were polar solvents (Hayouni
et al., 2007). These authors were able to check whether the water mixed with acetone
(alcohol) is the most efficient solvent extraction. Total polyphenol contents from
Juniperus berries were determined using the Folin-Ciocalteu method (Jayaprakasha,
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Singh, & Sakariah, 2001). Distilled water (7.9 mL) was added to 0.1 mL juniper berry
extract (1 g of filtered homogenised powder in 10 mL of methanol) and 0.5 mL
Folin-Ciocalteu reagent (1 : 1 with water). After 1 min, 1.5 mL of sodium carbonate
(1 M) was added, and the mixture was mixed and allowed to stand at room
temperature in the dark for 2 h. The absorbance was read at 765 nm, and the total
polyphenol concentration was calculated from a calibration curve, using gallic acid
as standard (50–500 mg L1). Results were expressed as mg gallic acid per 100 g of
dry weight basis (mg gallic acid per 100 g DW). All samples were analysed three times
and the results averaged.
The determination of flavonoids was performed according to the colorimetric
assay of Yong et al. (2008). Ethanol (3 : 7 in water) was added in 0.3 mL of each
extract. Then, 0.15 mL of NaNO2 (0.5 M) was added, followed by 0.15 mL of
AlCl3–6H2O (0.3 M). Test tubes were incubated at ambient temperature for 5 min,
and then 2 mL NaOH (1 M) was added in the mixture. The volume of reaction
mixture was made to 10 mL with distilled water. The mixture was vortexed and the
absorbance was measured at 506 nm. A calibration curve was prepared with rutin
(10–300 mg L1) and the results were expressed as mg rutin per 100 g of dry weight
basis (mg rutin per 100 g DW). All samples were analysed three times and the results
averaged.
Antioxidant capacities of juniper berries were determined by radical cation
(ABTSþ) and by DPPH. ABTSþ radical cation was produced by reacting 7 mM
ABTS solution with 2.45 mM potassium persulphate and allowing the mixture to
stand in the dark at room temperature before use. The ABTSþ solution was diluted
with ethanol to an absorbance of 0.70  0.02 at 734 nm. After addition of 25 mL of
sample or Trolox standard to 2 mL of diluted ABTSþ solution, absorbance at 734 nm
was measured at 5 min. Results were expressed as mM trolox equivalent antioxidant
capacity per 100 g dry weight (mM TEAC per 100 g DW; Re et al., 1999).
The capacity to scavenge DPPH free radicals was determined based on the
method described by Brandwilliams, Cuvelier, and Berset (1995). In 2 mL of
methanolic DPPH solution (100 mM), 25 mL of different juniper berry extracts was
added. After the reaction was allowed to take place in the dark for 30 min, and the
absorbance at 517 nm was recorded to determine the concentration of remaining
DPPH. Results were expressed as mM TEAC per 100 g DW.
 Natural Product Research 1741

3.4. Statistical analyses

All chemical compounds contents were carried out in duplicate or triplicate and
expressed as mean  SD. Statistical analyses were performed using STATISTICA
softwear (version 6.0). Differences were considered statistically significant at p50.05.

Acknowledgement
Authors thank Professor M. Zarrouk from ‘Borj-Cedria Science and Technology Park’
(Tunisia) for GC analysis.

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